Activatable protein containing protease-cleavable sequence and medical use thereof

Abstract

Provided are an activatable protein and a medical use thereof. Specifically, the present invention relates to an activatable protein containing a protease-cleavable sequence, a pharmaceutical composition thereof, a nucleic acid, a preparation method therefor, and a medical use thereof.

Description

Activated proteins containing protease-cleavable sequences and their pharmaceutical uses

[0001] This application claims priority to Chinese patent application CN 202510371185.5 filed on March 27, 2025; Chinese patent application CN202510801559.2 filed on June 16, 2025; Chinese patent application CN 202510852915.3 filed on June 24, 2025; and Chinese patent application CN 202610274759.1 filed on March 6, 2026. Technical Field

[0002] This disclosure pertains to the field of biotechnology, and more specifically, it relates to protease-cleavable sequences, activatable proteins containing protease-cleavable sequences, and their pharmaceutical uses. Background Technology

[0003] The statements herein are provided only as background information in connection with this disclosure and do not necessarily constitute prior art.

[0004] T-cell engagers (TCEs) act as linkers between T cells and tumor cells. By simultaneously binding to tumor-associated antigens (TAAs) and CD3 on T cells, they activate T cells, thereby achieving a killing effect on tumor cells. TCEs require low TAA expression levels; even low levels of TAA expression in normal tissues can lead to non-specific activation of T cells outside the tumor, causing toxicity such as cytokine storms (CRS). These toxicities limit the dosage increase of TCEs in the treatment of solid tumors, ultimately limiting their efficacy. TCE-activated antibody technology is an effective measure to improve TCE treatment of solid tumors (Janux presentation 20240126 & 20241202; Vir presentation 20250108). Protease-cleavable sequences are an important component of TCE-activated antibodies, and their activation efficiency is a crucial factor affecting the activity of TCE-activated antibodies (WO2022035866A1, US20190284283A1, US20200385469A1).

[0005] To ensure efficient activation of TCE-activated antibodies in various tumors, multiple types of protease substrates are combined to form protease-cleavable sequences, such as metalloproteinases (MMPs) and serine proteases (matriptase / uPA), which are widely upregulated in tumors. However, current protease-cleavable sequences still have shortcomings: they either have a bias towards MMP activation and thus limited activation efficiency by serine proteases; or they are non-selectively activated by multiple members of the MMP family, increasing the risk of peripheral non-specific activation. Summary of the Invention

[0006] This disclosure provides a protease-cleavable sequence, a polypeptide containing a protease-cleavable sequence, an activatable protein containing a protease-cleavable sequence, a conjugate, a nucleic acid encoded therefrom, a vector, a host cell, a pharmaceutical composition, a method for preparation thereof, and methods for treating, preventing or improving diseases or symptoms and related pharmaceutical uses thereof.

[0007] <Polypeptide (e.g., isolated polypeptide)>

[0008] In one aspect, this disclosure provides a polypeptide (e.g., an isolated polypeptide) comprising a protease-cleavable sequence comprising the amino acid sequence of SEQ ID NO: 9 (ASGRSTNA) or SEQ ID NO: 10 (LSGRSTNA).

[0009] In some embodiments, the polypeptide as described above (e.g., an isolated polypeptide) further comprises the amino acid sequence of SEQ ID NO: 11 (PLGLAG) of the protease-cleavable sequence.

[0010] In some embodiments, the polypeptide (e.g., an isolated polypeptide) as described in any of the preceding claims, wherein the protease-cleavable sequence further comprises a linker (or spacer sequence, hereinafter the same). In some embodiments, the polypeptide (e.g., an isolated polypeptide) as described in any of the preceding claims, wherein the linker is selected from GS, AS, GA, and AA. In some embodiments, the polypeptide (e.g., an isolated polypeptide) as described in any of the preceding claims, wherein the linker is at the C-terminus of SEQ ID NO: 9 or 10, or the linker is at the N-terminus of SEQ ID NO: 9 or 10. In some embodiments, the polypeptide (e.g., an isolated polypeptide) as described in any of the preceding claims, wherein the linker is at the N-terminus of SEQ ID NO: 11, or the linker is at the C-terminus of SEQ ID NO: 11. In some embodiments, the polypeptide (e.g., an isolated polypeptide) as described in any of the preceding claims, wherein the linker is located between SEQ ID NO: 9 and SEQ ID NO: 11; or the linker is located between SEQ ID NO: 10 and SEQ ID NO: 11. In some embodiments, the linker is at the C-terminus of SEQ ID NO: 9 or 10, and the linker is at the N-terminus of SEQ ID NO: 11. In some embodiments, the connector is at the N end of SEQ ID NO: 9 or 10, and the connector is at the C end of SEQ ID NO: 11.

[0011] In some embodiments, the polypeptide (e.g., an isolated polypeptide) as described in any of the preceding claims comprises a protease-cleavable sequence comprising an amino acid sequence that is identical to or has at least 70% (e.g., 75%, 77%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) sequence identity to SEQ ID NO: 2 or 8.

[0012] The amino acid sequence of SEQ ID NO: 8 is shown as X1SGRSTNAX2X3PLGLAG;

[0013] X1 can be A or L, preferably A;

[0014] X2 is either G or A, preferably G;

[0015] X3 can be S or A, preferably S.

[0016] Therefore, in some embodiments, the three variable residues in SEQ ID NO: 8 are selected from combinations of the following:

[0017] X1 = A, X2 = G, X3 = S;

[0018] X1 = A, X2 = G, X3 = A;

[0019] X1 = A, X2 = A, X3 = S;

[0020] X1 = A, X2 = A, X3 = A;

[0021] X1 = L, X2 = G, X3 = S;

[0022] X1 = L, X2 = G, X3 = A;

[0023] X1 = L, X2 = A, X3 = S;

[0024] X1 = L, X2 = A, X3 = A.

[0025] In some embodiments, the polypeptide (e.g., an isolated polypeptide) as described in any of the preceding claims, wherein the protease-cleavable sequence comprises the amino acid sequence of SEQ ID NO: 2, 1, 3, 4, 5, or 6. In some embodiments, the polypeptide (e.g., an isolated polypeptide) comprises a protease-cleavable sequence having 1, 2, or 3 amino acid substitutions, additions, or deletions relative to the amino acid sequences of SEQ ID NO: 2, 1, 3, 4, 5, or 6.

[0026] In some embodiments, the polypeptide (e.g., an isolated polypeptide) as described in any of the preceding embodiments, wherein the protease-cleavable sequence comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the protease-cleavable sequence consists of the amino acid sequence of SEQ ID NO: 2. In some embodiments, the protease-cleavable sequence has one amino acid substitution, addition, or deletion relative to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the protease-cleavable sequence has two amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the protease-cleavable sequence has three amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 2.

[0027] In some embodiments, the polypeptide (e.g., an isolated polypeptide) as described in any of the preceding embodiments, wherein the protease-cleavable sequence comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the protease-cleavable sequence consists of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the protease-cleavable sequence has one amino acid substitution, addition, or deletion relative to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the protease-cleavable sequence has two amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the protease-cleavable sequence has three amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 1.

[0028] Table 1. Protease-cleavable sequences

[0029] In some embodiments, the substitution, addition, or deletion of amino acids results in an amino acid sequence that is at least 70% identical (e.g., 75%, 77%, 80%, 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to any protein described herein. In some embodiments, the amino acid substitution is a conserved amino acid substitution. Among common amino acids, for example, a “conserved amino acid substitution” is described by substitution between amino acids within the following groups: (1) glycine, alanine, valine, leucine, and isoleucine; (2) phenylalanine, tyrosine, and tryptophan; (3) serine and threonine; (4) aspartic acid and glutamic acid; (5) glutamine and asparagine; and (6) lysine, arginine, and histidine.

[0030] In some embodiments, the protease-cleavable sequence comprises modified amino acids or non-natural amino acids, or modified non-natural amino acids, or combinations thereof. In some embodiments, the modified amino acids or modified non-natural amino acids comprise post-translational modifications. In some embodiments, the protease-cleavable sequence comprises modifications, including but not limited to acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of heme moieties, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine ​​residues, formation of pyroglutamic acid residues, formylation, γ-carboxylation, glycosylation, GPI anchoring, hydroxylation, iodination, methylation, myristylation, oxidation, proteolytic processing, phosphorylation, isopreneation, racemization, selenoylation, sulfation, and transfer RNA-mediated amino acid addition to the protein, such as argininoylation and ubiquitination. Modifications can be made anywhere the protease can cleave the sequence, including the peptide backbone or amino acid side chains.

[0031] In some embodiments, the polypeptide (e.g., an isolated polypeptide) as described in any of the preceding claims, wherein the protease-cleavable sequence is cleaved by the protease. In some embodiments, the polypeptide (e.g., an isolated polypeptide) as described in any of the preceding claims, wherein the protease is a protease expressed or overexpressed in the tumor microenvironment. In some embodiments, the protease is selected from one or more of metalloproteinases, serine proteases, cysteine ​​proteases, aspartic proteases, threonine proteases, glutamate proteases, gelatinases, and asparagine peptidases. In some embodiments, the protease is selected from one or more of metalloproteinases and serine proteases.

[0032] In some embodiments, the polypeptide (e.g., an isolated polypeptide) as described in any of the preceding claims, wherein the metalloproteinase is a matrix metalloproteinase. In some embodiments, the polypeptide (e.g., an isolated polypeptide) as described in any of the preceding claims, wherein the metalloproteinase is a matrix metalloproteinase selected from one or more of MMP2, MMP7, MMP9, MMP13, and MMP14; and / or

[0033] The serine protease mentioned therein is selected from one or more of Matriptase, uPA, and hepsin.

[0034] In some embodiments, the polypeptide (e.g., an isolated polypeptide) as described in any of the preceding claims, wherein the protease-cleavable sequence is cleaved by a variety of proteases. In some embodiments, the polypeptide (e.g., an isolated polypeptide) as described in any of the preceding claims, wherein the protease-cleavable sequence is cleaved by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more than 20 different proteases.

[0035] In some embodiments, the polypeptide (e.g., an isolated polypeptide) as described in any of the preceding embodiments is selected from one or more of MMP2, MMP7, MMP9, MMP11, MMP14, uPA, Matriptase, and pod protein.

[0036] In some embodiments, the polypeptide (e.g., an isolated polypeptide) as described in any of the preceding claims is selected from one, two, or three of MMP2, MMP7, MMP9, MMP11, MMP14, uPA, Matriptase, and pod proteins.

[0037] In one specific implementation, the polypeptide (e.g., an isolated polypeptide) as described in any of the preceding claims, wherein the protease is MMP9, uPA, and Matriptase.

[0038] In some embodiments, the polypeptide (e.g., an isolated polypeptide) as described in any of the preceding claims, wherein the protease-cleavable sequence has a property selected from one or more of the following:

[0039] The protease-cleavable sequence is cleaved by MMP9 at a rate of not less than 0.30 RFU / s (e.g., 0.35 RFU / s, 0.39 RFU / s, 0.40 RFU / s, 0.42 RFU / s, 0.47 RFU / s, 0.49 RFU / s, 0.50 RFU / s, 0.55 RFU / s, 0.58 RFU / s, 0.60 RFU / s, 0.70 RFU / s, 0.80 RFU / s, 0.90 RFU / s, 1.00 RFU / s, 1.10 RFU / s, 1.20 RFU / s, 1.30 RFU / s, or 1.37 RFU / s).

[0040] The protease-cleavable sequence is cleaved by Matriptase at a rate of not less than 0.10 RFU / s (e.g., 0.15 RFU / s, 0.18 RFU / s, 0.20 RFU / s, 0.30 RFU / s, 0.40 RFU / s, 0.49 RFU / s, 0.50 RFU / s, 0.60 RFU / s, 0.70 RFU / s, 0.75 RFU / s, 0.77 RFU / s, or 0.80 RFU / s);

[0041] The rate at which the protease-cleavable sequence is cleaved by uPA is not less than 0.10 RFU / s (e.g., 0.15 RFU / s, 0.20 RFU / s, 0.25 RFU / s, 0.29 RFU / s, 0.30 RFU / s, 0.33 RFU / s, 0.35 RFU / s, 0.37 RFU / s, or 0.38 RFU / s);

[0042] The rate at which the protease-cleavable sequence is cleaved by MMP14 is no higher than 0.30 RFU / s (e.g., 0.27 RFU / s, 0.25 RFU / s, 0.23 RFU / s, 0.20 RFU / s, 0.17 RFU / s, or 0.15 RFU / s).

[0043] In some embodiments, the polypeptide (e.g., an isolated polypeptide) as described in any of the preceding claims has an increased proteolytic rate compared to an uncleanable linker. In some embodiments, the polypeptide (e.g., an isolated polypeptide) as described in any of the preceding claims has an increased proteolytic rate of at least 5-fold (e.g., at least 8-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 75-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 120-fold) higher than an uncleanable linker.

[0044] In some embodiments, the polypeptide (e.g., an isolated polypeptide) as described in any of the preceding claims, wherein the protease is a matrix metalloproteinase (MMP) and / or a serine protease. In some embodiments, the isolated polypeptide as described in any of the preceding claims, wherein the matrix metalloproteinase is MMP2, MMP7, MMP9, MMP13, or MMP14, or combinations thereof. In some embodiments, the polypeptide (e.g., an isolated polypeptide) as described in any of the preceding claims, wherein the serine protease is uPA, Matriptase, or hepsin, or combinations thereof.

[0045] In some embodiments, the polypeptide (e.g., an isolated polypeptide) as described in any of the preceding claims has improved stability in cynomolgus monkey plasma compared to the control linker containing SEQ ID NO: 7. In some embodiments, the polypeptide (e.g., an isolated polypeptide) has at least a 2-fold (e.g., at least 3-fold, at least 5-fold, at least 8-fold, at least 10-fold) improved stability in cynomolgus monkey plasma compared to the stability of the control linker containing SEQ ID NO: 7.

[0046] In some embodiments, the polypeptide (e.g., isolated polypeptide) as described in any of the preceding claims has an increased protease cleavage rate compared to the control linker containing SEQ ID NO: 7. In some embodiments, the polypeptide (e.g., isolated polypeptide) as described in any of the preceding claims has a protease-cleavable sequence that is cleaved by MMP9 at a rate at least 0.5 times faster than the control linker containing SEQ ID NO: 7 (e.g., at least 0.8 times, at least 0.9 times, at least 1.0 times, at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2.0 times, at least 2.1 times, at least 2.2 times, at least 2.3 times, at least 2.4 times, at least 2.5 times, at least 2.6 times, at least 2.7 times, at least 2.8 times, or at least 2.9 times).

[0047] In some embodiments, the polypeptide (e.g., isolated polypeptide) as described in any of the preceding claims, wherein the protease-cleavable sequence disclosed herein is cleaved by Matriptase at a rate at least 2.0 times faster than the control linker containing SEQ ID NO: 7 is cleaved by Matriptase (e.g., at least 2.5 times, at least 3.0 times, at least 4.0 times, at least 5.0 times, at least 6.0 times, at least 7.0 times, at least 8.0 times, at least 9.0 times, at least 10.0 times, at least 11.0 times, or at least 11.4 times faster).

[0048] In some embodiments, the polypeptide (e.g., isolated polypeptide) as described in any of the preceding claims, wherein the protease-cleavable sequence disclosed herein is cleaved by uPA at a rate at least 1.2 times faster than the control linker containing SEQ ID NO: 7 is cleaved by uPA (e.g., at least 1.3 times, at least 1.5 times, at least 2.0 times, at least 2.5 times, at least 3.0 times, at least 3.5 times, at least 3.6 times, at least 3.7 times, at least 3.75 times, at least 3.8 times, at least 4.0 times, at least 4.5 times, at least 4.7 times, or at least 4.8 times).

[0049] In some embodiments, the polypeptide (e.g., isolated polypeptide) as described in any of the preceding claims, wherein the protease-cleavable sequence described herein is cleaved by MMP9 at a rate at least 1.5 times that of MMP14 (e.g., at least 1.8 times, at least 2.0 times, at least 2.1 times, at least 2.5 times, at least 3.0 times, at least 3.5 times, at least 4.0 times, at least 4.2 times, at least 4.5 times, at least 5.0 times, at least 5.5 times, at least 6.0 times, at least 6.5 times, at least 7.0 times, at least 7.5 times, at least 8.0 times, at least 8.5 times, at least 9.0 times, or at least 9.1 times).

[0050] In some embodiments, such as the polypeptide described in any of the preceding claims (e.g., isolated polypeptides), the cleavage rate of the protease-cleavable sequence is detected by fluorescence resonance energy transfer (FRET) technology.

[0051] In some embodiments, the polypeptide (e.g., isolated polypeptide) as described in any of the preceding embodiments, wherein the cleavage rate of the protease-cleavable sequence is determined according to the steps of Example 1.1.

[0052] In some embodiments, the polypeptide (e.g., isolated polypeptide) as described in any of the preceding claims, wherein the apparent cleavage rate (or Kcat / Km) of the protease-cleavable sequence disclosed herein cleaved by MMP9 is at least 1.2 times (e.g., at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2.0 times, at least 2.1 times, at least 2.2 times, at least 2.3 times, at least 2.4 times, or at least 2.5 times) of the apparent cleavage rate (or Kcat / Km) of the control linker containing SEQ ID NO: 7 cleaved by MMP9.

[0053] In some embodiments, the polypeptide (e.g., isolated polypeptide) as described in any of the preceding claims, wherein the apparent cleavage rate (or Kcat / Km) of the protease-cleavable sequence disclosed herein cleaved by Matriptase is at least 2.0 times (e.g., at least 2.3 times, at least 2.5 times, at least 3.0 times, at least 4.0 times, at least 5.0 times, at least 6.0 times, at least 7.0 times, at least 8.0 times, at least 9.0 times, at least 10.0 times, at least 11.0 times, at least 12.0 times, at least 13.0 times, at least 14.0 times, at least 14.5 times, at least 14.6 times, at least 14.7 times, at least 14.8 times, at least 15 times, at least 16 times, at least 18 times, at least 20 times, at least 22 times, or at least 25 times) of the apparent cleavage rate (or Kcat / Km) of the control linker containing SEQ ID NO: 7 cleaved by Matriptase.

[0054] In some embodiments, the polypeptide (e.g., isolated polypeptide) as described in any of the preceding claims, wherein the apparent cleavage rate (or Kcat / Km) of the protease-cleavable sequence disclosed herein cleaved by uPA is at least 0.5 times (e.g., at least 0.6 times, at least 1.0 times, at least 1.5 times, at least 2.0 times, at least 2.5 times, at least 3.0 times, at least 3.5 times, at least 4.0 times, at least 4.5 times, at least 5.0 times, at least 5.5 times, at least 6.0 times, at least 6.5 times, at least 7.0 times, at least 7.5 times, at least 8.0 times, at least 8.5 times, at least 9.0 times, or at least 9.5 times) of the control linker containing SEQ ID NO: 7.

[0055] In some embodiments, the polypeptide (e.g., isolated polypeptide) as described in any of the preceding claims, wherein the apparent cleavage rate (or Kcat / Km) of the protease-cleavable sequence described herein cleaved by MMP9 is at least 2.0 times (e.g., at least 3.0 times, at least 4.0 times, at least 5.0 times, at least 6.0 times, at least 7.0 times, at least 8.0 times, at least 9.0 times, at least 10.0 times, at least 11.0 times, at least 12.0 times, at least 13.0 times, at least 14.0 times, at least 15.0 times, at least 16.0 times, at least 17.0 times, at least 18.0 times, at least 19.0 times, at least 20.0 times, at least 21.0 times, at least 22.0 times, at least 23.0 times, at least 24.0 times, or at least 25.0 times) greater than the apparent cleavage rate (or Kcat / Km) of the MMP7 cleavage sequence.

[0056] In some embodiments, the polypeptide (e.g., isolated polypeptide) as described in any of the preceding claims, wherein the apparent cleavage rate (or Kcat / Km) of the protease-cleavable sequence described herein cleaved by MMP9 is at least 16.0 times (e.g., at least 17.0 times, at least 18.0 times, at least 19.0 times, at least 20.0 times, at least 22.0 times, at least 22.5 times, at least 23.0 times, at least 25.0 times, at least 30.0 times, at least 35.0 times, at least 40.0 times, at least 45.0 times, at least 50.0 times, at least 55.0 times, at least 56.0 times, at least 57.0 times, at least 58.0 times, at least 59.0 times, at least 60.0 times, at least 61.0 times, at least 61.5 times, at least 62.0 times, at least 65.0 times, or at least 70.0 times) of the apparent cleavage rate (or Kcat / Km) of the MMP14 cleavage sequence.

[0057] In some embodiments, for the polypeptide (e.g., an isolated polypeptide) as described in any of the preceding embodiments, the apparent cleavage rate (or Kcat / Km) of the protease-cleavable sequence is determined according to the steps of Example 1.3.

[0058] In some embodiments, the polypeptide (e.g., an isolated polypeptide) as described in any of the preceding claims, wherein the protease-cleavable sequence of the polypeptide as described in any of the preceding claims has a property selected from one or more of the following:

[0059] The protease-cleavable sequence was cleaved by MMP9 at a rate at least 0.5 times faster than the control linker containing SEQ ID NO: 7 was cleaved by MMP9.

[0060] The protease-cleavable sequence was cleaved by Matriptase at a rate at least 2.0 times faster than the control linker containing SEQ ID NO: 7 was cleaved by Matriptase.

[0061] The protease-cleavable sequence was cleaved by uPA at a rate at least 1.2 times faster than the control linker containing SEQ ID NO: 7 was cleaved by uPA.

[0062] The protease-cleavable sequence is cleaved by MMP9 at a rate at least 1.5 times that of MMP14.

[0063] The apparent cleavage rate (or Kcat / Km) of the protease-cleavable sequence cleaved by MMP9 was at least 1.2 times faster than the apparent cleavage rate (or Kcat / Km) of the control linker containing SEQ ID NO: 7 cleaved by MMP9.

[0064] The apparent cleavage rate (or Kcat / Km) of the protease-cleavable sequence cleaved by Matriptase is at least 2.0 times faster than the apparent cleavage rate (or Kcat / Km) of the control linker containing SEQ ID NO: 7 cleaved by Matriptase.

[0065] The apparent cleavage rate (or Kcat / Km) of the protease-cleavable sequence cleaved by uPA is at least 0.5 times faster than the apparent cleavage rate (or Kcat / Km) of the control linker containing SEQ ID NO: 7 cleaved by uPA.

[0066] The apparent cleavage rate (or Kcat / Km) of the protease-cleavable sequence by MMP9 is at least 2.0 times that by MMP7; and

[0067] The apparent cleavage rate (or Kcat / Km) of the protease-cleavable sequence cleaved by MMP9 is at least 16.0 times that of the apparent cleavage rate (or Kcat / Km) cleaved by MMP14.

[0068] <Protein-cleavable sequences>

[0069] In one aspect, this disclosure provides a protease-cleavable sequence comprising the amino acid sequence of SEQ ID NO: 9 (ASGRSTNA) or SEQ ID NO: 10 (LSGRSTNA).

[0070] In some embodiments, the protease-cleavable sequence as described above also includes the amino acid sequence of SEQ ID NO: 11 (PLGLAG).

[0071] In some embodiments, the protease-cleavable sequence as described in any of the preceding claims further includes a linker. In some embodiments, the protease-cleavable sequence as described in any of the preceding claims, wherein the linker is selected from GS, AS, GA, and AA. In some embodiments, the protease-cleavable sequence as described in any of the preceding claims, wherein the linker is at the C-terminus of SEQ ID NO: 9 or 10, or the linker is at the N-terminus of SEQ ID NO: 9 or 10. In some embodiments, the protease-cleavable sequence as described in any of the preceding claims, wherein the linker is at the N-terminus of SEQ ID NO: 11, or the linker is at the C-terminus of SEQ ID NO: 11. In some embodiments, the protease-cleavable sequence as described in any of the preceding claims, wherein the linker is located between SEQ ID NO: 9 and SEQ ID NO: 11; or the linker is located between SEQ ID NO: 10 and SEQ ID NO: 11. In some embodiments, the linker is at the C-terminus of SEQ ID NO: 9 or 10, and the linker is at the N-terminus of SEQ ID NO: 11. In some embodiments, the connector is at the N end of SEQ ID NO: 9 or 10, and the connector is at the C end of SEQ ID NO: 11.

[0072] In some embodiments, the protease-cleavable sequence as described in any of the preceding embodiments comprises an amino acid sequence that is identical to or has at least 70% (e.g., 75%, 77%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) sequence identity with SEQ ID NO: 2 or 8.

[0073] The amino acid sequence of SEQ ID NO: 8 is shown as X1SGRSTNAX2X3PLGLAG;

[0074] X1 can be A or L, preferably A;

[0075] X2 is either G or A, preferably G;

[0076] X3 can be S or A, preferably S.

[0077] In some embodiments, the protease-cleavable sequence as described in any of the preceding embodiments comprises the amino acid sequence of SEQ ID NO: 2, 1, 3, 4, 5, or 6. In some embodiments, the protease-cleavable sequence as described in any of the preceding embodiments consists of the amino acid sequence shown in SEQ ID NO: 2, 1, 3, 4, 5, or 6. In some embodiments, the protease-cleavable sequence comprises a protease-cleavable sequence having 1, 2, or 3 amino acid substitutions, additions, or deletions relative to the amino acid sequences of SEQ ID NO: 2, 1, 3, 4, 5, or 6.

[0078] In some embodiments, the protease-cleavable sequence as described in any of the preceding embodiments comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the protease-cleavable sequence consists of the amino acid sequence of SEQ ID NO: 2. In some embodiments, the protease-cleavable sequence has one amino acid substitution, addition, or deletion relative to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the protease-cleavable sequence has two amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the protease-cleavable sequence has three amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 2.

[0079] In some embodiments, the protease-cleavable sequence as described in any of the preceding embodiments comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the protease-cleavable sequence consists of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the protease-cleavable sequence has one amino acid substitution, addition, or deletion relative to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the protease-cleavable sequence has two amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the protease-cleavable sequence has three amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 1.

[0080] In some embodiments, the protease-cleavable sequence as described in any of the preceding embodiments is cleaved by a protease. In some embodiments, the protease-cleavable sequence as described in any of the preceding embodiments is wherein the protease is a protease expressed or overexpressed in the tumor microenvironment. In some embodiments, the protease is selected from one or more of metalloproteinases, serine proteases, cysteine ​​proteases, aspartic proteases, threonine proteases, glutamate proteases, gelatinases, and asparagine peptidases. In some embodiments, the protease is selected from one or more of metalloproteinases and serine proteases.

[0081] In some embodiments, the protease-cleavable sequence as described in any of the preceding embodiments is a matrix metalloproteinase. In some embodiments, the protease-cleavable sequence as described in any of the preceding embodiments is a matrix metalloproteinase selected from one or more of MMP2, MMP7, MMP9, MMP13, and MMP14; and / or

[0082] The serine protease mentioned therein is selected from one or more of Matriptase, uPA, and hepsin.

[0083] In some embodiments, the protease-cleavable sequence as described in any of the preceding embodiments is cleaved by a variety of proteases. In some embodiments, the protease-cleavable sequence as described in any of the preceding embodiments is cleaved by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more than 20 different proteases.

[0084] In some embodiments, the protease cleavable sequence as described in any of the preceding embodiments is selected from one or more of MMP2, MMP7, MMP9, MMP11, MMP14, uPA, Matriptase, and pod proteins.

[0085] In some embodiments, the protease-cleavable sequence as described in any of the preceding claims is provided, wherein the protease is selected from one, two, or three of MMP2, MMP7, MMP9, MMP11, MMP14, uPA, Matriptase, and pod proteins. In one specific embodiment, the protease-cleavable sequence as described in any of the preceding claims is provided, wherein the protease is MMP9, uPA, and Matriptase.

[0086] In some embodiments, the protease-cleavable sequence as described in any of the preceding embodiments, wherein the protease-cleavable sequence has one or more of the following characteristics:

[0087] The protease-cleavable sequence is cleaved by MMP9 at a rate of not less than 0.30 RFU / s (e.g., 0.35 RFU / s, 0.39 RFU / s, 0.40 RFU / s, 0.42 RFU / s, 0.47 RFU / s, 0.49 RFU / s, 0.50 RFU / s, 0.55 RFU / s, 0.58 RFU / s, 0.60 RFU / s, 0.70 RFU / s, 0.80 RFU / s, 0.90 RFU / s, 1.00 RFU / s, 1.10 RFU / s, 1.20 RFU / s, 1.30 RFU / s, or 1.37 RFU / s).

[0088] The protease-cleavable sequence is cleaved by Matriptase at a rate of not less than 0.10 RFU / s (e.g., 0.15 RFU / s, 0.18 RFU / s, 0.20 RFU / s, 0.30 RFU / s, 0.40 RFU / s, 0.49 RFU / s, 0.50 RFU / s, 0.60 RFU / s, 0.70 RFU / s, 0.75 RFU / s, 0.77 RFU / s, or 0.80 RFU / s);

[0089] The rate at which the protease-cleavable sequence is cleaved by uPA is not less than 0.10 RFU / s (e.g., 0.15 RFU / s, 0.20 RFU / s, 0.25 RFU / s, 0.29 RFU / s, 0.30 RFU / s, 0.33 RFU / s, 0.35 RFU / s, 0.37 RFU / s, or 0.38 RFU / s);

[0090] The rate at which the protease-cleavable sequence is cleaved by MMP14 is no higher than 0.30 RFU / s (e.g., 0.27 RFU / s, 0.25 RFU / s, 0.23 RFU / s, 0.20 RFU / s, 0.17 RFU / s, or 0.15 RFU / s).

[0091] In some embodiments, the protease-cleavable sequence as described in any of the preceding embodiments has an increased proteolytic rate compared to an uncleavable linker. In some embodiments, the protease-cleavable sequence as described in any of the preceding embodiments has an increased proteolytic rate of at least 5 times (e.g., at least 8 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 75 times, at least 80 times, at least 90 times, at least 100 times, at least 120 times) compared to an uncleavable linker. In some embodiments, the protease-cleavable sequence as described in any of the preceding embodiments, wherein the protease is a matrix metalloproteinase (MMP) and / or a serine protease. In some embodiments, the protease-cleavable sequence as described in any of the preceding embodiments, wherein the matrix metalloproteinase is MMP2, MMP7, MMP9, MMP13, or MMP14, or combinations thereof. In some embodiments, the protease can cleave the sequence as described in any of the preceding embodiments, wherein the serine protease is uPA, Matriptase, or hepsin, or combinations thereof.

[0092] In some embodiments, the protease-cleavable sequence as described in any of the preceding embodiments has improved stability in cynomolgus monkey plasma compared to the control linker containing SEQ ID NO: 7. In some embodiments, the protease-cleavable sequence as described in any of the preceding embodiments has at least a 2-fold (e.g., at least 3-fold, at least 5-fold, at least 8-fold, at least 10-fold) improved stability in cynomolgus monkey plasma compared to the stability of the control linker containing SEQ ID NO: 7.

[0093] In some embodiments, the protease-cleavable sequence as described in any of the preceding embodiments has an increased protease cleavage rate compared to the control linker containing SEQ ID NO: 7. In some embodiments, the protease-cleavable sequence as described in any of the preceding embodiments has a cleavage rate of at least 0.5 times faster with MMP9 than the control linker containing SEQ ID NO: 7 (e.g., at least 0.8 times, at least 0.9 times, at least 1.0 times, at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2.0 times, at least 2.1 times, at least 2.2 times, at least 2.3 times, at least 2.4 times, at least 2.5 times, at least 2.6 times, at least 2.7 times, at least 2.8 times, or at least 2.9 times).

[0094] In some embodiments, the protease-cleavable sequence as described in any of the preceding embodiments, wherein the protease-cleavable sequence disclosed herein is cleaved by Matriptase at a rate at least 2.0 times faster than the rate at which the control linker containing SEQ ID NO: 7 is cleaved by Matriptase (e.g., at least 2.5 times, at least 3.0 times, at least 4.0 times, at least 5.0 times, at least 6.0 times, at least 7.0 times, at least 8.0 times, at least 9.0 times, at least 10.0 times, at least 11.0 times, or at least 11.4 times).

[0095] In some embodiments, the protease-cleavable sequence as described in any of the preceding embodiments, wherein the protease-cleavable sequence disclosed herein is cleaved by uPA at a rate at least 1.2 times faster than the control linker containing SEQ ID NO: 7 is cleaved by uPA (e.g., at least 1.3 times, at least 1.5 times, at least 2.0 times, at least 2.5 times, at least 3.0 times, at least 3.5 times, at least 3.6 times, at least 3.7 times, at least 3.75 times, at least 3.8 times, at least 4.0 times, at least 4.5 times, at least 4.7 times, or at least 4.8 times).

[0096] In some embodiments, the protease-cleavable sequence as described in any of the preceding embodiments, wherein the protease-cleavable sequence described herein is cleaved by MMP9 at a rate at least 1.5 times that of MMP14 (e.g., at least 1.8 times, at least 2.0 times, at least 2.1 times, at least 2.5 times, at least 3.0 times, at least 3.5 times, at least 4.0 times, at least 4.2 times, at least 4.5 times, at least 5.0 times, at least 5.5 times, at least 6.0 times, at least 6.5 times, at least 7.0 times, at least 7.5 times, at least 8.0 times, at least 8.5 times, at least 9.0 times, or at least 9.1 times).

[0097] In some embodiments, such as the protease-cleavable sequence described in any of the preceding embodiments, the cleavage rate of said protease-cleavable sequence is detected by fluorescence resonance energy transfer (FRET).

[0098] In some embodiments, the protease-cleavable sequence as described in any of the preceding embodiments is used, wherein the cleavage rate of the protease-cleavable sequence is determined according to the steps of Example 1.1.

[0099] In some embodiments, the protease-cleavable sequence as described in any of the preceding embodiments, wherein the apparent cleavage rate (or Kcat / Km) of the protease-cleavable sequence disclosed herein being cleaved by MMP9 is at least 1.2 times (e.g., at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2.0 times, at least 2.1 times, at least 2.2 times, at least 2.3 times, at least 2.4 times, or at least 2.5 times) of the apparent cleavage rate (or Kcat / Km) of the control linker containing SEQ ID NO: 7 being cleaved by MMP9.

[0100] In some embodiments, the protease-cleavable sequence as described in any of the preceding embodiments, wherein the apparent cleavage rate (or Kcat / Km) of the protease-cleavable sequence disclosed herein cleaved by Matriptase is at least 2.0 times (e.g., at least 2.3 times, at least 2.5 times, at least 3.0 times, at least 4.0 times, at least 5.0 times, at least 6.0 times, at least 7.0 times, at least 8.0 times, at least 9.0 times, at least 10.0 times, at least 11.0 times, at least 12.0 times, at least 13.0 times, at least 14.0 times, at least 14.5 times, at least 14.6 times, at least 14.7 times, at least 14.8 times, at least 15 times, at least 16 times, at least 18 times, at least 20 times, at least 22 times, or at least 25 times) of the apparent cleavage rate (or Kcat / Km) of the control linker containing SEQ ID NO: 7 cleaved by Matriptase.

[0101] In some embodiments, the protease-cleavable sequence as described in any of the preceding embodiments, wherein the apparent cleavage rate (or Kcat / Km) of the protease-cleavable sequence disclosed herein cleaved by uPA is at least 0.5 times (e.g., at least 0.6 times, at least 1.0 times, at least 1.5 times, at least 2.0 times, at least 2.5 times, at least 3.0 times, at least 3.5 times, at least 4.0 times, at least 4.5 times, at least 5.0 times, at least 5.5 times, at least 6.0 times, at least 6.5 times, at least 7.0 times, at least 7.5 times, at least 8.0 times, at least 8.5 times, at least 9.0 times, or at least 9.5 times) of the apparent cleavage rate (or Kcat / Km) of the control linker containing SEQ ID NO: 7 cleaved by uPA.

[0102] In some embodiments, the protease-cleavable sequence as described in any of the preceding embodiments, wherein the apparent cleavage rate (or Kcat / Km) of the protease-cleavable sequence described herein by MMP9 is at least 2.0 times (e.g., at least 3.0 times, at least 4.0 times, at least 5.0 times, at least 6.0 times, at least 7.0 times, at least 8.0 times, at least 9.0 times, at least 10.0 times, at least 11.0 times, at least 12.0 times, at least 13.0 times, at least 14.0 times, at least 15.0 times, at least 16.0 times, at least 17.0 times, at least 18.0 times, at least 19.0 times, at least 20.0 times, at least 21.0 times, at least 22.0 times, at least 23.0 times, at least 24.0 times, or at least 25.0 times) of the apparent cleavage rate (or Kcat / Km) of the protease-cleavable sequence described herein.

[0103] In some embodiments, the protease-cleavable sequence as described in any of the preceding embodiments, wherein the apparent cleavage rate (or Kcat / Km) of the protease-cleavable sequence described herein by MMP9 is at least 16.0 times (e.g., at least 17.0 times, at least 18.0 times, at least 19.0 times, at least 20.0 times, at least 22.0 times, at least 22.5 times, at least 23.0 times, at least 25.0 times, at least 30.0 times, at least 35.0 times, at least 40.0 times, at least 45.0 times, at least 50.0 times, at least 55.0 times, at least 56.0 times, at least 57.0 times, at least 58.0 times, at least 59.0 times, at least 60.0 times, at least 61.0 times, at least 61.5 times, at least 62.0 times, at least 65.0 times, or at least 70.0 times) of the apparent cleavage rate (or Kcat / Km) of the protease-cleavable sequence described herein by MMP14.

[0104] In some embodiments, the protease-cleavable sequence as described in any of the preceding embodiments, wherein the apparent cleavage rate (or Kcat / Km) of the protease-cleavable sequence is determined according to the steps of Example 1.3.

[0105] In some embodiments, the protease-cleavable sequence as described in any of the preceding embodiments, wherein the protease-cleavable sequence has one or more of the following characteristics:

[0106] The protease-cleavable sequence was cleaved by MMP9 at a rate at least 0.5 times faster than the control linker containing SEQ ID NO: 7 was cleaved by MMP9.

[0107] The protease-cleavable sequence was cleaved by Matriptase at a rate at least 2.0 times faster than the control linker containing SEQ ID NO: 7 was cleaved by Matriptase.

[0108] The protease-cleavable sequence was cleaved by uPA at a rate at least 1.2 times faster than the control linker containing SEQ ID NO: 7 was cleaved by uPA.

[0109] The protease-cleavable sequence is cleaved by MMP9 at a rate at least 1.5 times that of MMP14.

[0110] The apparent cleavage rate (or Kcat / Km) of the protease-cleavable sequence cleaved by MMP9 was at least 1.2 times faster than the apparent cleavage rate (or Kcat / Km) of the control linker containing SEQ ID NO: 7 cleaved by MMP9.

[0111] The apparent cleavage rate (or Kcat / Km) of the protease-cleavable sequence cleaved by Matriptase is at least 2.0 times faster than the apparent cleavage rate (or Kcat / Km) of the control linker containing SEQ ID NO: 7 cleaved by Matriptase.

[0112] The apparent cleavage rate (or Kcat / Km) of the protease-cleavable sequence cleaved by uPA is at least 0.5 times faster than the apparent cleavage rate (or Kcat / Km) of the control linker containing SEQ ID NO: 7 cleaved by uPA.

[0113] The apparent cleavage rate (or Kcat / Km) of the protease-cleavable sequence by MMP9 is at least 2.0 times that by MMP7; and

[0114] The apparent cleavage rate (or Kcat / Km) of the protease-cleavable sequence cleaved by MMP9 is at least 16.0 times that of the apparent cleavage rate (or Kcat / Km) cleaved by MMP14.

[0115] <Active proteins containing protease-cleavable sequences>

[0116] In one aspect, this disclosure provides an activatable protein comprising a protease-cleavable sequence comprising the amino acid sequence of SEQ ID NO: 9 (ASGRSTNA) or SEQ ID NO: 10 (LSGRSTNA).

[0117] In some embodiments, the activatable protein as described above, wherein the protease-cleavable sequence further comprises the amino acid sequence of SEQ ID NO: 11 (PLGLAG).

[0118] In some embodiments, the activatable protein as described in any of the preceding claims, wherein the protease-cleavable sequence further comprises a linker (or spacer sequence, hereinafter the same). In some embodiments, the activatable protein as described in any of the preceding claims, wherein the linker is selected from GS, AS, GA, and AA. In some embodiments, the activatable protein as described in any of the preceding claims, the linker is located at the C-terminus of SEQ ID NO: 9 or 10, or the linker is located at the N-terminus of SEQ ID NO: 9 or 10. In some embodiments, the activatable protein as described in any of the preceding claims, the linker is located at the N-terminus of SEQ ID NO: 11, or the linker is located at the C-terminus of SEQ ID NO: 11. In some embodiments, the activatable protein as described in any of the preceding claims, the linker is located between SEQ ID NO: 9 and SEQ ID NO: 11; or the linker is located between SEQ ID NO: 10 and SEQ ID NO: 11. In some embodiments, the linker is located at the C-terminus of SEQ ID NO: 9 or 10, and the linker is located at the N-terminus of SEQ ID NO: 11. In some embodiments, the connector is at the N end of SEQ ID NO: 9 or 10, and the connector is at the C end of SEQ ID NO: 11.

[0119] In some embodiments, the activatable protein as described in any of the preceding claims comprises a protease-cleavable sequence comprising an amino acid sequence that is identical to or has at least 70% (e.g., 75%, 77%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) sequence identity to SEQ ID NO: 2 or 8.

[0120] The amino acid sequence of SEQ ID NO: 8 is shown as X1SGRSTNAX2X3PLGLAG;

[0121] X1 can be A or L, preferably A;

[0122] X2 is either G or A, preferably G;

[0123] X3 can be S or A, preferably S.

[0124] In some embodiments, the activatable protein as described in any of the preceding claims, wherein the protease-cleavable sequence comprises the amino acid sequence of SEQ ID NO: 2, 1, 3, 4, 5, or 6. In some embodiments, the activatable protein as described in any of the preceding claims, wherein the protease-cleavable sequence comprises the amino acid sequence of SEQ ID NO: 2, 1, 3, 4, 5, or 6. In some embodiments, the activatable protein comprises a protease-cleavable sequence having 1, 2, or 3 amino acid substitutions, additions, or deletions relative to the amino acid sequences of SEQ ID NO: 2, 1, 3, 4, 5, or 6.

[0125] In some embodiments, the activatable protein as described in any of the preceding embodiments, wherein the protease-cleavable sequence comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the protease-cleavable sequence consists of the amino acid sequence of SEQ ID NO: 2. In some embodiments, the protease-cleavable sequence has one amino acid substitution, addition, or deletion relative to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the protease-cleavable sequence has two amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the protease-cleavable sequence has three amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 2.

[0126] In some embodiments, the activatable protein as described in any of the preceding embodiments, wherein the protease-cleavable sequence comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the protease-cleavable sequence consists of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the protease-cleavable sequence has one amino acid substitution, addition, or deletion relative to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the protease-cleavable sequence has two amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the protease-cleavable sequence has three amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 1.

[0127] In some embodiments, as described in any of the preceding embodiments, the activatable protein contains a protease-cleavable sequence that can be cleaved by the protease. In some embodiments, as described in any of the preceding embodiments, the protease is a protease expressed or overexpressed in the tumor microenvironment. In some embodiments, the protease is selected from one or more of metalloproteinases, serine proteases, cysteine ​​proteases, aspartic proteases, threonine proteases, glutamate proteases, gelatinases, and asparagine peptidases. In some embodiments, the protease is selected from one or both of metalloproteinases and serine proteases.

[0128] In some embodiments, the activatable protein as described in any of the preceding embodiments, wherein the metalloproteinase is a matrix metalloproteinase. In some embodiments, the activatable protein as described in any of the preceding embodiments, wherein the metalloproteinase is a matrix metalloproteinase selected from one or more of MMP2, MMP7, MMP9, MMP13, and MMP14; and / or

[0129] The serine protease mentioned therein is selected from one or more of Matriptase, uPA, and hepsin.

[0130] In some embodiments, as described in any of the preceding embodiments, the activatable protein contains a protease-cleavable sequence that is cleaved by a plurality of proteases. In some embodiments, as described in any of the preceding embodiments, the protease-cleavable sequence contains a protease-cleavable sequence that is cleaved by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more than 20 different proteases.

[0131] In some embodiments, the activatable protein as described in any of the preceding embodiments is selected from one or more of MMP2, MMP7, MMP9, MMP11, MMP14, uPA, Matriptase, and pod proteins.

[0132] In some embodiments, the activatable protein as described in any of the preceding embodiments is selected from one, two, or three of MMP2, MMP7, MMP9, MMP11, MMP14, uPA, Matriptase, and pod proteins.

[0133] In one specific implementation, the activatable protein as described in any of the preceding claims, wherein the protease is MMP9, uPA, and Matriptase.

[0134] In some embodiments, the activatable protein as described in any of the preceding embodiments, wherein the protease-cleavable sequence has one or more properties selected from the following:

[0135] The protease-cleavable sequence is cleaved by MMP9 at a rate of not less than 0.30 RFU / s (e.g., 0.35 RFU / s, 0.39 RFU / s, 0.40 RFU / s, 0.42 RFU / s, 0.47 RFU / s, 0.49 RFU / s, 0.50 RFU / s, 0.55 RFU / s, 0.58 RFU / s, 0.60 RFU / s, 0.70 RFU / s, 0.80 RFU / s, 0.90 RFU / s, 1.00 RFU / s, 1.10 RFU / s, 1.20 RFU / s, 1.30 RFU / s, or 1.37 RFU / s).

[0136] The protease-cleavable sequence is cleaved by Matriptase at a rate of not less than 0.10 RFU / s (e.g., 0.15 RFU / s, 0.18 RFU / s, 0.20 RFU / s, 0.30 RFU / s, 0.40 RFU / s, 0.49 RFU / s, 0.50 RFU / s, 0.60 RFU / s, 0.70 RFU / s, 0.75 RFU / s, 0.77 RFU / s, or 0.80 RFU / s);

[0137] The rate at which the protease-cleavable sequence is cleaved by uPA is not less than 0.10 RFU / s (e.g., 0.15 RFU / s, 0.20 RFU / s, 0.25 RFU / s, 0.29 RFU / s, 0.30 RFU / s, 0.33 RFU / s, 0.35 RFU / s, 0.37 RFU / s, or 0.38 RFU / s);

[0138] The rate at which the protease-cleavable sequence is cleaved by MMP14 is no higher than 0.30 RFU / s (e.g., 0.27 RFU / s, 0.25 RFU / s, 0.23 RFU / s, 0.20 RFU / s, 0.17 RFU / s, or 0.15 RFU / s).

[0139] In some embodiments, the activatable protein as described in any of the preceding embodiments has an increased proteolytic rate compared to an uncleanable linker. In some embodiments, the activatable protein as described in any of the preceding embodiments has an increased proteolytic rate of at least 5-fold (e.g., at least 8-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 75-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 120-fold) higher proteolytic rate compared to an uncleanable linker.

[0140] In some embodiments, the activatable protein as described in any of the preceding embodiments, wherein the protease is a matrix metalloproteinase (MMP) and / or a serine protease. In some embodiments, the activatable protein as described in any of the preceding embodiments, wherein the matrix metalloproteinase is MMP2, MMP7, MMP9, MMP13, or MMP14, or combinations thereof. In some embodiments, the activatable protein as described in any of the preceding embodiments, wherein the serine protease is uPA, Matriptase, or hepsin, or combinations thereof.

[0141] In some embodiments, the activatable protein as described in any of the preceding embodiments exhibits improved stability in cynomolgus plasma compared to the control linker comprising SEQ ID NO: 7. In some embodiments, the activatable protein as described in any of the preceding embodiments exhibits at least a 2-fold (e.g., at least 3-fold, at least 5-fold, at least 8-fold, at least 10-fold) improved stability in cynomolgus plasma compared to the stability of the control linker comprising SEQ ID NO: 7.

[0142] In some embodiments, the activatable protein as described in any of the preceding embodiments has an increased protease cleavage rate of the protease-cleavable sequence disclosed herein compared to the control linker containing SEQ ID NO: 7. In some embodiments, the activatable protein as described in any of the preceding embodiments is cleaved by MMP9 at a rate at least 0.5 times faster than the control linker containing SEQ ID NO: 7 (e.g., at least 0.8 times, at least 0.9 times, at least 1.0 times, at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2.0 times, at least 2.1 times, at least 2.2 times, at least 2.3 times, at least 2.4 times, at least 2.5 times, at least 2.6 times, at least 2.7 times, at least 2.8 times, or at least 2.9 times).

[0143] In some embodiments, such as the activatable protein described in any of the preceding embodiments, the protease-cleavable sequence disclosed herein is cleaved by Matriptase at a rate at least 2.0 times faster than the control linker containing SEQ ID NO: 7 is cleaved by Matriptase (e.g., at least 2.5 times, at least 3.0 times, at least 4.0 times, at least 5.0 times, at least 6.0 times, at least 7.0 times, at least 8.0 times, at least 9.0 times, at least 10.0 times, at least 11.0 times, or at least 11.4 times).

[0144] In some embodiments, such as the activatable protein described in any of the preceding embodiments, the protease-cleavable sequence disclosed herein is cleaved by uPA at a rate at least 1.2 times faster than the control linker containing SEQ ID NO: 7 is cleaved by uPA (e.g., at least 1.3 times, at least 1.5 times, at least 2.0 times, at least 2.5 times, at least 3.0 times, at least 3.5 times, at least 3.6 times, at least 3.7 times, at least 3.75 times, at least 3.8 times, at least 4.0 times, at least 4.5 times, at least 4.7 times, or at least 4.8 times).

[0145] In some embodiments, such as the activatable protein described in any of the preceding embodiments, the protease-cleavable sequence described herein is cleaved by MMP9 at a rate at least 1.5 times that of MMP14 (e.g., at least 1.8 times, at least 2.0 times, at least 2.1 times, at least 2.5 times, at least 3.0 times, at least 3.5 times, at least 4.0 times, at least 4.2 times, at least 4.5 times, at least 5.0 times, at least 5.5 times, at least 6.0 times, at least 6.5 times, at least 7.0 times, at least 7.5 times, at least 8.0 times, at least 8.5 times, at least 9.0 times, or at least 9.1 times).

[0146] In some embodiments, such as the activatable protein described in any of the preceding embodiments, the cleavage rate of the protease-cleavable sequence is detected by fluorescence resonance energy transfer (FRET).

[0147] In some embodiments, such as the activatable protein described in any of the preceding embodiments, the cleavage rate of the protease-cleavable sequence is determined according to the steps of Example 1.1.

[0148] In some embodiments, the activatable protein as described in any of the preceding embodiments, wherein the apparent cleavage rate (or Kcat / Km) of the protease-cleavable sequence disclosed herein being cleaved by MMP9 is at least 1.2 times (e.g., at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2.0 times, at least 2.1 times, at least 2.2 times, at least 2.3 times, at least 2.4 times, or at least 2.5 times) of the apparent cleavage rate (or Kcat / Km) of the control linker containing SEQ ID NO: 7 being cleaved by MMP9.

[0149] In some embodiments, the activatable protein as described in any of the preceding embodiments, wherein the apparent cleavage rate (or Kcat / Km) of the protease-cleavable sequence disclosed herein cleaved by Matriptase is at least 2.0 times (e.g., at least 2.3 times, at least 2.5 times, at least 3.0 times, at least 4.0 times, at least 5.0 times, at least 6.0 times, at least 7.0 times, at least 8.0 times, at least 9.0 times, at least 10.0 times, at least 11.0 times, at least 12.0 times, at least 13.0 times, at least 14.0 times, at least 14.5 times, at least 14.6 times, at least 14.7 times, at least 14.8 times, at least 15 times, at least 16 times, at least 18 times, at least 20 times, at least 22 times, or at least 25 times) of the apparent cleavage rate (or Kcat / Km) of the control linker containing SEQ ID NO: 7 cleaved by Matriptase.

[0150] In some embodiments, the activatable protein as described in any of the preceding embodiments, wherein the apparent cleavage rate (or Kcat / Km) of the protease-cleavable sequence disclosed herein cleaved by uPA is at least 0.5 times (e.g., at least 0.6 times, at least 1.0 times, at least 1.5 times, at least 2.0 times, at least 2.5 times, at least 3.0 times, at least 3.5 times, at least 4.0 times, at least 4.5 times, at least 5.0 times, at least 5.5 times, at least 6.0 times, at least 6.5 times, at least 7.0 times, at least 7.5 times, at least 8.0 times, at least 8.5 times, at least 9.0 times, or at least 9.5 times) of the apparent cleavage rate (or Kcat / Km) of the control linker containing SEQ ID NO: 7 cleaved by uPA.

[0151] In some embodiments, the activatable protein as described in any of the preceding embodiments, wherein the apparent cleavage rate (or Kcat / Km) of the protease-cleavable sequence described herein by MMP9 is at least 2.0 times (e.g., at least 3.0 times, at least 4.0 times, at least 5.0 times, at least 6.0 times, at least 7.0 times, at least 8.0 times, at least 9.0 times, at least 10.0 times, at least 11.0 times, at least 12.0 times, at least 13.0 times, at least 14.0 times, at least 15.0 times, at least 16.0 times, at least 17.0 times, at least 18.0 times, at least 19.0 times, at least 20.0 times, at least 21.0 times, at least 22.0 times, at least 23.0 times, at least 24.0 times, or at least 25.0 times) greater than the apparent cleavage rate (or Kcat / Km) of MMP7.

[0152] In some embodiments, the activatable protein as described in any of the preceding embodiments, wherein the apparent cleavage rate (or Kcat / Km) of the protease-cleavable sequence described herein cleaved by MMP9 is at least 16.0 times (e.g., at least 17.0 times, at least 18.0 times, at least 19.0 times, at least 20.0 times, at least 22.0 times, at least 22.5 times, at least 23.0 times, at least 25.0 times, at least 30.0 times, at least 35.0 times, at least 40.0 times, at least 45.0 times, at least 50.0 times, at least 55.0 times, at least 56.0 times, at least 57.0 times, at least 58.0 times, at least 59.0 times, at least 60.0 times, at least 61.0 times, at least 61.5 times, at least 62.0 times, at least 65.0 times, or at least 70.0 times) greater than the apparent cleavage rate (or Kcat / Km) of the MMP14 cleavage sequence.

[0153] In some embodiments, such as the activatable protein described in any of the preceding embodiments, the apparent cleavage rate (or Kcat / Km) of the protease-cleavable sequence is determined according to the steps of Example 1.3.

[0154] In some embodiments, the activatable protein as described in any of the preceding embodiments, wherein the protease-cleavable sequence has one or more properties selected from the following:

[0155] The protease-cleavable sequence was cleaved by MMP9 at a rate at least 0.5 times faster than the control linker containing SEQ ID NO: 7 was cleaved by MMP9.

[0156] The protease-cleavable sequence was cleaved by Matriptase at a rate at least 2.0 times faster than the control linker containing SEQ ID NO: 7 was cleaved by Matriptase.

[0157] The protease-cleavable sequence was cleaved by uPA at a rate at least 1.2 times faster than the control linker containing SEQ ID NO: 7 was cleaved by uPA.

[0158] The protease-cleavable sequence is cleaved by MMP9 at a rate at least 1.5 times that of MMP14.

[0159] The apparent cleavage rate (or Kcat / Km) of the protease-cleavable sequence cleaved by MMP9 is at least 1.2 times that of the apparent cleavage rate (or Kcat / Km) of the control linker containing SEQ ID NO: 7 cleaved by MMP9.

[0160] The apparent cleavage rate (or Kcat / Km) of the protease-cleavable sequence cleaved by Matriptase is at least 2.0 times that of the apparent cleavage rate (or Kcat / Km) of the control linker containing SEQ ID NO: 7 cleaved by Matriptase.

[0161] The apparent cleavage rate (or Kcat / Km) of the protease-cleavable sequence cleaved by uPA is at least 0.5 times that of the apparent cleavage rate (or Kcat / Km) of the control linker containing SEQ ID NO: 7 cleaved by uPA.

[0162] The apparent cleavage rate (or Kcat / Km) of the protease-cleavable sequence by MMP9 is at least 2.0 times that by MMP7; and

[0163] The apparent cleavage rate (or Kcat / Km) of the protease-cleavable sequence cleaved by MMP9 is at least 16.0 times that of the apparent cleavage rate (or Kcat / Km) cleaved by MMP14.

[0164] In some embodiments, the activatable protein further comprises a flexible linker. In some embodiments, the flexible linker is further attached to one or both ends of the protease-cleavable sequence. The flexible linker at one end of the protease-cleavable sequence may be referred to as a first flexible linker, and the flexible linker at the other end may be referred to as a second flexible linker. In a particular embodiment, the protease-cleavable sequence and the flexible linker have any of the following general formulas:

[0165] (Protein-cleavable sequence)

[0166] (First flexible linker) - (Protein-cleavable sequence),

[0167] (Protein-cleavable sequence) - (Second flexible linker), and

[0168] (First flexible linker) - (Protein-cleavable sequence) - (Second flexible linker).

[0169] In some embodiments, the flexible linker is preferably a peptide linker. In some embodiments, a first flexible linker and a second flexible linker may be present or absent, and each independently contains the same or different flexible linkers containing at least one flexible amino acid (Gly or Ala, etc.). In some embodiments, the flexible linker comprises, for example, a sufficient number of residues (arbitrarily selected from amino acids Arg, Ile, Gln, Glu, Cys, Tyr, Trp, Thr, Val, His, Phe, Pro, Met, Lys, Gly, Ser, Asp, Asn, Ala, etc., particularly Gly, Ser, Asp, Asn and Ala, especially Gly and Ser, especially Gly, etc.) for the protease-cleavable sequence to obtain the desired protease accessibility.

[0170] In some embodiments, the flexible linkers adapted to both ends of the protease-cleavable sequence typically have the following characteristics: they improve the protease's access to the protease-cleavable sequence and increase the protease's cleavage efficiency. The flexible linkers can be selected from different lengths, such as 1 to 20 amino acids, 2 to 15 amino acids, or 3 to 12 amino acids, including 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids. In some embodiments, the flexible linker is a peptide linker of 1 to 7 amino acids.

[0171] In some embodiments, examples of the flexible connector include, but are not limited to, glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS:SEQ ID NO: 55)n, and (GGGS:SEQ ID NO: 36)n, where n is an integer of at least 1), alanine polymers (A)n (e.g., AA), glycine-alanine polymers (e.g., GA), alanine-serine polymers (e.g., AS), and other flexible connectors known in the conventional art. Glycine polymers and glycine-serine polymers are of particular interest because these amino acids are relatively unstructured and readily function as neutral bond chains between components.

[0172] Examples of flexible joints composed of glycine-serine polymers include, but are not limited to:

[0173] Ser;

[0174] Gly·Ser(GS);

[0175] Ser·Gly (SG);

[0176] Gly·Gly·Ser (GGS);

[0177] Gly·Ser·Gly(GSG);

[0178] Ser·Gly·Gly(SGG);

[0179] Gly·Ser·Ser(GSS);

[0180] Ser·Ser·Gly(SSG);

[0181] Ser·Gly·Ser(SGS);

[0182] Gly·Gly·Gly·Ser(GGGS,SEQ ID NO:36);

[0183] Gly·Gly·Ser·Gly(GGSG,SEQ ID NO:48);

[0184] Gly·Ser·Gly·Gly(GSGG,SEQ ID NO:49);

[0185] Ser·Gly·Gly·Gly(SGGG,SEQ ID NO:50);

[0186] Gly·Ser·Ser·Gly(GSSG,SEQ ID NO:51);

[0187] Gly·Gly·Gly·Gly·Ser(GGGGS,SEQ ID NO:37);

[0188] Gly·Gly·Gly·Ser·Gly(GGGSG,SEQ ID NO:52);

[0189] Gly·Gly·Ser·Gly·Gly(GGSGG,SEQ ID NO:53);

[0190] Gly·Ser·Gly·Gly·Gly(GSGGG,SEQ ID NO:54);

[0191] Gly·Ser·Gly·Gly·Ser(GSGGS,SEQ ID NO:55);

[0192] Ser·Gly·Gly·Gly·Gly(SGGGG,SEQ ID NO:56);

[0193] Gly·Ser·Ser·Gly·Gly(GSSGG,SEQ ID NO:57);

[0194] Gly·Ser·Gly·Ser·Gly (GSGSG, SEQ ID NO: 58);

[0195] Ser·Gly·Gly·Ser·Gly (SGGSG, SEQ ID NO: 59);

[0196] Gly·Ser·Ser·Ser·Gly (GSSSG, SEQ ID NO: 60);

[0197] Gly·Gly·Gly·Gly·Gly·Ser (GGGGGS, SEQ ID NO: 61);

[0198] Ser·Gly·Gly·Gly·Gly·Gly (SGGGGG, SEQ ID NO: 62);

[0199] Gly·Gly·Gly·Gly·Gly·Gly·Ser (GGGGGGS, SEQ ID NO: 63);

[0200] Ser·Gly·Gly·Gly·Gly·Gly·Gly (SGGGGGG, SEQ ID NO: 64);

[0201] (Gly·Gly·Gly·Gly·Ser(GGGGS, SEQ ID NO:37))n;

[0202] (Ser·Gly·Gly·Gly·Gly(SGGGG, SEQ ID NO:56))n.

[0203] <Antigen-binding domain that specifically binds to the target antigen>

[0204] In some embodiments, the activatable protein, as described in any of the preceding embodiments, further comprises an antigen-binding domain that specifically binds to the target antigen.

[0205] In some embodiments, as described in any of the preceding embodiments, the antigen-binding domain that specifically binds to the target antigen is Fab, scFv, dAb (e.g., VHH, VH, or VL), Fd, Fv, dsFv, scFab, Fab′, or F(ab′)2. In some embodiments, as described in any of the preceding embodiments, the antigen-binding domain that specifically binds to the target antigen is Fab.

[0206] In some embodiments, as described in any of the preceding embodiments, the activatable protein wherein the antigen-binding domain that specifically binds to the target antigen is located at the C-terminus or N-terminus of the protease-cleavable sequence. In some embodiments, as described in any of the preceding embodiments, the antigen-binding domain that specifically binds to the target antigen is located at the C-terminus of the protease-cleavable sequence.

[0207] In some embodiments, as described in any of the preceding embodiments, the target antigen is an effector cell antigen or a tumor-associated antigen. In some embodiments, as described in any of the preceding embodiments, the target antigen is an effector cell antigen. In some embodiments, as described in any of the preceding embodiments, the target antigen is selected from T-cell antigens, B-cell antigens, and NK-cell antigens.

[0208] In some embodiments, such as the activatable protein described in any of the preceding embodiments, the target antigen is a T cell surface antigen.

[0209] In some implementations, the activatable protein as described in any of the preceding embodiments is CD3.

[0210] <Mask Peptide>

[0211] In some embodiments, the activatable protein, as described in the preceding one, also includes a masking peptide.

[0212] In some embodiments, such as the activatable protein described in any of the preceding embodiments, wherein the masking peptide inhibits the binding of the antigen-binding domain of the target antigen to the target antigen.

[0213] In some embodiments, as described in any of the preceding embodiments, the activatable protein wherein the masking peptide inhibits the binding of the antigen-binding domain to CD3. In some embodiments, as described in any of the preceding embodiments, the activatable protein wherein the masking peptide inhibits the binding of the antigen-binding domain to the N-terminus of CD3ε.

[0214] In some embodiments, of the activatable protein as described in any of the preceding claims, the masking peptide binds to the antigen-binding domain via ionic interactions, electrostatic interactions, hydrophobic interactions, π-stacking interactions, and hydrogen bonding interactions, or combinations thereof. In some embodiments, of the activatable protein as described in any of the preceding claims, the masking peptide comprises a peptide sequence of at least 1 to no more than 40 amino acids. In some embodiments, of the activatable protein as described in any of the preceding claims, the masking peptide comprises a peptide sequence of at least 1 to no more than 30 amino acids. In some embodiments, of the activatable protein as described in any of the preceding claims, the masking peptide comprises a peptide sequence of at least 1 to no more than 20 amino acids. In some embodiments, of the activatable protein as described in any of the preceding claims, the masking peptide comprises a peptide sequence of at least 1 to no more than 15 amino acids. In some embodiments, of the activatable protein as described in any of the preceding claims, the masking peptide comprises a peptide sequence of at least 5 to no more than 10 amino acids. In some embodiments, of the activatable protein as described in any of the preceding claims, the masking peptide is a peptide sequence comprising 8 amino acids.

[0215] In some embodiments, as described in any of the preceding embodiments, the activatable protein, wherein the masking peptide comprises a cyclic peptide or a linear peptide. In some embodiments, as described in any of the preceding embodiments, the activatable protein, wherein the masking peptide comprises a cyclic peptide. In some embodiments, as described in any of the preceding embodiments, the activatable protein, wherein the masking peptide comprises a linear peptide.

[0216] In some embodiments, the activatable protein as described in any of the preceding embodiments, the masking peptide comprises SEQ ID NO: 26, or an amino acid sequence having at least 80% (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with it.

[0217] In some embodiments, such as the activatable protein described in any of the preceding embodiments, the masking peptide is a masking peptide sequence derived from WO2016179003A1, WO2023104190A1, or WO2024259378A1.

[0218] In some embodiments, as described in any of the preceding embodiments, the activatable protein has the masking peptide at the N-terminus or C-terminus of the protease-cleavable sequence. In some embodiments, as described in any of the preceding embodiments, the activatable protein has the masking peptide at the N-terminus of the protease-cleavable sequence.

[0219] In some embodiments, such as the activatable protein of any of the preceding claims, wherein the masking peptide is operatively linked to the N-terminus of the protease-cleavable sequence, and the antigen-binding domain specifically binding to the target antigen is operatively linked to the C-terminus of the protease-cleavable sequence; or

[0220] The masking peptide is operatively linked to the C-terminus of the protease-cleavable sequence, and the antigen-binding domain that specifically binds to the target antigen is operatively linked to the N-terminus of the protease-cleavable sequence.

[0221] In one specific embodiment, the activatable protein as described in any of the preceding claims, wherein the masking peptide is operatively linked to the N-terminus of the protease-cleavable sequence, and the antigen-binding domain that specifically binds to the target antigen is operatively linked to the C-terminus of the protease-cleavable sequence.

[0222] <Half-life extension>

[0223] In some embodiments, the activatable protein, as described in the preceding one, also includes a half-life extension portion.

[0224] In some embodiments, such as the activatable protein described in any of the preceding embodiments, the extended half-life portion is operatively linked to the N-terminus or C-terminus of the protease-cleavable sequence. Furthermore, the extended half-life portion does not interfere with the masking effect of the masking peptide or the cleavage activity of tumor microenvironment proteases.

[0225] In some embodiments, of the activatable protein as described in any of the preceding claims, the masking peptide is further operably linked to the half-life extension portion. In some embodiments, of the activatable protein as described in any of the preceding claims, the half-life extension portion is operably linked to the N-terminus or C-terminus of the protease-cleavable sequence via the masking peptide. In some embodiments, of the activatable protein as described in any of the preceding claims, the half-life extension portion is operably linked to the N-terminus of the protease-cleavable sequence via the masking peptide, and the antigen-binding domain specifically binding to the target antigen is operably linked to the C-terminus of the protease-cleavable sequence.

[0226] In some embodiments, such as the activatable protein described in any of the preceding embodiments, wherein the extended half-life portion binds to a serum protein or a fragment thereof, a circulating immunoglobulin or a fragment thereof, or CD35 / CR1; or wherein the extended half-life portion is a natural peptide, a synthetic peptide, an engineered scaffold, or an engineered large-volume serum protein.

[0227] In some embodiments, the circulating immunoglobulin or fragment thereof, as described above, comprises IgG1, IgG2, IgG3, IgG4, slgA, IgM, or IgD, or fragment thereof.

[0228] In some embodiments, such as the activatable protein described in any of the preceding embodiments, wherein the half-life extension portion binds to a serum protein or a fragment thereof, or CD35 / CR1; or wherein the half-life extension portion is a natural peptide, a synthetic peptide, an engineered scaffold, or an engineered large-volume serum protein.

[0229] In some embodiments, the engineered scaffold includes sdAb, scFv, Fab, VHH, type III fibronectin domains, immunoglobulin-like scaffolds, DARPin, cysteine ​​knot peptides, lipid carrier proteins, triple-helix bundle scaffolds, protein G-associated albumin-binding modules, or DNA or RNA aptamer scaffolds. In some embodiments, the bulk serum protein is albumin, transferrin, IgG1, IgG2, IgG4, IgG3, IgA monomer, factor XIII, fibrinogen, IgE, or pentamer IgM.

[0230] In some embodiments, the activatable protein as described in any of the preceding claims, wherein the serum protein comprises albumin, thyroxine-binding protein, thyroxine transporter, 1-acid glycoprotein, transferrin, transferrin receptor or its transferrin-binding moiety, or fibrinogen. In some embodiments, the activatable protein as described in any of the preceding claims, wherein the serum protein is albumin. In some embodiments, the activatable protein as described in any of the preceding claims, wherein the serum protein is human serum albumin (HSA).

[0231] In some embodiments, the activatable protein as described in any of the preceding embodiments, wherein the extended half-life portion binds to a serum protein or a fragment thereof, or the extended half-life portion is a serum protein or a fragment thereof.

[0232] In some embodiments, such as the activatable protein described in any of the preceding embodiments, wherein the extended half-life portion specifically binds to human serum albumin (HSA).

[0233] In some embodiments, such as the activatable protein described in any of the preceding embodiments, the half-life extension portion is a half-life extension domain.

[0234] In some embodiments, such as the activatable protein described in any of the preceding embodiments, the extended half-life portion is an anti-HSA antibody or its antigen-binding fragment.

[0235] In some embodiments, as described in any of the preceding embodiments, the activated protein wherein the extended half-life portion is an immunoglobulin single variable domain (dAb), scFv, Fd, Fv, dsFv, Fab, scFab, Fab′, or F(ab′)2. In some embodiments, as described in any of the preceding embodiments, the activated protein wherein the immunoglobulin single variable domain is VHH.

[0236] In some embodiments, the activatable protein as described in any of the preceding embodiments, wherein the VHH is camel-derived, humanized, reverse-mutated, affinity-matured, T-cell epitope-removed, antibody deamidated, and / or antibody isomerized.

[0237] In some embodiments, the activatable protein as described in any of the preceding embodiments, wherein the extended half-life portion comprises CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 of the extended half-life portion comprises CDR1, CDR2 and CDR3 of SEQ ID NO: 12, respectively.

[0238] In some embodiments, as described in any of the preceding embodiments, the CDR1, CDR2, and CDR3 of the extended half-life portion are defined according to a numbering rule selected from Kabat, IMGT, Chothia, AbM, and Contact. In some embodiments, as described in any of the preceding embodiments, the CDR1, CDR2, and CDR3 of the extended half-life portion are defined according to the Kabat numbering rule. In some embodiments, as described in any of the preceding embodiments, the CDR1, CDR2, and CDR3 of the extended half-life portion are defined according to the IMGT numbering rule. In some embodiments, as described in any of the preceding embodiments, the CDR1, CDR2, and CDR3 of the extended half-life portion are defined according to the Chothia numbering rule. In some embodiments, as described in any of the preceding embodiments, the CDR1, CDR2, and CDR3 of the extended half-life portion are defined according to the AbM numbering rule. In some implementations, such as the activatable protein described in any of the preceding embodiments, the CDR1, CDR2, and CDR3 of the extended half-life portion are defined according to the Contact numbering rules.

[0239] In some embodiments, the activatable protein as described in any of the preceding embodiments, wherein CDR1 comprises the amino acid sequence of SEQ ID NO: 13, CDR2 comprises the amino acid sequence of SEQ ID NO: 14, and CDR3 comprises the amino acid sequence of SEQ ID NO: 15.

[0240] In some implementations, such as the activatable protein described in any of the preceding embodiments, the CDR1, CDR2, and CDR3 of the extended half-life portion are defined according to the Kabat numbering rules.

[0241] In some embodiments, the activatable protein as described in any of the preceding embodiments, the extended half-life portion comprises SEQ ID NO: 12, or an amino acid sequence having at least 80% (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with it.

[0242] <Active Proteins>

[0243] In some embodiments, the activatable protein as described in any of the preceding embodiments comprises:

[0244] (a1) Specifically binds to the first antigen-binding domain of CD3;

[0245] (b1) A second antigen-binding domain that specifically binds to tumor-associated antigens (TAAs);

[0246] (c1) The extended half-life portion;

[0247] (d1) A masking peptide that inhibits the binding of the first antigen-binding domain to CD3;

[0248] (e1) Protease-cleavable sequence.

[0249] In some embodiments, as described in any of the preceding embodiments, the first antigen-binding domain is Fab, scFv, dAb (e.g., VHH, VH, or VL), Fd, Fv, dsFv, scFab, Fab′, or F(ab′)2. In some embodiments, as described in any of the preceding embodiments, the first antigen-binding domain is Fab.

[0250] In some embodiments, as described in any of the preceding embodiments, the activatable protein wherein the second antigen-binding domain is an immunoglobulin single variable domain (e.g., VHH), scFv, Fd, Fv, dsFv, Fab, scFab, Fab′, or F(ab′)2. In some embodiments, the second antigen-binding domain is VHH.

[0251] In some embodiments, the activatable protein as described in any of the preceding embodiments comprises:

[0252] (a1) Specifically binds to the first antigen-binding domain of CD3, wherein the first antigen-binding domain is Fab;

[0253] (b1) Specifically binds to the second antigen-binding domain of TAA, wherein the second antigen-binding domain is VHH;

[0254] (c1) A half-life extension portion that specifically binds to HSA, wherein the half-life extension portion is VHH;

[0255] (d1) A masking peptide that inhibits the binding of the first antigen-binding domain to CD3;

[0256] (e1) Protease-cleavable sequence;

[0257] The half-life extension portion is operatively linked to the N-terminus of the Fab of the first antigen-binding domain via a masking peptide and a protease-cleavable sequence.

[0258] In some embodiments, the activatable protein as described in any of the preceding embodiments comprises:

[0259] (a1) Specifically binds to the first antigen-binding domain of CD3, wherein the first antigen-binding domain is Fab;

[0260] (b1) Specifically binds to the second antigen-binding domain of TAA, wherein the second antigen-binding domain is VHH;

[0261] (c1) A half-life extension portion that specifically binds to HSA, wherein the half-life extension portion is VHH;

[0262] (d1) A masking peptide that inhibits the binding of the first antigen-binding domain to CD3;

[0263] (e1) Protease-cleavable sequence;

[0264] The activatable protein, from the N-terminus to the C-terminus, includes a VHH that specifically binds to HSA, a masking peptide, a protease-cleavable sequence, a Fab that specifically binds to CD3, and a VHH that specifically binds to TAA.

[0265] In some embodiments, the activatable protein as described in any of the preceding embodiments comprises:

[0266] (a1) A first antigen-binding domain that specifically binds to CD3, wherein the first antigen-binding domain is Fab, and the Fab comprises a light chain variable region (VL), a light chain constant region (CL), a heavy chain variable region (VH), and a heavy chain constant region 1 (CH1).

[0267] (b1) A second antigen-binding domain that specifically binds to tumor-associated antigens (TAAs), wherein the second antigen-binding domain is VHH;

[0268] (c1) A half-life extension portion that specifically binds to HSA, wherein the half-life extension portion is VHH;

[0269] (d1) A masking peptide that inhibits the binding of the first antigen-binding domain to CD3;

[0270] Preferably, the masking peptide inhibits the binding of the first antigen-binding domain to the N-terminus of CD3ε;

[0271] (e1) Protease-cleavable sequence;

[0272] The half-life extension portion is operatively linked to the N-terminus of the VH or VL of the first antigen-binding domain via a masking peptide and a protease-cleavable sequence.

[0273] In some embodiments, the activatable protein as described in any of the preceding embodiments comprises:

[0274] (a1) A first antigen-binding domain that specifically binds to CD3, wherein the structure of the first antigen-binding domain is Fab, and the Fab includes a light chain variable region (VL), a light chain constant region (CL), a heavy chain variable region (VH), and a heavy chain constant region 1 (CH1).

[0275] (b1) A second antigen-binding domain that specifically binds to tumor-associated antigens (TAAs), wherein the structure of the second antigen-binding domain is VHH;

[0276] (c1) A half-life extension portion that specifically binds to HSA, wherein the structure of the half-life extension portion is VHH;

[0277] (d1) A masking peptide that inhibits the binding of the first antigen-binding domain to CD3;

[0278] Preferably, the masking peptide inhibits the binding of the first antigen-binding domain to the N-terminus of CD3ε;

[0279] (e1) Protease-cleavable sequence;

[0280] The extended half-life portion is operatively linked to the N-terminus of the VL of the first antigen-binding domain via a masking peptide and a protease-cleavable sequence, and the second antigen-binding domain is operatively linked to the C-terminus of CH1, the C-terminus of CL, or the N-terminus of VH of the first antigen-binding domain; or

[0281] The extended half-life portion is operatively linked to the N-terminus of the VH of the first antigen-binding domain via a masking peptide and a protease-cleavable sequence, and the second antigen-binding domain is operatively linked to the C-terminus of CH1, the C-terminus of CL, or the N-terminus of VL of the first antigen-binding domain.

[0282] In some embodiments, the activatable protein as described in any of the preceding embodiments comprises a first strand having the structure shown in formula (a) and a second strand having the structure shown in formula (b), wherein the structures shown in formulas (a) and (b) are arranged from the N-terminus to the C-terminus, wherein:

[0283] (a)[α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[connector 3]-[CL];

[0284] (b)[α-CD3-VH]-[linker 4]-[CH1]-[linker 2]-[α-TAA-VHH];

[0285] Wherein, linker 1, linker 2, linker 3 and linker 4 are the same or different peptide linkers, or linker 3 and linker 4 are not present; linker 1 contains a masking peptide and a protease-cleavable sequence sequentially from the N-terminus to the C-terminus, wherein the masking peptide is as defined in the preceding item, and the protease-cleavable sequence is as defined in the preceding item.

[0286] The CL and CH1 are as defined in the preceding item; the α-HSA-VHH is as defined in the preceding item as the VHH that specifically binds the extended half-life portion of HSA; the α-CD3-VH and α-CD3-VL are as defined in the preceding item as the heavy chain variable region and light chain variable region of the first antigen-binding domain that specifically binds CD3; the α-TAA-VHH is as defined in the preceding item as the VHH that specifically binds the second antigen-binding domain of TAA.

[0287] In some embodiments, the activatable protein as described in any of the preceding embodiments comprises a first strand having the structure shown in formula (a) and a second strand having the structure shown in formula (b), wherein the structures shown in formulas (a) and (b) are arranged from the N-terminus to the C-terminus, wherein:

[0288] (a)[α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL];

[0289] (b)[α-CD3-VH]-[CH1]-[connector 2]-[α-TAA-VHH];

[0290] Wherein, linker 1 and linker 2 are the same or different peptide linkers, or linker 2 is not present; linker 1 contains, from N-terminus to C-terminus, a masking peptide and a protease-cleavable sequence, wherein the masking peptide is as defined in the preceding item, and the protease-cleavable sequence is as defined in the preceding item.

[0291] The CL and CH1 are as defined in the preceding item; the α-HSA-VHH is as defined in the preceding item as the VHH that specifically binds the extended half-life portion of HSA; the α-CD3-VH and α-CD3-VL are as defined in the preceding item as the heavy chain variable region and light chain variable region of the first antigen-binding domain that specifically binds CD3; the α-TAA-VHH is as defined in the preceding item as the VHH that specifically binds the second antigen-binding domain of TAA.

[0292] In some embodiments, the activatable protein as described in any of the preceding embodiments comprises a first strand having the structure shown in formula (a) and a second strand having the structure shown in formula (b), wherein the structures shown in formulas (a) and (b) are arranged from the N-terminus to the C-terminus, wherein:

[0293] (a) [α-HSA-VHH]-[masking peptide]-[protease-cleavable sequence]-[α-CD3-VL]-[CL];

[0294] (b)[α-CD3-VH]-[CH1]-[connector 2]-[α-TAA-VHH];

[0295] Wherein the linker 2 is an uncleavable linker, the masking peptide is as defined in the preceding item, and the protease cleavable sequence is as defined in the preceding item;

[0296] The CL and CH1 are as defined in the preceding item; the α-HSA-VHH is as defined in the preceding item as the VHH that specifically binds the extended half-life portion of HSA; the α-CD3-VH and α-CD3-VL are as defined in the preceding item as the heavy chain variable region and light chain variable region of the first antigen-binding domain that specifically binds CD3; the α-TAA-VHH is as defined in the preceding item as the VHH that specifically binds the second antigen-binding domain of TAA.

[0297] In some embodiments, as described in any of the preceding embodiments, the linker 2, linker 3, and linker 4 are non-cleavable linkers. In some embodiments, as described in any of the preceding embodiments, the linker 2, linker 3, and linker 4 have an amino acid sequence selected from SEQ ID NO: 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, and 47.

[0298] In some embodiments, the activatable protein as described in any of the preceding claims further comprises one or more identical or different antigen-binding domains that specifically bind to the target antigen. In some embodiments, the activatable protein as described in any of the preceding claims contains an antigen-binding domain that specifically binds to the target antigen as defined in any of the preceding claims. In some embodiments, the activatable protein as described in any of the preceding claims contains an antigen-binding domain that specifically binds to the target antigen that specifically binds to tumor-associated antigens and / or effector cell antigens.

[0299] In some embodiments, the activatable protein as described in any of the preceding claims comprises a first chain and a second chain. In some embodiments, the activatable protein as described in any of the preceding claims, wherein the first chain and the second chain form a heterodimer.

[0300] In some embodiments, as described in any of the preceding embodiments, the activatable protein wherein the first and second chains are linked by disulfide bonds in CH1 and CL to form a heterologous protein. In some embodiments, as described in any of the preceding embodiments, the positions of CH1 and CL may be interchanged.

[0301] In other embodiments, the activatable protein as described in any of the preceding embodiments comprises a first strand having the structure shown in formula (a') and a second strand having the structure shown in formula (b'), wherein the structures shown in formula (a') and formula (b') are arranged from the N-terminus to the C-terminus, wherein:

[0302] (a')[α-HSA-VHH]-[masking peptide]-[protease-cleavable sequence]-[α-CD3-VL]-[CH1];

[0303] (b')[α-CD3-VH]-[CL]-[connector 2]-[α-TAA-VHH];

[0304] The linker 2, the masking peptide, the protease-cleavable sequence, the CL, CH1, the α-HSA-VHH, the α-CD3-VH and α-CD3-VL, and the α-TAA-VHH are as defined in any of the preceding items.

[0305] In some embodiments, such as the activatable protein described in the preceding claim, the sequences of the first strand and the second strand are different. In some embodiments, such as the activatable protein described in the preceding claim, the first strand and the second strand are used only to distinguish amino acid sequences and do not limit the positional relationship of the polypeptide and protein. When either one is the first strand, the other is the second strand.

[0306] In some embodiments, the activatable protein as described in any of the preceding claims is an activatable antibody. In some embodiments, the activatable protein as described in any of the preceding claims is a multispecific activatable antibody. In some embodiments, the activatable protein as described in any of the preceding claims is a trispecific activatable antibody.

[0307] In some embodiments, the activatable protein as described in any of the preceding embodiments does not contain an Fc domain. In some embodiments, the activatable protein as described in any of the preceding embodiments does not contain a CH2 domain. In some embodiments, the activatable protein as described in any of the preceding embodiments does not contain a CH3 domain.

[0308] In some embodiments, such as the activatable protein described in any of the preceding embodiments, the inhibition of antigen-binding activity of the antigen-binding domain (e.g., the first antigen-binding domain) by the masking peptide is weaker when the protease-cleavable sequence is cleaved by the protease than when the inhibition of antigen-binding activity of the antigen-binding domain (e.g., the first antigen-binding domain) by the masking peptide is in the state where the protease-cleavable sequence is not cleaved.

[0309] In some embodiments, such as the activatable protein described in any of the preceding embodiments, the protease-cleavable sequence of the activatable protein has a shorter half-life after being cleaved by the protease than before cleavage.

[0310] In some embodiments, such as the activatable protein described in any of the preceding embodiments, wherein the antigen-binding domain (e.g., a first antigen-binding domain) is released from the activatable protein after the protease-cleavable sequence of the activatable protein is cleaved by a protease, and wherein the antigen-binding domain (e.g., the first antigen-binding domain) has higher antigen-binding activity in its released state from the activatable protein than in its unreleased state from the activatable protein.

[0311] In some embodiments, such as the activatable protein described in any of the preceding embodiments, the antigen-binding activity of the antigen-binding domain (e.g., the first antigen-binding domain) is inhibited by association of the masking peptide with the antigen-binding domain (e.g., the first antigen-binding domain).

[0312] In some embodiments, such as the activatable protein described in any of the preceding embodiments, the protease-cleavable sequence is cleaved by the protease such that the antigen-binding domain (e.g., a first antigen-binding domain) is released from the masking peptide or / and the association between the masking peptide and the antigen-binding domain (e.g., the first antigen-binding domain) is canceled.

[0313] In another aspect, this disclosure provides a conjugate comprising an activatable protein and a payload or a pharmaceutically acceptable salt thereof as described in any of the preceding claims.

[0314] In some embodiments, such as the conjugates described above, the activatable protein and the payload or a pharmaceutically acceptable salt thereof are connected via a connector.

[0315] In another aspect, this disclosure provides a protease-cleavable sequence, a polypeptide, or a pharmaceutically acceptable salt thereof, including a solvate (e.g., a hydrate, a non-aqueous solvate) or a non-solvent of the protease-cleavable sequence, the polypeptide, or a pharmaceutically acceptable salt thereof as described in any of the preceding claims.

[0316] In another aspect, this disclosure provides a pharmaceutical composition comprising a polypeptide as described in any of the preceding claims, an activatable protein as described in any of the preceding claims, or a conjugate as described in any of the preceding claims, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0317] In another aspect, this disclosure provides one or more nucleic acid molecules (e.g., isolated nucleic acid molecules) that encode a protease-cleavable sequence as described in any of the preceding claims, or an activatable protein as described in any of the preceding claims.

[0318] In another respect, this disclosure provides one or more carriers that contain nucleic acid molecules as described in the previous one.

[0319] In another aspect, this disclosure provides one or more host cells that contain nucleic acid molecules as described in the previous one.

[0320] In another aspect, this disclosure provides a method for preparing a protease-cleavable sequence or an activatable protein as described in any of the preceding claims, the method comprising expressing one or more nucleic acid molecules (e.g., isolated nucleic acid molecules) as described in any of the preceding claims, or culturing one or more host cells as described in any of the preceding claims to produce the protease-cleavable sequence or the activatable protein.

[0321] In another aspect, this disclosure provides a method for treating, preventing, or improving a disease or condition, comprising administering to a subject in need a therapeutically effective amount of a polypeptide, an activatable protein, a conjugate, or a pharmaceutical composition as described in any of the preceding claims.

[0322] In another aspect, this disclosure provides use in the preparation of a medicament for treating, preventing, or improving a disease or condition, comprising administering to a subject a therapeutically effective amount of a polypeptide, an activatable protein, a conjugate, or a pharmaceutical composition as described in any of the preceding claims.

[0323] In another aspect, this disclosure provides a polypeptide, an activatable protein, a conjugate, or a pharmaceutical composition as described in any of the preceding claims, for use as a medicament.

[0324] In some embodiments, the disease or condition is a tumor, autoimmune disease, inflammatory disease, infection, cardiovascular disease, endocrine and metabolic disease, chronic respiratory disease, digestive disease, urinary disease, rheumatoid and osteoarthritis, or neurological and psychiatric disease (such as neurodegenerative diseases). In some embodiments, the disease or condition is a tumor, autoimmune disease, or inflammatory disease. In some embodiments, the disease or condition is a tumor. In some embodiments, the tumor is a solid tumor. In some embodiments, the disease or condition is associated with abnormal expression of TAA.

[0325] In some embodiments, the activatable protein comprising the protease-cleavable sequence disclosed herein has an increased rate of proteolysis, improved serum stability, improved in vitro tumor cell killing, improved or equivalent plasma stability in cynomolgus monkeys, improved or equivalent pharmacokinetic parameters in cynomolgus monkeys, improved hepatotoxicity levels in cynomolgus monkeys, and good drug-likeness.

[0326] Increased MMP9 secretion induced by T cell activation has the potential to promote the activation of the cascade of activatable antibodies. MMP9 upregulation induced by TCE administration can achieve the cascade activation of activatable antibodies, thereby enhancing drug efficacy. However, members of the MMP family share high homology in their catalytic domains and substrate similarity, and exhibit different normal tissue expression profiles. These characteristics increase the risk of extratumor activation of protease-cleavable sequences. Improving the MMP9 specificity of cleavable sequences is one strategy to reduce their extratumor non-specific activation. Therefore, MMP7 and MMP14 can be selected as representatives of the other two phylogenetic branches (PNAS. 2014, 7, 111(40): E4148-55) in the MMPs phylogenetic tree to evaluate the selectivity of protease-cleavable sequences for MMP family proteases. Attached Figure Description

[0327] Figure 1 is a schematic diagram of the structure of F1_B, where the black solid line represents the linker and the light gray rectangle on the black solid line represents the CD3 masking peptide. Detailed Implementation

[0328] Terminology (Definition)

[0329] To facilitate understanding of this disclosure, certain technical and scientific terms are described below. Unless otherwise expressly defined in this disclosure, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0330] The singular forms “a,” “an,” and “the” used in the specification and claims include plural references unless the context clearly indicates otherwise.

[0331] Unless the context clearly requires otherwise, the words “comprising,” “having,” “including,” etc., in the patent specification and claims should be understood as “including but not limited to,” rather than as exclusive or exhaustive.

[0332] The term "and / or" implies both "and" and "or". For example, the phrase "A, B and / or C" is intended to cover each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0333] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem., 243, p3558 (1968).

[0334] The term "CD3" refers to an antigen expressed on T cells as a portion of the multimolecular T cell receptor (TCR), composed of homodimers or heterodimers formed from two of the following four receptor chains: CD3-ε (CD3E or CD3 epsilon), CD3-δ (CD3D), CD3-ζ, and CD3-γ. Human CD3-ε (hCD3ε) contains the amino acid sequence described in UniProtKB / Swiss-Prot: P07766.2. Human CD3-δ (hCD3δ) contains the amino acid sequence described in UniProtKB / Swiss-Prot: P04234.1. Therefore, unless explicitly stated as originating from a non-human species, such as "mouse CD3," "monkey CD3," etc., the term "CD3" refers to human CD3.

[0335] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., the α-carbon bound to hydrogen, carboxyl, amino, and R groups), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. These analogs have modified R groups (e.g., ortholeucine) or modified peptide backbones but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are chemical compounds that have a structure different from the general chemical structure of amino acids but function in a manner similar to naturally occurring amino acids.

[0336] The term "amino acid mutation" includes amino acid substitution (also known as amino acid replacement), deletion, insertion, and modification. Any combination of substitution, deletion, insertion, and modification can be performed to achieve the final construct, provided that the final construct possesses the desired properties, such as reduced or absent binding to Fc receptors. Amino acid sequence deletions and insertions include deletions and insertions at the amino and / or carboxyl ends of the polypeptide chain. A specific amino acid mutation can be an amino acid substitution. In some embodiments, an amino acid mutation is a non-conservative amino acid substitution, i.e., replacing one amino acid with another amino acid that has a different structure and / or chemical properties. Amino acid substitution includes substitution by non-naturally occurring amino acids or by derivatives of 20 naturally occurring amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. Methods other than genetic engineering that alter amino acid side chain groups, such as chemical modification, are also expected to be available. Various names may be used in this disclosure to refer to the same amino acid mutation. In this disclosure, the amino acid residue at a specific site may be represented by the format "position + amino acid residue". For example, 102S indicates that the amino acid residue at position 102 is S. C102S indicates that the amino acid residue at position 102 has mutated from C to S. When the residue at a specific site is defined in the claim using the format "position + amino acid residue", the original residue at that site does not limit the scope of protection.

[0337] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably in this document.

[0338] The term “antibody” is used in the broadest sense and covers a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), activatable antibodies, antibody fragments, and antigen-binding fragments (or antigen-binding portions), as long as they exhibit the desired antigen-binding activity.

[0339] The term "antigen-binding molecule" is used in the broadest sense to encompass molecules that specifically bind antigens, including but not limited to antibodies, other peptides with antigen-binding activity, and antibody fusion proteins formed by the fusion of both, as well as any molecule containing the aforementioned activatable proteins, antibodies, peptides, and antibody fusion proteins, provided they exhibit the desired antigen-binding activity. The antigen-binding molecules disclosed herein comprise a variable region (VH) and a variable region (VL), which together constitute the antigen-binding domain. The antigen-binding molecules disclosed herein comprise a single variable domain of an immunoglobulin (e.g., a single-domain antibody, nanobody, VHH). Exemplarily, the antigen-binding molecules in this disclosure are multispecific antibodies or multispecific activatable antibodies (e.g., trispecific antibodies or trispecific activatable antibodies, pentaspecific antibodies or pentaspecific activatable antibodies).

[0340] The terms “activatable protein,” “activatable antibody,” “protease-activated antibody,” and “intact activatable antibody” are used interchangeably herein to refer to recombinant “masked” or “masked” binding compounds designed to exhibit antibody-like binding specificity to biological targets only after activation by exposure to certain proteases. Structurally, an activatable protein comprises at least: an antigen-binding domain, a masking portion (e.g., a masking peptide), and a cleavable portion (e.g., a protease-cleavable sequence).

[0341] The term "natural antibody" refers to naturally occurring immunoglobulin molecules. For example, natural IgG antibodies are heterotetraglycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH, also known as the variable heavy domain or heavy chain variable region), followed by a heavy chain constant region. The natural IgG heavy chain constant region typically contains three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL, also known as the variable light domain or light chain variable domain), followed by a constant light domain (light chain constant region, CL).

[0342] The term "operably linked" refers to a functional relationship between two or more peptide or polypeptide domains or nucleic acid (e.g., DNA) segments. In this disclosure, the term "operably linked" means linking two or more amino acid segments to produce a functional polypeptide. For example, in the context of antigen-binding molecules of this disclosure, individual antigen-binding domains may be linked directly or via peptide linkers. In the context of nucleic acids encoding fusion proteins, such as polypeptide chains of antigen-binding molecules of this disclosure, "operably linked" means linking two nucleic acids such that the amino acid sequences encoded by the two nucleic acids remain within the frame.

[0343] The term "association" in this disclosure refers to the state in which two or more polypeptide regions interact with each other. Typically, hydrophobic bonds, hydrogen bonds, ionic bonds, etc., are formed between the intended polypeptide regions to form an associated product. As a common example of association, antibodies, represented by natural antibodies, are known to retain the paired structure of the heavy chain variable region (VH) and the light chain variable region (VL) through non-covalent bonds between them. In some embodiments of this disclosure, the masking peptide associates with an antigen-binding domain.

[0344] The term "release" refers to the separation of two domains of a polypeptide.

[0345] The term "variable region" or "variable domain" in an antibody refers to the domain in the antibody heavy or light chain involved in antibody binding to the antigen. In this disclosure, the antibody heavy chain variable region (VH) and light chain variable region (VL) each contain four conserved frame regions (FRs) and three complementarity-determining regions (CDRs). The term "complementarity-determining region" or "CDR" refers to the region within the variable domain that primarily facilitates antigen binding; "frame" or "FR" refers to the variable domain residues other than the CDR residues. The VH contains three CDR regions: HCDR1, HCDR2, and HCDR3; the VL contains three CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus (also known as the N-terminus) to the carboxyl terminus (also known as the C-terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0346] The amino acid sequence boundaries of CDRs can be determined using various well-known schemes, such as the "Kabat" numbering rule, the "Chothia" numbering rule, the "ABM" numbering rule, the "contact" numbering rule, and the ImMunoGenTics (IMGT) numbering rule. The correspondence between various numbering systems is well known to those skilled in the art and is exemplified as shown in Table 2 below.

[0347] Table 2. Relationship between CDR numbering systems

[0348] Unless otherwise stated, the variable regions and CDRs in this disclosure embodiment are governed by the "Kabat" numbering rule. Although the Kabat numbering rule is used in specific implementations to define amino acid residues, corresponding technical solutions using other numbering systems are considered equivalent.

[0349] The term "antibody fragment" refers to a molecule that is distinct from the intact antibody but contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, dsFv, Fab, Fab′, Fab′-SH, Fd, F(ab′)2, and single-domain antibodies (sdAbs, such as VH, VL, VHH, or V). HH ), single-chain Fab (scFab), biantibody, linear antibody, single-chain antibody (e.g., scFv, sc(Fv)2); and multispecific antibodies formed from antibody fragments.

[0350] "Antigen-binding fragments" encompass full-length antibodies, Fab, modified Fab, Fab', Fab'-SH, modified Fab', F(ab')2, Fv, dsFv, Fab-Fv, Fab-dsFv, Fd, single-domain antibodies (sdAb, such as VH or VL or VHH), single-chain Fab (scFab), single-chain antibodies (such as scFv, sc(Fv)2), biantibodies, linear antibodies, bivalent or trivalent or quadrivalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody, and epitope-binding fragments of any of the above (see, for example, Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). Methods for generating and preparing these antigen-binding fragments are well known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

[0351] "Immunoglobulin single variable domain" is generally used to refer to an immunoglobulin variable domain (which can be a heavy chain or light chain domain, including VH, VHH, or VL domains) that can form a functional antigen-binding site without interacting with other variable domains (e.g., without the VH / VL interaction required between the VH and VL domains of a conventional four-chain monoclonal antibody). Examples of "immunoglobulin single variable domain" include nanobodies (including VHH, humanized VHH, and / or camelified VH, such as camelified human VH), IgNAR, domains, and (single-domain) antibodies (such as dAbs) that are VH domains or derived from VH domains. TM ) and antibodies that are VL domains or derived from VL domains (such as dAbs) TM Immunoglobulin single variable domains based on and / or derived from heavy chain variable domains (such as VH or VHH domains) are generally preferred. A specific example of an immunoglobulin single variable domain is the “VHH domain” (or simply “VHH”) as defined below.

[0352] The antigen-binding site of a "single-domain antibody" is located on and formed by a single variable domain of an immunoglobulin. This distinguishes a "single-domain antibody" from "conventional" immunoglobulins or fragments thereof (such as Fab, scFv, etc.) (where two immunoglobulin variable domains, specifically two variable domains, interact to form the antigen-binding site). Typically, in conventional immunoglobulins, the heavy chain variable domain (VH) and the light chain variable domain (VL) interact to form the antigen-binding site. In this case, the complementarity-determining regions (CDRs) of both VH and VL contribute to the antigen-binding site; a total of six CDRs are involved in the formation of the antigen-binding site. Conversely, the binding site of a single-domain antibody is formed by a single VH, VHH, or VL domain. Therefore, the antigen-binding site of a single variable domain of an immunoglobulin is formed by no more than three CDRs.

[0353] The term "VHH domain," also known as heavy chain single-domain antibody, VHH, V HThe H domain, VHH antibody fragment, VHH antibody, and nanobody are variable domains of antigen-binding immunoglobulins called "heavy chain antibodies" (i.e., "antibodies lacking light chains") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)). The term "VHH" is used to distinguish the variable domain from the heavy chain variable domain (referred to herein as the "VH domain" or "VH") present in conventional 4-chain antibodies and the light chain variable domain (referred herein as the "VL domain" or "VL") present in conventional 4-chain antibodies. The VHH domain can specifically bind epitopes in the absence of other antigen-binding domains (unlike the VH or VL domains in conventional 4-chain antibodies, where the epitope is recognized by both the VL and VH domains).

[0354] The VHH domain is a small, stable, and highly efficient antigen recognition unit formed by a single immunoglobulin domain. In some cases, the VHH is derived from naturally occurring antibodies lacking the light chain found in camels or cartilaginous fish, or from synthetic and non-immunogenic VHHs that can be constructed accordingly. In some cases, the VHH is a natural VHH, such as a VHH derived from camels, or a recombinant protein containing a heavy-chain variable domain. In some embodiments, the VHH is derived from species selected from camels, llamas, vicuñas, guanacos, and cartilaginous fish (e.g., but not limited to sharks). In some embodiments, the VHH is derived from alpacas (e.g., but not limited to Huacaya alpaca or Suri alpaca). In this disclosure, the terms VHH domain, VHH, VHH antibody fragment, VHH antibody, and "nanobody" and "single-domain antibody" are used interchangeably and refer to an immunoglobulin single variable domain having a FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 structure and specifically binding to an epitope without the presence of another immunoglobulin variable domain.

[0355] VHHs include, but are not limited to, natural antibodies produced by camelids, or antibodies produced by camelids that have been humanized, or those obtained through phage display technology. The total number of amino acid residues in a VHH will typically be in the range of 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein. Methods for obtaining VHHs that bind to specific antigens or epitopes have previously been disclosed in the following literature: R. van der Linden et al., Journal of Immunological Methods, 240(2000)185-195; Li et al., J Biol Chem., 287(2012)13713-13721; Deffar et al., African Journal of Biotechnology Vol.8(12), pp.2645-2652, 17June, 2009 and WO94 / 04678.

[0356] In this disclosure, the term "α-HSA-VHH" refers to a VHH antibody that binds to HSA. The term "α-CD3-VL" refers to the light chain variable region of an anti-CD3 antibody. The term "α-CD3-VH" refers to the heavy chain variable region of an anti-CD3 antibody. The term "α-TAA-VHH" refers to a VHH antibody that binds to TAA. The term "α-target A-VHH" refers to a VHH antibody that binds to target A.

[0357] As is known in the art regarding VH and VHH domains, the total number of amino acid residues in each CDR may differ and may not correspond to the total number of amino acid residues indicated by the Kabat number (i.e., one or more positions according to the Kabat number may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat number). This means that, in general, the Kabat number may or may not correspond to the actual number of amino acid residues in the actual sequence. Other numbering systems or encoding rules include Chothia, IMGT, and AbM.

[0358] The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of an antibody heavy chain, including both native and engineered Fc regions. In some embodiments, the Fc region comprises two identical or different subunits. Suitable Fc regions for the antibodies described herein include the Fc regions of human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. In some embodiments, the boundaries of the Fc region may also vary, for example, by deleting the C-terminal lysine (residue 447 according to the EU numbering system) or by deleting both the C-terminal glycine and lysine (residues 446 and 447 according to the EU numbering system). Unless otherwise stated, the Fc region is numbered according to the EU numbering system, also known as the EU index.

[0359] The Fc region can be appropriately obtained by partially digesting IgG monoclonal antibodies with proteolytic enzymes such as pepsin, followed by eluting the components adsorbed on the protein A or protein G column. As the proteolytic enzyme, any enzyme capable of restrictively digesting full-length antibodies to produce Fab and F(ab')2 by appropriately setting the enzyme reaction conditions such as pH is acceptable; there is no particular limitation, and examples include pepsin and papain.

[0360] In this disclosure, the term "Fc region" or "Fc domain" refers to an antibody region that contains at least a CH2 domain and a CH3 domain. In this disclosure, the term "CH2 region" or "CH2 domain" is intended to refer to the CH2 region of an immunoglobulin. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to the EU numbering system (according to the IMGT website). However, the CH2 region can also be any other antibody isotype as described in this disclosure.

[0361] In this disclosure, the terms “CH3 region,” “CH3 domain,” or “CH3 structural domain” are intended to refer to the CH3 region of an immunoglobulin. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to the EU numbering system (according to the IMGT website). However, the CH3 region can also be any other antibody isotype as described in this disclosure.

[0362] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a specific source or species, while the remaining portion of the heavy and / or light chain is derived from another different source or species.

[0363] The terms "human antibody," "fully human antibody," and "completely human antibody" are used interchangeably, referring to antibodies whose variable and constant regions are human sequences. This term encompasses antibodies derived from human genes but with sequence alterations, such as reduced potential immunogenicity, increased affinity, or the elimination of cysteine ​​residues or glycosylation sites that might cause undesirable folding. This term also covers antibodies recombined in non-human cells (which may confer glycosylations not characteristic of human cells). The term also includes antibodies generated in transgenic mice containing some or all human immunoglobulin heavy and light chain loci. The meaning of "human antibody" explicitly excludes humanized antibodies containing non-human antigen-binding residues.

[0364] The term "pre-existing antidrug antibody" or "pre-ADA" refers to ADA already present in a subject or individual to whom a drug (e.g., a protein or peptide drug, more specifically, an antibody drug) will be administered. Pre-existing antidrug antibodies may be present in a subject or individual being used for the first time in an experiment (i.e., a subject or individual to whom the drug has never been previously administered). In some embodiments, the C-terminus of the protein described in this disclosure, the nanobody (VHH), is modified to reduce its binding to pre-ADA or ADA, such as that present in serum. Reduced binding to pre-ADA or ADA means that the molecule binds to pre-ADA (or ADA) with reduced affinity or reduced avidity.

[0365] In some embodiments, the C-terminal modification includes C-terminal modifications derived from WO2023093899A1 (included herein by reference in its entirety) and CN202111429892.3, and the patents that have priority to these patents (included herein by reference in its entirety). Other techniques used in this disclosure for C-terminal modification to reduce antibody binding to pre-ADA are also well known in the art, such as WO2012175741A3 (Ablynx), WO2013024059A3 (GSK), and WO2015173325A2 (Ablynx).

[0366] The term "affinity" refers to the overall strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Unless otherwise specified, as used herein, binding "affinity" refers to internal binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its ligand Y can typically be represented by the dissociation constant (KD). Affinity can be measured using conventional methods known in the art, including those described herein.

[0367] As used herein, the term "kassoc" or "ka" refers to the association rate of a specific antibody-antigen interaction, and the term "kdis" or "kd" refers to the dissociation rate of a specific antibody-antigen interaction. The term "KD" refers to the dissociation constant, which is derived from the ratio of kd to ka (i.e., kd / ka) and expressed as a molar concentration (M). The KD value of an antibody can be determined using methods known in the art. For example, it can be measured using a biosensing system such as a system for measuring surface plasmon resonance (e.g., Biacore), or by measuring affinity in solution using solution equilibrium titration (SET).

[0368] The term "surface plasmon resonance" refers to the optical phenomenon of analyzing real-time interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIAcore™ system (Biacore LifeSciences division of GE Healthcare, Piscataway, NJ).

[0369] The term "effector function" refers to biological activities attributable to the antibody's Fc region (either the native Fc region or the Fc region with amino acid sequence mutations) and that vary across antibody isotypes. Examples of antibody effector functions include, but are not limited to: C1q binding and complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0370] The term "effect cell antigen" refers to molecules expressed by effector cells, including but not limited to cell surface molecules such as proteins, glycoproteins, or lipoproteins. Exemplary effector cell antigens include proteins of the CD3 complex or T cell receptors (TCRs), CD4, CD8, CD25, CD38, CD69, CD45RO, CD57, CD95, CD107, and CD154, as well as effector molecules such as cytokines that are associated with, bound to, expressed within, or expressed and released by effector cells.

[0371] The term "monoclonal antibody" refers to a group of substantially homogeneous antibodies, meaning that the antibody molecules contained in this group have the same amino acid sequence, except for the possible small number of naturally occurring mutations. In contrast, polyclonal antibody formulations typically contain multiple different antibodies with different amino acid sequences in their variable structural domains, and they generally specifically target different epitopes. "Monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. In some embodiments, the antibodies provided in this disclosure are monoclonal antibodies.

[0372] The term "antigen" refers to a molecule or molecular part that can be bound by antigen-binding proteins, including, for example, antibodies. An antigen may have one or more epitopes that can interact with different antigen-binding proteins, such as antibodies.

[0373] The term "epitope" refers to a region on an antigen that is capable of specifically binding to an antibody or its antigen-binding fragment. Epitopes can be formed from a continuous string of amino acids (linear epitopes) or contain discontinuous amino acids (conformal epitopes), for example, due to the folding of the antigen (i.e., the tertiary folding of an antigen as a protein). The difference between conformational and linear epitopes is that in the presence of a denaturing solvent, the antibody loses binding to the conformational epitope. An epitope contains at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind to a specific epitope (i.e., those that bind the same epitope) can be performed using methods routine in the art, such as, but not limited to, alanine scanning, peptide blotting, peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen (see Prot. Sci. 9 (2000) 487-496), and cross-blocking.

[0374] The terms "capable of specific binding," "specific binding," or "binding" refer to the ability of an antibody to bind to a specific antigen or epitope with a higher affinity than to other antigens or epitopes. In some embodiments, the KD of antibody binding to an antigen is 10% or less (e.g., 1%) of the KD of the antibody binding to a nonspecific antigen (e.g., BSA, casein). KD can be measured using known methods, such as by... Surface plasmon resonance assays are used to measure this. However, antibodies that specifically bind to antigens or epitopes within antigens may be cross-reactive to other related antigens, for example, to corresponding antigens from other species (homologous) (such as humans or monkeys, such as the cynomolgus (cyno), chimpanzee (chimp), or common marmoset (marmoset)).

[0375] The term "non-binding" means that the antibody cannot be detected to bind to an antigen or its epitope in the manner described above specific binding.

[0376] The terms “antibody-dependent cell cytotoxicity,” “antibody-dependent cell-mediated cytotoxicity,” or “ADCC” refer to mechanisms that induce cell death that rely on the interaction between antibody-coated target cells and lytic effector cells (such as natural killer (NK) cells, monocytes, macrophages, and neutrophils) via Fcγ receptors (FcγR) expressed on the effector cells. For example, NK cells express FcγRIIIa, while monocytes express FcγRI, FcγRII, and FcγRIIIa. The ADCC activity of the antibodies described herein can be assessed in vitro using cells expressing the antigen as target cells and NK cells as effector cells. Cell lysis is detected based on the release of markers (e.g., radioactive substrates, fluorescent dyes, or native intracellular proteins) from lysed cells.

[0377] The term "antibody-dependent phagocytosis (ADCP)" refers to the mechanism by which antibody-coated target cells are eliminated through internalization by phagocytes (such as macrophages or dendritic cells).

[0378] The term "complement-dependent cytotoxicity" or "CDC" refers to a mechanism that induces cell death in which the Fc effector domain of a target-binding antibody binds to and activates the complement component C1q. C1q then activates the complement cascade, leading to target cell death. Activation of complement can also result in the deposition of complement components on the surface of target cells, which promote CDC by binding to complement receptors (e.g., CR3) on leukocytes.

[0379] The terms “tumor microenvironment” (TME), “cancer microenvironment”, and “tumor environment” are used interchangeably, possessing the same properties and meanings and encompassing the microenvironment in which tumors develop. While the normal cellular microenvironment can inhibit the growth of malignant cells, changes occurring in the tumor microenvironment can synergistically support cell proliferation.

[0380] Furthermore, tumor-secreted proteins alter the microenvironment by contributing growth factors and proteases that degrade the extracellular matrix and influence cell motility and adhesion. Stromal cells secrete ECM proteins, cytokines, growth factors, proteases, protease inhibitors, and endoglucosidases such as heparanase. Matrix metalloproteinases (MMPs) are important secreted proteins closely associated with cancer development. Tumor-associated epithelial cells express MMPs at higher levels compared to normal epithelial cells. In some embodiments, the tumor microenvironment contains enhanced protease activity compared to the non-tumor environment.

[0381] The term "C-terminus" of a polypeptide, such as carboxyl-terminus, C-terminus, C-tail, C-terminus, C-terminus, or COOH-terminus, is the end of an amino acid chain (protein or polypeptide) terminated by a free carboxyl group (-COOH). When a protein is translated from messenger RNA, it is produced from the N-terminus to the C-terminus. The convention for writing peptide sequences is to place the C-terminus on the right and write the sequence from N-terminus to C-terminus. In some embodiments, the C-terminus of a polypeptide includes up to the last amino acid residue of the polypeptide, which contributes its amino group to form a peptide bond with the carboxyl group of its adjacent amino acid residue.

[0382] The term "N-terminus" of a polypeptide, such as amino-terminus, NH2-terminus, N-terminus, N-terminus, N-terminus, or amine-terminus, is the start of a protein or polypeptide and refers to the free amino group (-NH2) located at the end of the polypeptide. Normally, the amino group bonds to another carboxyl group in the protein to make it chained, but since only one of the two regions at the end of a protein is chained, the free amino group refers to the N-terminus. As mentioned above, by convention, peptide sequences in LTR language are written from N-terminus to C-terminus, from left to right. This associates the translation direction with the text direction (because when a protein is translated from messenger RNA, it is produced from N-terminus to C-terminus—an amino acid is added to the carbonyl terminus). In some embodiments, the N-terminus of the polypeptide contains the first amino acid of the polypeptide, which contributes its carboxyl group to form a peptide bond with the amino group of its adjacent amino acid residue.

[0383] The term "nucleic acid" is used interchangeably with the term "polynucleotide" in this disclosure and refers to deoxyribonucleotides or ribonucleotides and their polymers in single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, or non-naturally occurring, have similar binding properties to a reference nucleic acid, and are metabolized in a manner similar to that of a reference nucleotide. Examples of such analogs include, but are not limited to, thiophosphates, aminophosphates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide-nucleic acids (PNAs). "Isolated" nucleic acid refers to a nucleic acid molecule that has been separated from its components in its natural environment. Isolated nucleic acids include nucleic acid molecules contained in cells that typically contain such molecules but are present outside the chromosome or at a chromosomal location different from their natural chromosomal location. Isolated nucleic acids encoding polypeptides or fusion proteins refer to one or more nucleic acid molecules encoding polypeptides or fusion proteins, including one or more such nucleic acid molecules in a single or separate vector, and one or more such nucleic acid molecules present at one or more locations in the host cell. Unless otherwise stated, a particular nucleic acid sequence also implicitly encompasses variants of its conserved modifications (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly stated sequences. Specifically, as detailed below, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of bases and / or deoxyinosine residues.

[0384] The term "sequence identity" refers to the degree (percentage) to which two sequences share the same amino acids / nucleic acids at equivalent positions; wherein, when performing optimal alignment of two sequences, gaps are introduced where necessary to obtain the maximum percentage of sequence identity, and no conserved substitutions are considered part of the sequence identity. To determine the percentage of sequence identity, alignment can be performed using techniques known in the art, such as publicly available computer software, such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0385] The term "vector" refers to a polynucleotide molecule capable of transporting another polynucleotide linked to it. One type of vector is a "plasmid," which is a circular double-stranded DNA loop in which an additional DNA segment can be attached. Another type of vector is a viral vector, such as an adeno-associated virus vector (AAV or AAV2), in which an additional DNA segment can be attached to the viral genome. Some vectors are capable of autonomous replication in the host cells to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and attachable mammalian vectors). Other vectors (e.g., non-attached mammalian vectors) can integrate into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome. The term "expression vector" or "expression construct" refers to a vector capable of transforming host cells and containing a nucleic acid sequence that directs and / or controls (alongside the host cell) the expression of one or more heterologous coding regions operatively linked to it. Expression constructs can include, but are not limited to, sequences that affect or control transcription, translation, and, in the presence of introns, influence RNA splicing of coding regions operatively linked to them.

[0386] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their derived progeny, regardless of passage number. Progeny may not be identical to parental cells in their nucleic acid contents and may contain mutations. This disclosure includes mutant progeny with the same function or biological activity as screened or selected in the initially transformed cells. Host cells include prokaryotic and eukaryotic host cells, wherein eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cattle, horse, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.Fungal cells include yeast and filamentous fungal cells, including, for example, *Pichia pastoris*, *Pichia finlandica*, *Pichia trehalophila*, *Pichia koclamae*, *Pichia membranaefaciens*, *Pichia minuta* (Ogataea minuta, *Pichia lindneri*), *Pichia xiaopuntiae*, *Pichia thermotolerans*, *Pichia salictaria*, *Pichia guercuum*, *Pichia pijperi*, *Pichia stiptis*, *Pichia methanolica*, *Pichia* genus, *Saccharomyces cerevisiae*, *Saccharomyces* genus, and *Hansenula*. Kluyveromyces polymorpha, Kluyveromyces lactis, Candida albicans, Aspergillus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa.

[0387] The host cells disclosed herein cannot develop into complete plants or animal individuals.

[0388] As used in this disclosure, the terms “cell,” “cell line,” and “cell culture” are used interchangeably, and all such names include progeny. Therefore, the terms “transformer” and “transformed cell” include primary subject cells and cultures derived therefrom, regardless of the number of passages. It should also be understood that, due to intentional or unintentional mutations, not all progeny will have identical DNA contents. This includes mutant progeny that have the same function or biological activity as the original transformed cells from which they were selected.

[0389] "Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, including the circumstances in which the event or circumstances may or may not occur.

[0390] The term "pharmaceutical composition" refers to a mixture containing one or more antigen-binding molecules described herein along with other chemical components, such as physiological / pharmaceutical carriers and excipients.

[0391] The term "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical formulation that is distinct from the active ingredient and non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0392] The terms “subject” or “individual” include both humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates, sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless otherwise specified, the terms “patient” or “subject” are used interchangeably in this disclosure. In some embodiments, the individual or subject is a human being.

[0393] "Administration" or "giving," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refers to the contact between an exogenous drug, therapeutic agent, diagnostic agent, or composition and the animal, human, subject, cell, tissue, organ, or biological fluid.

[0394] The term "sample" refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within the subject's body. Exemplary samples include biological fluids such as blood, serum and serous fluid, plasma, lymph, urine, saliva, cystic fluid, tears, excretions, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites, pleura, pericardium, peritoneum, fluids in the abdominal cavity and other body cavities, fluids collected by bronchoalveolar lavage fluid, synovial fluid, liquid solutions in contact with the subject or biological sources, such as cell and organ culture media (including cell or organ conditioned media), lavage fluids, tissue biopsy samples, fine-needle aspiration, surgically removed tissue, organ cultures, or cell cultures.

[0395] "Treatment" and "treatment" (and their grammatical variations) refer to clinical interventions that attempt to alter the natural processes of the individual being treated, and can be implemented for prevention or during a clinicopathological process. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing / decreasing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and resolving or improving prognosis. In some implementations, antibodies disclosed herein are used to delay disease onset or slow disease progression.

[0396] The terms "recurrence," "relapse," and "relapsed" refer to the recovery of cancer or disease after a clinical assessment of disease resolution. A diagnosis of distant metastasis or local recurrence can be considered a recurrence.

[0397] The terms "refractory" or "resistant" refer to cancers or diseases that do not respond to treatment.

[0398] An "effective dose" is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate such symptoms and / or underlying causes, prevent the occurrence of symptoms and / or underlying causes, and / or improve or mitigate damage caused by or associated with a disease state (e.g., lung disease). In some embodiments, an effective dose is a therapeutically effective dose or a preventatively effective dose. A "therapeuticly effective dose" is an amount sufficient to treat a disease state or symptom, particularly a state or symptom associated with that disease state, or otherwise prevent, inhibit, delay, or reverse the progression of the disease state or any other undesirable symptom associated with that disease. A "preventatively effective dose" is an amount that, when administered to a subject, will have a predetermined preventative effect, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or related symptoms. A complete therapeutic or preventative effect may not occur after the administration of a single dose, but may occur after the administration of a series of doses. Therefore, a therapeutically or preventatively effective dose may be administered in a single or multiple-dose manner. "Therapeutic effective dose" and "preventive effective dose" can vary depending on a number of factors, such as an individual's disease state, age, sex, and weight, as well as the ability of the treatment or combination of treatments to elicit the desired response in the individual. Exemplary indicators of an effective treatment or combination of treatments include, for example, improved health status in the patient.

[0399] antibody structure

[0400] In some implementations, the antibodies provided in this disclosure are full-length antibodies.

[0401] In some implementations, the antibodies provided in this disclosure are antigen-binding fragments.

[0402] In some implementations, the antibodies provided in this disclosure are antibody fragments.

[0403] In some embodiments, the antibody fragment is a Fab, Fab′, Fab′-SH, or F(ab′)2 fragment, particularly a Fab fragment. “Fab” is a monovalent fragment consisting of VL, VH, CL, and CH1 domains. A “Fab fragment” can be generated by cleavage of an antibody with papain. “Fab′” contains VL, CL, VH, and CH1, and also contains a region between the CH1 and CH2 domains, allowing interchain disulfide bonds to form between the two heavy chains of two Fab′ fragments to form an F(ab′)2 molecule. “Fab′-SH” is a Fab′ fragment in which the cysteine ​​residues in the constant region have free thiol groups. “F(ab′)2” is a divalent fragment comprising two Fab fragments linked by disulfide bonds in the hinge region.

[0404] In other embodiments, the antibody fragment is a biantibody, triantibody, or tetraantibody. A biantibody is an antibody fragment having two antigen-binding sites. In some embodiments, the antibody fragment contains linked VH and VL domains within the same polypeptide chain (VH-VL). By using a short linker that prevents pairing between two domains on the same chain, these domains are forced to pair with complementary domains on another chain, thereby creating two antigen-binding sites, the two antigens of which may be the same or different. In some embodiments, the antibody fragment contains a Fab domain and a VHH domain, thereby creating two or more antigen-binding sites.

[0405] In other embodiments, the antibody fragment is a single-chain Fab fragment. A “single-chain Fab fragment” or “scFab” is a polypeptide consisting of VH, CH1, VL, CL, and a linker, wherein the antibody domain and the linker have one of the following sequences in the N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL. In some embodiments, the linker is a polypeptide having at least 30 amino acids. In other embodiments, the linker is a polypeptide having between 32 and 50 amino acids. The single-chain Fab fragment is stabilized via a native disulfide bond between CL and CH1. Additionally, these single-chain Fab molecules can be further stabilized by inserting cysteine ​​residues (e.g., at position 44 in the heavy chain variable region and position 100 in the light chain variable region, according to Kabat numbering) to create interchain disulfide bonds.

[0406] In other embodiments, the antibody fragment is an Fv fragment composed of the VH and VL domains of a single arm of the antibody.

[0407] In other embodiments, the antibody fragment is a single-chain variable fragment (scFv). An “scFv” is a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light and heavy chain variable regions are sequentially linked by a short, flexible peptide linker, capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, in this disclosure, the scFv may have VL and VH variable regions in any order; for example, relative to the N-terminus and C-terminus of the polypeptide, the scFv may comprise a VL-linker-VH or may comprise a VH-linker-VL.

[0408] In other embodiments, the antibody fragment is dsFv, which is obtained by linking polypeptides in which one amino acid residue in each VH and VL is replaced by a cysteine ​​residue via disulfide bonds between cysteine ​​residues. The amino acid residues to be replaced by cysteine ​​residues can be selected based on prediction of the antibody's three-dimensional structure using known methods (Protein Engineering. 7:697 (1994)).

[0409] In other embodiments, the antibody fragment is a single-domain antibody (dAb). A single-domain antibody is an antibody fragment containing all or part of the heavy chain variable domain or all or part of the light chain variable domain. In some embodiments, a single-domain antibody refers to a single-domain antibody that does not contain a light chain and can specifically bind to an epitope in the absence of other antigen-binding domains. A single-domain antibody is a small, stable, and highly efficient antigen recognition unit formed by a single immunoglobulin domain.

[0410] In some embodiments, the antibodies provided in this disclosure are chimeric antibodies. In some embodiments, the chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In some embodiments, the chimeric antibody is a "class-switched" antibody, wherein the class or subclass has been changed from the class or subclass of the parent antibody.

[0411] In some embodiments, the antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce its immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody contains one or more variable regions, wherein the CDR or a portion thereof is derived from the non-human antibody, and the FR or a portion thereof is derived from the human antibody. Optionally, the humanized antibody may also contain a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody may be replaced with corresponding residues from the non-human antibody (e.g., an antibody providing the CDR sequence).

[0412] Humanized antibodies and their generation methods are reviewed in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); US Patent Nos. 5,821,337,7,527,791,6,982,321 and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describes specificity-determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describes “resurfuacing”); Dall'Acqua et al., Methods 36:43-60 (2005) (describes “FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer 83:252-260 (2000) (describes the “guided selection” method for FR shuffling).

[0413] Human frame regions that can be used for humanization include, but are not limited to: frame regions selected using a "best-fit" method (see, for example, Sims et al., J. Immunol. 151:2296 (1993)); frame regions of the common sequence of human antibodies derived from specific subgroups of light chain variable regions or heavy chain variable regions (see, for example, Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al., J. Immunol., 151:2623 (1993)); mature human (somatic mutant) frame regions or human germline frame regions (see, for example, Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); and the frame regions obtained by screening FR libraries (see, for example, Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271: 22611-22618 (1996)).

[0414] Variants of activatable proteins

[0415] In some embodiments, amino acid sequence variants of the activatable protein provided in this disclosure are included. For example, it may be desirable to improve the binding affinity and / or other biological properties of the activatable protein. Amino acid sequence variants of the activatable protein can be prepared by introducing suitable modifications into the nucleotide sequence encoding the activatable protein, or by peptide synthesis. Such modifications include, for example, deletion, and / or insertion, and / or substitution of residues within the amino acid sequence of the activatable protein. Any combination of deletion, insertion, and substitution can be performed to obtain the final construct, provided that the final construct possesses the desired characteristics, such as antigen-binding properties.

[0416] Replace, insert, and delete variants

[0417] In some embodiments, antibody variants with one or more amino acid substitutions are provided. Substitution mutagenesis sites of interest include CDR and FR. Conserved substitutions are shown in Table 3 under the heading “Preferred Substitutions.” More substantial variations are provided in Table 3 under the heading “Exemplary Substitutions” and are further described below with reference to the amino acid side chain categories. Amino acid substitutions can be introduced into the antibody of interest, and the product can be screened for desired activities, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0418] Table 3. Substitution of amino acids

[0419] Based on common side-chain characteristics, amino acids can be grouped as follows:

[0420] (1) Hydrophobic: Leucine, Met, Ala, Val, Leu, Ile;

[0421] (2) Neutral and hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0422] (3) Acidic: Asp, Glu;

[0423] (4) Alkaline: His, Lys, Arg;

[0424] (5) Residues that affect chain orientation: Gly, Pro;

[0425] (6) Aromatic: Trp, Tyr, Phe.

[0426] Non-conservative replacement would require replacing a member of one of these categories with a member of another category.

[0427] One class of substitution variants involves replacing one or more CDR residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further research will have alterations (e.g., improvements) to certain biological properties (e.g., increased affinity, decreased immunogenicity) relative to the parent antibody, and / or will substantially retain certain biological properties of the parent antibody. An exemplary substitution variant is an affinity-matured antibody, which can be conveniently generated, for example, using phage display-based affinity maturation techniques (such as those described herein). In short, one or more CDR residues are mutated, and the variant antibody is displayed on a phage and screened for specific biological activities (e.g., binding affinity). CDRs can be altered (e.g., substituted), for example, to improve antibody affinity. Such alterations can be made to CDR “hotspots,” residues encoded by codons that undergo mutations at a high frequency during somatic maturation, and / or residues that contact the antigen, while testing the binding affinity of the resulting variant VH or VL. In some implementations of affinity maturation, diversity is introduced into the selected variant gene for maturation using any of a variety of methods, such as error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis. A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves CDR-directed approaches, where several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scan mutagenesis or modeling. In particular, HCDR3 and LCDR3 are frequently targeted.

[0428] In some embodiments, substitution, insertion, or deletion can occur within one or more CDRs, as long as such changes do not materially reduce the antibody's ability to bind to the antigen. For example, conserved changes (e.g., conserved substitutions, as provided herein) can be made to CDRs that do not materially reduce binding affinity. Such changes can, for example, be external to the antigen-contacting residues in the CDR. In some embodiments of the variant VH and VL sequences provided above, each CDR is either unchanged or contains no more than one, two, or three amino acid substitutions.

[0429] One method for identifying residues or regions in an antibody that can serve as mutagenic targets is called "alanine scan mutagenesis." In this method, a residue or target group of residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., Ala or polyalanine) to determine if the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Furthermore, the contact points between the antibody and antigen can be identified by studying the crystal structure of the antigen-antibody complex. These contact residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants can be screened to determine if they contain the desired properties.

[0430] Amino acid sequence insertions include fusion of the amino and / or carboxyl ends of peptides ranging in length from 1 residue to 100 or more residues, and intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of the N- or C-terminus of the antibody with an enzyme or a peptide that extends the serum half-life of the antibody.

[0431] Recombination method

[0432] Activatable proteins can be produced using recombinant methods. For these methods, one or more isolated nucleic acids encoding the activatable protein are provided.

[0433] In some embodiments, this disclosure provides isolated nucleic acids encoding activatable proteins as described in any of the preceding embodiments. Such nucleic acids can be derived from independent polypeptide chains encoding any of the foregoing (e.g., polypeptide chains containing any CDR, VH, and / or VL, heavy chains, and / or light chains as described in this disclosure). In another aspect, this disclosure provides one or more vectors (e.g., expression vectors) containing such nucleic acids. In yet another aspect, this disclosure provides host cells containing such nucleic acids. In some embodiments, a method for preparing a polypeptide or fusion protein is provided, wherein the method includes culturing a host cell containing nucleic acid encoding the polypeptide or fusion protein, as provided above, under conditions suitable for expression, and optionally recovering the activatable protein from the host cell (or host cell culture medium).

[0434] To recombinantly generate an activatable protein, the nucleic acid encoding the protein is isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using standard procedures, or generated through recombinant methods or obtained through chemical synthesis.

[0435] Suitable host cells for cloning or expressing vectors encoding activatable proteins include prokaryotic or eukaryotic cells as described herein. For example, they can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. After expression, the vector can be separated from the bacterial cell paste in a soluble fraction and further purified.

[0436] Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding fusion proteins, including fungal and yeast strains. Suitable host cells for expressing fusion proteins can also be derived from multicellular organisms (invertebrates and vertebrates); examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of fall armyworm (Spodoptera frugiperda) cells; plant cell cultures can also be used as hosts, such as US5959177, US 6040498, US6420548, US 7125978, and US6417429; and vertebrate cells, such as mammalian cell lines adapted for growth in suspension, can also be used as hosts. Other examples of suitable mammalian host cell lines include SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (293 or 293T cells); young hamster kidney cells (BHK); mouse seltoli cells (TM4 cells); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumors (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells. Other suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells; and myeloma cell lines such as Y0, NSO, and Sp2 / 0. For reviews of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0437] Measurement

[0438] The activatable proteins described herein can be identified, screened, or characterized by their physical / chemical properties and / or biological activities using a variety of assays known in the art. In one aspect, the activity of the activatable proteins disclosed herein can be tested, for example, by known methods such as ELISA, Western blotting, etc.

[0439] Treatment methods and routes of administration

[0440] Any activatable proteins provided in this disclosure can be used in treatment methods. In another aspect, the activatable proteins provided in this disclosure are used in the manufacture or preparation of pharmaceuticals. In some embodiments, the disease is a tumor, an autoimmune disease, or an inflammatory disease. In some embodiments, the tumor is a solid tumor.

[0441] In another aspect, pharmaceutical compositions comprising the said activatable protein are provided, for example, for any of the pharmaceutical uses or therapeutic methods described above. In one embodiment, the pharmaceutical composition comprises any activatable protein provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition further comprises at least one additional therapeutic agent.

[0442] The proteins disclosed herein can be used alone or in combination with other agents for treatment. For example, the activatable proteins disclosed herein can be administered in combination with at least one other therapeutic agent.

[0443] The activatable proteins (and any other therapeutic agents) disclosed herein may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, and, if local treatment is required, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Administration may be carried out via any suitable route, such as by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. Various dosing schedules are considered herein, including, but not limited to, single or multiple administrations at multiple time points, bolus administration, and pulsatile infusion.

[0444] The activatable proteins disclosed herein will be formulated, administered, and applied in accordance with good medical practice. Factors considered in this context include the specific condition being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to a medical practitioner. The activatable proteins may be formulated with or without one or more agents currently used for the prevention or treatment of the stated condition. The effective amount of such other agents depends on the amount present in the pharmaceutical composition, the type of condition or treatment, and other factors. These are generally used at the same dosage and route of administration as described herein, or at approximately 1% to 99% of the dosage described herein, or at other dosages, and in any route determined empirically / clinically as appropriate.

[0445] For the prevention or treatment of disease, the appropriate dosage of the activatable protein disclosed herein (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of therapeutic molecule, the severity and course of the disease, whether it is administered for preventive or therapeutic purposes, previous treatments, the patient's clinical history and response to the therapeutic molecule, and the judgment of the attending physician. The therapeutic molecule is appropriately administered to the patient either once or after a series of treatments.

[0446] Products

[0447] In another aspect of this disclosure, an article of manufacture (such as a medicine box) is provided, comprising materials that can be used to treat, prevent, and / or diagnose the aforementioned conditions. The article of manufacture comprises a container and a label or package insert on or in conjunction with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed from various materials such as glass or plastic. The container contains a composition, alone or in combination with another composition, that is effective in treating, preventing, and / or diagnosing the condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper puncturable by a hypodermic needle). At least one active agent in the composition is an activatable protein of this disclosure. The label or package insert indicates that the use of the composition is for the treatment of a selected condition. Furthermore, the article of manufacture may comprise: (a) a first container containing a composition, wherein the composition contains an activatable protein of this disclosure; and (b) a second container containing a composition, wherein the composition contains an additional therapeutic agent. The article of manufacture in embodiments of this disclosure may further comprise a package insert indicating that the composition can be used to treat a specific condition. Alternatively, or additionally, the article may further comprise a second (or third) container containing a pharmaceutically acceptable buffer solution. From a commercial and user perspective, it may further include other materials as desired, including additional buffers, diluents, filters, needles, and syringes.

[0448] Example

[0449] The present disclosure is further described below with reference to examples and test cases, but these examples and test cases are not intended to limit the scope of the disclosure. Experimental methods in the examples and test cases of this disclosure that do not specify specific conditions are generally performed under conventional conditions, such as those described in Cold Spring Harbor's Antibody Technology Manual or Molecular Cloning Manual; or under conditions recommended by the raw material or commercial manufacturer. Reagents whose specific source is not specified are commercially available, conventional reagents.

[0450] Example 1. Activity evaluation of protease cleavable sequences

[0451] Example 1.1. Cleavage rate of protease-cleavable sequences

[0452] The cleavable sequences of the proteases to be evaluated were chemically synthesized. Dabcyl was modified at the N-terminus, and EDANS was modified at the C-terminus. The cleavage rates of different enzymes on the cleavable sequences were evaluated using fluorescence resonance energy transfer (FRET) technology. 100 μM peptides were added to reaction systems containing 5 nM MMP9 (R&D, 911-MP-010), 10 nM Matriptase (R&D, 3946-SEB-010), and 10 nM uPA (R&D, 1310-SE-010), respectively. Fluorescence signals were dynamically acquired using a FlexStation3 microplate reader under excitation conditions of 340 nm and emission conditions of 490 nm. The initial cleavage rates were calculated, and the results are shown in Table 4.

[0453] Table 4. Enzyme cleavage rates of different enzymes on proteases

[0454] The results showed that the rates at which the protease-cleavable sequences disclosed herein were cleaved by the three proteases were significantly different from those of the control linker Ref1: compared with the control linker Ref1, L-1, L-3, L-4, L-5 and L-6 were cleaved by Matriptase and uPA at a faster rate, while L-2 was cleaved by MMP9 and Matriptase at a faster rate.

[0455] Example 1.2. Evaluation of the selectivity of protease-cleavable sequences for MMP family proteases by cleavage rate

[0456] The cleavable sequences of the proteases to be evaluated were chemically synthesized. Dabcyl was modified at the N-terminus, and EDANS was added to the C-terminus. The cleavage rates of the cleavable sequences by different MMP families were evaluated using fluorescence resonance energy transfer (FRET) technology. 100 μM of the peptide was added to reaction systems containing 5 nM MMP9 (R&D, 911-MP-010) and 10 nM MMP14 (R&D, 914-MPN-010), respectively. Fluorescence signals were dynamically acquired using a FlexStation 3 microplate reader under excitation conditions of 340 nm and emission conditions of 490 nm, and the initial cleavage rates were calculated. The ratio of the initial cleavage rates of MMP9 to MMP14 was used to characterize protease selectivity. The results are shown in Table 5.

[0457] Table 5. Selectivity of protease-cleavable sequences for other proteases in the MMP family

[0458] The results showed that the selectivity of L-1, L-2, L-3, L-4, L-5 and L-6 for MMP14 was comparable to or better than that of Ref1.

[0459] Example 1.3. Kcat / Km of protease-cleavable sequences

[0460] The cleavable sequences of the proteases to be evaluated were chemically synthesized. Each sequence was modified with Dabcyl at the N-terminus and with EDANS glutamate at the C-terminus. The cleavage rates of different enzymes on the cleavable sequences were evaluated using fluorescence resonance energy transfer (FERT) technology, and Kcat / Km (reflecting the efficiency of the enzyme-catalyzed reaction) was calculated based on the cleavage rate.

[0461] The initial cleavage rates of various sequences at different concentrations (100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.13 μM, and 1.56 μM) were sequentially measured using 5 nM MMP9 (R&D, 911-MP-010), 10 nM Matriptase (R&D, 3946-SEB-010), and 10 nM uPA (R&D, 1310-SE-010). Vmax and Km were calculated by fitting Michaelis-Menten curves using Graphpad Prism 10 software. Kcat / Km = Vmax ÷ enzyme concentration ÷ Km. Using Ref1 as a reference, its corresponding Kcat / Km value was set to 1. The fold change of other sequences relative to Ref1 was calculated, and the results are shown in Table 6.

[0462] Table 6. Comparison of Kcat / Km folds of cleavable sequences by different enzymes

[0463] The results showed that, compared with Ref1, L-1 was more efficiently cleaved by Matriptase and uPA, while L-2 was more efficiently cleaved by MMP9 and Matriptase.

[0464] Example 1.4. Evaluation of the selectivity of protease cleavable sequences for MMP family proteases using Kcat / Km.

[0465] The cleavable sequences of the proteases to be evaluated were chemically synthesized. Each sequence was modified with Dabcyl at the N-terminus and with EDANS glutamate at the C-terminus. The cleavage rates of different enzymes on the cleavable sequences were evaluated using fluorescence resonance energy transfer (FERT) technology, and Kcat / Km (reflecting the efficiency of the enzyme-catalyzed reaction) was calculated based on the cleavage rate.

[0466] The initial reaction rates of each sequence catalyzed by 5 nM MMP9 (R&D, 911-MP-010), 10 nM MMP7 (R&D, 907-MP-010), and 10 nM MMP14 (R&D, 914-MPN-010) at multiple concentrations (100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.13 μM, and 1.56 μM) were sequentially measured. The Kcat / Km ratio of each sequence cleaved by different enzymes was calculated as described in Example 1.3. The ratio of MMP9 Kcat / Km to MMP7 or MMP14 was calculated to characterize the selectivity of each sequence for other proteins in the MMPs family. The results are shown in Table 7.

[0467] Table 7. Selectivity of protease-cleavable sequences for MMP family proteases

[0468] The results showed that L-1 and L-2 were more selective than Ref1 for MMP7 and MMP14.

[0469] Example 2. Validation of the activity of protease-cleavable sequences on activatable antibodies

[0470] Example 2.1. Expression and purification of activatable antibodies

[0471] The preparation of the activated antibodies disclosed herein was performed using the Expi-293F expression system. Nucleotide sequences encoding the first and second strands of each activated antibody were synthesized and inserted into the vector pcDNA3.4 to obtain the corresponding recombinant plasmids. These recombinant plasmids were used for cloning and / or expression in host cells. Expi-293F cells (Thermo Fisher Scientific) were passaged and cultured in OPM-293CD05 medium (OPM, Cat#81075). Cell density was measured one day prior to transfection, and the cell density was adjusted to 1.6 × 10⁶ cells / day using fresh cell culture medium. 6 cells / mL, cell density before transfection was 3 × 10⁶. 6 -3.5×10 6The transfection plasmid volume was 1 μg per 1 mL of cells. The ratio of plasmid to Polyethylenimine Max (Mw 40,000*)-High Potency Linear PEI (Polysciences, Cat#24765-1) was 1:3. The transfection system was 10% of the expression system. For example, in a 100 mL transfection system, after filtering the plasmid, it was adjusted to 5 mL using Opit-MEM (gbico, Cat#11058-021). 300 μL (1 mg / mL) of PEI was then added to 4.7 mL of the solution. Mix Opit-MEM thoroughly and incubate at room temperature for 5 minutes. Add the prepared PEI to the plasmid system, mix thoroughly, and incubate at room temperature for 15 minutes. Slowly add the PEI-plasmid complex to the Expi-293F cell suspension and mix well. Incubate on a shaker. 24 hours after transfection, add 5% OPM-293ProFeed (OPM, Cat#F081918) at a volume ratio. 6 days after transfection, collect the supernatant and analyze it using KappaSelect. TM Affinity column (Cytiva, Cat#17545801) and CaptureSelect TM IgG-CH1 (Thermo, Cat#1943462010) purified antibody.

[0472] KappaSelect TM Affinity purification: Before purification, equilibrate the chromatography column with 10-20 column volumes of 1×PBS (Sangon Biotech, Cat#SD8117) until UV light returns to baseline; load the collected culture supernatant onto the chromatography column, and wash the column with 1×PBS until UV light returns to baseline to remove non-specifically bound proteins; rinse the chromatography column with elution buffer (0.1M glycine hydrochloride buffer, pH 3.0) and collect the eluent; then neutralize to neutral with 1M Tris-HCl (Sangon Biotech, Cat#B548139-0500) buffer for further purification.

[0473] CH1 affinity purification: Before purification, equilibrate the chromatography column with 10 column volumes of 1×PBS (Sangon Biotech, Cat#SD8117) until UV light returns to baseline; load the neutralized KappaSelect purification eluent onto the chromatography column, and wash the column with 10 column volumes of 1×PBS until UV light returns to baseline to remove non-specifically binding proteins; wash the chromatography column with CH1 eluent (20 mM acetic acid + 150 mM sodium chloride, pH 3.5) and collect the eluent; transfer the eluent to an Amicon Ultra-15 Centrifugal Filter (Merck, Cat#UFC901024), add 30 mM CH3COONa, pH 5.0 to the maximum mark, centrifuge at 3500 rpm for 10 minutes at 4°C, repeat this operation three times to complete the buffer exchange and concentration, transfer the concentrated sample to a 15 mL centrifuge tube, and adjust the concentration to 1-2 mg / mL. After sterilization using a 0.22 μm syringe filter in a biosafety cabinet, the sample was transferred to a 1.5 mL centrifuge tube. 100 μL of the solution was then subjected to SDS-PAGE and SEC analysis. Further qualitative and quantitative analysis of the activated antibodies was performed using a combination of capillary electrophoresis (CE-SDS) and liquid chromatography-mass spectrometry (LC-MS).

[0474] Example 2.2. Structure of an Activated Antibody

[0475] The structure of the EGFR / CD3-activatable antibody disclosed herein is F1_B. F1_B is an asymmetric structure, comprising a first chain and a second chain, as shown in Figure 1. Details are as follows:

[0476] First chain: [α-HSA-VHH]-[connector 1]-[α-CD3-VL]-[CL];

[0477] Second chain: [α-CD3-VH]-[CH1]-[linker 2]-[α-EGFR-VHH];

[0478] Linker 1 is a cleavable linker, which contains a CD3 masking peptide and a protease cleavable sequence from the N-terminus to the C-terminus; linker 2 is an uncleavable linker; the structures of the first and second chains are both arranged from the N-terminus to the C-terminus.

[0479] The various parts of the activatable antibody disclosed herein are interconnected by linkers, which are divided into non-cleavable linkers and cleavable linkers.

[0480] Table 8. Indivisible Connective Subsequences

[0481] The variable region sequence of the anti-HSA single-domain antibody was derived from WHO INN PL105 (2011), and the specific sequence is as follows:

[0482] Table 9. CDR sequences of anti-HSA single-domain antibodies

[0483] Note: The CDRs in the table are CDRs determined according to the Kabat numbering system.

[0484] The variable region sequence of the anti-CD3 antibody is derived from patent PCT / CN2024 / 141016, and the specific sequence is as follows:

[0485] >82_VH_H7

[0486] >82_VL_L8 S27eP,L94V

[0487] >CL_kappa

[0488] CH1_IgG1

[0489] The CDR sequences of anti-CD3 antibodies are shown in the table below:

[0490] Table 10. CDR of anti-CD3 antibodies

[0491] Note: The CDRs in the table are CDRs determined according to the Kabat numbering system.

[0492] CD3 antibody masking peptide

[0493] The 9G8 variable region sequence of the anti-EGFR single-domain antibody is derived from Structural Evaluation of EGFR Inhibition Mechanisms for Nanobodies / VHH Domains (Schmitz KR, et al. structure. 2013; 21:1214-1224), and the specific sequence is as follows:

[0494] >α-EGFR-VHH

[0495] The sequence of the EGFR / CD3 antibody:

[0496] The first chain of act.EGFR-CD3

[0497] The second chain of act.EGFR-CD3

[0498] The first strand of EGFR-CD3-L1

[0499] The second strand of EGFR-CD3-L1

[0500] The first strand of EGFR-CD3-L2

[0501] The second strand of EGFR-CD3-L2

[0502] The first chain of EGFR-CD3-NCL

[0503] The second chain of EGFR-CD3-NCL

[0504] Table 11. Antibody Control Table

[0505] Note: act.EGFR-CD3 is an EGFR / CD3 antibody, indicating the activated antibody; EGFR-CD3-L1 and EGFR-CD3-L2 indicate EGFR / CD3-activated antibodies; EGFR-CD3-NCL is an EGFR / CD3 antibody containing an uncleavable linker with a CD3 masking peptide.

[0506] Example 2.3. Evaluation of the activation level of activatable antibodies

[0507] Activated antibodies containing protease-cleavable sequences were added at a working concentration of 5 μM to reaction systems containing 5 nM or 10 nM MMP9 (R&D, 911-MP-010), 10 nM Matriptase (R&D, 3946-SEB-010), and 10 nM uPA (R&D, 1310-SE-010), respectively, and reacted at 37°C for 2 h, 2 h, 2 h, and overnight, respectively. After denaturation, activated light chains, intact light chains, and heavy chains of different molecular weights were separated by SDS-PAGE. After Coomassie brilliant blue staining, images were taken using the ChemiDoc™ MP imaging system (BioRad), and the intensity (Int) of each band was analyzed using Image Lab software (BioRad). The gray values ​​of the intact light chain were normalized using the gray values ​​of the heavy chain in each lane (normalized Int = intact light chain Int ÷ heavy chain Int). The activation level (%) of the antibody after protease treatment was calculated as (normalized Int before activation - normalized Int after activation) ÷ normalized Int before activation × 100. The results are shown in Table 12.

[0508] Table 12. Activation level (%) of antibodies that can be activated after protease treatment

[0509] The results showed that the activatable antibodies containing the protease-cleavable sequences L-1 and L-2 could be effectively activated by the three proteases.

[0510] Example 2.4 Killing activity of activated antibodies after in vitro activation

[0511] Activated antibodies containing protease-cleavable sequences were added at a working concentration of 5 μM to reaction systems containing 10 nM MMP9 (R&D, 911-MP-010) and 20 nM Matriptase (R&D, 3946-SEB-010), respectively. After overnight incubation at 37°C, the cytotoxic function of PBMCs was assessed. HT-29Red-Fluc (PerkinElmer, BW124353-V) was added to 96-well plates at a ratio of 10,000 cells per well, and PBMCs were added at a ratio of effector cells to target cells of 5:1. Then, serially diluted antibodies were added, and the plates were incubated in a 5% CO2 cell culture incubator for 72 hours. After incubation, the level of PBMC-mediated tumor cell killing by activated antibodies was detected using the ONE-Glo™ luciferase Assay System (Promega, E6120), thereby determining the cytotoxic activity of activated antibodies with different protease-cleavable sequences after in vitro activation.

[0512] Table 13. Activity of activatable antibodies in mediating PBMC-mediated tumor cell killing in vitro.

[0513] The results are shown in Table 13. Compared with antibodies with non-cleavable linkers (NCLs), the activatable antibodies with protease-cleavable sequences L-1 and L-2 showed stronger activity in the PBMC killing system after in vitro activation.

[0514] Although the invention has been described in detail with the aid of accompanying drawings and examples for clarity of understanding, these descriptions and examples should not be construed as limiting the scope of this disclosure. All patent and scientific literature disclosures cited herein are clearly and fully incorporated by reference.

Claims

1. A protease-cleavable sequence comprising an amino acid sequence that is identical to or has at least 70% sequence identity with SEQ ID NO: 2 or 8.

2. The protease-cleavable sequence according to claim 1, comprising the amino acid sequence shown in SEQ ID NO: 2, 1, 3, 4, 5 or 6; Preferably, the protease-cleavable sequence comprises the amino acid sequence shown in SEQ ID NO: 2 or 1.

3. The protease-cleavable sequence according to claim 1 or 2, wherein the protease is expressed or overexpressed in the tumor microenvironment; Preferably, the protease is selected from one or more of metalloproteinases and serine proteases; More preferably, the metalloproteinase is a matrix metalloproteinase, which is selected from one or more of MMP2, MMP7, MMP9, MMP13, and MMP14; and / or The serine protease is selected from one or more of uPA, Matriptase, and hepsin.

4. The protease-cleavable sequence according to any one of claims 1 to 3, having one or more of the following characteristics: The rate at which the protease-cleavable sequence is cleaved by MMP9 is not less than 0.30 RFU / s; The rate at which the protease-cleavable sequence is cleaved by Matriptase is not less than 0.10 RFU / s; The rate at which the protease-cleavable sequence is cleaved by uPA is not less than 0.10 RFU / s; The rate at which the protease-cleavable sequence is cleaved by MMP14 is no higher than 0.30 RFU / s; The protease-cleavable sequence is cleaved by MMP9 at a rate at least 1.5 times that of MMP14. The Kcat / Km of the protease-cleavable sequence cleaved by MMP9 is at least 2.0 times that of the Kcat / Km cleaved by MMP7; The protease-cleavable sequence has a Kcat / Km cleaved by MMP9 that is at least 16.0 times larger than the Kcat / Km cleaved by MMP14.

5. An activatable protein comprising a protease-cleavable sequence according to any one of claims 1 to 4.

6. The activatable protein according to claim 5, further comprising at least one antigen-binding domain that specifically binds to the target antigen.

7. The activatable protein according to claim 6, wherein the antigen-binding domain that specifically binds to the target antigen is Fab, scFv, dAb, Fd, Fv, dsFv, scFab, Fab′ or F(ab′)2.

8. The activatable protein according to claim 6 or 7, wherein the antigen-binding domain that specifically binds to the target antigen is located at the C-terminus or N-terminus of the protease-cleavable sequence; Preferably, the antigen-binding domain that specifically binds to the target antigen is located at the C-terminus of the protease-cleavable sequence.

9. The activatable protein according to any one of claims 6 to 8, wherein the target antigen is an effector cell antigen or a tumor-associated antigen; Preferably, the target antigen is an effector cell antigen; More preferably, the target antigen is a T cell surface antigen; Most preferably, the target antigen is CD3.

10. The activatable protein according to any one of claims 6 to 9, further comprising a masking peptide; Preferably, the masking peptide inhibits the binding of the antigen-binding domain of the specifically binding target antigen to the target antigen.

11. The activatable protein of claim 10, wherein the masking peptide is located at the N-terminus or C-terminus of the protease-cleavable sequence; Preferably, the masking peptide is located at the N-terminus of the protease-cleavable sequence.

12. The activatable protein according to claim 10 or 11, wherein: The masking peptide is operatively linked to the N-terminus of the protease-cleavable sequence, and the antigen-binding domain that specifically binds to the target antigen is operatively linked to the C-terminus of the protease-cleavable sequence. or The masking peptide is operatively linked to the C-terminus of the protease-cleavable sequence, and the antigen-binding domain that specifically binds to the target antigen is operatively linked to the N-terminus of the protease-cleavable sequence.

13. The activatable protein according to any one of claims 5 to 12, further comprising a half-life extension portion; Preferably, the extended half-life portion binds to serum protein or a fragment thereof, or the extended half-life portion is serum protein or a fragment thereof; More preferably, the extended half-life portion specifically binds to human serum albumin (HSA); More preferably, the extended half-life portion is an anti-HSA antibody or its antigen-binding fragment.

14. The activatable protein according to claim 13, wherein the extended half-life portion is an immunoglobulin single variable domain, scFv, Fd, Fv, dsFv, Fab, scFab, Fab′ or F(ab′)2.

15. The activatable protein according to any one of claims 5 to 14, comprising: (a1) Specifically binds to the first antigen-binding domain of CD3; (b1) A second antigen-binding domain that specifically binds to tumor-associated antigens (TAAs); (c1) The extended half-life portion that specifically binds to HSA; (d1) A masking peptide that inhibits the binding of the first antigen-binding domain to CD3; (e1) The protease-cleavable sequence according to any one of claims 1 to 4.

16. A pharmaceutical composition comprising: The activatable protein according to any one of claims 5 to 15, and One or more pharmaceutically acceptable carriers, diluents or excipients.

17. A nucleic acid molecule encoding a protease-cleavable sequence according to any one of claims 1 to 4 or an activatable protein according to any one of claims 5 to 15.

18. A host cell comprising the nucleic acid molecule as described in claim 17.

19. A method for preparing a protease-cleavable sequence as claimed in any one of claims 1 to 4, or an activatable protein as claimed in any one of claims 5 to 15, the method comprising expressing a nucleic acid molecule as claimed in claim 17, or culturing a host cell as claimed in claim 18, to produce the protease-cleavable sequence or the activatable protein.

20. A method of treating, preventing, or improving a disease or condition, comprising administering to a subject in need a therapeutically effective amount of an activatable protein as described in any one of claims 5 to 15, or a pharmaceutical composition as described in claim 16; Preferably, the disease or condition is a tumor, an autoimmune disease, or an inflammatory disease; More preferably, the tumor is a solid tumor.

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