Masked antibodies, libraries, and methods of use

Abstract

A library containing synthetic polynucleotides encoding masked binding polypeptides is provided herein. Masked binding polypeptides and polypeptide libraries containing such masking binding polypeptides are further provided herein. Vectors, vector libraries, cells, kits, and methods for preparing and using masked polypeptide libraries are also provided herein.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of priority from International Application PCT / CN2023 / 087999, filed on 13 April 2023, the entire contents of which are incorporated herein by reference. Submission of sequence listings in ASCII text files.

[0002] The contents of the electronic sequence list (695402001741seqlist.xml, size: 99,142 bytes, creation date: April 4, 2024) are incorporated herein by reference in their entirety.

[0003] This disclosure relates to polynucleotides and polynucleotide libraries useful for screening and / or identifying one or more masked conjugated polypeptides (e.g., masked antibodies), as well as polypeptides and polypeptide libraries useful for screening and / or identifying masked peptides (e.g., masked antibodies), related cells, methods, and kits. [Background technology]

[0004] Masked binding polypeptides, such as masked antibodies, can exhibit an "activatable" structure in which the antigen-binding portion of that structure is less adept at binding to its target in the presence of one or more specific proteases, for example, when the masking peptide is not cleaved. Therefore, these masked binding polypeptides provide antigen-specific binding proteins that can bind to targets in specific circumstances (in the presence of an antigen-rich and / or protease-rich tumor microenvironment (TME); antigens such as CTLA-4 are upregulated in lymphoma nodes of the TME, and MMP2 / 9 and other proteases are also upregulated in the TME).

[0005] While numerous masked binding polypeptides have been developed, the process for developing such proteins is ineffective due to flaws in the design and screening processes of the masking peptides. Masked antibodies are typically designed and screened to select masking peptides with a high on-rate and low off-rate for binding to the antibody-binding site of the target antigen. As a result, masking peptides that still exhibit self-inhibitory properties towards cleaved antibodies are often identified, as low off-rate masking peptides adhere to the antibody-binding site and block binding to the target antigen. The potent self-inhibitory binding of masking peptides can reduce the efficiency with which antibodies bind to their targets in vivo. Furthermore, antibodies selected from these libraries often bind at high levels to antigens located in non-target tissues or to normal tissues via peripheral circulation, resulting in significant off-target effects that compromise safety. In addition, existing masked antibodies are often designed to be rapidly cleaved in vivo, and the cleaved, unmasked antibodies circulate in the bloodstream, potentially leading to toxicity associated with antibody binding to antigens in non-target tissues.

[0006] Therefore, there is a need for new concepts, improved methods, and products that can help identify dynamic peptides, rather than self-inhibitory ones, such as masked antibodies or other masked binding polypeptides with several desirable attributes. [Overview of the project]

[0007] To meet the above and other needs, this specification discloses a library of polynucleotides useful, for example, for screening and / or identifying masked conjugated polypeptides (e.g., masked antibodies). The libraries described herein provide masked antibodies with desirable stability, structural and chemical diversity, as well as downstream development potential.

[0008] In one embodiment, the disclosure provides a library of polynucleotides encoding masked binding polypeptides (e.g., masked antibodies) that are effective, stable, and have a desirable therapeutic index. As described herein, the disclosed library is designed to identify masked binding polypeptides (e.g., masked antibodies) that can bind to a target antigen enriched or upregulated within the tumor microenvironment (TME) without being inhibited by a masking peptide attached to the antibody binding site. Thus, the masked binding polypeptides (e.g., masked antibodies) preferentially accumulate in regions where the concentration of the target antigen is sufficiently high and strongly bind to the unmasked antibody after cleavage of the masking peptide. For example, in some embodiments, the masking peptide (e.g., the masking peptide of the masked antibody) has a high dissociation rate constant (koff) from the antibody binding site and is not self-inhibitory. Furthermore, the masked binding polypeptides (e.g., masked antibodies) do not accumulate. In regions where the concentration of the target antigen is low (e.g., a target in normal tissue), off-target effects are reduced and the therapeutic window of the masked binding polypeptides (e.g., masked antibodies) generated from the disclosed library is improved.

[0009] Therefore, masked antibodies generated from the library accumulate in areas of high antigen concentration. In these high-antigen areas, significant cleavage of the masking units occurs, resulting in the generation of unmasked antibodies with high affinity for the target antigen. Conversely, in tissues with relatively low concentrations of the target antigen (e.g., blood), masked antibodies generated from the library do not accumulate. Furthermore, the high off-rate prevents masked antibodies from attaching to antigens in low-concentration microenvironments, mitigating potential off-target effects.

[0010] The polynucleotide libraries of this disclosure are designed to encode masked binding polypeptides (e.g., masked antibodies) having masking sequences (masking units) that can confer binding of the masked polypeptide to the target antigen without significant adhesion to the target antigen in normal tissue. Optimal balance can be achieved by appropriately selecting the masking units encoded by the library, as disclosed herein. In particular, the structured peptide libraries of this disclosure are designed to balance the composition of charged and polar amino acids and hydrophobic amino acids within the masking units, thereby enabling competitive binding between the antigen and the dynamic masking peptide through concentration-dependent antigen binding for transient activation.

[0011] In some embodiments, the library is designed to enrich glycine and proline residues within the loop region of a polypeptide (e.g., an antibody). Antibodies encoded by such a library can fold into structured loops to effectively shield the binding of the masked antibody to the target antigen without excessive self-inhibition or stickiness, and competitive binding between the antigen and the masked antibody is prevented by the masking peptide.

[0012] In some embodiments, the library was designed to avoid high-risk post-translational modification (PTM) sites, including free cysteine, glycosylation sites, deamidation sites, aspartate isomerization sites, and oxidation sites. For example, in certain embodiments, NG, M, W, and NX[S / T] sites within identified masking units were removed.

[0013] In some embodiments, a library of polynucleotides encodes masked antibodies. Specifically, in some embodiments, a library comprising polynucleotides, provided herein, wherein the polynucleotides in the library encode at least two, at least three, at least four, at least five, or at least ten antibodies, each having a unique masking peptide (MP), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), the MP being ligated to the N-terminus of the VH or VL of the antibody, the MP comprising a masking unit (MU) and a linkage unit (LU), the MU being of formula (I): The formula includes an amino acid sequence following X1X2CX3(Xm)nX4X5CX6X7, where: n is between 2 and 8. X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y, and X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. Each Xm is an amino acid independently selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X4 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X6 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X7 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine. In some embodiments, n is 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 3-4, 3-5, 3-6, 3-7, 3-8, 4-5, 4-6, 4-7, 4-8, 5-6, 5-7, 5-8, 6-7, 6-8, or 2-8. In a particular embodiment, n is 2, 3, 4, 5, 6, 7, or 8.

[0014] In another embodiment, a library comprising polynucleotides, provided herein, wherein the polynucleotides in the library encode at least two, at least three, at least four, at least five, or at least ten antibodies, each having a unique masking peptide (MP), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), the MP being ligated to the N-terminus of the VH or VL of the antibody, the MP comprising a masking unit (MU) and a linkage unit (LU), the MU comprising an amino acid sequence according to formula (II): X1X2CX3X4X5X6X7CX8X9, where: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X4 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, E, F, G, H, K, L, P, Q, R, S, T, V, and Y. X6 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X7 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X8 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X9 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine. In some embodiments, X1 is an amino acid selected from the group consisting of A, F, H, and V. X2 is an amino acid selected from the group consisting of A, L, and P. X3 is an amino acid selected from the group consisting of A, G, L, and R. X4 is an amino acid selected from the group consisting of E, G, K, and P. X5 is an amino acid selected from the group consisting of F, K, L, and V. X6 is an amino acid selected from the group consisting of F, L, P, and S. X7 is an amino acid selected from the group consisting of F, P, and Y. X8 is an amino acid selected from the group consisting of G, I, L, and P, and, X9 is an amino acid selected from the group consisting of E, Q, T, and V. In some embodiments, the polynucleotides in the library encode a masking peptide (MP), the MP comprising a masking unit (MU) and a linkage unit (LU). In some embodiments, the MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-10.

[0015] In some embodiments, a library comprising polynucleotides, provided herein, wherein the polynucleotides in the library encode at least two, at least three, at least four, at least five, or at least ten antibodies, each having a unique masking peptide (MP), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), the MP being ligated to the N-terminus of the VH or VL of the antibody, the MP comprising a masking unit (MU) and a linkage unit (LU), the MU comprising an amino acid sequence according to formula (III): X1X2CX3X4X5X6X7X8CX9X10, where: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X4 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X6 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, R, S, T, V, and Y. X7 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X8 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X9 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X10 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine. In some embodiments, X1 is an amino acid selected from the group consisting of A, F, H, N, P, S, and Y. X2 is an amino acid selected from the group consisting of A, L, P, S, and V. X3 is an amino acid selected from the group consisting of A, I, K, P, R, and V. X4 is an amino acid selected from the group consisting of A, G, L, P, V, and Y. X5 is an amino acid selected from the group consisting of F, I, L, P, R, and S. X6 is an amino acid selected from the group consisting of F, K, L, P, and Y. X7 is an amino acid selected from the group consisting of I, N, P, S, and V. X8 is an amino acid selected from the group consisting of A, F, L, and Y. X9 is an amino acid selected from the group consisting of G, K, Q, S, and V, and X10 is an amino acid selected from the group consisting of G, Q, R, S, T, and V. In some embodiments, the polynucleotides in the library encode a masking peptide (MP), which includes a masking unit (MU) and a linkage unit (LU). In some embodiments, the MU includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-19.

[0016] In some embodiments, a library comprising polynucleotides, provided herein, wherein the polynucleotides in the library encode at least two, at least three, at least four, at least five, or at least ten antibodies, each having a unique masking peptide (MP), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), the MP being linked to the N-terminus of the VH or VL of the antibody, the MP comprising a masking unit (MU) and a linkage unit (LU), the MP comprising a masking unit (MU) and a linkage unit (LU), the MU comprising an amino acid sequence according to formula (IV): X1X2CX3X4X5X6X7X8X9CX10X11, where: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X4 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X6 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X7 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, R, S, T, V, and Y. X8 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X9 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X10 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X11 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine. In some embodiments, X1 is an amino acid selected from the group consisting of A, H, L, P, S, and T. X2 is an amino acid selected from the group consisting of A, D, F, L, and Y. X3 is an amino acid selected from the group consisting of E, L, P, and R. X4 is an amino acid selected from the group consisting of A, E, K, P, and R. X5 is an amino acid selected from the group consisting of E, F, G, and L. X6 is an amino acid selected from the group consisting of A, F, P, T, and Y. X7 is an amino acid selected from the group consisting of A, P, S, T, and V. X8 is an amino acid selected from the group consisting of A, N, P, and S. X9 is an amino acid selected from the group consisting of V and Y. X10 is an amino acid selected from the group consisting of I, P, and R, and, X11 is an amino acid selected from the group consisting of E, G, I, P, and V. In some embodiments, a library comprising polynucleotides is provided herein, wherein the polynucleotides in the library encode a masking peptide (MP), and the MP comprises a masking unit (MU) and a linkage unit (LU). In some embodiments, the MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-27.

[0017] In some embodiments, a library comprising polynucleotides, provided herein, wherein the polynucleotides in the library encode at least two, at least three, at least four, at least five, or at least ten antibodies, each having a unique masking peptide (MP), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), the MP being ligated to the N-terminus of the VH or VL of the antibody, the MP comprising a masking unit (MU) and a linkage unit (LU), the MU comprising an amino acid sequence according to formula (V):X1X2CX3X4X5X6X7X8X9X10CX11X12, wherein: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X4 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X6 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X7 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X8 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, R, S, T, V, and Y. X9 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X10 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X11 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X12 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine. In some embodiments, X1 is an amino acid selected from the group consisting of A, F, P, S, and Y. X2 is an amino acid selected from the group consisting of H, L, P, S, and V. X3 is an amino acid selected from the group consisting of E, G, K, P, Q, and R. X4 is an amino acid selected from the group consisting of A, F, H, P, R, V, and Y. X5 is an amino acid selected from the group consisting of A, D, F, G, V, and Y. X6 is an amino acid selected from the group consisting of D, F, H, L, P, and V. X7 is an amino acid selected from the group consisting of H, K, P, R, and V. X8 is an amino acid selected from the group consisting of A, F, K, L, and P. X9 is an amino acid selected from the group consisting of A, D, F, L, and P. X10 is an amino acid selected from the group consisting of A, F, V, and Y. X11 is an amino acid selected from the group consisting of G, I, K, L, and R, and, X12 is an amino acid selected from the group consisting of A, E, K, P, R, and T. In some embodiments, polynucleotides in the library encode a masking peptide (MP), where the MP comprises a masking unit (MU) and a linkage unit (LU). In some embodiments, the MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 29-32 and 112-114.

[0018] In some embodiments of the above-described embodiments, the masking unit (MU) does not contain the amino acid sequence NG, DG, NXS, or NXT, where X is any amino acid. In some embodiments of the above-described embodiments, the masking peptide (MP) further comprises an N-terminal unit (NU) ligated to the N-terminus of the MU. In certain embodiments, the N-terminal unit is about 1 to 12 amino acid residues long. In certain embodiments, the N-terminal unit includes E, EVG, EVGSY (SEQ ID NO: 33), EVGVLDV (SEQ ID NO: 86), or EVGAESGVK (SEQ ID NO: 88).

[0019] In some embodiments of the above-described aspects, the LU does not include a cleavage site. In certain embodiments, the LU includes a linker. In certain embodiments, the linker includes or consists of an amino acid sequence selected from the group consisting of GS, GGS, GGG, SGRG (SEQ ID NO: 104), GGGGS (SEQ ID NO: 105), GGGGT (SEQ ID NO: 36), SGGS (SEQ ID NO: 37), GGSG (SEQ ID NO: 106), GGSGG (SEQ ID NO: 107), GGSSG (SEQ ID NO: 108), GSGGG (SEQ ID NO: 109), GGGSG (SEQ ID NO: 110), GSSSG (SEQ ID NO: 111), GSGGS (SEQ ID NO: 77), SGGG (SEQ ID NO: 79), GGGS (SEQ ID NO: 80), and GGGGSGGSGGGS (SEQ ID NO: 81). In certain embodiments, the LU includes the amino acid sequence of SEQ ID NO: 81.

[0020] In some embodiments of the aforementioned aspects, the LU includes a first cutting portion (C1). In a particular embodiment, the first cleavage site (C1) is a protease cleavage site of a protease selected from the group consisting of urokinase-type plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, tobacco etch virus (TEV) protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE. In certain embodiments, the first cleavage site (C1) includes an amino acid sequence selected from the group consisting of SGRSA (SEQ ID NO: 34) and PLGLAG (SEQ ID NO: 35). In some embodiments, the LU further includes a second cleavage site (C2). In a particular embodiment, the second cleavage site (C2) is a protease cleavage site of a protease selected from the group consisting of urokinase-type plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, tobacco etch virus (TEV) protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE. In certain embodiments, the second cleavage site (C2) includes an amino acid sequence selected from the group consisting of SGRSA (SEQ ID NO: 34) and PLGLAG (SEQ ID NO: 35). In certain embodiments, the first and second cleavage sites are the same. In certain embodiments, the first and second cleavage sites are different.In some embodiments, the LU further includes a first linker (L1). In certain embodiments, the first linker (L1) includes an amino acid sequence selected from the group consisting of GS, GGS, GGG, SGRG (SEQ ID NO: 104), GGGGS (SEQ ID NO: 105), GGGGT (SEQ ID NO: 36), SGGS (SEQ ID NO: 37), GGSG (SEQ ID NO: 106), GGSGG (SEQ ID NO: 107), GGSSG (SEQ ID NO: 108), GSGGG (SEQ ID NO: 109), GGGSG (SEQ ID NO: 110), GSSSG (SEQ ID NO: 111), GSGGS (SEQ ID NO: 77), SGGG (SEQ ID NO: 79), GGGS (SEQ ID NO: 80), and / GGGGSGGSGGGS (SEQ ID NO: 81). In certain embodiments, the LU further includes a second linker (L2). In a particular embodiment, the second linker (L2) contains an amino acid sequence selected from the group consisting of GS, GGS, GGG, SGRG (SEQ ID NO: 104), GGGGS (SEQ ID NO: 105), GGGGT (SEQ ID NO: 36), SGGS (SEQ ID NO: 37), GGSG (SEQ ID NO: 106), GGSGG (SEQ ID NO: 107), GGSSG (SEQ ID NO: 108), GSGGG (SEQ ID NO: 109), GGGSG (SEQ ID NO: 110), GSSSG (SEQ ID NO: 111), GSGGS (SEQ ID NO: 77), SGGG (SEQ ID NO: 79), GGGS (SEQ ID NO: 80), and GGGGSGGSGGGS (SEQ ID NO: 81). In some embodiments, LU comprises the following from the N-terminus to the C-terminus: 1) a first cleavage site (C1) and a first linker (L1); 2) a first cleavage site (C1), a first linker (L1), a second cleavage site (C2), and a second linker (L2); 3) a first linker (L1), a first cleavage site (C1), and a second linker (L2); or 4) a first linker (L1), a first cleavage site (C1), a second linker (L2), and a second cleavage site (C2). In some embodiments, LU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 38-42 and 119.

[0021] In some embodiments of the above-described embodiments, the masking peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-65, 67-75, 83-85, 87, and 116-118. In some embodiments of the above-described embodiments, the MP is ligated to the N-terminus of VL. In some embodiments of the above-described embodiments, the MP is ligated to the N-terminus of VH.

[0022] In some embodiments of the aforementioned models, each antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), with the MP ligated to the N-terminus of the VH or VL of the antibody. In certain embodiments, the antibody comprises an scFv containing the VH and VL. In certain embodiments, the antibody comprises a Fab containing the VH and VL. In certain embodiments, the antibody comprises an antibody heavy chain and an antibody light chain. In certain embodiments, the MP ligated to the N-terminus of the VL of the antibody.

[0023] In some embodiments of the aforementioned model, each antibody comprises a heavy chain variable region (VH), and the MP is ligated to the N-terminus of the antibody's VH. In a particular embodiment, the antibody comprises a VHH single-domain antibody or a VHH-Fc antibody containing VH, and the MP is ligated to the N-terminus of the antibody's VH.

[0024] In some embodiments of the aforementioned aspects, the polynucleotide encoding the polypeptide is located in a vector. In certain embodiments, the vector is an expression vector or a presentation vector. In some embodiments, the polynucleotide encoding the polypeptide is located in a host cell. In certain embodiments, the cell is a bacterial cell, a yeast cell, an insect cell, or a mammalian cell. In some embodiments of the aforementioned aspects, the MU in the library is 10 9 from 10 14 It exhibits a wide range of diversity.

[0025] In other embodiments, this specification provides libraries comprising antibodies encoded by polynucleotides in any of the libraries described above. In certain embodiments, each polypeptide is displayed on a cell surface or phage surface. In certain embodiments, the cell is a bacterial cell, a yeast cell, an insect cell, or a mammalian cell.

[0026] In other embodiments, this specification provides a method for producing antibodies, comprising culturing host cells expressing one of the aforementioned libraries containing antibodies under conditions suitable for antibody production. In certain embodiments, the method for producing antibodies further comprises recovering the antibodies produced by the cells. In certain embodiments, the method for producing antibodies further comprises testing the antibodies for their ability to maintain a masked phenotype while remaining soluble.

[0027] In some embodiments, the Specified Method provides a method for screening masked antibodies that bind to a target using any of the aforementioned libraries, the method comprising: a) contacting the expressed antibody of the library with the target to determine a first binding affinity or lack of detectable binding to the target; b) contacting the control antibody with the target to determine a second binding affinity; and c) selecting an expressed antibody that has a first binding affinity lower than the second binding affinity or does not bind detectably to the target, the first and second binding affinities being measured as KD, EC50, or IC50. In certain embodiments, the LU includes at least a first cleavage site (C1). In certain embodiments, the control antibody is the expressed antibody of the library after the LU has been cleaved. In certain embodiments, the expressed antibody is selected if its binding affinity after the LU has been cleaved is at least 2 times, at least 5 times, at least 10 times, at least 50 times, at least 100 times, at least 500 times, or at least 1000 times the binding affinity of the expressed antibody before the LU was cleaved.

[0028] In other embodiments, this specification provides a method for using any of the aforementioned libraries to screen for masked antibodies that bind to a target, the method comprising: a) contacting an expressed antibody from the library having the masking peptide with a first cell expressing a target antigen to determine the binding EC50; b) contacting a control antibody lacking the masking peptide with a first cell expressing a target antigen to determine the binding EC50; c) contacting an expressed antibody from the library with a second cell expressing a target antigen, the second cell expressing a lower level of the target antigen than the first cell; d) contacting a control antibody lacking the masking peptide with a second cell to determine the binding EC50; e) determining the ratio of the EC50 from step a) to the EC50 from step b) as a first masking efficiency; f) determining the ratio of the EC50 from step c) to the EC50 from step d) as a second masking efficiency; and g) selecting an expressed antibody having a second masking efficiency higher than the first masking efficiency. In certain embodiments, step (g) includes selecting an expressed antibody having a second masking efficiency that is at least 10%, at least 50%, at least 2x, at least 3x, at least 4x, at least 5x, or at least 10x higher than a first masking efficiency. In certain embodiments, the second masking efficiency is at least 50%, at least 2x, at least 3x, at least 4x, at least 5x, or at least 10x higher than the first masking efficiency. In certain embodiments, the control antibody is an antibody having the same antigen-binding domain as the expressed antibody in the library. In certain embodiments, the control antibody is the parent antibody. In certain embodiments, the LU does not contain a cleavage site. In certain embodiments, the LU contains at least a first cleavage site (C1). In certain embodiments, the control antibody is the expressed antibody in the library after the LU has been cleaved. In certain embodiments, the method includes cleaving the LU to produce the control antibody.

[0029] In some embodiments, methods for identifying masked antibodies capable of binding to an antigen in a concentration-dependent manner are provided herein, comprising: a) contacting a masked antibody having a masking peptide with a first cell expressing a target antigen to determine its binding EC50, wherein the masking peptide comprises a masking unit (MU) and a linkage unit (LU) from the N-terminus to the C-terminus; b) contacting a control antibody lacking the masking peptide with a first cell expressing a target antigen to determine its binding EC50; and c) contacting a first cell expressing a target antigen with the masked antibody... d) Contact a second cell to determine its binding EC50, the second cell expressing a lower level of the target antigen than the first cell; d) Contact a control antibody lacking the masking peptide to the second cell to determine its binding EC50; e) Determine the ratio of the EC50 from step a) to the EC50 from step b) as the first masking efficiency; f) Determine the ratio of the EC50 from step c) to the EC50 from step d) as the second masking efficiency; and g) Identify a concentration-dependent antigen-binding antibody if the second masking efficiency is higher than the first masking efficiency. In certain embodiments, step (g) includes identifying a concentration-dependent antigen-binding antibody if the second masking efficiency is at least 10%, at least 50%, at least 2 times, at least 3 times, at least 4 times, at least 5 times, or at least 10 times higher than the first masking efficiency. In certain embodiments, the control antibody is an antibody having the same antigen-binding domain as the masked antibody. In certain embodiments, the control antibody is the parent antibody. In certain embodiments, the LU does not contain a cleavage site. In certain embodiments, the LU contains at least a first cleavage site (C1). In certain embodiments, the control antibody is the masked antibody after the LU has been cleaved. In certain embodiments, the method includes cleaving the LU to produce the control antibody. In certain embodiments, the second masking efficiency is at least 50%, at least 2, at least 3, at least 4, at least 5, or at least 10 times higher than the first masking efficiency.

[0030] In some embodiments, the Specified herein provides antibodies encoded by one or more polynucleotides from any of the aforementioned libraries.

[0031] In some embodiments, this specification provides a kit that includes one of the libraries described above.

[0032] In some embodiments, any of the aforementioned libraries comprises cells, and at least two, at least three, at least four, at least five, or at least ten of the cells in the library contain the polynucleotides of the aforementioned library.

[0033] In some embodiments, a masked antibody comprising a masking peptide (MP) and an antibody is provided herein, wherein the antibody comprises an antibody light chain variable region (VL) and / or an antibody heavy chain variable region (VH), the MP is linked to the N-terminus of the VH or VL, and the MP comprises a masking unit (MU) and a linkage unit (LU) from the N-terminus to the C-terminus; the MU comprises an amino acid sequence according to formula (I): X1X2CX3(Xm)nX4X5CX6X7, where n is 2 to 8, where: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y, and X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, each Xm is independently an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y, X4 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y, X5 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y, X6 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and X7 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and C is cysteine. In some embodiments, n is 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 3-4, 3-5, 3-6, 3-7, 3-8, 4-5, 4-6, 4-7, 4-8, 5-6, 5-7, 5-8, 6-7, 6-8, or 2-8. In a particular embodiment, n is 2, 3, 4, 5, 6, 7, or 8.

[0034] In some embodiments, a masked antibody comprising a masking peptide (MP) and an antibody is provided herein, wherein the antibody comprises an antibody light chain variable region (VL) and / or an antibody heavy chain variable region (VH), the MP is linked to the N-terminus of the VH or VL, and the MP comprises a masking unit (MU) and a linkage unit (LU) from the N-terminus to the C-terminus; the MU comprises an amino acid sequence according to formula (II): X1X2CX3X4X5X6X7CX8X9, where: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X4 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, E, F, G, H, K, L, P, Q, R, S, T, V, and Y. X6 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X7 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X8 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and Y. X9 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine. In some embodiments, MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 10.

[0035] In some embodiments, a masked antibody comprising a masking peptide (MP) and an antibody, provided herein, wherein the antibody comprises an antibody light chain variable region (VL) and / or an antibody heavy chain variable region (VH), the MP is ligated to the N-terminus of the VH or VL, and the MP comprises a masking unit (MU) and a linkage unit (LU) from the N-terminus to the C-terminus; the MU comprises an amino acid sequence according to formula (III): X1X2CX3X4X5X6X7X8CX9X10, where: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X4 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X6 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, R, S, T, V, and Y. X7 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X8 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X9 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X10 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and C is cysteine. In some embodiments, X1 is an amino acid selected from the group consisting of A, F, H, N, P, S, and Y. X2 is an amino acid selected from the group consisting of A, L, P, S, and V. X3 is an amino acid selected from the group consisting of A, I, K, P, R, and V. X4 is an amino acid selected from the group consisting of A, G, L, P, V, and Y. X5 is an amino acid selected from the group consisting of F, I, L, P, R, and S. X6 is an amino acid selected from the group consisting of F, K, L, P, and Y. X7 is an amino acid selected from the group consisting of I, N, P, S, and V. X8 is an amino acid selected from the group consisting of A, F, L, and Y. X9 is an amino acid selected from the group consisting of G, K, Q, S, and V, and, X10 is an amino acid selected from the group consisting of G, Q, R, S, T, and V. In some embodiments, MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-19.

[0036] In some embodiments, a masked antibody comprising a masking peptide (MP) and an antibody is provided herein, wherein the antibody comprises an antibody light chain variable region (VL) and / or an antibody heavy chain variable region (VH), the MP is linked to the N-terminus of the VH or VL, and the MP comprises a masking unit (MU) and a linkage unit (LU) from the N-terminus to the C-terminus; the MU comprises an amino acid sequence according to formula (IV): X1X2CX3X4X5X6X7X8X9CX10X11, where: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X4 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X6 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X7 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, R, S, T, V, and Y. X8 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X9 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X10 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X11 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine. In some embodiments, X1 is an amino acid selected from the group consisting of A, H, L, P, S, and T. X2 is an amino acid selected from the group consisting of A, D, F, L, and Y. X3 is an amino acid selected from the group consisting of E, L, P, and R. X4 is an amino acid selected from the group consisting of A, E, K, P, and R. X5 is an amino acid selected from the group consisting of E, F, G, and L. X6 is an amino acid selected from the group consisting of A, F, P, T, and Y. X7 is an amino acid selected from the group consisting of A, P, S, T, and V. X8 is an amino acid selected from the group consisting of A, N, P, and S. X9 is an amino acid selected from the group consisting of V and Y. X10 is an amino acid selected from the group consisting of I, P, and R, and, X11 is an amino acid selected from the group consisting of E, G, I, P, and V. In some embodiments, MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-27.

[0037] In some embodiments, a masked antibody comprising a masking peptide (MP) and an antibody, provided herein, wherein the antibody comprises an antibody light chain variable region (VL) and / or an antibody heavy chain variable region (VH), the MP is linked to the N-terminus of the VH or VL, and the MP comprises a masking unit (MU) and a linkage unit (LU) from the N-terminus to the C-terminus; the MU comprises an amino acid sequence according to formula (V): X1X2CX3X4X5X6X7X8X9X10CX11X12, where: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X4 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X6 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X7 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X8 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, R, S, T, V, and Y. X9 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X10 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X11 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X12 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine. In some embodiments, X1 is an amino acid selected from the group consisting of A, F, P, S, and Y. X2 is an amino acid selected from the group consisting of H, L, P, S, and V. X3 is an amino acid selected from the group consisting of E, G, K, P, Q, and R. X4 is an amino acid selected from the group consisting of A, F, H, P, R, V, and Y. X5 is an amino acid selected from the group consisting of A, D, F, G, V, and Y. X6 is an amino acid selected from the group consisting of D, F, H, L, P, and V. X7 is an amino acid selected from the group consisting of H, K, P, R, and V. X8 is an amino acid selected from the group consisting of A, F, K, L, and P. X9 is an amino acid selected from the group consisting of A, D, F, L, and P. X10 is an amino acid selected from the group consisting of A, F, V, and Y. X11 is an amino acid selected from the group consisting of G, I, K, L, and R, and, X12 is an amino acid selected from the group consisting of A, E, K, P, R, and T. In some embodiments, MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-32 and 112-114.

[0038] In some embodiments, in any of the aforementioned masked antibodies, MU does not contain the amino acid sequence NG, DG, NXS, or NXT, and X is any amino acid. In some embodiments, MP further includes an N-terminal unit (NU) ligated to the N-terminus of MU. In certain embodiments, the N-terminal unit is about 1 to 12 amino acid residues long. In certain embodiments, the N-terminal unit includes E, EVG, EVGSY (SEQ ID NO: 33), EVGVLDV (SEQ ID NO: 86), or EVGAESGVK (SEQ ID NO: 88). In some embodiments, the masked antibody is an activatable antibody. In some embodiments, LU does not contain a cleavage site. In certain embodiments, LU includes a linker. In certain embodiments, the linker includes an amino acid sequence selected from the group consisting of GS, GGS, GGG, SGRG (SEQ ID NO: 104), GGGGS (SEQ ID NO: 105), GGGGT (SEQ ID NO: 36), SGGS (SEQ ID NO: 37), GGSG (SEQ ID NO: 106), GGSGG (SEQ ID NO: 107), GGSSG (SEQ ID NO: 108), GSGGG (SEQ ID NO: 109), GGGSG (SEQ ID NO: 110), GSSSG (SEQ ID NO: 111), GSGGS (SEQ ID NO: 77), SGGG (SEQ ID NO: 79), GGGS (SEQ ID NO: 80), and GGGGSGGSGGGS (SEQ ID NO: 81). In certain embodiments, the linker includes the amino acid sequence of SEQ ID NO: 81. In some embodiments, the LU includes a first cleavage site (C1).In a particular embodiment, the first cleavage site (C1) is a protease cleavage site of a protease selected from the group consisting of urokinase-type plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, tobacco etch virus (TEV) protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE. In certain embodiments, the first cleavage site (C1) includes an amino acid sequence selected from the group consisting of SGRSA (SEQ ID NO: 34) and PLGLAG (SEQ ID NO: 35). In some embodiments, the LU further includes a second cleavage site (C2). In a particular embodiment, the second cleavage site (C2) is a protease cleavage site of a protease selected from the group consisting of urokinase-type plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, tobacco etch virus (TEV) protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE. In certain embodiments, the second cleavage site (C2) includes an amino acid sequence selected from the group consisting of SGRSA (SEQ ID NO: 34) and PLGLAG (SEQ ID NO: 35). In certain embodiments, the first and second cleavage sites are the same. In certain embodiments, the first and second cleavage sites are different.

[0039] In some embodiments, in any of the masked antibodies described above, the LU further comprises a first linker (L1). In some embodiments, the first linker (L1) comprises an amino acid sequence selected from the group consisting of GS, GGS, GGG, SGRG (SEQ ID NO: 104), GGGGS (SEQ ID NO: 105), GGGGT (SEQ ID NO: 36), SGGS (SEQ ID NO: 37), GGSG (SEQ ID NO: 106), GGSGG (SEQ ID NO: 107), GSGSG (SEQ ID NO: 108), GSGGG (SEQ ID NO: 109), GGGSG (SEQ ID NO: 110), GSSSG (SEQ ID NO: 111), GSGGS (SEQ ID NO: 77), SGGG (SEQ ID NO: 79), GGGS (SEQ ID NO: 80), and GGGGSGGSGGGS (SEQ ID NO: 81). In certain embodiments, the LU further comprises a second linker (L2). In certain embodiments, the second linker (L2) contains an amino acid sequence selected from the group consisting of GS, GGS, GGG, SGRG (SEQ ID NO: 104), GGGGS (SEQ ID NO: 105), GGGGT (SEQ ID NO: 36), SGGS (SEQ ID NO: 37), GGSG (SEQ ID NO: 106), GGSGG (SEQ ID NO: 107), GGSSG (SEQ ID NO: 108), GSGGG (SEQ ID NO: 109), GGGSG (SEQ ID NO: 110), GSSSG (SEQ ID NO: 111), GSGGS (SEQ ID NO: 77), SGGG (SEQ ID NO: 79), GGGS (SEQ ID NO: 80), and GGGGSGGSGGGS (SEQ ID NO: 81). In some embodiments, the LU extends from the N-terminus to the C-terminus: 1) First cutting section (C1) and first linker (L1); 2) First cut section (C1), first linker (L1), second cut section (C2), and second linker (L2); 3) First linker (L1), first cut section (C1), and second linker (L2); or 4) Including a first linker (L1), a first cut section (C1), a second linker (L2), and a second cut section (C2). In some embodiments, LU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 38-42 and 119.

[0040] In some embodiments, in any of the aforementioned masked antibodies, the masking peptide contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-65, 67-75, 83-85, 87, and 116-118. In some embodiments, the MP is ligated to the N-terminus of VL. In some embodiments, the MP is ligated to the N-terminus of VH.

[0041] In some embodiments, in any of the masked antibodies described above, each antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the MP is ligated to the N-terminus of the VH or VL of the antibody. In certain embodiments, the antibody comprises an scFv containing the VH and VL. In certain embodiments, the antibody comprises a Fab containing the VH and VL. In certain embodiments, the antibody comprises an antibody heavy chain and an antibody light chain. In certain embodiments, the MP is ligated to the N-terminus of the VL of the antibody.

[0042] In some embodiments, in any of the masked antibodies described above, the antibody comprises a VHH single-domain antibody or a VHH-Fc antibody containing VH, wherein the MP is ligated to the N-terminus of VH in the antibody.

[0043] In some embodiments, in any of the aforementioned polynucleotides, the polynucleotide encodes the aforementioned masked antibody. In certain embodiments, the expression vector containing the polynucleotide is operably ligated to a promoter. In certain embodiments, the host cell contains the expression vector. In certain embodiments, the host cell is a bacterial cell, a yeast cell, an insect cell, or a mammalian cell. In certain embodiments, the method for producing the masked antibody comprises culturing the host cell under conditions suitable for producing the masked antibody. In certain embodiments, the method for producing the masked antibody further comprises recovering the antibody produced by the cell.

[0044] The masked binding polypeptides (e.g., masked antibodies) identified from the libraries of this disclosure possess advantageous properties. Firstly, autoinhibition upon activation (e.g., cleavage of the masking unit) is minimized by using a masking peptide with a relatively high off-rate to the antibody binding site to prevent effective binding between the antigen and antibody during peptide cleavage. Secondly, concentration-dependent binding of the masked polypeptide (e.g., masked antibody) to an antigen (e.g., TME) localized around the target region increases the local concentration of the masked antibody in the target region. In the case of a masking peptide with a cleavable linker, the masked antibody undergoes time-dependent cleavage of the masking peptide for persistent activation because the target region (e.g., TME) contains an enriched protease that enables such cleavage. Thirdly, kinetic control of peptide cleavage by the enzyme-rich microenvironment of the target region (e.g., TME) is also an important attribute. The kinetics of masking peptide cleavage in the microenvironment can be regulated, for example, by the selection of different protease cleavage sites within the masking peptide (e.g., protease sites present at varying levels within the TME) and by changes in the number of protease cleavage sites within the masking peptide linkage unit (e.g., one protease cleavage site and two protease cleavage sites). This regulation may result in a "sustained-release" effect of masked antibody activation localized within the TME.

[0045] In some embodiments, after selecting a masked binding polypeptide (e.g., a masked antibody) from a disclosed library, additional sequences of the masking peptide can be modified to adjust and optimize the folding, chemical, manufacturing, and control (CMC) solubility, thermal and cyclic stability, PK, and anti-drug antibody (ADA) related to the immunogenicity of the masking peptide in the context of the antibody binding site. For example, both the linker sequence and the N-terminal peptide (e.g., the amino acid residue bound to the N-terminus of the masking peptide) can be modified to control these factors.

[0046] In another embodiment, the disclosure provides masked antibodies generated from libraries disclosed herein. In some embodiments, the masked antibodies have MPs comprising masking units (MUs) and linkage units (LUs), as disclosed herein. In other embodiments, the LUs are cleaved after administration of the masked antibody. In other embodiments, the LUs are not cleaved after administration of the masked antibody. It will be understood that cleavable antibodies are administered at higher concentrations than non-cleavable antibodies.

[0047] In another embodiment, masked polypeptides (e.g., masked antibodies) may be selected by comparing their half-maximal effective concentration (EC50) and / or masking efficiency in environments with different antigen concentrations (e.g., environments with high antigen concentration and environments with low antigen concentration). In certain embodiments, masked polypeptides (e.g., masked antibodies) are selected that have a lower EC50 (i.e., increased potency) in regions with higher antigen concentrations than in regions with lower antigen concentrations. In other embodiments, masked antibodies are selected that have a lower masking efficiency in regions with higher antigen concentrations than in regions with lower antigen concentrations. In some embodiments, such concentration-dependent selection of masked polypeptides (e.g., masked antibodies) is performed after pre-selecting two or more masked polypeptides (e.g., masked antibodies) from a library. In some embodiments, the library is a library as disclosed herein. In other such embodiments, the library is not a library as disclosed herein.

[0048] In some embodiments, the masking efficiency of a masked polypeptide (e.g., a masked antibody) is about 2 to 50 times lower in an antigen-enriched environment (e.g., TME) than in a normal level of antigen (e.g., healthy tissue). In some embodiments, the masking efficiency of a masked polypeptide (e.g., a masked antibody) is about 3 to 20 times lower in an antigen-enriched environment (e.g., TME) than in a normal level of antigen (e.g., healthy tissue). In some embodiments, the masking efficiency of a masked polypeptide (e.g., a masked antibody) is about 4 to 10 times lower in an antigen-enriched environment (e.g., TME) than in a normal level of antigen (e.g., healthy tissue).

[0049] It should be understood that one, some, or all of the characteristics of the various embodiments described above and herein may be combined to form other embodiments of the present disclosure. These and other embodiments of the present disclosure will be apparent to those skilled in the art. These and other embodiments of the present disclosure will be further described by embodiments for carrying out subsequent inventions. [Brief explanation of the drawing]

[0050] [Figure 1A-1C] This shows the concentration-dependent binding of a masked anti-HER2 antibody to cells expressing various concentrations of HER2 antigen. Figure 1A shows binding to SCOV3 cells with high HER2 expression levels and high HER2 antigen concentrations. Figure 1B shows binding to MCF7 cells with low HER2 expression levels and low HER2 antigen concentrations. Figure 1C shows binding to A549 cells with low HER2 expression levels and low HER2 antigen concentrations.

[0051] [Figure 2] This shows in vitro time-dependent cleavage of isolated anti-CTLA4 masked antibodies TY24652 and TY26294 by MMP-9.

[0052] [Figure 3A-3D] This shows the in vivo time-dependent cleavage of anti-CTLA4 masked antibodies TY24652 and TY26294 in a mouse H22 tumor model. Mice were administered 5 mg / kg of each antibody. Fresh tissue was isolated from mice at 24 and 96 hours post-administration and analyzed for cleaved and intact antibodies by Western blotting. Figure 3A shows Western blots of cleaved and complete antibodies from tissue collected 24 hours post-administration. Figure 3B is a bar graph showing the relative amounts of complete and cleaved antibodies from tissue collected within 24 hours post-administration. Figure 3C shows Western blots of cleaved and complete antibodies from tissue collected 96 hours post-administration. Figure 3D is a bar graph showing the relative amounts of complete and cleaved antibodies from tissue collected 96 hours post-administration.

[0053] [Figure 4A-4C] This study demonstrates the in vivo time-dependent cleavage of the anti-CTLA4 masked antibody TY26294 in a mouse SHP-77 xenograft model. Mice were administered a single intravenous dose of 10 mg / kg of TY26294, and plasma and tumor samples were collected at various time points to analyze the presence of cleaved and complete antibodies. Figure 4A shows the complete and cleaved forms of TY26294 in plasma samples over time. Figure 4B shows the complete and cleaved forms of TY26294 in SHP-77 tumor samples over time. Figure 4C shows a comparison of TY26294 cleavage in tumors and plasma. [Modes for carrying out the invention]

[0054] This specification describes an improved library for selecting masked antigen-binding polypeptides (e.g., masked antibodies) useful for therapeutic treatments with a superior safety profile. In the library described herein, the masking peptides linked to the antigen-binding polypeptides (e.g., masked antibodies) are designed and executed using masking sequences (masking units) with relatively high off-rates, enabling concentration-dependent binding and reduced self-inhibition, optimized cleavable and non-cleavable linking units and N-terminal peptides, and good developability. The improved masking units incorporate several unique functionalities, making the library described herein a powerful tool for identifying masking units of target antibodies with improved efficacy and safety profiles.

[0055] First, amino acids with charged side chains (D / E / H / K / R) were enriched into the masking unit design. These charged polar amino acids, along with amino acids with polar side chains (S / T / N / Q / Y), constitute the majority of the masking unit residues in the library composition. Thus, the identified masking units provide the masked antigen-binding domain with a balanced ratio of charge and distribution of polar amino acid residues and hydrophobic amino acids. Second, the masking units contain a loop region surrounded by two cysteine ​​residues that form an intramolecular disulfide bond. Importantly, glycine and proline residues, which are crucial to the peptide backbone structure, are intentionally enriched in the loop region for optimal loop formation. Thus, the identified masking units can fold into a structured loop to effectively shield the binding of the antigen and masked antibody without excessive self-inhibition or stickiness, preventing competitive binding of the masking peptide to the antigen and masked antibody. Thirdly, the library was designed to avoid high-risk post-translational modification (PTM) sites such as free cysteine ​​(except for the two cysteine ​​adjacent to the loop region), glycosylation sites, deamidation sites, aspartate isomerization sites, and oxidation sites. For example, the NG, M, W, and NX[S / T] sites were removed from the identified masking units. These design considerations allow for the selection of masked antibodies with less "sticky" self-inhibitory masking units compared to previous masking antibodies. The selected antibodies can undergo concentration-dependent competitive binding of the antigen to the antigen-binding domain (ABD) in the TME, for example, in high concentrations of the antigen, without cleavage-dependent removal of the masking peptide from the antibody. Thus, the selected masked antibodies are enriched and transiently activated in the TME, while masked antibodies present in other tissues (e.g., bloodstream) remain masked by the masking units. In the case of masked antibodies with cleavable linkers, enrichment and transient activation are followed by time-dependent permanent activation by proteases in the TME. This can be adjusted by modifying the N-terminus and cleavable linker sequence of the masked antibody.These characteristics allow for the generation of masked antibodies that exhibit high binding affinity within the TME, resulting in a high antitumor effect, while simultaneously minimizing toxic effects from antibody binding outside the TME. I. Definition

[0056] Before describing this disclosure in detail, it should be understood that this disclosure is not limited to any particular composition or biological system, and is, needless to say, diverse. It should also be understood that the terms used herein are intended solely to describe specific embodiments and are not intended to be limiting.

[0057] As used herein, unless the context makes clear, the singular forms “a,” “an,” and “the” include multiple references. For example, a reference to “molecule” may include two or more such combinations, etc.

[0058] As used herein, the term “approximately” refers to the normal margin of error for each value, as readily understood by those skilled in the art. References to “approximately” values ​​or parameters herein include (and describe) embodiments relating to the value or parameter itself.

[0059] It should be understood that the aspects and embodiments of the disclosure described herein include "including," "consisting of," and "essentially consisting of."

[0060] As used herein, the term "and / or" is intended to include phrases such as "A and / or B" as encompassing both A and B, A or B, A (alone), and B (alone). Similarly, as used herein, the term "and / or" is intended to include phrases such as "A, B, and / or C" as encompassing each of the following embodiments: 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).

[0061] The term "amino acid" refers to natural and synthetic amino acids, as well as amino acid analogs and amino acid mimes that function similarly to natural amino acids. Natural amino acids are those encoded by the genetic code, as well as those that have been later modified, such as hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. The term "amino acid analog" refers to compounds that have the same basic chemical structure as natural amino acids, but whose C-terminal carboxyl group, N-terminal amino group, or side-chain functional group is chemically modified to a different functional group. The term "amino acid mime" refers to chemical compounds that have a different structure from the general chemical structure of amino acids, but that function similarly to natural amino acids.

[0062] Where used herein, the 20 conventional amino acids and their abbreviations follow conventional usage. For example, see Immunology-ASynthesis (2nd Edition, E.S. Goluband, D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)).

[0063] The terms “polypeptide,” “protein,” and “peptide” are used interchangeably herein and may refer to polymers of two or more amino acids.

[0064] As used interchangeably herein, “polynucleotide” or “nucleic acid” refers to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or analogs thereof, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase or by synthetic reactions. Polynucleotides may include modified nucleotides, such as methylated nucleotides and their analogs. Modifications to the nucleotide structure, where present, may be conjugated before or after the assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may include post-synthesis modifications, such as conjugation to labels. Other types of modifications include, for example, "caps" that replace one or more naturally occurring nucleotides with analogs, internucleotide modifications such as those by non-charged bonds (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and those by charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), such as those involving suspension portions of proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those by insertors (e.g., acridine, psoralen, etc.), those involving chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.), those involving alkylating agents, those by modifying bonds (e.g., alpha-anomeric nucleic acids, etc.), and the unmodified form of polynucleotides. Furthermore, any of the hydroxyl groups normally present in the sugar may be substituted with, for example, a phosphonic acid group or a phosphate group, protected with a standard protecting group, or activated to prepare additional binding to additional nucleotides, or conjugated to a solid or semi-solid support. The 5' and 3' terminal OH groups can be phosphorylated or substituted with amines or organic capping groups of 1 to 20 carbon atoms. Other hydroxyls can be derivatized with standard protecting groups.Polynucleotides may also contain analogous forms of ribose or deoxyribose sugars commonly known in the art, such as 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-, or 2'-azidol-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xylose, or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acrylic acid analogs, and basic nucleoside analogs such as methylriboside. One or more phosphodiester bonds may be replaced with alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which the phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H, or a substituted or unsubstituted alkyl (1-20C) (optionally including an ether (-O-) linkage), aryl, alkenyl, cycloalkyl, cycloalkenyl, or aralgyl. Not all links in the polynucleotide are to be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[0065] The term “isolated nucleic acid” refers to a nucleic acid molecule of genomic, cDNA, or synthetic origin, or a combination thereof, that has been isolated from other nucleic acid molecules present in the nucleic acid’s natural source. For example, with respect to genomic DNA, the term “isolated” includes nucleic acid molecules that have been isolated from the chromosome to which the genomic DNA is naturally bound. Preferably, “isolated” nucleic acids do not contain sequences naturally adjacent to the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid of interest).

[0066] As used herein, “library” refers to a set of two or more entities that share a common class. For example, a library containing polynucleotides may refer to a set of two or more polynucleotides. The term “library” is used most broadly herein and specifically encompasses sublibraries that may or may not be combined.

[0067] As used herein, “unique” refers to a member of a set that is distinct from the other members of the set. For example, a unique masked antibody in a library may refer to a masked antibody that has a specific mask peptide sequence not shared by other masked antibodies in the library. In practice, it should be understood that a “unique” member of a library’s physical realization may exist in two or more copies. For example, a library may contain multiple “unique” masked antibodies such that one or more of the “unique” masked antibody molecules occur in two or more copies.

[0068] As used herein, “variety” refers to variety and / or heterogeneity. For example, antibody diversity in a library refers to a wide variety of antibodies present in the library, each with its own unique sequence, or to a variety of masked antibodies in the library, each with its own unique sequence within the masking peptide.

[0069] The term "antibody" is used in its broadest sense herein and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, masked antibodies (e.g., activatable or deactivatable antibodies), multispecific antibodies (e.g., bispecific antibodies), and antibody fragments or single-domain antibodies of human and natural origin (e.g., single-strand variable fragments or scFv or VHH single-domain fragments, etc.), as long as they exhibit the desired biological activity.

[0070] In some embodiments, the term “antibody” refers to an antigen-binding protein (i.e., immunoglobulin) having a basic four-polypeptide chain structure consisting of two identical heavy (H) chains and two identical light (L) chains. Each L chain is linked to an H chain by one disulfide covalent bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each heavy chain has a variable region (abbreviated herein as VH) at its N-terminus, followed by a constant region. The heavy chain constant region consists of three domains CH1, CH2, and CH3. Each light chain has a variable region (abbreviated herein as VI) at its N-terminus, followed by a constant region at its opposite end. The light chain constant region consists of one domain CL. VL aligns with VH, and CL aligns with the first constant domain (CH1) of the heavy chain. The pairing of VH and VL together forms a single antigen-binding site. IgM antibodies consist of five basic heterotetrameric units and an additional polypeptide called a J chain, and thus contain 10 antigen-binding sites. However, secreted IgA antibodies can polymerize to form multivalent aggregates containing 2 to 5 basic quad units and J chains.

[0071] The VH and VL regions can be further subdivided into hypervariable regions called hypervariable regions (HVRs) based on structural and sequence analysis. Within the HVRs are scattered more conserved regions called framework regions (FWs) (see, e.g., Chenetal. (1999) J. Mol. Biol. (1999) 293, 865-881). Each VH and VL consists of three HVRs and four FWs, sequenced from the amino terminus to the carboxyl terminus in the following order: FW-1_HVR-1_FW-2_HVR-2_FW-3_HVR-3_FW4. Throughout this disclosure, the three HVRs of the heavy chain are referred to as HVR-H1, HVR-H2, and HVR-H3. Similarly, the three HVRs of the light chain are referred to as HVR-L1, HVR-L2, and HVR-L3.

[0072] Table I below provides exemplary CDR definitions according to various algorithms known in the art. Table 1. CDR definition TIFF2026515742000001.tif50170 1 The residue numbering follows the nomenclature of Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., US Dept. of Health and Human Services, “Sequences of proteins of immunological interest” (1991). 2 Residue numbering follows the nomenclature of Chothia et al., J. Mol. Biol. 196:901-917(1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948(1997). 3 Residue numbering follows the nomenclature of MacCallum et al., J. Mol. Biol. 262:732-745(1996); Abhinandan and Martin, Mol. Immunol.,45:3832-3839(2008). 4 Residue numbering follows the nomenclature of Lefranc MP et al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plueckthun, J. Mol. Biol., 309:657-670 (2001). 5 Residue numbering follows the nomenclature of Honegger and Plueckthun, J. Mol. Biol., 309:657-670 (2001).

[0073] The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of immunoglobulins to various cells of the immune system (e.g., effector cells) and to host tissues or factors, including the first component (C1q) of the classical complement system. In the light and heavy chains, the variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 10 or more amino acids (see, for example, Fundamental Immunology Ch.7 (Paul, W., ed., 2nd ed. Raven Press, NY) (1989)).

[0074] Light chains (L chains) derived from any vertebrate species can be assigned to one of two distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Antibodies can be assigned to different classes or isotypes depending on the amino acid sequence of the constant domain of their heavy chain (CH). There are five classes of antibodies: IgA, IgD, IgE, IgG, and IgM, each having heavy chains named α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu), respectively. The IgG class of antibodies can be further classified into four subclasses, IgG1, IgG2, IgG3, and IgG4, based on the gamma heavy chains Y1-Y4.

[0075] In this specification, the terms “antigen-binding fragment,” “antigen-binding moiety,” or “antigen-binding domain” of an antibody, as used interchangeably, refer to one or more portions of an antibody that possess the ability to bind to the antigen to which the antibody binds. Examples of antibody "antigen-binding fragments" include (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab′)2 fragments, which are bivalent fragments containing two Fab fragments linked by disulfide crosslinks in a hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VL and VH domains of a single arm of the antibody; (v) scFv fragments consisting of VL and VH domains of a single arm of the antibody fused via a linker; (vi) dAb fragments consisting of a VH domain (Wardetal., Nature 341:544-546 (1989)), also known as VHH monovariate domains; (vii) VHH-Fc antibodies, which consist of a VH domain fused to an Fc domain; and (viii) isolated complementarity-determining regions (CDRs).

[0076] The term “masked antibody” refers to an antibody or its antigen-binding fragment that contains a masking peptide that interferes with, inhibits, reduces, prevents, inhibits, or competes with the antibody’s antigen-binding domain for binding to its target. Masked antibodies can be produced by conjugating a masking peptide to the antigen-binding domain of an antibody. In some embodiments, a masked antibody or its antigen-binding fragment exhibits a first binding affinity to a target when in an inactive state (e.g., inhibited or masked by the masking peptide) and a second binding affinity to a target when in an activated state (e.g., not inhibited or masked by the masking peptide (e.g., the masking peptide has been cleaved from the antibody)), where the second binding affinity is greater than the first binding affinity. Such embodiments may be called “activatable antibodies.”

[0077] Activatable antibodies can be activated in various ways. For example, an activatable antibody may contain a cleavable linker that, when cleaved, leads to the activation of the antibody. Thus, an activatable masked antibody may be produced by linking a masking peptide containing an activatable component (e.g., a cleavable site) to the antigen-binding domain of the antibody. In another example, a masked activatable antibody is not cleavable, but instead may be activated in a concentration-dependent manner, for example, under conditions of high concentration of the target antigen. Masked antibodies and activatable antibodies are described, for example, in U.S. Patent Publication 2019 / 0241886 and U.S. Patent Publication 2021 / 0207126, the contents of which are incorporated herein by reference in their entirety.

[0078] A "masking peptide" refers to a peptide that inhibits the binding of an antigen-binding domain to a target antigen, and typically contains a masking unit (MU) and a linkage unit (LU) from the N-terminus to the C-terminus. The C-terminus of a masking peptide is usually bound to the N-terminus, or to the VH or VL end of the antigen-binding domain. In some embodiments, the masking peptide or a portion thereof (e.g., the MU) very efficiently interferes with or inhibits the binding of the antigen-binding domain to its target, resulting in extremely low and / or below-detection binding of the antigen-binding domain to its target (e.g., binding is undetectable by ELISA or flow cytometry assays). In other embodiments, the masking peptide or a portion thereof (e.g., the MU) binds weakly to the antigen-binding domain of an antibody, thus allowing detectable binding of the antigen-binding domain to its target under certain conditions (e.g., binding may be detected in ELISA or flow cytometry assays where the antigen is present at high concentrations). The masked antibodies or polypeptides described herein may include, for example, one or more linkers located within the LU, between MU and LU, between LU and VH or VL, or between VH and the hinge region of Fc. The masking peptide may further include an N-terminal unit consisting of 1 to 12 amino acids at its N-terminus.

[0079] The LU of a masking peptide typically contains at least one linker and may or may not contain at least one cleavage site. Cleavage sites generally include cleavable amino acid sequences, e.g., amino acid sequences that function as substrates for enzymes, and / or cysteine-cysteine ​​pairs capable of forming a reducible disulfide bond. Therefore, when terms such as “cleaved,” “cleavable,” and “cleaved” are used in relation to cleavage sites, these terms include not only enzymatic cleavage by proteases, for example, but also the breakdown of the disulfide bond between cysteine-cysteine ​​pairs by reduction of the disulfide bond, which may result from exposure to a reducing agent. The amino acid sequence of the cleavage site may overlap with or be contained within the MU. Masked antibodies or masked polypeptides may contain cleavage sites configured to mediate the activation of the antibody or polypeptide. For example, if the cleavage sites of an activatable masked antibody having a cleavable masking peptide are intact (e.g., including a cysteine-cysteine ​​disulfide bond that is not cleaved and / or reduced by the corresponding enzyme), the masking peptide or any part thereof may interfere with or inhibit the binding of the antigen-binding domain to its target.

[0080] In some cases, the LU of a masking peptide may be non-cleavable and may not contain cleavable sites. Activatable masked antibodies with LUs lacking cleavable sites may be activated by high antigen concentrations, for example, within the tumor microenvironment (TME), if the tumor expresses high levels of the antigen. This can lead to transient activation of the antibody. For example, an antibody may be concentration-dependently activated within a tumor or tissue with high expression of the target antigen, and then inactivated when it moves from the tumor or tissue to an environment with lower antigen concentrations (e.g., entering the circulatory system).

[0081] The term "masking efficiency" refers to the efficiency with which a masking peptide inhibits the binding of an antigen-binding domain to a target antigen. Masking efficiency can be measured as a difference or ratio of properties (e.g., binding affinity to the target antigen) or activity (e.g., inhibition of binding between the target antigen and ligand) between a masked antibody (e.g., an activatable antibody) having an antigen-binding domain and a masking peptide, and a corresponding unmasked antibody ("parent antibody") having the same antigen-binding domain but lacking the masking peptide. Masking efficiency can also be measured as a difference or ratio of the binding affinity of a masked antibody or masked polypeptide containing an antigen-binding domain, and the binding affinity of an unmasked antibody or unmasked polypeptide containing an antigen-binding domain (e.g., the masking peptide is cleaved from the antibody or is transiently activated by competitive binding between the antigen and the masked antibody). For example, masking efficiency can be measured by dividing the EC50 or KD of a masked antibody bound to an inactivated (e.g., inhibited, masked, and / or uncleaved) target antigen by the EC50 or KD of an unmasked antibody bound to an activated (e.g., uninhibited, unmasked, and / or cleaved) target antigen, or by the EC50 or KD of a parent antibody (e.g., not linked to a masking peptide) bound to the target antigen. The EC50 value can be measured by an ELISA assay or a JurkatNFAT reporter assay, for example, as described in U.S. Patent Application Publication No. 20210207126A1. The KD value can be measured, for example, using surface plasmon resonance.

[0082] The term "competing for binding" refers to the interaction of two antibodies in binding to a target. A first antibody competes with a second antibody for binding if, in the presence of the second antibody, the binding of the first antibody to its congener epitope is detected to a noticeable degree compared to the binding of the first antibody in the absence of the second antibody. An alternative, though possible, is that the binding of the second antibody to its epitope is also detected to a noticeable degree in the presence of the first antibody. That is, the first antibody can inhibit the binding of the second antibody to its respective epitope without the second antibody inhibiting the first antibody's binding to that epitope. However, if each antibody detectably inhibits the binding of the other antibody to its congener epitope, whether to the same degree, more, or less, the antibodies are said to "cross-compete" with each other for binding to their respective epitopes.

[0083] The term "epitope" refers to the portion of an antigen to which an antibody (or its antigen-binding fragment) binds. Epitopes can be formed from both adjacent amino acids or non-adjacent amino acids that are paralleled by tertiary folding of the protein. Epitopes formed from adjacent amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes can contain a variety of amino acids in their unique spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, X-ray crystallography, two-dimensional nuclear magnetic resonance, deuterium and hydrogen exchange combined with mass spectrometry, or site-directed mutagenesis, or all methods used in combination with computational models of the complex structure of the antigen and its bound antibody and its variants (see, e.g., EpitopeMappingProtocolsinMethodsinMolecularBiology, Vol.66, GEMorris, Ed. (1996)). Once the desired epitope of an antigen is determined, an antibody against that epitope can be generated, for example, using the techniques described herein. Furthermore, antibody generation and characterization can elucidate information about the desired epitope. This information allows for competitive screening of antibodies for binding to the same epitope. An approach to achieve this is to conduct cross-competition studies to discover antibodies that bind competitively to each other; that is, antibodies compete for binding to the antigen. A high-throughput process for "binning" antibodies based on this cross-competition is described in PCT Publication WO03 / 48731.

[0084] The term "germline" refers to the nucleotide sequences of antibody genes and gene segments that are passed from parent to offspring via germ cells. Germline sequences are distinct from nucleotide sequences that encode antibodies in mature B cells, which are altered by recombination and high-frequency mutation events during the maturation process of B cells.

[0085] The term “glycosylation site” refers to an amino acid residue recognized by eukaryotic cells as a site for the attachment of a sugar residue. Typical amino acids to which carbohydrates such as oligosaccharides attach are asparagine (N-linked), serine (O-linked), and threonine (O-linked) residues. Specific attachment sites are typically signaled by an amino acid sequence, referred to herein as the “glycosylation site sequence.” The glycosylation site sequence for N-linked glycosylation is -Asn-X-Ser- or -Asn-X-Thr-, where X can be any conventional amino acid other than proline. The terms “N-linked” and “O-linked” refer to chemical groups that function as attachment sites between a sugar molecule and an amino acid residue. N-linked sugars attach via an amino group. O-linked sugars attach via a hydroxyl group. The term “glycan occupancy” refers to the presence of a carbohydrate moiety linked to the glycosylation site (i.e., the glycan moiety is occupied). If there are at least two potential glycosylation sites on the polypeptide, then either zero (occupation of 0-glycan sites), one (occupation of 1-glycan sites), or both (occupation of 2-glycan sites) of these sites may be occupied by the carbohydrate moiety.

[0086] The term “host cell” refers to a cell line that can be manipulated to produce a target protein, protein fragment, or peptide. Host cells include, but are not limited to, cultured cells, such as mammalian cultured cells derived from rodents (rats, mice, guinea pigs, or hamsters) such as CHO, BHK, NSO, SP2 / 0, YB2 / 0; human cells (e.g., HEK293F cells, HEK293T cells; or human tissue or hybridoma cells, yeast cells, insect cells (e.g., S2 cells), bacterial cells (e.g., E. coli cells), and cells contained within transgenic animals or cultured tissues. The term encompasses not only specific target cells but also their offspring. Such offspring may not be identical to the parent cells because certain modifications can occur in subsequent generations due to either mutation or environmental influences, but they still fall within the scope of the term “host cell.”

[0087] A "human antibody" is an antibody produced by a human or human cell, or derived from a non-human source that utilizes the human antibody repertoire, possessing an amino acid sequence that corresponds to another human antibody coding sequence. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0088] The term "humanized antibody" refers to a chimeric antibody that contains amino acid residues derived from a human antibody sequence. Humanized antibodies may contain some or all of the CDRs or HVRs from non-human animals or synthetic antibodies, but the antibody framework and constant region contain amino acid residues derived from a human antibody sequence.

[0089] The term “exemplary antibody” refers to any one of the antibodies described herein. These antibodies may be of any class (e.g., IgA, IgD, IgE, IgG, and IgM). Thus, each antibody identified above encompasses antibodies of all five classes having the same amino acid sequence for the VL and VH regions. Furthermore, antibodies of the IgG class may be of any subclass (e.g., IgG1, IgG2, IgG3, and IgG4). Thus, each antibody identified above in the IgG subclass encompasses antibodies of all four subclasses having the same amino acid sequence for the VL and VH regions. The amino acid sequences of the heavy chain constant regions of human antibodies in the five classes and four IgG subclasses are known in the art.

[0090] An “isolated” antibody or conjugated molecule is one that has been isolated from its natural environment. In some embodiments, the antibody is purified to a purity of 95% or greater than 99%, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For an overview of methods for assessing antibody purity, see, for example, Flatmanetal., J. Chromatogr. B848:79-87 (2007).

[0091] The term "ka" refers to the association rate constant of a particular antibody-antigen interaction, while the term "kd" refers to the dissociation rate constant of a particular antibody-antigen interaction.

[0092] The term "KD" refers to the parallel dissociation constant of a particular antibody-antigen interaction. It is derived from the kd to ka ratio (i.e., kd / ka) and expressed as molar concentration (M). KD is used as a measure of the affinity of an antibody to its binding partner. A smaller KD indicates a stronger binding of the antibody or a higher affinity between the antibody and the antigen. For example, an antibody with a nanomolar (nM) dissociation constant will bind more strongly to a particular antigen than an antibody with a micromolar (μM) dissociation constant. The KD value of an antibody can be determined using methods well established in the art. One method for determining the KD of an antibody is by using ELISA. For example, an assay procedure using ELISA.

[0093] The term "mammal" refers to all animal species in the mammalian class. Examples of mammals include humans; laboratory animals such as rats, mice, hamsters, rabbits, non-human primates, and guinea pigs; domesticated animals such as cats, dogs, cows, sheep, goats, horses, and pigs; and captured wild animals such as lions, tigers, and elephants.

[0094] In relation to specific disease conditions in mammals, the terms “prevent” or “prevention” refer to preventing or delaying the onset of the disease, or preventing the manifestation of its clinical or asymptomatic symptoms.

[0095] As used herein, “sequence identity” between two polypeptide sequences refers to the percentage of amino acids that are identical between the sequences. The amino acid sequence identity of polypeptides can be conventionally determined using known computer programs such as Bestfit, FASTA, or BLAST (see, for example, Pearson, MethodsEnzymol. 183:63-98 (1990); Pearson, MethodsMol.Biol. 132:185-219 (2000); Altschuletal., J.Mol.Biol. 215:403-410 (1990); Altschuletal., NucleicAcidsRes. 25:3389-3402 (1997)). When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference amino acid sequence, the parameters are set such that the percentage of identity is calculated over the entire length of the reference amino acid sequence, and a difference in homology of up to 5% of the total number of amino acid residues in the reference sequence is permitted. This aforementioned method for determining the percentage of identity between polypeptides is applicable to all proteins, fragments, or variants thereof disclosed herein.

[0096] As used herein, the terms “binding,” “binding,” “specifically binding to,” or “specific to” refer to measurable and reproducible interactions, such as binding between a target and an antibody, which determines the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody that binds to a target (which may be an epitope), or specifically binds to it, is an antibody that binds to this target more easily and / or for a longer period of time, with higher affinity or binding strength, than an antibody that binds to other targets. In one embodiment, the degree of antibody binding to an unrelated target is less than about 10% of the antibody binding to the target, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM. In certain embodiments, the antibody specifically binds to an epitope on a protein that is conserved between proteins of different species. In another embodiment, specific binding may include, but does not require, exclusive binding.

[0097] The terms “to treat,” “to treat,” or “treatment” refer to causing a desirable or beneficial effect in a mammal with respect to a particular disease condition in a mammal. Desirable or beneficial effects may include a reduction in the frequency or severity of one or more symptoms of the disease (i.e., tumor growth and / or metastasis, or other effects mediated by the number and / or activity of immune cells), or the prevention or inhibition of further progression of the disease, condition, or disorder. In the context of treating cancer in mammals, desirable or beneficial effects may include the inhibition of further growth or metastasis of cancer cells, the death of cancer cells, the inhibition of cancer recurrence, the reduction of cancer-related pain, or the improvement of the mammal’s viability. Effects may be subjective or objective. For example, if the animal is a human, the human may report improved vitality or viability, or reduced pain, as subjective symptoms of improvement or treatment response. Alternatively, a clinician may report a reduction in tumor size or load based on physical examination, laboratory values, tumor markers, or radiographic findings. Some clinical signs that clinicians may observe regarding treatment response include normalization of laboratory values ​​such as white blood cell count, red blood cell count, platelet count, erythrocyte sedimentation rate, and various enzyme levels. Furthermore, clinicians may observe a decrease in detectable tumor markers. Alternatively, objective improvement can be assessed using other tests, such as ultrasound imaging, magnetic resonance imaging, and positron emission tomography.

[0098] The term "vector" refers to a nucleic acid molecule capable of transporting foreign nucleic acid molecules. Foreign nucleic acid molecules are ligated into the vector nucleic acid molecule by recombination techniques such as ligation or recombination. This allows the foreign nucleic acid molecule to be grown, selected, further manipulated, or expressed in a host cell or organism. Vectors can be plasmids, phages, transposons, cosmids, chromosomes, viruses, or virions. Some types of vectors can be integrated into the host cell's genome upon introduction, thereby replicating alongside the host genome (e.g., non-episomal mammalian vectors). Other types of vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors with bacterial replication and episomal mammalian vectors). Another specific type of vector capable of directing the expression of expressible foreign nucleic acids to which they are operably ligated is commonly referred to as an "expression vector." Expression vectors generally have a regulatory sequence that drives the expression of expressible foreign nucleic acids. Simpler vectors, known as "transcription vectors," are transcriptionally capable but not translatable; these can replicate in target cells but cannot be expressed. The term “vector” encompasses all types of vectors, regardless of their function. Vectors that can direct the expression of expressible nucleic acids to which they are functionally linked are typically referred to as “expression vectors.” Other examples of “vectors” may include presentation vectors (e.g., vectors that direct the expression and presentation of encoded polypeptides on the surface of viruses or cells (such as bacterial cells, yeast cells, insect cells, and / or mammalian cells)).

[0099] As used herein, “subject,” “patient,” or “individual” may refer to a human or a non-human animal. “Non-human animal” may refer to any animal not classified as human, such as domesticated animals, livestock, or zoo animals, sports animals, pet animals (e.g., dogs, horses, cats, cows, etc.), as well as animals used in research. Research animals may refer to, but are not limited to, nematodes, arthropods, vertebrates, mammals, frogs, rodents (e.g., mice or rats), fish (e.g., zebrafish or pufferfish), birds (e.g., chickens), dogs, cats, and non-human primates (e.g., rhesus macaques, crab-eating macaques, chimpanzees, etc.). In some embodiments, the subject, patient, or individual is a human.

[0100] “Effective dose” means the minimum effective amount in terms of dosage and duration required to achieve one or more desired or indicated effects, including therapeutic or preventive outcomes. An effective dose may be provided in one or more doses. For the purposes of this disclosure, an effective dose of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve a preventive or therapeutic treatment. As understood in clinical contexts, an effective dose of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition (e.g., an effective dose when administered as monotherapy or in combination therapy). Thus, “effective dose” may be considered in the context of administration of one or more therapeutic agents, and a single agent may be considered to be given in an effective dose if, when combined with one or more other agents, a desired outcome can or does not occur. II. Libraries and Library Generation

[0101] Certain aspects of this disclosure relate to libraries for screening antibodies having masking peptides (e.g., masked antibodies). In some embodiments, the library comprises a plurality of polynucleotides, each encoding an antibody (e.g., masked antibody) linked to a masking peptide. In some embodiments, the library comprises a plurality of antibodies (e.g., a plurality of masked antibodies), each linked to a masking peptide. In some embodiments, antibodies (e.g., masked antibodies) having masking peptides in the library are displayed on cell surfaces and phage surfaces. The masking peptide may include a masking unit (MU) and a linkage unit (LU) from the N-terminus to the C-terminus. One or more masking units in the library may include a unique sequence such as that shown in formulas (I), (II), (III), (IV), or (V) described in Example 1. In some embodiments, the masking peptide further comprises an N-terminal unit linked to the N-terminus of the MU. In some embodiments, the LU includes one or more linkers. In some embodiments, the LU includes one or more cleavage sites, e.g., protease cleavage sites. In some embodiments, the LU further comprises one or more linker sequences in addition to one or more cleavage sites. In some embodiments, the LU comprises one or more linkers and does not contain cleavage sites. In some embodiments, the antibody comprises an antibody heavy chain variable domain (VH) and / or an antibody light chain variable domain (VL). In some embodiments, the masking peptide is ligated to the N-terminus of the antibody's VH or VL. The libraries described herein are useful for screening and / or identifying one or more masked antibodies. Also provided herein are libraries of host cells or phages presenting antibodies having masking peptides (e.g., masked antibodies).

[0102] In one embodiment, a library comprising polynucleotides, provided herein, wherein the polynucleotides in the library encode at least two, at least three, at least four, at least five, or at least ten antibodies, each having a unique masking peptide (MP), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), the MP being ligated to the N-terminus of the VH or VL of the antibody, the MP comprising a masking unit (MU) and a linkage unit (LU), the MU comprising an amino acid sequence according to formula (I): X1X2CX3(Xm)nX4X5CX6X7, where: n is between 2 and 8. X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y, and X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. Each Xm is an amino acid independently selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X4 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X6 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X7 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, S,T,andV,and C is cysteine. In some embodiments, n is 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 3-4, 3-5, 3-6, 3-7, 3-8, 4-5, 4-6, 4-7, 4-8, 5-6, 5-7, 5-8, 6-7, 6-8, or 2-8. In a particular embodiment, n is 2, 3, 4, 5, 6, 7, or 8.

[0103] In another embodiment, a library comprising polynucleotides provided herein, wherein the polynucleotides in the library encode at least two, at least three, at least four, at least five, or at least ten antibodies, each having a unique masking peptide (MP), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), the MP being ligated to the N-terminus of the VH or VL of the antibody, the MP comprising a masking unit (MU) and a linkage unit (LU), the MU comprising an amino acid sequence according to formula (II): X1X2CX3X4X5X6X7CX8X9, where: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X4 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, E, F, G, H, K, L, P, Q, R, S, T, V, and Y. X6 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X7 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X8 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X9 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine. In some embodiments, X1 is an amino acid selected from the group consisting of A, F, H, and V. X2 is an amino acid selected from the group consisting of A, L, and P, X3 is an amino acid selected from the group consisting of A, G, L, and R, X4 is an amino acid selected from the group consisting of E, G, K, and P, X5 is an amino acid selected from the group consisting of F, K, L, and V, X6 is an amino acid selected from the group consisting of F, L, P, and S, X7 is an amino acid selected from the group consisting of F, P, and Y, X8 is an amino acid selected from the group consisting of G, I, L, and P, and, X9 is an amino acid selected from the group consisting of E, Q, T, and V. In some embodiments, MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-10. In some embodiments, the MU in the library is about 10 8 to 5x10 9 (e.g., 10 9 to 5x10 9 ) and has diversity up to.

[0104] In another aspect, a library comprising polynucleotides, wherein the polynucleotides in the library provided herein each encode at least two, at least three, at least four, at least five, or at least ten antibodies having a unique masking peptide (MP), each of the antibodies comprising a heavy chain variable region (VH) and / or a light chain variable region (VL), the MP is linked to the N-terminus of the VH or VL of the antibody, the MP comprises a masking unit (MU) and a linkage unit (LU), and the MU comprises an amino acid sequence according to formula (III): X1X2CX3X4X5X6X7X8CX9X10, wherein: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y, X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y, X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X4 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X6 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, R, S, T, V, and Y. X7 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X8 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X9 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X10 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine. In some embodiments, X1 is an amino acid selected from the group consisting of A, F, H, N, P, S, and Y. X2 is an amino acid selected from the group consisting of A, L, P, S, and V. X3 is an amino acid selected from the group consisting of A, I, K, P, R, and V. X4 is an amino acid selected from the group consisting of A, G, L, P, V, and Y. X5 is an amino acid selected from the group consisting of F, I, L, P, R, and S. X6 is an amino acid selected from the group consisting of F, K, L, P, and Y. X7 is an amino acid selected from the group consisting of I, N, P, S, and V. X8 is an amino acid selected from the group consisting of A, F, L, and Y. X9 is an amino acid selected from the group consisting of G, K, Q, S, and V, and, X10 is an amino acid selected from the group consisting of G, Q, R, S, T, and V. In some embodiments, MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-19. In some embodiments, the MU in the library is approximately 10 9 from 10 11 (For example, 10 10 ~10 11 It has a wide range of diversity, up to )

[0105] In another embodiment, a library comprising polynucleotides, provided herein, wherein the polynucleotides in the library encode at least two, at least three, at least four, at least five, or at least ten antibodies, each having a unique masking peptide (MP), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), the MP being ligated to the N-terminus of the VH or VL of the antibody, the MP comprising a masking unit (MU) and a linkage unit (LU), the MU comprising an amino acid sequence according to formula (IV): X1X2CX3X4X5X6X7X8X9CX10X11, wherein: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X4 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X6 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X7 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X8 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X9 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X10 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X11 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine. In some embodiments, X1 is an amino acid selected from the group consisting of A, H, L, P, S, and T. X2 is an amino acid selected from the group consisting of A, D, F, L, and Y. X3 is an amino acid selected from the group consisting of E, L, P, and R. X4 is an amino acid selected from the group consisting of A, E, K, P, and R. X5 is an amino acid selected from the group consisting of E, F, G, and L. X6 is an amino acid selected from the group consisting of A, F, P, T, and Y. X7 is an amino acid selected from the group consisting of A, P, S, T, and V. X8 is an amino acid selected from the group consisting of A, N, P, and S. X9 is an amino acid selected from the group consisting of V and Y. X10 is an amino acid selected from the group consisting of I, P, and R, and, X11 is an amino acid selected from the group consisting of E, G, I, P, and V. In some embodiments, MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-27. In some embodiments, the MU in the library is approximately 10 11 From 5x10 12 (For example, 1012 ~5x10 12 It has a wide range of diversity, up to )

[0106] In another embodiment, a library comprising polynucleotides, provided herein, wherein the polynucleotides in the library encode at least two, at least three, at least four, at least five, or at least ten antibodies, each having a unique masking peptide (MP), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), the MP being ligated to the N-terminus of the VH or VL of the antibody, the MP comprising a masking unit (MU) and a linkage unit (LU), the MU comprising an amino acid sequence according to formula (V):X1X2CX3X4X5X6X7X8X9X10CX11X12, where: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X4 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X6 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X7 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X8 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, R, S, T, V, and Y. X9 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X10 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X11 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X12 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine. In some embodiments, X1 is an amino acid selected from the group consisting of A, F, P, S, and Y. X2 is an amino acid selected from the group consisting of H, L, P, S, and V. X3 is an amino acid selected from the group consisting of E, G, K, Q, P, and R. X4 is an amino acid selected from the group consisting of A, F, H, P, R, V, and Y. X5 is an amino acid selected from the group consisting of A, D, F, G, V, and Y. X6 is an amino acid selected from the group consisting of D, F, H, L, P, and V. X7 is an amino acid selected from the group consisting of H, K, P, R, and V. X8 is an amino acid selected from the group consisting of A, F, K, L, and P. X9 is an amino acid selected from the group consisting of A, D, F, L, and P. X10 is an amino acid selected from the group consisting of A, F, V, and Y. X11 is an amino acid selected from the group consisting of G, I, K, L, and R, and, X12 is an amino acid selected from the group consisting of A, E, K, P, R, and T. In some embodiments, MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 29-32 and 112-114. In some embodiments, the MU in the library is approximately 10 12 From 5x10 13 (For example, 10 13 ~2x10 13 It has a wide range of diversity, up to )

[0107] In another embodiment, a library comprising polynucleotides, provided herein, wherein the polynucleotides in the library encode at least two, at least three, at least four, at least five, or at least ten antibodies, each having a unique masking peptide (MP), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) that specifically bind to CTLA4 in the absence of the MP, the MP being ligated to the N-terminus of the VH or VL of the antibody, the MP comprising a masking unit (MU) and a linkage unit (LU), the MU comprising an amino acid sequence according to formula (V):X1X2CX3X4X5X6X7X8X9X10CX11X12, where: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X4 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X6 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X7 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X8 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, R, S, T, V, and Y. X9 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X10 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X11 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X12 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine. In some embodiments, X1 is an amino acid selected from the group consisting of P, S, and Y. X2 is an amino acid selected from the group consisting of H, P, and S. X3 is an amino acid selected from the group consisting of E, K, Q, and R. X4 is an amino acid selected from the group consisting of P, R, V, and Y. X5 is an amino acid selected from the group consisting of A, F, G, and Y. X6 is an amino acid selected from the group consisting of L, P, and V. X7 is an amino acid selected from the group consisting of H, K, P, and R. X8 is an amino acid selected from the group consisting of A, K, L, and P. X9 is an amino acid selected from the group consisting of D, F, L, and P. X10 is an amino acid selected from the group consisting of A, F, and Y. X11 is an amino acid selected from the group consisting of I, K, L, and R, and, X12 is an amino acid selected from the group consisting of E, K, and R. In some embodiments, MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 29-32. In some embodiments, the MU in the library is approximately 10 12 From 5x10 13 (For example, 10 13 ~2x10 13 It has a wide range of diversity, up to )

[0108] In another embodiment, a library comprising polynucleotides, provided herein, wherein the polynucleotides in the library encode at least two, at least three, at least four, at least five, or at least ten antibodies, each having a unique masking peptide (MP), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) that specifically bind to CD137 in the absence of the MP, the MP being ligated to the N-terminus of the VH or VL of the antibody, the MP comprising a masking unit (MU) and a linkage unit (LU), the MU comprising an amino acid sequence according to formula (V):X1X2CX3X4X5X6X7X8X9X10CX11X12, where: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X4 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X6 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X7 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X8 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, R, S, T, V, and Y. X9 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X10 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X11 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X12 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine. In some embodiments, X1 is an amino acid selected from the group consisting of A, F, and S. X2 is an amino acid selected from the group consisting of L and V. X3 is an amino acid selected from the group consisting of G and P. X4 is an amino acid selected from the group consisting of A, F, and H. X5 is an amino acid selected from the group consisting of D and V. X6 is an amino acid selected from the group consisting of D, F, and H. X7 is an amino acid selected from the group consisting of H and V. X8 is an amino acid selected from the group consisting of F and L. X9 is an amino acid selected from the group consisting of A and F. X10 is an amino acid selected from the group consisting of F and V. X11 is an amino acid selected from the group consisting of G, I, and R, and, X12 is an amino acid selected from the group consisting of A, P, and T. In some embodiments, MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 112-114. In some embodiments, the MU in the library is approximately 10 12 From 5x10 13 (For example, 10 13 ~2x10 13 It has a wide range of diversity, up to )

[0109] Furthermore, this specification also provides a library of antibodies containing masking peptides.

[0110] In one embodiment, the Specified provides a library comprising at least two, at least three, at least four, at least five, or at least ten antibodies, each having a unique masking peptide (MP), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), the MP being ligated to the N-terminus of the VH or VL of the antibody, the MP comprising a masking unit (MU) and a linkage unit (LU), the MU comprising an amino acid sequence according to formula (I): X1X2CX3(Xm)nX4X5CX6X7, where: n is 2 to 8. X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y, and X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. Each Xm is an amino acid independently selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X4 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X6 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X7 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine. In some embodiments, n is 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 3-4, 3-5, 3-6, 3-7, 3-8, 4-5, 4-6, 4-7, 4-8, 5-6, 5-7, 5-8, 6-7, 6-8, or 2-8. In a particular embodiment, n is 2, 3, 4, 5, 6, 7, or 8.

[0111] In another embodiment, the Specified provides a library comprising at least two, at least three, at least four, at least five, or at least ten antibodies, each having a unique masking peptide (MP), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), the MP being ligated to the N-terminus of the VH or VL of the antibody, the MP comprising a masking unit (MU) and a linkage unit (LU), the MU comprising an amino acid sequence according to formula (II): X1X2CX3X4X5X6X7CX8X9, where: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X4 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, E, F, G, H, K, L, P, Q, R, S, T, V, and Y. X6 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X7 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X8 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X9 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine. In some embodiments, X1 is an amino acid selected from the group consisting of A, F, H, and V. X2 is an amino acid selected from the group consisting of A, L, and P. X3 is an amino acid selected from the group consisting of A, G, L, and R. X4 is an amino acid selected from the group consisting of E, G, K, and P. X5 is an amino acid selected from the group consisting of F, K, L, and V. X6 is an amino acid selected from the group consisting of F, L, P, and S. X7 is an amino acid selected from the group consisting of F, P, and Y. X8 is an amino acid selected from the group consisting of G, I, L, and P, and, X9 is an amino acid selected from the group consisting of E, Q, T, and V. In some embodiments, MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-10. In some embodiments, the MU in the library is approximately 10 8 From 5x10 9 (For example, 10 9 ~5x10 9 It has a wide range of diversity, up to )

[0112] In another embodiment, the Specified provides a library comprising at least two, at least three, at least four, at least five, or at least ten antibodies, each having a unique masking peptide (MP), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), the MP being ligated to the N-terminus of the VH or VL of the antibody, the MP comprising a masking unit (MU) and a linkage unit (LU), the MU comprising an amino acid sequence according to formula (III): X1X2CX3X4X5X6X7X8CX9X10, where: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X4 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X6 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, R, S, T, V, and Y. X7 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X8 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X9 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X10 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine. In some embodiments, X1 is an amino acid selected from the group consisting of A, F, H, N, P, S, and Y. X2 is an amino acid selected from the group consisting of A, L, P, S, and V. X3 is an amino acid selected from the group consisting of A, I, K, P, R, and V. X4 is an amino acid selected from the group consisting of A, G, L, P, V, and Y. X5 is an amino acid selected from the group consisting of F, I, L, P, R, and S. X6 is an amino acid selected from the group consisting of F, K, L, P, and Y. X7 is an amino acid selected from the group consisting of I, N, P, S, and V. X8 is an amino acid selected from the group consisting of A, F, L, and Y. X9 is an amino acid selected from the group consisting of G, K, Q, S, and V, and, X10 is an amino acid selected from the group consisting of G, Q, R, S, T, and V. In some embodiments, MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-19. In some embodiments, the MU in the library is approximately 109 from 10 11 (For example, 10 10 ~10 11 It has a wide range of diversity, up to )

[0113] In another embodiment, the Specified provides a library comprising at least two, at least three, at least four, at least five, or at least ten antibodies, each having a unique masking peptide (MP), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), the MP being ligated to the N-terminus of the VH or VL of the antibody, the MP comprising a masking unit (MU) and a linkage unit (LU), the MU comprising an amino acid sequence according to formula (IV): X1X2CX3X4X5X6X7X8X9CX10X11, where: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X4 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X6 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X7 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, R, S, T, V, and Y. X8 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X9 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X10 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, X11 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine. In some embodiments, X1 is an amino acid selected from the group consisting of A, H, L, P, S, and T, X2 is an amino acid selected from the group consisting of A, D, F, L, and Y, X3 is an amino acid selected from the group consisting of E, L, P, and R, X4 is an amino acid selected from the group consisting of A, E, K, P, and R, X5 is an amino acid selected from the group consisting of E, F, G, and L, X6 is an amino acid selected from the group consisting of A, F, P, T, and Y, X7 is an amino acid selected from the group consisting of A, P, S, T, and V, X8 is an amino acid selected from the group consisting of A, N, P, and S, X9 is an amino acid selected from the group consisting of V, and Y, X10 is an amino acid selected from the group consisting of I, P, and R, and, X11 is an amino acid selected from the group consisting of E, G, I, P, and V. In some embodiments, MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 21 - 27. In some embodiments, the MU within the library has a diversity of about 10 11 to 5x10 12 (e.g., 10 12 to 5x10 12 ) up to.

[0114] In another embodiment, the Specified provides a library comprising at least two, at least three, at least four, at least five, or at least ten antibodies, each having a unique masking peptide (MP), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), the MP being ligated to the N-terminus of the VH or VL of the antibody, the MP comprising a masking unit (MU) and a linkage unit (LU), the MU comprising an amino acid sequence according to formula (V):X1X2CX3X4X5X6X7X8X9X10CX11X12, where: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X4 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X6 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X7 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X8 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, R, S, T, V, and Y. X9 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X10 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X11 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X12 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine. In some embodiments, X1 is an amino acid selected from the group consisting of A, F, P, S, and Y. X2 is an amino acid selected from the group consisting of H, L, P, S, and V. X3 is an amino acid selected from the group consisting of E, G, K, Q, P, and R. X4 is an amino acid selected from the group consisting of A, F, H, P, R, V, and Y. X5 is an amino acid selected from the group consisting of A, D, F, G, V, and Y. X6 is an amino acid selected from the group consisting of D, F, H, L, P, and V. X7 is an amino acid selected from the group consisting of H, K, P, R, and V. X8 is an amino acid selected from the group consisting of A, F, K, L, and P. X9 is an amino acid selected from the group consisting of A, D, F, L, and P. X10 is an amino acid selected from the group consisting of A, F, V, and Y. X11 is an amino acid selected from the group consisting of G, I, K, L, and R, and, X12 is an amino acid selected from the group consisting of A, E, K, P, R, and T. In some embodiments, MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 29-32 and 112-114. In some embodiments, the MU in the library is approximately 10 12 From 5x10 13 (For example, 10 13 ~2x10 13 It has a wide range of diversity, up to )

[0115] In another embodiment, the Specified provides a library comprising at least two, at least three, at least four, at least five, or at least ten antibodies, each having a unique masking peptide (MP), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) that specifically bind to CTLA4 in the absence of the MP, the MP being ligated to the N-terminus of the VH or VL of the antibody, the MP comprising a masking unit (MU) and a linkage unit (LU), the MU comprising an amino acid sequence according to formula (V):X1X2CX3X4X5X6X7X8X9X10CX11X12, where: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X4 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X6 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X7 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X8 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, R, S, T, V, and Y. X9 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X10 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X11 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X12 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine. In some embodiments, X1 is an amino acid selected from the group consisting of P, S, and Y, X2 is an amino acid selected from the group consisting of H, P, and S, X3 is an amino acid selected from the group consisting of E, K, Q, and R, X4 is an amino acid selected from the group consisting of P, R, V, and Y, X5 is an amino acid selected from the group consisting of A, F, G, and Y, X6 is an amino acid selected from the group consisting of L, P, and V, X7 is an amino acid selected from the group consisting of H, K, P, and R, X8 is an amino acid selected from the group consisting of A, K, L, and P, X9 is an amino acid selected from the group consisting of D, F, L, and P, X10 is an amino acid selected from the group consisting of A, F, and Y, X11 is an amino acid selected from the group consisting of I, K, L, and R, and, X12 is an amino acid selected from the group consisting of E, K, and R. In some embodiments, MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-32. In some embodiments, the MU within the library has a diversity of about 10 12 to 5x10 13 (e.g., 10 13 to 2x10 13 ).

[0116] In another embodiment, the Specified provides a library comprising at least two, at least three, at least four, at least five, or at least ten antibodies, each having a unique masking peptide (MP), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) that specifically bind to CD137 in the absence of the MP, the MP being ligated to the N-terminus of the VH or VL of the antibody, the MP comprising a masking unit (MU) and a linkage unit (LU), the MU comprising an amino acid sequence according to formula (V):X1X2CX3X4X5X6X7X8X9X10CX11X12, where: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X4 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X6 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X7 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X8 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, R, S, T, V, and Y. X9 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X10 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X11 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X12 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine. In some embodiments, X1 is an amino acid selected from the group consisting of A, F, and S. X2 is an amino acid selected from the group consisting of L and V. X3 is an amino acid selected from the group consisting of G and P. X4 is an amino acid selected from the group consisting of A, F, and H. X5 is an amino acid selected from the group consisting of D and V. X6 is an amino acid selected from the group consisting of D, F, and H. X7 is an amino acid selected from the group consisting of H and V. X8 is an amino acid selected from the group consisting of F and L. X9 is an amino acid selected from the group consisting of A and F. X10 is an amino acid selected from the group consisting of F and V. X11 is an amino acid selected from the group consisting of G, I, and R, and, X12 is an amino acid selected from the group consisting of A, P, and T. In some embodiments, MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 112-114. In some embodiments, the MU in the library is approximately 10 12 From 5x10 13 (For example, 10 13 ~2x10 13 It has a wide range of diversity, up to )

[0117] In some embodiments of the libraries described herein, MU does not contain amino acid sequences of M, NG, DG, NXS, NXT, or any combination thereof.

[0118] In some embodiments of the libraries described herein, the masking peptide (MP) is ligated to the N-terminus of the VL. In other embodiments of the libraries described herein, the MP is ligated to the N-terminus of the VL. In some embodiments of the libraries described herein, each antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the MP is ligated to the N-terminus of the VH or VL of the antibody. In some embodiments of the libraries described herein, each antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the MP is ligated to the N-terminus of the VL of the antibody. In some embodiments of the libraries described herein, each antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the MP is ligated to the N-terminus of the VH of the antibody.

[0119] In some embodiments of the libraries described herein, each antibody comprises an scFv including a heavy chain variable region (VH) and a light chain variable region (VL). In certain embodiments, the MP is ligated to the VL of the scFv. In certain embodiments, the MP is ligated to the VH of the scFv.

[0120] In some embodiments of the libraries described herein, each antibody comprises a Fab including a heavy chain variable region (VH) and a light chain variable region (VL). In certain embodiments, the MP is ligated to the VL of the Fab. In certain embodiments, the MP is ligated to the VH of the Fab.

[0121] In some embodiments of the libraries described herein, each antibody comprises a heavy chain variable region (VH), and the MP is ligated to the N-terminus of the VH of the antibody. In some variations, the antibody does not contain a light chain variable region (VL). In certain embodiments, the antibody is a VHH single-domain antibody (also called a nanobody) comprising a heavy chain variable region (VH), wherein the MP is ligated to the N-terminus of the VH of the VHH, here. In certain embodiments, the antibody is a VHH-Fc antibody comprising a heavy chain variable region (VH), wherein the MP is ligated to the N-terminus of the VH of the VHH-Fc, here.

[0122] For some purposes, the Library of this Disclosure may have at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 250, at least 500, and at least 10 3 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , at least 10 12 , at least 10 13 , at least 10 14 , at least 10 15 , at least 10 16 , at least 10 17 , at least 10 18 , or at least 10 19 It contains multiple polynucleotides, each encoding a unique masking unit. For some purposes, the Library of this Disclosure may have at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 250, at least 500, and at least 10 3 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , at least 10 12 , at least 10 13 , at least 10 14 , at least 10 15 , at least 1016 , at least 10 17 , at least 10 18 , or at least 10 19 This product contains multiple antibodies, each having a masking peptide containing a unique masking unit.

[0123] In some embodiments, the library of the Disclosure includes one or more vectors (e.g., expression vectors and / or display vectors) containing one or more polynucleotides (e.g., synthetic polynucleotides) of the Disclosure. In some embodiments, each antibody having a masking peptide in the library is fused with all or part of a protein (e.g., a viral coat protein, a bacterial surface protein, a yeast surface protein, an insect cell surface protein, a mammalian cell surface protein) (i.e., a fusion protein is produced). In some embodiments, the fusion protein is displayed on the surface of a particle or host cell. In some embodiments, the library of the Disclosure includes host cells and particles (e.g., phages) that display antibodies having the masking peptide of the Disclosure.

[0124] For example, a method for preparing a library of the present disclosure is further provided herein by providing and assembling polynucleotide sequences (e.g., synthetic polynucleotides) of the library of the present disclosure. Polynucleotides encoding antibodies having masking peptides as described herein can be cloned into any vector suitable for the expression of part or all of the polypeptide sequence. In some embodiments, the polynucleotide is cleaned into a vector that enables the production of part or all of a polypeptide fused to all or part of a protein (e.g., viral coat protein, bacterial surface protein, yeast surface protein, insect cell surface protein, mammalian cell surface protein) (i.e., creation of a fusion protein) and presented on the surface of a particle or cell. Several types of vectors are available, and for example, phagemide vectors may be used to carry out the present disclosure. As is known to those skilled in the art, phagemide vectors generally contain a variety of components, including a promoter, signal sequence, phenotypic selection gene, replication origin, and other necessary components. In some embodiments, polynucleotides encoding a polypeptide region can be cloned into a vector for expression in bacterial cells for bacterial presentation or in yeast cells for yeast presentation. Exemplary vectors are described in U.S. PG Publication No. US20160145604. In some embodiments, the vector is a presentation vector comprising a polynucleotide encoding an amino acid sequence to be presented on a surface (e.g., the surface of a phage, bacterium, yeast, insect, or mammalian cell) from 5' to 3', a restriction site, a second polynucleotide encoding a surface peptide that can be presented on the surface, and a second restriction site. In some embodiments, the second polynucleotide encodes a phage coat protein, a yeast outer wall protein (e.g., Aga2), a bacterial outer membrane protein, a cell surface tether domain, or an adapter, or a truncated or derivative thereof. In some embodiments, the surface peptide is for phage presentation, yeast presentation, bacterial presentation, insect presentation, or mammalian presentation, or shuttle presentation between them.In some embodiments, when expressed, the amino acid sequence and surface peptide are presented as a fusion protein on the surface. In some embodiments, the vector further includes a fusion tag on the 5' side of the first restriction site or the 3' side of the second restriction site.

[0125] Certain aspects of this disclosure relate to a population of cells containing the vector(s) described herein. Antibodies having a masking peptide encoded by a polynucleotide generated by any of the methods described herein or other preferred methods can be expressed and screened to identify masked antibodies having the desired structure and / or activity. Protein expression can be carried out, for example, using cell-free extracts (e.g., ribosome presentation), phage presentation, prokaryotic cells (e.g., bacterial presentation), or eukaryotic cells (e.g., yeast presentation). In some embodiments, the cells are bacterial cells, yeast cells, insect cells, or mammalian cells (e.g., Chinese hamster ovary (CHO) cells). Methods for transfecting bacterial cells, yeast cells, or mammalian cells are known in the art and are described in the references cited herein. The expression of proteins (e.g., from the libraries of this disclosure) in these cell types, as well as the screening of the masked antibodies of interest, are described in more detail below.

[0126] Alternatively, polynucleotides can be expressed in E. coli expression systems, such as those described by Pluckthun and Skerra (Meth. Enzymol., 1989, 178:476; Biotechnology, 1991, 9:(273)). Mutant proteins can be expressed for secretion in culture medium and / or bacterial cytoplasm, as described by Better and Horwitz, Meth. Enzymol., 1989, 178:476. In some embodiments, polynucleotides can be expressed in E. coli expression systems, such as those described by Pluckthun and Skerra (Meth. Enzymol., 1989, 178:476). The lipeptides are bound to the 3' end of signal-encoding sequences, such as ompA, phoA, or pelB signal sequences (Leietal., J. Bacteriol., 1987, 169:4379). These gene fusions are assembled into disisstronic constructs so that they can be expressed from a single vector and secreted into the periplasmic region of E. coli, where they can be refolded and recovered in their active form (Skerraetal., Biotechnology, 1991, 9:273).

[0127] In other embodiments, the polypeptide sequences of this disclosure are expressed on the membrane surface of a prokaryote, for example, E. coli, using the secretion signaling and lipidization moieties described, for example, US20040072740, US20030100023, and US20030036092.

[0128] Alternatively, the polypeptide sequences of this disclosure may be expressed and screened by fixed periplasmic expression (APEx2-hybrid surface presentation), for example, as described in Jeongetal., PNAS, 2007, 104:8247, or by other fixation methods, for example, as described in Mazoretal., Nature Biotechnology, 2007, 25:563.

[0129] Mammalian cells, such as myeloma cells (e.g., NS / O cells), hybridoma cells, Chinese hamster ovary (CHO) cells, and human embryonic kidney (HEK) cells, may also be used for the expression of polypeptides of this disclosure. Polypeptides expressed in mammalian cells (e.g., activatable binding polypeptides such as masked antibodies) may be designed to be secreted into the culture medium or expressed on the cell surface.

[0130] In other embodiments, polypeptides or antibodies (e.g., masked antibodies) can be selected using mammalian cell presentation (Hoetal., PNAS, 2006, 103:9637). In some embodiments, as described above and illustrated below, polypeptides or antibodies are selected after the production of some or all polypeptides or antibodies fused to all or some of the viral coat proteins (i.e., to create a fusion protein), and can then be presented on the surface of particles or cells, for example, using phage presentation.

[0131] Certain aspects of this disclosure relate to non-human animals comprising the polynucleotides or polynucleotide libraries of this disclosure. For example, the non-human animals of this disclosure may be modified so that their genomes contain polynucleotides encoding polypeptides or antibodies having the mask peptides of this disclosure. In some embodiments, transgenic animals (e.g., mice) express polypeptides or antibodies encoded by polynucleotides. Techniques for modifying the genomes of non-human animals are known in the art (e.g., the methods used to produce Xenomouse®).

[0132] In some embodiments, the masking peptides in the library described herein further comprise an N-terminal unit. In certain embodiments, the N-terminal unit comprises or consists of 1 to 12 amino acid residues. In some embodiments, the N-terminal unit is ligated to the N-terminus of the masking unit. In some embodiments, the N-terminal unit comprises amino acid E, or the peptides EVG, EVGSY (SEQ ID NO: 33), EVGVLDV (SEQ ID NO: 86), or EVGAESGVK (SEQ ID NO: 88). In certain embodiments, the N-terminal unit comprises amino acid E, or the peptides EVG, EVGSY (SEQ ID NO: 33), EVGVLDV (SEQ ID NO: 86), or EVGAESGVK (SEQ ID NO: 88).

[0133] In some embodiments of the libraries described herein, a linkage unit (LU) comprises one or more linkers. For example, glycine polymer (G)n (wherein n is an integer of at least 1 (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, etc.)); GS, GGS, GGG, SGRG (SEQ ID NO: 104), GGGGS (SEQ ID NO: 105), GGGGT (SEQ ID NO: 36), SGGS (SEQ ID NO: 37), GGSG (SEQ ID NO: 106), GGSGG (SEQ ID NO: 107), GGSSG (SEQ ID NO: 108), GSGGG (SEQ ID NO: 109), GGGSG (SEQ ID NO: 110), GSSSG (SEQ ID NO: 108), GSGGG (SEQ ID NO: 109), GGGSG (SEQ ID NO: 110), GSSSG (SEQ ID NO: 108), GSGGG (SEQ ID NO: 109), GGGSG (SEQ ID NO: 110), GSSSG (SEQ ID NO: 108), GGS8), GGSGG (SEQ ID NO: 110), GSSSG (SEQ ID NO: 108), GGSGG (S Any suitable linker known in the art (e.g., a flexible linker) may be used, including glycine-serine polymers (GS) n (wherein n is an integer of at least 1 (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, etc.), such as glycine-alanine polymers; alanine-serine polymers, etc. The linker sequence may be of any length, such as about 1 amino acid (e.g., glycine or serine) to about 20 amino acids (e.g., 20 amino acid glycine polymer or glycine-serine polymer), about 1 amino acid to about 15 amino acids, about 3 amino acids to about 12 amino acids, about 4 amino acids to about 10 amino acids, about 5 amino acids to about 9 amino acids, about 6 amino acids to about 8 amino acids, etc. In some embodiments, the linker is of any length of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

[0134] In some embodiments of the libraries described herein, a linkage unit (LU) comprises one or more linkers as described herein, but does not include a cleavage site. In some embodiments, the linkage unit comprises a non-cleavable linker. In certain embodiments, the non-cleavable linker comprises the amino acid sequence of SEQ ID NO: 81. In certain embodiments, the masking peptide comprises the amino acid sequence of SEQ ID NO: 82.

[0135] In some embodiments, a linkage unit (LU) in the library described herein includes one or more cleavage sites. In some embodiments, the LU includes at least a first cleavage site (CS1) (e.g., a first protease cleavage site). In some embodiments, the LU further includes a second cleavage site (CS2). In some embodiments, the first and / or second cleavage sites are protease cleavage sites. In some embodiments, the first and second cleavage sites are the same. In some embodiments, the first and second cleavage sites are different. Any suitable protease cleavage site that is recognized and / or cleaved by any protease known in the art (e.g., a protease known to coexist with a polypeptide target containing a cleavage site) may be used, including, for example, urokinase-type plasminogen activator (uPA), matrix metalloproteinases (e.g., MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-19, MMP-20, MMP-23, MMP-24, MMP-26, and / or MMP-27), tobacco etch virus (TEV) protease, plasmin, thrombin, PSA, PSMA, ADAMS / ADAMTS (e.g., ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMMDEC1, ADAMTS1, ADAMTS4, and / or ADAMTS5), caspases (e.g., caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase Catheses (e.g., cathese-13 and / or caspase-14), aspartate proteases (e.g., RACE and / or renin), aspartate cathepsins (e.g., cathepsin D and / or cathepsin E), cysteine ​​cathepsins (e.g., cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2 and / or cathepsin X / Z / P), cysteine ​​proteinases (e.g.,Cruzipain, regmain, and / or Otubain-2), KLK (e.g., KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, and / or KLK14), metalloproteinases (e.g., meprin, neprilysin, PSMA, and / or BMP-1), serine proteases (e.g., activated protein C, cathepsin A, cathepsin G, chymase, and / or coagulation factor proteases (e.g., FVIIa, FIXa, FXa, FXIa, F XIIa)) Includes protease cleavage sites recognized and / or cleaved by elastase, granzyme B, guanidinobenzoatase, HtrA1, human neutrophil elastase, lactoferrin, malapsin, NS3 / 4A, PACE4, tPA, tryptase, type II transmembrane serine proteases (TTSPs) (e.g., DESC1, DPP-4, FAP, hepsin, matryptase-2, MT-SP1 / matryptase, TMPRSS2, TMPRSS3, and / or TMPRSS4). In some embodiments, the first protease cleavage site is a cleavage site of a protease selected from uPA, MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, TEV protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE. In some embodiments, the first protease cleavage site is a cleavage site of a protease selected from uPA, MMP-2, MMP-9, and / or TEV proteases. In some embodiments, the protease cleavage site includes the amino acid sequence SGRSA (SEQ ID NO: 34), the amino acid sequence PLGLAG (SEQ ID NO: 35), or a combination thereof.

[0136] In some embodiments, the LU further includes a first linker (L1). In some embodiments, the first linker (L1) is located at the C-terminus of a first cleavage site (CS1) (e.g., a first protease cleavage site). In some embodiments, the LU includes a (CS1)-L1 structure from the N-terminus to the C-terminus. In some embodiments, the LU further includes a second linker (L2). In some embodiments, L2 is located at the C-terminus of a second cleavage site (CS2). In some embodiments, the LU includes a (CS1)-L1-(CS2)-L2 structure from the N-terminus to the C-terminus. In some embodiments, the first linker (L1) is located at the N-terminus of the first cleavage site (CS1). In certain embodiments, the LU includes a L1-(CS1)-L2 structure from the N-terminus to the C-terminus. In certain embodiments, the LU includes a L1-(CS1)-L2-(CS2) structure from the N-terminus to the C-terminus. In some embodiments, the LU of this disclosure comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 38-42, 81, and 119.

[0137] In some embodiments, the masking peptide of the Disclosure comprises a MU-(CS1)-L1 structure from the N-terminus to the C-terminus. In some embodiments, the masking peptide of the Disclosure comprises a (MU)-(CS1)-L1-(CS2)-L2 structure from the N-terminus to the C-terminus. In some embodiments, the masking peptide of the Disclosure comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 43-65 and 67.

[0138] In some embodiments, the masking peptide of the Disclosure comprises a MU-L1-(CS1)-L2 structure from the N-terminus to the C-terminus. In some embodiments, the masking peptide of the Disclosure comprises a MU-L1-(CS1)-L2-(CS2) structure from the N-terminus to the C-terminus. In some embodiments, the masking peptide of the Disclosure comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 68-75, 83-85, 87, and 116-119.

[0139] In some embodiments, the Disclosure relates to a polynucleotide library encoding one or more antibodies or antibody libraries, wherein each antibody in the library includes an antigen-binding domain (ABD). In some embodiments, the ABD includes an antibody light chain variable region and / or an antibody heavy chain variable region. In some embodiments, the ABD includes both an antibody light chain variable region and an antibody heavy chain variable region. In some embodiments, the ABD includes an antibody heavy chain variable region but does not include an antibody light chain variable region. In some embodiments, the ABD of the Disclosure includes an antibody light chain variable region and / or an antibody heavy chain variable region having specificity for any target, including, for example, CTLA4, CD137, PD-1, PD-L1, PD-L2, LAG3, TIM3, B7-H3, OX40, CD3, CD19, CD20, CD40, CD95, CD120a, BTLA, VISTA, ICOS, BCMA, Her1, Her2, Her3, and / or B7-H4.

[0140] In some embodiments, the antibody comprises a full-length antibody light chain and a full-length antibody heavy chain. The antibody light chain may be a kappa or lambda light chain. The antibody heavy chain may be any class, such as IgG, IgM, IgE, IgA, or IgD. In some embodiments, the antibody heavy chain is an IgG class, such as IgG1, IgG2, IgG3, or IgG4 subclass. The antibody heavy chains described herein may be converted from one class or subclass to another using methods known in the art.

[0141] One or more of the antibodies described herein may incorporate an HVR sequence (e.g., one, two, or three heavy chain variable region HVR sequences and / or one, two, or three light chain variable region HVR sequences), a heavy chain variable region sequence and / or light chain variable region sequence of any of the antibodies described in PCT application PCT / CN2017 / 098333 (which is incorporated herein by reference in its entirety), PCT application PCT / CN2017 / 098299 (which is incorporated herein by reference in its entirety), PCT application PCT / CN2017 / 098332 (which is incorporated herein by reference in its entirety), and / or simultaneously, any of the heavy chain variable region sequences and / or light chain variable region sequences of any of the antibodies described in U.S. Patent Application Publication 2021 / 0206855 (which is incorporated herein by reference in its entirety).

[0142] One or more of the antibodies described herein may incorporate an HVR sequence (e.g., one, two, or three heavy chain variable region HVR sequences and / or one, two, or three light chain variable region HVR sequences), a heavy chain variable region sequence, and / or a light chain variable region sequence of any of the antibodies described herein (e.g., anti-CTLA4 antibody, anti-CD137 antibody).

[0143] In some embodiments, the antibody is an anti-CTLA4 antibody comprising (a) an antibody light chain variable region comprising HVR-L1 comprising the amino acid sequence RASQSVRGRFLA (SEQ ID NO: 95), HVR-L2 comprising the amino acid sequence DASNRATGI (SEQ ID NO: 96), and / or HVR-L3 comprising the amino acid sequence YCQQSSSWPPT (SEQ ID NO: 97); and (b) an antibody heavy chain variable region comprising HVR-H1 comprising the amino acid sequence YSISSGYHWSWI (SEQ ID NO: X98), HVR-H2 comprising the amino acid sequence LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 99), and / or HVR-H3 comprising the amino acid sequence ARSYVYFDY (SEQ ID NO: 100). In some embodiments, the antibody includes an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 93, or an amino acid sequence having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence of SEQ ID NO: 93; and / or an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 94, or an amino acid sequence having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence of SEQ ID NO: 94. In some embodiments, the antibody includes an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 93, or an amino acid sequence having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence of SEQ ID NO: 93; and an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 94, or an amino acid sequence having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence of SEQ ID NO: 94. In some embodiments, the antibody includes an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 93, and an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 94.

[0144] Exemplary anti-CTLA4 antibody light chain variable region (SEQ ID NO: 93): DIQLTQSPSSLSASVGDRVTITCRASQSVRGRFLAWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSSSWPPTFGQGTKVEIKR

[0145] Exemplary anti-CTLA4 antibody heavy chain variable region (SEQ ID NO: 94): EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWSWIRQAPGKGLEWLARIDWDDDKYYSTSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARSYVYFDYWGQGTLVTVSS III. Screening Method for Masked Antibodies

[0146] In some embodiments, this specification provides a method for screening a library of polynucleotides and polypeptides described herein with respect to masked antibodies.

[0147] In some embodiments, a method is provided for using a polynucleotide or polypeptide library described herein as a masked antibody that binds to a target, the method comprising: a) contacting an expressed antibody from the library having a masking peptide with the target to determine a first binding affinity or lack of detectable binding to the target; b) contacting a control antibody lacking a masking peptide with the target to determine a second binding affinity; and c) selecting an expressed antibody that has a first binding affinity lower than the second binding affinity or does not bind detectably to the target, the first and second binding affinities being measured as KD, EC50, or IC50. In some embodiments, the masking peptide comprises a masking unit (MU) and a linkage unit from the N-terminus to the C-terminus. In some embodiments, the LU comprises at least a first cleavage site (C1). In certain embodiments, if the LU of the library comprises at least a first cleavage site (L1), the control antibody is the expressed antibody from the library after the LU has been cleaved. In some embodiments, an expressed antibody is selected if the binding affinity of the expressed antibody in the library after the LU is cleaved is at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, or at least 5000 times the binding affinity of the expressed antibody before the LU is cleaved. In some embodiments, step (c) includes selecting an expressed antibody that does not bind detectably to the target, for example, under the same contact conditions used to determine the second binding affinity in step (b). In some embodiments, this method includes calculating the masking efficiency by determining the ratio of the first binding affinity in step a) to the second binding affinity in step b).In a particular embodiment, a library-expressed antibody is selected if its masking efficiency is at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500, or at least 5000.

[0148] In some embodiments, a method is provided for screening masked antibodies that bind to a target using a polynucleotide library or polypeptide library described herein, comprising the following steps: a) contacting an expressed antibody from the library having a masking peptide with a first cell expressing a target antigen to determine the binding affinity (e.g., binding EC50); b) contacting a control antibody lacking a masking peptide with a first cell expressing a target antigen until a binding affinity (e.g., binding EC50) is achieved; c) contacting the expressed antibody from the library with the target antigen d) Contacting a second cell expressing the antibody to determine its binding affinity (e.g., binding EC50) (where the second cell expresses a lower level of the target antigen than the first cell); e) Contacting a control antibody lacking the masking peptide to the second cell to determine its binding affinity (e.g., binding EC50); f) Determining the ratio of the binding affinity from step a) to the binding affinity from step b) as the first masking efficiency; g) Determining the ratio of the binding affinity from step c) to the binding affinity from step d) as the second masking efficiency; g) Selecting an expressed antibody having a second masking efficiency higher than the first masking efficiency. In certain embodiments, the first and second binding affinities are measured as KD, EC50, or IC50. In some embodiments, step (g) includes selecting an expressed antibody having a second masking efficiency that is at least 5%, at least 10%, not 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 100 times, at least 200 times, at least 300 times, at least 400 times, at least 500 times, or at least 1000 times higher than a first masking efficiency. In some embodiments, the control antibody is an antibody having the same antigen-binding domain as the expressed antibody in the library. In certain embodiments, the control antibody is the parent antibody.In some embodiments, the masking peptide includes a masking unit (MU) and a linkage unit (LU) from the N-terminus to the C-terminus. In some embodiments, the LU does not contain a cleavage site. In other embodiments, the LU contains at least a first cleavage site (C1). In some embodiments, the LU contains at least a first cleavage site, and the control antibody is the expressed antibody in the library after the LU has been cleaved. In a particular embodiment in which the LU contains at least a first cleavage site, the method includes cleaving the LU to produce the control antibody. In some embodiments, the second masking efficiency is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least twice, at least three times, at least four times, at least five times, at least ten times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 100 times, at least 200 times, at least 300 times, at least 400 times, at least 500 times, at least 100 times, at least 200 times, at least 300 times, at least 400 times, at least 500 times, or at least 1000 times higher than the first masking efficiency.

[0149] In some embodiments, a method is provided for screening masked antibodies that bind to a target using a polynucleotide library or polypeptide library described herein, comprising the steps of: a) contacting an expressed antibody from the library having the masking peptide with a first cell expressing the target antigen to determine its binding EC50; b) contacting a control antibody lacking the masking peptide with a first cell expressing the target antigen to determine its binding EC50; c) applying the expressed antibody from the library to a target d) Contact a second cell expressing the target antigen to determine the binding EC50, the second cell expressing the target antigen at a lower level than the first cell; d) Contact a control antibody lacking the masking peptide to the second cell to determine the binding EC50; e) Determine the ratio of the EC50 from step a) to the EC50 from step b) as the first masking efficiency; f) Determine the ratio of the EC50 from step c) to the EC50 from step d) as the second masking efficiency; and g) Select an expressed antibody having a second masking efficiency higher than the first masking efficiency. In some embodiments, step (g) includes selecting an expressed antibody having a second masking efficiency that is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 100 times, at least 200 times, at least 300 times, at least 400 times, at least 500 times, or at least 1000 times higher than a first masking efficiency. In some embodiments, the control antibody is an antibody having the same antigen-binding domain as the expressed antibody in the library. In certain embodiments, the control antibody is the parent antibody. In some embodiments, the masking peptide includes a masking unit (MU) and a linkage unit from the N-terminus to the C-terminus. In some embodiments, the LU does not include a cleavage site. In other embodiments, the LU includes at least a first cleavage site (C1).In some embodiments, the LU includes at least a first cleavage site, and the control antibody is the antibody expressed in the library after the LU has been cleaved. In certain embodiments where the LU includes at least a first cleavage site, the method includes cleaving the LU to produce the control antibody. In some embodiments, the second masking efficiency is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least twice, at least three times, at least four times, at least five times, at least ten times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 100 times, at least 200 times, at least 300 times, at least 400 times, at least 500 times, or at least 1000 times higher than the first masking efficiency.

[0150] In some embodiments, this specification provides a method for identifying a masked antibody capable of binding to an antigen in a concentration-dependent manner, the method comprising the steps of: a) contacting the masked antibody having the masking peptide with a first cell expressing a target antigen to determine its binding affinity (e.g., binding EC50); b) contacting a control antibody lacking the masking peptide with a first cell expressing a target antigen to determine its binding affinity (e.g., binding EC50); c) contacting the masked antibody with a second cell expressing a target antigen to determine its binding affinity (e.g., binding EC50); d) determine the binding affinity (e.g., binding EC50) of the second cells, which express a lower level of the target antigen than the first cells; d) contact the second cells with a control antibody lacking the masking peptide to determine the binding affinity (e.g., binding EC50); e) determine the ratio of the binding affinity from step a) to the binding affinity from step b) as the first masking efficiency; f) determine the ratio of the binding affinity from step c) to the binding affinity from step d) as the second masking efficiency; and g) if the second masking efficiency is higher than the first masking efficiency, identify a concentration-dependent antigen-binding antibody. In certain embodiments, the binding affinity is measured as KD, EC50, or IC50. In some embodiments, step (g) includes identifying a concentration-dependent antigen-binding antibody if the second masking efficiency is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 100 times, at least 200 times, at least 300 times, at least 400 times, at least 500 times, or at least 1000 times higher than the first masking efficiency. In some embodiments, the control antibody is an antibody having the same antigen-binding domain as the masked antibody. In some embodiments, the control antibody is a parent antibody. In some embodiments, the masking peptide includes a masking unit (MU) and a linkage unit (LU) from the N-terminus to the C-terminus.In some embodiments, the LU does not contain a cleavage site. In other embodiments, the LU contains at least a first cleavage site (C1). In some embodiments, the LU contains at least a first cleavage site (C1), and the control antibody is a masked antibody after the LU has been cleaved. In a particular embodiment in which the LU contains at least a first cleavage site (C1), the method comprises cleaving the LU to produce the control antibody. In some embodiments, the second masking efficiency is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 100 times, at least 200 times, at least 300 times, at least 400 times, at least 500 times, or at least 1000 times higher than the first masking efficiency.

[0151] In some embodiments, this specification provides a method for identifying a masked antibody capable of binding to an antigen in a concentration-dependent manner, the method comprising the steps of: a) contacting the masked antibody having the masking peptide with a first cell expressing a target antigen to determine the binding EC50; b) contacting a control antibody lacking the masking peptide with a first cell expressing a target antigen to determine the binding EC50; c) contacting the masked antibody with a second cell expressing a target antigen to determine the binding EC50. g) The second cells express a lower level of the target antigen than the first cells; d) A control antibody lacking the masking peptide is brought into contact with the second cells to determine the binding EC50; e) The ratio of the EC50 from step a) to the EC50 from step b) is determined as the first masking efficiency; f) The ratio of the EC50 from step c) to the EC50 from step d) is determined as the second masking efficiency; and g) If the second masking efficiency is higher than the first masking efficiency, a concentration-dependent antigen-binding antibody is identified. In some embodiments, step (g) includes identifying a concentration-dependent antigen-binding antibody if the second masking efficiency is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 100 times, at least 200 times, at least 300 times, at least 400 times, at least 500 times, or at least 1000 times higher than the first masking efficiency. In some embodiments, the control antibody is an antibody having the same antigen-binding domain as the masked antibody. In some embodiments, the control antibody is a parent antibody. In some embodiments, the masking peptide includes a masking unit (MU) and a linkage unit (LU) from the N-terminus to the C-terminus. In some embodiments, the LU does not include a cleavage site. In other embodiments, the LU includes at least a first cut portion (C1).In some embodiments, the LU includes at least a first cleavage site (C1), and the control antibody is a masked antibody after the LU has been cleaved. In certain embodiments where the LU includes at least a first cleavage site (C1), the method includes cleaving the LU to produce the control antibody. In some embodiments, the second masking efficiency is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least twice, at least three times, at least four times, at least five times, at least ten times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 100 times, at least 200 times, at least 300 times, at least 400 times, at least 500 times, or at least 1000 times higher than the first masking efficiency.

[0152] In some embodiments, if the linkage unit (LU) of the library contains at least one cleavage site, the method includes a) contacting the expressed antibody or protein of the library with the target before the linkage unit (LU) is cleaved, b) contacting the expressed antibody or protein of the library with the target after the LU has been cleaved, and c) selecting one or more expressed antibodies or proteins after the cleavage of the LU that have a higher binding affinity to the target than before the cleavage of the cleavage site.

[0153] In other embodiments, if the linking units (LUs) of the library do not contain at least one cleavage site (e.g., are non-cleavable), the method includes a) contacting the expressed antibody of the library with a first culture medium, cells, or tissue expressing a target; b) contacting the expressed antibody of the library with a second culture medium, cells, or tissue, wherein the second culture medium, cells, or tissue has a lower concentration of the target compared to the first culture medium, cells, or tissue; and c) selecting an expressed antibody that binds more to the first culture medium, cells, or tissue compared to the second culture medium, cells, or tissue. In some embodiments, if the linkage units (LUs) of the library do not contain at least one cleavage site (e.g., are non-cleavable), the method includes: a) contacting the expressed antibody of the library with a first medium, cell, or tissue expressing the target; b) contacting the parent antibody lacking a masking peptide with a first medium, cell, or tissue expressing the target; c) contacting the expressed antibody of the library with a second medium, cell, or tissue (where the second medium, cell, or tissue has a lower concentration of the target compared to the first medium, cell, or tissue); d) contacting the parent antibody lacking a masking peptide with a second medium, cell, or tissue expressing the target; and e) selecting an expressed antibody that (i) has lower binding to both the first and second mediums, cells, or tissues compared to the parent antibody, and (ii) has higher binding to the first medium, cell, or tissue compared to the second medium, cell, or tissue.

[0154] In some embodiments, the masked antibodies or proteins of the Disclosure are context-dependent (e.g., activated in specific contexts, such as a protease-rich tumor microenvironment (TME) or a TME with a high concentration of the target antigen, and can only bind to those targets). In some embodiments, the masked antibodies of the Disclosure offer improved safety compared to more conventional unmasked antibodies (e.g., exhibit reduced toxicity, do not induce significant changes in the weight of many organs, and do not alter liver histopathology, hematology, and / or blood biochemistry). In some embodiments, the masked antibodies of the Disclosure have improved pharmacokinetic properties (e.g., longer in vivo half-life) compared to more conventional unmasked antibodies.

[0155] In some embodiments, the masked antibody or protein of the Disclosure comprises (e.g., from the N-terminus to the C-terminus) a) a masking peptide comprising a masking unit (MU) and a linkage unit (LU), and b) an antigen-binding domain (ABD) comprising an antibody heavy chain variable region (VH) and / or an antibody light chain variable region (VL). In some embodiments, the masked antibody or protein of the Disclosure comprises (e.g., from the N-terminus to the C-terminus) a) a masking peptide comprising an N-terminal unit, a masking unit (MU), and a linkage unit (LU), and b) an antigen-binding domain (ABD) comprising an antibody heavy chain variable region (VH) and / or an antibody light chain variable region (VL). In some embodiments, the masking unit (MU) binds to the ABD, reducing or inhibiting the binding of the masked antibody to its target compared to the binding of the corresponding antibody or protein lacking the MU to its target, and / or compared to the binding of the ABD to its target. In some embodiments, the masking units (MUs) are present in a quantity of at least about 2.0 (e.g., at least about 2.0, at least about 3.0, at least about 4.0, at least about 5.0, at least about 6.0, at least about 7.0, at least about 8.0, at least about 9.0, at least about 10, at least about 25, at least about 50, less) before activation (e.g., removing the MUs from the antibody by cleaving the LUs, or contacting the masked antibody with a culture medium, cells, tissue, or tumor containing a high concentration of the target antigen). It has a masking efficiency of at least approximately 75, at least approximately 100, at least approximately 150, at least approximately 200, at least approximately 300, at least approximately 400, at least approximately 500, at least approximately 600, at least approximately 700, at least approximately 800, at least approximately 900, at least approximately 1000, at least approximately 1500, at least approximately 2000, at least approximately 2500, at least approximately, at least approximately 3000, at least approximately 3500, at least approximately 4000, at least approximately 4500, or at least approximately 5000).

[0156] In some embodiments, masking efficiency is measured as the difference in affinity of a masked antibody containing masking units (MUs) for binding to the target antigen compared to the affinity of an antibody lacking MUs for binding to the target antigen before activation (e.g., before removing MUs from the antibody by cleavage of LUs, or before contacting the masked antibody with a culture medium, cells, tissue, or tumor containing a high concentration of the target antigen), or as the difference in affinity of a masked antibody containing MUs (before activation) to the target antigen compared to the affinity of an antibody after activation (e.g., after removing MUs from the antibody by cleavage of LUs, or after contacting the masked antibody with a culture medium, cells, tissue, or tumor containing a high concentration of the target antigen). In some embodiments, masking efficiency is measured by dividing the EC50 of binding of the masked antibody containing masking units (MUs) (before activation, e.g., before removing the MUs from the antibody by cleavage of the LU, or before contacting the masked antibody with a medium, cells, tissue, or tumor containing a high concentration of the target antigen) by the EC50 of the parent antibody (e.g., an unmasked antibody having the same ABD as the masking peptide but lacking masking units). In some embodiments, EC50 is measured by ELISA. In some embodiments, the masking units (MUs) bind to the ABD, inhibiting the masked polypeptide from binding to the target.

[0157] In some embodiments, the antibodies of this disclosure generally exhibit, when activated (e.g., when MU is removed from the antibody by cleavage of LU, or when the antibody comes into contact with a culture medium, cells, tissue, or tumor containing a high concentration of the target antigen), a binding affinity of the antibody to the target is at least approximately twice as high (e.g., at least approximately 2 times, at least approximately 2.5 times, at least approximately 3 times, at least approximately 3.5 times, at least approximately 4 times, at least approximately 4.5 times, at least approximately 5 times, at least approximately 5.5 times, at least approximately 6 times, at least approximately 6.5 times, at least approximately 7 times, at least approximately 7.5 times, at least approximately 8 times, at least approximately 8 times) compared to antibodies with an unactivated masking peptide. If the antibody increases by 5 times, at least about 9 times, at least about 9.5 times, at least about 10 times, at least about 25 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 250 times, at least about 500 times, at least about 750 times, or at least about 1000 times, at least about 2000 times, at least about 2500 times, at least about 3000 times, at least about 3500 times, at least about 4000 times, at least about 4500 times, at least about 5000 times, at least about 5500 times, at least about 6000 times, at least about 6500 times, or at least about 7000 times or more, it is generally considered to be a masked antibody or an activatable antibody.In some embodiments, the polypeptides of the Disclosure, after activation (e.g., removal of MU from an antibody by cleavage of LU, or contact of the antibody with a medium, cells, tissue, or tumor having a high concentration of the target antigen), have an EC50 of at least approximately 2 times (e.g., at least approximately 2 times, at least approximately 2.5 times, at least approximately 3 times, at least approximately 3.5 times, at least approximately 4 times, at least approximately 4.5 times, at least approximately 5 times, at least approximately 5.5 times, at least approximately 6 times, at least approximately 6.5 times, at least approximately 7 times, at least approximately 7.5 times, at least approximately 8 times, at least approximately 8.5 times, at least approximately 9 times, and less An antibody is generally considered "activatable" if its polypeptide EC50 decreases by approximately 9.5 times, at least approximately 10 times, at least approximately 25 times, at least approximately 50 times, at least approximately 75 times, at least approximately 100 times, at least approximately 250 times, at least approximately 500 times, at least approximately 750 times, or at least approximately 1000 times, at least approximately 2000 times, at least approximately 2500 times, at least approximately 3000 times, at least approximately 3500 times, at least approximately 4000 times, at least approximately 4500 times, at least approximately 5000 times, at least approximately 5500 times, at least approximately 6000 times, at least approximately 6500 times, or at least approximately 7000 times or more. In some embodiments, an antibody of the present disclosure is generally considered "activatable" if, after activation (e.g., removal of MU from the antibody by cleavage of LU, or contact of the antibody with a medium, cells, tissue, or tumor having a high concentration of the target antigen), the polypeptide EC50 decreases by at least approximately 2 times. EC50 can be measured, for example, by an ELISA or FACS assay.

[0158] In some embodiments, when a masking unit is bound to the antigen-binding domain of the masked antibody, the KD of the antibody against its target is approximately twice (e.g., approximately 2, approximately 2.5, approximately 3, approximately 3.5, approximately 4, approximately 4.5, approximately 5, approximately 5.5, approximately 6, approximately 6.5, approximately 7, approximately 7.5, approximately 8, approximately 8.5, approximately 9, approximately 9.5, approximately 10, approximately 25, approximately 50, approximately 75, approximately 100, approximately 250, approximately 500, approximately 750, or approximately 1000 times or more) the KD of the antibody when the masking unit is bound to the antigen-binding domain (e.g., after removing the MU from the antibody via cleavage of the LU, or after contacting the antibody with a medium, cells, tissue, or tumor having a high concentration of the target antigen). In some embodiments, when a masking unit binds to the antigen-binding domain of a masked antibody, the KD of the antibody against its target becomes approximately twice the KD of the parent antibody (e.g., an unmasked antibody having the same antigen-binding domain (ABD) as the masking peptide but lacking a masking unit) (e.g., approximately 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 25, 50, 75, 100, 250, 500, 750, 1000, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, or more than 7000).

[0159] In some embodiments, the masking unit sterically prevents the binding of the masked antibody to its target and / or allosterically prevents the binding of the masked antibody to its target. In some embodiments, the masking unit does not contain the amino acid sequence of the natural binding partner of the antigen-binding domain of the masked antibody.

[0160] In some embodiments, the dissociation constant of the masking unit of the antigen-binding domain (ABD) is greater than the dissociation constant of the masked antibody against the target (in the case of the active form, for example, after removing MU from the antibody by cleavage of LU, or after contacting the antibody with a medium, cells, tissue, or tumor containing a high concentration of the target antigen). In some embodiments, the dissociation constant of the masking unit for the antigen-binding domain is about twice the dissociation constant of the masked antibody against the target (e.g., about 2 times, about 2.5 times, about 3 times, about 3.5 times, about 4 times, about 4.5 times, about 5 times, about 5.5 times, about 6 times, about 6.5 times, about 7 times, about 7.5 times, about 8 times, about 8.5 times, about 9 times, about 9.5 times, about The polymerision ratios are approximately 10x, 25x, 50x, 75x, 100x, 250x, 500x, 750x, 1000x, 2000x, 2500x, 3000x, 3500x, 4000x, 4500x, 5000x, 5500x, 6000x, 6500x, or 7000x or more (when in active form). In some embodiments, the dissociation constant of the masking unit of the antigen-binding domain is approximately equal to the dissociation constant of the masked antibody against the target (in activated form). In some embodiments, the masking unit (MU) binds to the antigen-binding domain and prevents the polypeptide from binding to the target only when the polypeptide is not activated (e.g., the MU has not been removed from the antibody by cleavage of the LU, or the antibody has not been in contact with a medium, cells, tissue, or tumor containing a high concentration of the target antigen). In some embodiments, activation induces cleavage of the polypeptide within the cleavage site of the linkage unit. In some embodiments, activation induces a structural change in the polypeptide (e.g., substitution of the masking unit (MU)) so that the masking peptide no longer interferes with the binding of the polypeptide to its target.

[0161] The masked antibodies described herein may be further modified. In some embodiments, the masked antibodies are ligated to additional molecular entities. Examples of additional molecular entities include pharmaceuticals, peptides or proteins, detection agents or labels, and antibodies.

[0162] In some embodiments, the masked antibodies of this disclosure are linked to pharmaceuticals. Examples of pharmaceuticals include cytotoxic agents or other cancer treatments, and radioisotopes. Specific examples of cytotoxic agents include taxol, cytochalasin B, gramicidin D, ethidium bromine, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, cortisine, doxorubicin, daunorubicin, dihydroxyanthracine dione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, and their analogs or homologs. Other therapeutic agents include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechloretamine, thiotepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclotosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mitramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine). Examples of radioisotopes that can be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine-131, indium-111, yttrium-90, and lutetium-177. Methods for linking polypeptides to pharmaceuticals are known in the art, including the use of various linker technologies. Examples of linker types include hydrazone, thioether, ester, disulfide, and peptide-containing linkers.For further considerations regarding linkers and methods for linking therapeutic agents to antibodies, see, for example, Saito et al., Adv. DrugDeliv. Rev. 55:199-215 (2003), Trail, et al., CancerImmunol.Immunother. 52:328-337 (2003), Payne, CancerCell3:207-212 (2003), Allen, Nat. Rev. Cancer2:750-763 (2002), Pastan and Kreitman, Curr. Opin. Investig. Drugs3:1089-1091 (2002), and Senter and Springer (2001) Adv. DrugDeliv. Rev. 53:247-264. IV. Masked Antibodies

[0163] Other aspects of this disclosure relate to masked antibodies (e.g., activatable antibodies) selected from the libraries described herein, and derivatives of masked antibodies. In some embodiments, the masked antibody comprises an antibody and a masking peptide (MP). The masking peptide may include a masking unit (MU) and a linkage unit (LU) from the N-terminus to the C-terminus. The MU may include or be composed of the amino acid sequence shown in formula (I), (II), (III), (IV), or (V) described in Example 1. In some embodiments, the masking peptide further comprises an N-terminal unit bound to the N-terminus of the MU. In some embodiments, the LU includes one or more linkers. In some embodiments, the LU includes one or more cleavage sites, e.g., protease cleavage sites. In some embodiments, the LU further includes one or more linker sequences in addition to one or more cleavage sites. In some embodiments, the LU includes one or more linkers and does not include cleavage sites. In some embodiments, the antibody includes an antibody heavy chain variable domain (VH) and / or an antibody light chain variable domain (VL). In some embodiments, the masking peptide is ligated to the N-terminus of the VH or VL of the antibody. In some embodiments, the masked antibody comprises a full-length antibody light chain and / or a full-length antibody heavy chain. In some embodiments, the masked antibody comprises a Fab fragment. In some embodiments, the masked antibody comprises a single-strand variable fragment (scFv). In other embodiments, the masked antibody comprises a VHH single-domain fragment. In certain embodiments, the masked antibody comprises a VHH-Fc antibody. In some embodiments, the masked antibody is expressed on a cell surface (e.g., a display on yeast or mammalian cells). In some embodiments, the masked antibody is an activatable antibody.

[0164] In one embodiment, a masked antibody is provided comprising an antibody and a masking peptide (MP), wherein the antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), the MP is linked to the N-terminus of the VH or VL of the antibody, the MP comprises a masking unit (MU) and a linkage unit (LU), the MU comprises an amino acid sequence according to formula (I): X1X2CX3(Xm)nX4X5CX6X7, where: n is between 2 and 8. X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y, and X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. Each Xm is an amino acid independently selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X4 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X6 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X7 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine. In some embodiments, n is 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 3-4, 3-5, 3-6, 3-7, 3-8, 4-5, 4-6, 4-7, 4-8, 5-6, 5-7, 5-8, 6-7, 6-8, or 2-8. In a particular embodiment, n is 2, 3, 4, 5, 6, 7, or 8.

[0165] In one embodiment, a masked antibody is provided comprising an antibody and a masking peptide (MP), wherein the antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), the MP is linked to the N-terminus of the VH or VL of the antibody, the MP comprises a masking unit (MU) and a linkage unit (LU), the MU comprises an amino acid sequence according to formula (II): X1X2CX3X4X5X6X7CX8X9, where: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X4 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, E, F, G, H, K, L, P, Q, R, S, T, V, and Y. X6 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X7 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X8 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X9 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine. In some embodiments, X1 is an amino acid selected from the group consisting of A, F, H, and V. X2 is an amino acid selected from the group consisting of A, L, and P. X3 is an amino acid selected from the group consisting of A, G, L, and R. X4 is an amino acid selected from the group consisting of E, G, K, and P. X5 is an amino acid selected from the group consisting of F, K, L, and V. X6 is an amino acid selected from the group consisting of F, L, P, and S. X7 is an amino acid selected from the group consisting of F, P, and Y. X8 is an amino acid selected from the group consisting of G, I, L, and P, and, X9 is an amino acid selected from the group consisting of E, Q, T, and V. In some embodiments, MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 10.

[0166] In one embodiment, a masked antibody is provided comprising an antibody and a masking peptide (MP), wherein the antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), the MP is linked to the N-terminus of the VH or VL of the antibody, the MP comprises a masking unit (MU) and a linkage unit (LU), the MU comprises an amino acid sequence according to formula (III): X1X2CX3X4X5X6X7X8CX9X10, where: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X4 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X6 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, R, S, T, V, and Y. X7 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X8 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X9 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X10 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine. In some embodiments, X1 is an amino acid selected from the group consisting of A, F, H, N, P, S, and Y. X2 is an amino acid selected from the group consisting of A, L, P, S, and V. X3 is an amino acid selected from the group consisting of A, I, K, P, R, and V. X4 is an amino acid selected from the group consisting of A, G, L, P, V, and Y. X5 is an amino acid selected from the group consisting of F, I, L, P, R, and S. X6 is an amino acid selected from the group consisting of F, K, L, P, and Y. X7 is an amino acid selected from the group consisting of I, N, P, S, and V. X8 is an amino acid selected from the group consisting of A, F, L, and Y. X9 is an amino acid selected from the group consisting of G, K, Q, S, and V, and, X10 is an amino acid selected from the group consisting of G, Q, R, S, T, and V. In some embodiments, MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-19.

[0167] In one embodiment, a masked antibody is provided comprising an antibody and a masking peptide (MP), wherein the antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), the MP is linked to the N-terminus of the VH or VL of the antibody, the MP comprises a masking unit (MU) and a linkage unit (LU), the MU comprising an amino acid sequence according to formula (IV): X1X2CX3X4X5X6X7X8X9CX10X11, where: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X4 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X6 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X7 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, R, S, T, V, and Y. X8 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X9 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X10 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X11 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine. In some embodiments, X1 is an amino acid selected from the group consisting of A, H, L, P, S, and T. X2 is an amino acid selected from the group consisting of A, D, F, L, and Y. X3 is an amino acid selected from the group consisting of E, L, P, and R. X4 is an amino acid selected from the group consisting of A, E, K, P, and R. X5 is an amino acid selected from the group consisting of E, F, G, and L. X6 is an amino acid selected from the group consisting of A, F, P, T, and Y. X7 is an amino acid selected from the group consisting of A, P, S, T, and V. X8 is an amino acid selected from the group consisting of A, N, P, and S. X9 is an amino acid selected from the group consisting of V and Y. X10 is an amino acid selected from the group consisting of I, P, and R, and, X11 is an amino acid selected from the group consisting of E, G, I, P, and V. In some embodiments, MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-27.

[0168] In one embodiment, a masked antibody is provided comprising an antibody and a masking peptide (MP), wherein the antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), the MP is ligated to the N-terminus of the VH or VL of the antibody, the MP comprises a masking unit (MU) and a linkage unit (LU), the MU comprises an amino acid sequence according to formula (V): X1X2CX3X4X5X6X7X8X9X10CX11X12, where: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X4 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X6 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X7 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X8 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, R, S, T, V, and Y. X9 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X10 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X11 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X12 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine. In some embodiments, X1 is an amino acid selected from the group consisting of A, F, P, S, and Y. X2 is an amino acid selected from the group consisting of H, L, P, S, and V. X3 is an amino acid selected from the group consisting of E, G, K, Q, P, and R. X4 is an amino acid selected from the group consisting of A, F, H, P, R, V, and Y. X5 is an amino acid selected from the group consisting of A, D, F, G, V, and Y. X6 is an amino acid selected from the group consisting of D, F, H, L, P, and V. X7 is an amino acid selected from the group consisting of H, K, P, R, and V. X8 is an amino acid selected from the group consisting of A, F, K, L, and P. X9 is an amino acid selected from the group consisting of A, D, F, L, and P. X10 is an amino acid selected from the group consisting of A, F, V, and Y. X11 is an amino acid selected from the group consisting of G, I, K, L, and R, and, X12 is an amino acid selected from the group consisting of A, E, K, P, R, and T. In some embodiments, MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-32 and 112-114.

[0169] In one embodiment, a masked antibody comprising an antibody and a masking peptide (MP) is provided, wherein the antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) that specifically bind to CTLA4 in the absence of the MP, the MP being ligated to the N-terminus of the VH or VL of the antibody, the MP comprising a masking unit (MU) and a linkage unit (LU), the MU comprising an amino acid sequence according to formula (V):X1X2CX3X4X5X6X7X8X9X10CX11X12, where: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X4 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X6 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X7 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X8 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, R, S, T, V, and Y. X9 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X10 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X11 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X12 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine. In some embodiments, X1 is an amino acid selected from the group consisting of P, S, and Y. X2 is an amino acid selected from the group consisting of H, P, and S. X3 is an amino acid selected from the group consisting of E, K, Q, and R. X4 is an amino acid selected from the group consisting of P, R, V, and Y. X5 is an amino acid selected from the group consisting of A, F, G, and Y. X6 is an amino acid selected from the group consisting of L, P, and V. X7 is an amino acid selected from the group consisting of H, K, P, and R. X8 is an amino acid selected from the group consisting of A, K, L, and P. X9 is an amino acid selected from the group consisting of D, F, L, and P. X10 is an amino acid selected from the group consisting of A, F, and Y. X11 is an amino acid selected from the group consisting of I, K, L, and R, and, X12 is an amino acid selected from the group consisting of E, K, and R. In some embodiments, MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 29 to 32.

[0170] In one embodiment, a masked antibody is provided comprising an antibody and a masking peptide (MP), wherein the antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) that specifically bind to CD137 in the absence of the MP, the MP being ligated to the N-terminus of the VH or VL of the antibody, the MP comprising a masking unit (MU) and a linkage unit (LU), the MU comprising an amino acid sequence according to formula (V):X1X2CX3X4X5X6X7X8X9X10CX11X12, where: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X4 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X6 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X7 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X8 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, R, S, T, V, and Y. X9 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X10 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X11 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X12 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine. In some embodiments, X1 is an amino acid selected from the group consisting of A, F, and S. X2 is an amino acid selected from the group consisting of L and V. X3 is an amino acid selected from the group consisting of G and P. X4 is an amino acid selected from the group consisting of A, F, and H. X5 is an amino acid selected from the group consisting of D and V. X6 is an amino acid selected from the group consisting of D, F, and H. X7 is an amino acid selected from the group consisting of H and V. X8 is an amino acid selected from the group consisting of F and L. X9 is an amino acid selected from the group consisting of A and F. X10 is an amino acid selected from the group consisting of F and V. X11 is an amino acid selected from the group consisting of G, I, and R, and, X12 is an amino acid selected from the group consisting of A, P, and T. In some embodiments, MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 112-114.

[0171] In some embodiments of the masked antibodies described herein, MU does not contain the amino acid sequence of M, NG, DG, NXS, NXT, or any combination thereof.

[0172] In some embodiments of the masked antibody described herein, the masking peptide (MP) is ligated to the N-terminus of the VL. In other embodiments of the masked antibody described herein, the MP is ligated to the N-terminus of the VL. In some embodiments of the masked antibody described herein, the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the MP is ligated to the N-terminus of the VH or VL of the antibody. In some embodiments of the masked antibody described herein, the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the MP is ligated to the N-terminus of the VL of the antibody. In some embodiments of the masked antibody described herein, each antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the MP is ligated to the N-terminus of the VH of the antibody.

[0173] In some embodiments of the masked antibodies described herein, the antibody comprises an scFv including a heavy chain variable region (VH) and a light chain variable region (VL). In certain embodiments, the MP is linked to the VL of the scFv. In certain embodiments, the MP is linked to the VH of the scFv.

[0174] In some embodiments of the masked antibodies described herein, the antibody comprises a Fab including a heavy chain variable region (VH) and a light chain variable region (VL). In certain embodiments, the MP is linked to the VL of the Fab. In certain embodiments, the MP is linked to the VH of the Fab.

[0175] In some embodiments of the masked antibodies described herein, the antibody comprises a heavy chain variable region (VH), and the MP is ligated to the N-terminus of the VH of the antibody. In some variations, the antibody does not contain a light chain variable region (VL). In certain embodiments, the antibody comprises a heavy chain variable region (VH), forming a VHH single-domain antibody (also called a nanobody), with the MP ligated to the N-terminus of the VH of the VHH. In certain embodiments, the antibody comprises a heavy chain variable region (VH), forming a VHH-Fc antibody, with the MP ligated to the N-terminus of the VH of the VHH-Fc antibody.

[0176] In some embodiments, the Specified provides polynucleotides encoding the masked antibodies of the Disclosure. In some embodiments, the Specified provides vectors (e.g., expression vectors and / or display vectors) comprising one or more polynucleotides (e.g., synthetic polynucleotides) encoding the masked antibodies of the Disclosure. In some embodiments, the antibody is fused with all or part of a protein (e.g., a viral coat protein, a bacterial surface protein, a yeast surface protein, an insect cell surface protein, a mammalian cell surface protein) (i.e., a fusion protein is produced). In some embodiments, the fusion protein is displayed on the surface of a particle or host cell. In some embodiments, the Specified provides host cells and particles (e.g., phages) displaying the masked antibodies described herein.

[0177] Furthermore, this specification provides methods for preparing and assembling polynucleotides (e.g., synthetic polynucleotides) encoding the masked antibodies of this disclosure. Polynucleotides encoding antibodies having the masking peptide described herein can be cloned into any suitable vector for expressing part or all of the polypeptide sequence of the masked antibody. In some embodiments, the polynucleotide enables the production of part or all of the masked antibody fused to all or part of a protein (e.g., viral coat protein, bacterial surface protein, yeast surface protein, insect cell surface protein, mammalian cell surface protein) (i.e., producing a fusion protein) and displayed on the surface of a particle or cell. Several types of vectors are available and can be used in the implementation of this disclosure (e.g., phagemide vectors). Phagemide vectors generally contain a variety of components, including a promoter, signal sequence, phenotypic selection gene, origin of replication site, and other necessary components known to those skilled in the art. In some embodiments, the polynucleotide encoding the masked antibody can be cloned into a vector to be expressed in bacterial cells in the case of bacterial display, or in yeast cells in the case of yeast display. Exemplary vectors are described above as described herein and in U.S. Patent Publication No. US20160145604.

[0178] Certain aspects of this disclosure relate to cell populations comprising one or more vectors encoding one or more polynucleotides encoding the masked antibodies described herein. The masked antibodies described herein can be expressed and screened to identify masked antibodies having a desired structure and / or activity. Expression of masked antibodies can be carried out, for example, using cell-free extracts (e.g., ribosome display), phage display, prokaryotic cells (e.g., bacterial display), or eukaryotic cells (e.g., yeast display). In some embodiments, the cells are bacterial cells, yeast cells, insect cells, or mammalian cells (e.g., Chinese hamster ovary (CHO) cells). Methods for transfecting bacterial cells, yeast cells, or mammalian cells are known in the art and are described in the references cited herein. Expression of proteins in these cell types (e.g., from the libraries of this disclosure) and screening of the masked antibodies of interest are described in further detail below.

[0179] Alternatively, polynucleotides can be expressed in E. coli expression systems as described by Pluckthun and Skerra (Meth. Enzymol., 1989, 178:476; Biotechnology, 1991, 9:273). Mutant proteins can be expressed to be secreted into the culture medium and / or bacterial cytoplasm, as described by Better and Horwitz, Meth. Enzymol., 1989, 178:476. In some embodiments, masked antibodies are ligated to the 3' end of a sequence encoding a signal sequence, such as ompA, phoA, or pelB signal sequences (Leietal., J. Bacteriol., 1987, 169:4379). These gene fusions are assembled into a disistronic structure so that they can be expressed from a single vector, secreted to the periphery of E. coli cells, where they refold and are recovered in an active form (Skerra et al., Biotechnology, 1991, 9:273).

[0180] In other embodiments, the masked antibodies of this disclosure are expressed on the membrane surface of a prokaryote, such as Escherichia coli, using secretion signaling and lipidization moieties, for example, as described in US20040072740, US20030100023, and US20030036092.

[0181] Alternatively, the masked antibodies of this disclosure can be expressed and screened by anchored periplasmic expression (APEx2-hybrid surface display), as described, for example, Jeongetal., PNAS, 2007, 104:8247, or by other anchoring methods, as described, for example, Mazoretal., Nature Biotechnology, 2007, 25:563.

[0182] Higher eukaryotic cells, such as mammalian cells, such as myeloma cells (e.g., NS / O cells), hybridoma cells, Chinese hamster ovary (CHO) cells, and human embryonic kidney (HEK) cells, can also be used for the expression of the masked antibodies of this disclosure. Masked antibodies expressed in mammalian cells can be designed to be secreted into the culture medium or expressed on the cell surface.

[0183] Certain aspects of this disclosure relate to non-human animals comprising the polynucleotides or polynucleotide libraries of this disclosure. For example, the non-human animals of this disclosure may have their genomes modified to include polynucleotides encoding the masked antibodies of this disclosure. In some embodiments, transgenic animals (e.g., mice) express the masked antibodies described herein. Techniques for modifying the genomes of non-human animals are known in the art (e.g., methods used to generate Xenomouse™).

[0184] In some embodiments of the masked antibodies described herein, the masking peptide further comprises an N-terminal unit. In certain embodiments, the N-terminal unit comprises or consists of 1 to 12 amino acid residues. In some embodiments, the N-terminal unit is ligated to the N-terminus of the masking unit. In some embodiments, the N-terminal unit comprises amino acid E, or the peptides EVG, EVGSY (SEQ ID NO: 33), EVGVLDV (SEQ ID NO: 86), or EVGAESGVK (SEQ ID NO: 88). In certain embodiments, the N-terminal unit comprises amino acid E, or the peptides EVG, EVGSY (SEQ ID NO: 33), EVGVLDV (SEQ ID NO: 86), or EVGAESGVK (SEQ ID NO: 88).

[0185] In some embodiments of the masked antibodies described herein, the linkage unit (LU) comprises one or more linkers. For example, glycine polymer (G)n (wherein n is an integer of at least 1 (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, etc.)); GS, GGS, GGG, SGRG (SEQ ID NO: 104), GGGGS (SEQ ID NO: 105), GGGGT (SEQ ID NO: 36), SGGS (SEQ ID NO: 37), GGSG (SEQ ID NO: 106), GGSGG (SEQ ID NO: 107), GSGSG (SEQ ID NO: 108), GSGGG (SEQ ID NO: 109), GGGSG (SEQ ID NO: 110), GSSSG (SEQ ID NO: 108), GSGGG (SEQ ID NO: 109), GGGSG (SEQ ID NO: 110), GSSSG (SEQ ID NO: 104), GGSSG (SEQ ID NO: 107), GGSSG (SEQ ID NO: 108), GSGGG (SEQ ID NO: 109), GGGSG (SEQ ID NO: 110), GSSSG (SEQ ID NO: 104), GGS10), GGSSG (SEQ ID NO: 104), GGSSG (SEQ ID NO: 104), GGSSG Any suitable linker known in the art (e.g., a flexible linker) may be used, including glycine-serine polymers (GS) n (wherein n is an integer of at least 1 (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, etc.), such as glycine-alanine polymers; alanine-serine polymers, etc. The linker sequence may be of any length, such as about 1 amino acid (e.g., glycine or serine) to about 20 amino acids (e.g., 20 amino acid glycine polymer or glycine-serine polymer), about 1 amino acid to about 15 amino acids, about 3 amino acids to about 12 amino acids, about 4 amino acids to about 10 amino acids, about 5 amino acids to about 9 amino acids, about 6 amino acids to about 8 amino acids, etc. In some embodiments, the linker is of any length of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

[0186] In some embodiments of the masked antibodies described herein, the linkage unit (LU) comprises one or more linkers as described herein and does not contain cleavage sites. In some embodiments, the linkage unit comprises a non-cleavable linker. In certain embodiments, the non-cleavable linker comprises the amino acid sequence of SEQ ID NO: 81.

[0187] In some embodiments of the masked antibodies described herein, the linkage unit (LU) includes one or more cleavage sites. In some embodiments, the LU includes at least a first cleavage site (CS1) (e.g., a first protease cleavage site). In some embodiments, the LU further includes a second cleavage site (CS2). In some embodiments, the first and / or second cleavage sites are protease cleavage sites. In some embodiments, the first and second cleavage sites are the same. In some embodiments, the first and second cleavage sites are different. Any suitable protease cleavage site that is recognized and / or cleaved by any protease known in the art (e.g., a protease known to coexist with a polypeptide target containing a cleavage site) may be used, including, for example, urokinase-type plasminogen activator (uPA), matrix metalloproteinases (e.g., MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-19, MMP-20, MMP-23, MMP-24, MMP-26, and / or MMP-27), tobacco etch virus (TEV) protease, plasmin, thrombin, PSA, PSMA, ADAMS / ADAMTS (e.g., ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMMDEC1, ADAMTS1, ADAMTS4, and / or ADAMTS5), caspases (e.g., caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase Catheses (e.g., cathese-13 and / or caspase-14), aspartate proteases (e.g., RACE and / or renin), aspartate cathepsins (e.g., cathepsin D and / or cathepsin E), cysteine ​​cathepsins (e.g., cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2 and / or cathepsin X / Z / P), cysteine ​​proteinases (e.g.,Cruzipain, regmain, and / or Otubain-2), KLK (e.g., KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, and / or KLK14), metalloproteinases (e.g., meprin, neprilysin, PSMA, and / or BMP-1), serine proteases (e.g., activated protein C, cathepsin A, cathepsin G, chymase, and / or coagulation factor proteases (e.g., FVIIa, FIXa, FXa, FXIa, F XIIa)) Includes protease cleavage sites recognized and / or cleaved by elastase, granzyme B, guanidinobenzoatase, HtrA1, human neutrophil elastase, lactoferrin, malapsin, NS3 / 4A, PACE4, tPA, tryptase, type II transmembrane serine proteases (TTSPs) (e.g., DESC1, DPP-4, FAP, hepsin, matryptase-2, MT-SP1 / matryptase, TMPRSS2, TMPRSS3, and / or TMPRSS4). In some embodiments, the first protease cleavage site is a cleavage site of a protease selected from uPA, MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, TEV protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE. In some embodiments, the first protease cleavage site is a cleavage site of a protease selected from uPA, MMP-2, MMP-9, and / or TEV proteases. In some embodiments, the protease cleavage site includes the amino acid sequence SGRSA (SEQ ID NO: 34), the amino acid sequence PLGLAG (SEQ ID NO: 35), or a combination thereof.

[0188] In some embodiments of the masked antibodies described herein, the LU further comprises a first linker (L1). In some embodiments, the first linker (L1) is located at the C-terminus of the first cleavage site (CS1) (e.g., the first protease cleavage site). In some embodiments, the LU includes a (CS1)-L1 structure from the N-terminus to the C-terminus. In some embodiments, the LU further includes a second linker (L2). In some embodiments, L2 is located at the C-terminus of the second cleavage site (CS2). In some embodiments, the LU includes a (CS1)-L1-(CS2)-L2 structure from the N-terminus to the C-terminus. In some embodiments, the first linker (L1) is located at the N-terminus of the first cleavage site (CS1). In certain embodiments, the LU includes a L1-(CS1)-L2 structure from the N-terminus to the C-terminus. In certain embodiments, the LU includes a L1-(CS1)-L2-(CS2) structure from the N-terminus to the C-terminus. In some embodiments, the LU of this disclosure comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 38-42, 81, and 119.

[0189] In some embodiments of the masked antibodies described herein, the masking peptide comprises a MU-(CS1)-L1 structure from the N-terminus to the C-terminus. In some embodiments, the masking peptide of the Disclosure comprises a (MU)-(CS1)-L1-(CS2)-L2 structure from the N-terminus to the C-terminus. In some embodiments, the masking peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 43-65 and 67.

[0190] In some embodiments, the masking peptide comprises a MU-L1-(CS1)-L2 structure from the N-terminus to the C-terminus. In some embodiments, the masking peptide comprises a MU-L1-(CS1)-L2-(CS2) structure from the N-terminus to the C-terminus. In some embodiments, the masking peptide of the present disclosure comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 68-75, 83-85, 87, and 116-118.

[0191] In some embodiments, the masked antibody includes an antigen-binding domain (ABD). In some embodiments, the ABD includes an antibody light chain variable region and / or an antibody heavy chain variable region. In some embodiments, the ABD includes both an antibody light chain variable region and an antibody heavy chain variable region. In some embodiments, the ABD includes an antibody heavy chain variable region but does not include an antibody light chain variable region. In some embodiments, the ABD of the present disclosure includes an antibody light chain variable region and / or an antibody heavy chain variable region having specificity for any target, including, for example, CTLA4, CD137, PD-1, PD-L1, PD-L2, LAG3, TIM3, B7-H3, OX40, CD3, CD19, CD20, CD40, CD95, CD120a, BTLA, VISTA, ICOS, BCMA, Her1, Her2, Her3, and / or B7-H4.

[0192] In some embodiments, the masked antibody comprises a full-length antibody light chain and a full-length antibody heavy chain. The antibody light chain may be a kappa or lambda light chain. The antibody heavy chain may be any class, such as IgG, IgM, IgE, IgA, or IgD. In some embodiments, the antibody heavy chain is an IgG class, such as IgG1, IgG2, IgG3, or IgG4 subclass. The antibody heavy chains described herein may be converted from one class or subclass to another using methods known in the art.

[0193] The masked antibodies described herein may incorporate the HVR sequences (e.g., one, two, or three heavy chain variable region HVR sequences and / or one, two, or three light chain variable region HVR sequences), heavy chain variable region sequences, and / or light chain variable region sequences) of any antibody described in PCT application number PCT / CN2017 / 098333 (which is incorporated herein by reference in its entirety), PCT application number PCT / CN2017 / 098299 (which is incorporated herein by reference in its entirety), PCT application number PCT / CN2017 / 098332 (which is incorporated herein by reference in its entirety), and / or U.S. Patent Application Publication 2021 / 0206855 (which is incorporated herein by reference in its entirety).

[0194] The masked antibodies described herein may incorporate the HVR sequence (e.g., one, two, or three heavy chain variable region HVR sequences and / or one, two, or three light chain variable region HVR sequences), heavy chain variable region sequence, and / or light chain variable region sequence of any of the antibodies described herein (e.g., anti-CTLA4 antibody, anti-CD137 antibody).

[0195] In some embodiments, the masked antibody is an anti-CTLA4 antibody comprising (a) an antibody light chain variable region comprising HVR-L1 comprising the amino acid sequence RASQSVRGRFLA (SEQ ID NO: 95), HVR-L2 comprising the amino acid sequence DASNRATGI (SEQ ID NO: 96), and / or HVR-L3 comprising the amino acid sequence YCQQSSSWPPT (SEQ ID NO: 97); and (b) an antibody heavy chain variable region comprising HVR-H1 comprising the amino acid sequence YSISSGYHWSWI (SEQ ID NO: X98), HVR-H2 comprising the amino acid sequence LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 99), and / or HVR-H3 comprising the amino acid sequence ARSYVYFDY (SEQ ID NO: 100). In some embodiments, the masked antibody includes an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 93, or an amino acid sequence having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence of SEQ ID NO: 93; and / or an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 94, or an amino acid sequence having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence of SEQ ID NO: 94. In some embodiments, the masked antibody includes an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 93, or an amino acid sequence having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence of SEQ ID NO: 93; and an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 94, or an amino acid sequence having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence of SEQ ID NO: 94. In some embodiments, the antibody includes an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 93, and an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 94.

[0196] Exemplary anti-CTLA4 antibody light chain variable region (SEQ ID NO: 93): DIQLTQSPSSLSASVGDRVTITCRASQSVRGRFLAWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSSSWPPTFGQGTKVEIKR

[0197] Exemplary anti-CTLA4 antibody heavy chain variable region (SEQ ID NO: 94): EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWSWIRQAPGKGLEWLARIDWDDDKYYSTSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARSYVYFDYWGQGTLVTVSS

[0198] The masked antibodies of the disclosure can be produced, for example, using recombinant methods and compositions as described in U.S. Patent No. 4,816,567. In some embodiments, isolated nucleic acids encoding any of the masked antibodies are provided. Such nucleic acids may encode amino acid sequences including VL and / or VH of the antibody (e.g., the light chain and / or heavy chain of the antibody). In some embodiments, one or more vectors (e.g., expression vectors) containing such nucleic acids are provided herein. In some embodiments, host cells containing such nucleic acids are provided. In one embodiment, the host cell contains (e.g., is transformed by) one or two vectors encoding the masked antibodies described herein. In some embodiments, the host cell is a eukaryote, e.g., yeast cell, insect cell, Chinese hamster ovary (CHO) cell or lymphocyte cell (e.g., Y0, NS0, Sp20 cell), plant cell, or bacterial cell. In some embodiments, a method is provided for producing a masked antibody, which comprises culturing host cells containing nucleic acids encoding the masked antibody provided above under conditions suitable for the expression of the masked antibody, and, if necessary, recovering the masked antibody from the host cell (or host cell culture medium).

[0199] In any of the embodiments described above, the masked antibody may be an antibody that binds to a specific target, including but not limited to CTLA4, CD137, PD-1, PD-L1, PD-L2, LAG3, TIM3, B7-H3, OX40, CD3, CD19, CD20, CD40, CD47, CD95, CD120a, BTLA, VISTA, ICOS, BCMA, Her1, Her2, Her3, Nectin-4, PSMA, FOLR-1, MUC16, GPC3, MUC1, KLK2, Claudin-18.2, and / or B7-H4. In some embodiments, the masked antibody is not an anti-CD47 antibody.

[0200] In some embodiments, the masking peptide does not have any of the following sequences: TIFF2026515742000002.tif18170V.Composition

[0201] In other embodiments, the Disclosure provides compositions comprising one or more of the masked antibodies described herein. In some embodiments, the composition is a pharmaceutical composition comprising a polypeptide (e.g., a masked conjugated polypeptide, e.g., a masked antibody) and a pharmaceutically acceptable carrier. The composition can be prepared by conventional methods known in the art.

[0202] The term "pharmaceutically acceptable carrier" refers to any inert substance suitable for use in formulations for the delivery of polypeptides (e.g., masked antibodies). Carriers may include antifouling agents, binders, coatings, disintegrants, fillers or diluents, preservatives (such as antioxidants, antimicrobials, or antifungals), sweeteners, absorption retarders, wetting agents, emulsifiers, and buffers. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (such as glycerol, propylene glycol, and polyethylene glycol), glucose, vegetable oils (such as olive oil), saline, buffers, buffered saline, and isotonic agents such as sugars, polyhydric alcohols, sorbitol, and sodium chloride.

[0203] The composition may be in any preferred form, such as liquid, semi-solid, and solid dosage forms. Examples of liquid dosage forms include solutions (e.g., injectable and injectable solutions), microemulsions, liposomes, dispersions, or suspensions. Examples of solid dosage forms include tablets, pills, capsules, microcapsules, and powders. A particular form of composition suitable for delivering polypeptides (e.g., masked conjugated polypeptides, e.g., masked antibodies) is a sterile liquid, such as a solution, suspension, or dispersion for injection or infusion. A sterile solution can be prepared by incorporating the required amount of polypeptide (e.g., masked conjugated polypeptides, e.g., masked antibodies) into a suitable carrier, followed by sterile microfiltration. A dispersion is prepared by incorporating polypeptides (e.g., masked conjugated polypeptides, e.g., masked antibodies) into a sterile vehicle containing a basic dispersion medium and other carriers. In the case of sterile powders for the preparation of sterile liquids, the preparation method includes vacuum drying and freeze-drying to obtain a powder in which the active ingredient is supplemented with any additional desired components from its pre-sterilized filtered solution. Various dosage forms of the composition can be prepared by conventional techniques known in the art.

[0204] The relative amount of polypeptide (e.g., masked antibody) contained in a composition will vary depending on many factors, including the specific polypeptide and carrier used, the dosage form, and the desired release and pharmacodynamic properties. The amount of polypeptide (e.g., masked conjugated polypeptide, e.g., masked antibody) in a single dosage form is generally the amount that produces a therapeutic effect, but may be less. Generally, this amount is in the range of about 0.01 percent to about 99 percent, about 0.1 percent to about 70 percent, or about 1 percent to about 30 percent relative to the total weight of the dosage form.

[0205] In addition to polypeptides (e.g., masked antibodies), one or more additional therapeutic agents may be included in the composition. Examples of additional therapeutic agents are described below herein. A suitable amount of additional therapeutic agent to be included in the composition can be easily selected by those skilled in the art and will vary depending on many factors, such as the specific drug and carrier used, the dosage form, and the desired release and pharmacodynamic properties. The amount of additional therapeutic agent included in a single dosage form is generally the amount of the drug that produces the therapeutic effect, but may be less.

[0206] Any polypeptide (e.g., masked antibody) and / or composition (e.g., pharmaceutical composition) described herein may be used in the preparation of pharmaceuticals (e.g., pharmaceuticals for use in subjects requiring the treatment of cancer or the delay of cancer progression). VI. Kit

[0207] In another embodiment, a kit is provided comprising a library of cells or phages exhibiting an antibody having a polynucleotide, polypeptide, or masking peptide of the Disclosure, or a polypeptide or antibody having a masking peptide of the Disclosure. In some embodiments, the kit further comprises a package insert containing instructions for using the library, for example, to express, modify, screen, or otherwise use, to identify a masked antibody of interest. In some embodiments, the kit further comprises one or more buffers for storing, transferring, administering, or otherwise using, for example, one or more polynucleotides (e.g., synthetic polynucleotides). In some embodiments, the kit further comprises one or more containers for storing one or more polynucleotides. In some embodiments, the kit further comprises one or more vectors for transfection, for example, with one or more polynucleotides of a host cell.

[0208] In another embodiment, kits comprising polypeptides and / or compositions described herein are provided herein. In some embodiments, the kit further includes a package insert containing instructions for use of a selected masked conjugated polypeptide (e.g., a masked antibody), an antibody, and / or a composition. In some embodiments, the kit further includes one or more buffers for storing, transferring, administering, or otherwise using, for example, a masked conjugated polypeptide (e.g., a masked antibody), and / or a composition. In some embodiments, the kit further includes one or more containers for storing or administering a masked conjugated polypeptide, an antibody, and / or a composition.

[0209] The foregoing description shall be deemed sufficient to enable those skilled in the art to practice the present disclosure. The following examples are presented for illustrative purposes only and are not intended in any way to limit the scope of the present disclosure. In fact, in addition to the modifications shown and described herein, various modifications of the present disclosure will be apparent to those skilled in the art from the foregoing description and will fall within the scope of the appended claims. [Examples]

[0210] Example 1: Method for identifying masking peptides of masked antibodies

[0211] As described above, there is a need for improved methods and products useful for identifying masking peptides (MPs) of masked antibodies. A novel set of masking unit (MU) libraries was designed based on data analysis of antibody structural motifs and an accumulated SAFEbody collection. In the library design, antibody stability, structural and chemical diversity, and downstream development potential were considered. Accordingly, this specification describes an improved library of masking peptides designed and executed using masking units with good development potential. Several unique features were incorporated into the improved masking unit library that were strongly enhanced in identifying masking units of target antibodies with good developability: 1) The library was designed to balance chemical diversity in the MU region. Amino acids with charged side chains, such as E / D / H / K / R, are preferred (enriched) in the MU library. Along with amino acids with polar side chains, such as S / T / N / Q / Y, these charged polar amino acids constitute the majority of the library composition. 2) The peptide library was designed to include stable motifs, which are indicated by their prevalence in data analysis. Key residues for early chain compression during folding, such as glycine and proline, are intentionally retained in loop regions for faster loop formation. 3) The library was designed to avoid high-risk post-translational modification (PTM) sites such as free cysteine, glycosylation sites, deamidation sites, aspartate isomerization sites, and oxidation sites. For example, NG and DGNX[S / T] sites were excluded in the new MU library.

[0212] This specification provides a library (SAFEbody masking unit library) comprising polynucleotides encoding polypeptides, wherein the polypeptide comprises a masking peptide (MP) and an antigen-binding domain (ABD); the MP comprises a masking unit (MU) and a linkage unit (LU) from the N-terminus to the C-terminus; the MP is bound to the N-terminus of the ABD; and the MU comprises an amino acid sequence according to formula (I), in which. X1 X2 CX3 (Xm) n X4 X5 CX6 X7 The above n is 2 to 8, and here X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. Each Xm is independently an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y, X4 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y, X5 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y, X6 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, X7 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, Said C represents the amino acid cysteine.

[0213] Four exemplary constrained peptide libraries (CPLs) were designed (Table 2A) to identify potential masking units (MUs). The diversity of the libraries (possible peptide combinations) is in the range of approximately 5x10 9 to 2x10 13 . Their compositions are described in Table 2B below. Table 2A: Diversity of Library Design Masking Units TIFF2026515742000003.tif29170 1 X represents an amino acid; the subscript indicates the number of amino acids at each position. 2 C represents one cysteine amino acid residue. Table 2B: Amino Acid Composition at Each Position of the Masking Unit (MU) Library TIFF2026515742000004.tif187170 1 X represents an amino acid; the subscript indicates the number of amino acids at each position. 2 C represents one cysteine amino acid residue.

[0214] For optimization, two cleavage peptide sequences were introduced into the construct's linkage units following these masking unit sequences. The protease recognition sites introduced during library construction were SGRSA (SEQ ID NO: 34) for protease urokinase-type plasminogen activator (uPA) and PLGLAG (SEQ ID NO: 35) for protease matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). These recognition sites have been used by many groups in the in vivo tumor cell-specific activation of targeted drugs (see, for example, Keetal. (1997) JBiolChem 272(33):20456-62; Gerspachetal. (2006) CancerImmunolImmunother 55(12):1590-600; and Jiangetal. (2004) ProcNatlAcadSciUSA101(51): 17867-72).

[0215] The masking peptide further contains an N-terminal unit fused to the C-terminus of the masking unit. In this example, EVGSY (SEQ ID NO: 33) was used as the N-terminal unit.

[0216] To identify masking peptide sequences that can effectively mask the parental antibody, the N-terminus of the light chain variable domain of the antigen-binding domain of the anti-CTLA4scFv parental antibody was fused to the C-terminus of the masking peptide. In this example, a yeast library was constructed that displayed fusion proteins on the yeast cell surface, with each masking unit from the improved peptide library directly fused to the N-terminus of the light chain of the parental antigen-binding domain. The yeast library was then subjected to several FACS-based screenings. First, yeast clones with low antigen binding were enriched, then the enriched yeast clones were treated with proteases to remove the masking units, and clones with high antigen binding were selected. After 4-5 selections, plasmids were extracted from these clones, and the masking unit sequences were confirmed by DNA sequencing. The enrichment of specific amino acids in optimizing the masking units from the four libraries is shown in Table 3. Exemplary masking units obtained from library screening are shown in Tables 4A-D. Table 5 shows exemplary masking polypeptide sequences after library selection, with the N-terminal unit fused to the masking unit and linkage unit from N-terminus to C-terminus. Table 3: Concentration of specific amino acids after screening in each masking unit library. TIFF2026515742000005.tif233170 Table 4A: Exemplary arrangement of masking units in Library 1 TIFF2026515742000006.tif29170 Table 4B: Exemplary arrangement of masking units in Library 2 TIFF2026515742000007.tif51170 Table 4C: Exemplary arrangement of masking units in Library 3 TIFF2026515742000008.tif46170 Table 4D: Exemplary arrangement of masking units in Library 4 TIFF2026515742000009.tif29170 Table 5: Examples of masking peptides TIFF2026515742000010.tif151170 1The N-terminal unit EVGSY (SEQ ID NO: 33) of the underlined part is added to the N-terminus of each masking unit. 2 The bold parts are invariant cleavage sites such as SGRSA (SEQ ID NO: 34) and PLGLAG (SEQ ID NO: 35), and the italic parts are mutant linker sequences such as GGGGT (SEQ ID NO: 36) and SGGS (SEQ ID NO: 37). 3 The linkage unit sequence SGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 38) is added to the C-terminus of each masking unit.

[0217] By optimizing the N-terminal unit sequence and the linkage unit, the masking efficiency can be further improved. In Tables 6A - 6C, the identified masking unit sequence (BC3855) is combined with different N-terminal units (E, EVG, EVGSY (SEQ ID NO: 33), EVGVLDV (SEQ ID NO: 86), EVGAESGVK (SEQ ID NO: 88), or EVGSY (SEQ ID NO: 33)) and various linkage units: SGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 38), SGRGPLGLAGGS (SEQ ID NO: 39), SGGGPLGLAGGS (SEQ ID NO: 40), GGGGPLGLAGGS (SEQ ID NO: 41), GGGPLGLAGGGS (SEQ ID NO: 42), and GGGGSGGSGGGS (SEQ ID NO: 81). The resulting properties vary for each construct. Exemplary masking peptides with various compositions of the linkage unit are shown in Tables 6A - 6C. Table 6A. Sequences of the N-terminal unit, masking unit, linker, and cleavage site of an antibody with a C1-L1-C2-L2 configured linkage unit. TIFF2026515742000011.tif22170 Table 6B. Sequences of the N-terminal unit, masking unit, linker, and cleavage site of an antibody with an L1-C1-L2 configured linkage unit. TIFF2026515742000012.tif51170 Table 6C. Sequences of the N-terminal unit, masking unit, and linker of an antibody with a linkage unit without a cleavage site. TIFF2026515742000013.tif26170

[0218] Using the above library, anti-CTLA4 masking peptides were screened. Recombinant human CTLA4-Fc was diluted to 1 μg / mL in PBS and spread overnight on a Maxisorp plate at 4°C. The plate was blocked at 37°C for 1 hour with PBS supplemented with 3% nonfat milk. After washing, 100 μL of 3-fold serially diluted antibody was added to each well. After incubation at 37°C for 1 hour, the plate was washed four times and 100 μL of HRP-conjugated anti-human IgG (Fab-specific) (1:6000 dilution) was added to each well. The plate was incubated at 37°C for 1 hour, washed four times, then 50 μL of TMB substrate solution was added to each well and the plate was incubated at room temperature. After stopping the reaction with 50 μL of H2SO4 per well, the absorbance at 450 nm was measured. EC50 was evaluated by fitting ELISA data using an asymmetric sigmoid (5-parameter logistic equation) model in GraphPadPrism6 software. Two calculated masking efficiencies were obtained for each of the activatable antibodies TY24463, TY24464, TY24649, and TY24652 by performing one experiment, and for TY24148, TY24466, and TY24465 by performing two experiments. The masking efficiency for each activatable antibody was calculated by dividing the EC50 of the activatable antibody binding by the EC50 of the parent antibody (TY21580). As shown in Table 7, all activatable antibodies showed a dramatic decrease in binding to their antigen compared to the parent antibody, and the calculated masking efficiencies ranged from 773 to 6925. The differences in masking efficiency are likely due to variations in EC50 value measurements and data fitting, and the masking efficiency of each activatable antibody is likely within the calculated range (e.g., the masking efficiency of the activatable antibody TY22465 is 3749–5097). These results indicate that several masking peptides identified from CPL maintained their masking efficiency in mammalian cells and when expressed as part of a complete IgG molecule.Furthermore, masking efficiency varies based on the density of immobilized antigens within the cell line used or their respective densities. Higher antigen concentrations result in lower masking efficiency. Table 7. Examples of optimized masking peptides obtained by modifying the N-terminal and linkage units surrounding the BC3855 masking unit. TIFF2026515742000014.tif113170 1 The N-terminal unit (E or EVGSY) is underlined. 2 The bolded text indicates invariant cleavage sites such as SGRSA (SEQ ID NO: 34) and PLGLAG (SEQ ID NO: 35). 3 Italicized text indicates variant linker sequences such as GGGGT (sequence number 36) and SGGS (sequence number 37). HMW: High molecular weight LMW: low molecular weight ME: Masking efficiency

[0219] Purified activatable antibodies were treated with proteases that recognize cleavage sequences, and then tested to determine whether the removal of masking peptides restored their activity. For example, 20 μg of TY22404 (0.5 mg / mL) was treated with 1 μg of recombinant human uPA (Acrobiosystems, #PLU-H5229) in reaction buffer (50 mM Tris HCl, 0.01% Tween 20, pH 8.5), or TY22404 was treated with 5 or 10 units of recombinant human MMP-9 (BioVision, #7867-500) in reaction buffer (50 mM Tris, 150 mM NaCl, 5 mM CaCl2, 20 μM ZnCl2, pH 7.5). The reaction was carried out at 37°C for 21 hours. The masking peptides were confirmed to be removed from the light chain by SDS-PAGE analysis. Next, the masking efficiency was measured by ELISA as described above. Example 2: Effect of N-terminal peptide on masking efficiency of masking peptide

[0220] Of the three units of a masking peptide (MP), the masking unit (MU) determines its ability to mask the parent antibody, but both the N-terminal unit and the linkage unit (LU) influence the masking efficiency, and the N-terminal unit in particular can be an effective multiplier for extending the range of masking efficiency of a specific masking unit. A set of short peptides was selected as a universal N-terminal unit applicable to any masking peptide to tune the masking efficiency. In Table 8, this set of N-terminal units was tested with TY24652, and its masking efficiency was measured and benchmarked. Different selections of N-terminal units can also be used to fine-tune properties of the masked antibody, such as PI and charge distribution. Table 8. Additional masking peptide sequences by various N-terminal peptides. TIFF2026515742000015.tif63170 Example 3: Concentration-dependent binding activity - Binding to HER2+ tumor cell lines

[0221] Here, we used flow cytometry to measure the concentration-dependent binding activity of parental and masked anti-HER2 antibodies TY23477 and TY24925 against HER2+ tumor cell lines with different antigen expression levels. While both TY23477 and TY24925 antibodies possess cleavable linkage units, these units were not cleaved in the in vitro assay. Therefore, these results demonstrate concentration-dependent binding of the antibodies even in the absence of cleavage-dependent removal of the masking peptide.

[0222] Briefly, SKOV3, MCF7, or A549 cells were seeded at 1.0 × 10⁵ / well in 96-well plates and incubated with serially diluted test antibodies in 1% FBS / 1640 buffer at 4°C for 30 minutes. The cells were then washed twice with DPBS and further incubated with secondary APC anti-human IgGFc antibody at 4°C for 30 minutes. Finally, the cells were washed twice with DPBS and suspended in FACS buffer for flow cytometry analysis. MFI values ​​versus concentrations were then analyzed using FlowJo, and the data were further fitted using 4-parameter nonlinear regression to obtain EC50 values ​​using GraphPadPrism software.

[0223] As shown in Figures 1A-1C and Table 9A, both masked antibodies, TY23477 and TY24925, showed reduced EC50 binding to tumor cell lines compared to the parent antibody trastuzumab. The masking efficiency, as measured by comparing the EC50s of the masked antibodies with that of the parent antibody, was lower on SKOV3 cells, which have higher antigen expression levels, than the masking efficiency in MCF7 and A549 cells. The masking peptides of the anti-HER2 masked antibodies are shown in Table 9B. Table 9A. Concentration-dependent binding of masked anti-HER2 antibodies to cell lines with different antigen expression levels. TIFF2026515742000016.tif42170 Table 9B. Masking peptides of masked anti-HER2 antibodies TIFF2026515742000017.tif18170

[0224] The concentration-dependent binding between the antigen and the masked antibody suggests that competitive binding between the masked antibody and the antigen functions as a mechanism for enriching the masked antibody around the antigen-containing region, such as CTLA-4, which is upregulated by tumor induction. These data highlight the design of dynamic rather than sticky masking peptides with relatively high off-rates for the innovative masking technique of the present invention. This differs significantly from other self-suppressive masking techniques. The sequence composition and structural features of the masking peptide library are discussed in detail herein. Example 4: Time-dependent cutting Time-dependent cleavage of masked CTLA4 antibodies with purified enzymes

[0225] Time-dependent cleavage of TY24652 and TY26294 was evaluated. Recombinant human MMP-9 (proprietary) was first activated with APMA (100 μg / mLMMP-9 and 1 mMAPMA were incubated at 37°C for 24 hours). TY24652 or TY26294 was mixed with activated MMP9 and reacted in 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij35 (w / v), pH 7.5 buffer at 37°C for 900 seconds, 1800 seconds, 3600 seconds, 7200 seconds, and 14400 seconds. After division, the ratio of cleaved LC to total LC was analyzed using reduced SDS-PAGE. The masking peptides of the anti-CTLA4 antibodies used in this study are shown in Table 10. Table 10. Masking peptides of masked anti-CTLA4 antibodies TIFF2026515742000018.tif22170

[0226] As shown in Figure 2, both TY24652 and TY26294 were disconnected in a time-dependent manner. Completed disconnections occurred between 2 hours and 4 hours. Quantification of cleaved antibodies from a mouse H22 tumor model using conventional Western blotting.

[0227] Here, time-dependent in vivo cleavage of masked antibodies TY22404 and TY24652 in mouse H22 tumor models or in plasma and liver was measured using conventional Western blotting (WB).

[0228] Fresh tissues obtained from mice administered TY22404 and TY24652 (5 mg / kg) were homogenized into powder on ice using a handheld electric homogenizer (WUXIWoxin). The homogenized tissues were then resuspended in RIPA lysis buffer (CST) containing the protease inhibitor cocktail, using the tissue homogenate-to-protease inhibitor ratios based on the product description of the protease inhibitor cocktail. Antibodies were captured from the tissue homogenates by incubation at room temperature for 3 hours on a rotator using AmMag® Protein A magnetic beads (GenScript). Purified antibodies were separated on a 12% SDS-PAGE gel (Beyotime) with reduced denaturation conditions and subsequently transferred to a PVDF membrane (Millipore). Masked and cleaved antibody signals were detected using peroxidase Afinipure mouse anti-goat IgG (H+L) antibody (JacksonImmunoResearch). The blots were scanned using an AmershamImager600 (GEHealthcareLifeSciences).

[0229] As shown in Figures 3A–3D, these results indicate that the masked antibodies TY22404 and TY24652 are selectively cleaved over time (24 hours and 96 hours) in mouse H22 tumor sites, liver, and plasma. Furthermore, based on band intensity, the cleavage rate at 96 hours post-treatment is higher than that at 24 hours post-treatment. In addition, TY22404 is more readily cleaved than TY24652, and it is shown that the rate of cleavage-based activation of the masked antibody can be adjusted using the number of cleavage sites (one for TY24652 and two for TY22404). Plasma and tumor PK data of TY26294 in the SHP-77 xenograft model

[0230] TY26294 is an anti-CD47 antibody containing the masking peptide sequence LTVDYFCDIDPLYCNAGGGPLGLAGSGGS (SEQ ID NO: 92). CB17SCID mice with SHP-77 tumors were administered a single intravenous dose of TY26294 at 10 mg / kg. Plasma and tumor samples were collected at different time points. Concentrations of complete and cleaved forms of TY26294 were analyzed using established ELISA methods.

[0231] As shown in Figures 4A-4C, these results indicate that the masked antibody TY26294 is selectively cleaved in mouse SHP-77 tumor sites and plasma over time. Example 5: Identification of masking peptides for antibodies targeting human CD137

[0232] The constraint peptide library described in Example 1 was used to screen for masking units against a VHH antibody targeting human CD137. The cleaved peptide sequence was introduced into the construct's linkage unit following the masking unit sequence. Specifically, the protease recognition sites PLGLAG (SEQ ID NO: 35) of protease matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) were used. This recognition site has been used by many groups in the in vivo tumor cell-specific activation of targeted drugs (see, for example, Keetal. (1997) JBiolChem 272(33): 20456-62; Gerspachetal. (1997) JBiolChem 272(33): 20456-62; Gerspachetal. (2006) CancerImmunolImmunother 55(12): 1590-600; and Jiangetal. (2004) ProcNatlAcadSciUSA 101(51): 17867-72).

[0233] To identify masking peptide sequences that can effectively mask parental antibodies, the C-terminus of the masking peptide was fused to the N-terminus of a CD137-binding VHH single-domain antibody. The VH domain of the VHH single-domain antibody is the CD137-targeting VH domain of INBRX-105. Each masking unit from improved peptide library 4 (Table 2B) was directly fused to the N-terminus of a VHH antibody, and a yeast library was constructed to screen for masking peptides by displaying the fusion protein on the surface of yeast cells. Subsequently, the yeast library underwent several FACS-based screenings. First, yeast clones with low antigen binding were enriched, then the enriched yeast clones were treated with proteases to remove the masking units, and clones with high antigen binding were selected.

[0234] Yeast cells induced in glucose-free medium (1x10 8 The cells were harvested, washed once with PBSA buffer, and incubated with different concentrations of biotinylated antigens at room temperature for 1 hour. After incubation with the antigens, the yeast cells were washed with PBSA buffer and treated with streptavidin fluorescent dye (phycoerythrin (PE)-streptavidin; 1:500 dilution, eBioscience#2-4317-87) or goat anti-HumanFcAlexaFluor® 647 conjugate (Jackson#109-606-098) at 4°C for 30 minutes. The cells were then washed twice with PBSA buffer and sorted by optical density of 2–3 OD / mL (MoFloXDP). The concentration of biotinylated CD137-HisFc was 2 nM, and cells with low antigen binding were collected. After the initial selection, the collected yeast cells were treated with TEV protease (2 μg / OD cells, Genscript) at 30°C for 30 minutes, and activation of the target antibody by protease cleavage was confirmed. After multiple selections, single clones were classified on selection medium and grown individually, and activation of antigen binding via cleavage was confirmed. Exemplary masking peptides identified in the screening are shown in Table 11A, and their masking peptide units and invariant cleavage peptide sequences are listed in Table 11B. Table 11A: Exemplary masking peptides for masked anti-CD137VHH antibodies TIFF2026515742000019.tif26170 1 The underlined N-terminal unit EVGSY (SEQ ID NO: 33) is added to the N-terminus of each masking unit. 2 The bolded text indicates the invariant cleavage site PLGLAG (sequence number 35), while the italicized text indicates variant linker sequences such as GGG and SGGS (sequence number 37). 3 The linkage unit sequence GGGPLGLAGSGGS (sequence number 119) is added to the C-terminus of each masking unit. Table 11B. Sequence of N-terminal unit, masking unit, linker, and cleavage site of anti-CD137VHH antibody TIFF2026515742000020.tif44170

[0235] To test the masking efficiency of the identified masking units, selected activatable anti-CD137VHH single-domain antibodies were fused to human Fc domains. The sequences of VHH single-domain antibodies without masking peptides fused to the Fc domain are shown in Table 12. The masking efficiency of the exemplary activatable antibody A was measured by an ELISA-based assay. Recombinant human CD137-His was diluted to 2 μg / mL in PBS and coated onto Maxisorp plates, which were left overnight at 4°C. The plates were blocked at 37°C for 1 hour in PBS supplemented with 3% non-fat milk. After washing, 100 μL of 3-fold serial dilutions of the antibody were added to each well. After incubation at 37°C for 1 hour, the plates were washed four times, and 100 μL of HRP-conjugated anti-human IgG (Fc-specific) (1033 ng / mL) was added to each well. After incubation at 37°C for 1 hour and washing four times, 50 μL of TMB substrate solution was added to each well, and the plates were incubated at room temperature. After stopping the reaction with 50 μL of H2SO4 per well, absorbance at 450 nm was measured. EC50 was evaluated by fitting the ELISA data using a sigmoid (4-parameter logistic equation) model in GraphPadPrism software. Table 12. Anti-CD137VHH-FC antibody TIFF2026515742000021.tif49170

[0236] The masking efficiency of the activatable antibody TY28851 was calculated by dividing the EC50 of TY28851 binding to CD137 by the EC50 of the parental VHH-Fc antibody (TY28974, SEQ ID NO: 115) binding to CD137. TY28851 showed a masking efficiency of 43. These results demonstrate the remarkable effectiveness of the library described herein for selecting antibodies masked with high masking efficiency across multiple targets and antibody formats.

Claims

1. A library comprising polynucleotides, wherein the polynucleotides in the library encode at least two, at least three, at least four, at least five, or at least ten antibodies, each having a unique masking peptide (MP), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), the MP being ligated to the N-terminus of the VH or VL of the antibody, the MP comprising a masking unit (MU) and a linkage unit (LU), the MU comprising an amino acid sequence according to formula (I): X1X2CX3(Xm)nX4X5CX6X7, where n is 2 to 8, where: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y, and X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. Each Xm is an amino acid independently selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X4 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X6 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X7 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. C is cysteine.

2. A library comprising polynucleotides, wherein the polynucleotides in the library encode at least two, at least three, at least four, at least five, or at least ten antibodies, each having a unique masking peptide (MP), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), the MP being ligated to the N-terminus of the VH or VL of the antibody, the MP comprising a masking unit (MU) and a linkage unit (LU), the MU comprising an amino acid sequence according to formula (II): X1X2CX3X4X5X6X7CX8X9, where: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X4 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, E, F, G, H, K, L, P, Q, R, S, T, V, and Y. X6 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X7 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X8 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X9 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine.

3. The above, X1 is an amino acid selected from the group consisting of A, F, H, and V. X2 is an amino acid selected from the group consisting of A, L, and P. X3 is an amino acid selected from the group consisting of A, G, L, and R. X4 is an amino acid selected from the group consisting of E, G, K, and P. X5 is an amino acid selected from the group consisting of F, K, L, and V. X6 is an amino acid selected from the group consisting of F, L, P, and S. X7 is an amino acid selected from the group consisting of F, P, and Y. X8 is an amino acid selected from the group consisting of G, I, L, and P, and, X9 is an amino acid selected from the group consisting of E, Q, T, and V, and is the library according to claim 2.

4. The library according to claim 3, wherein the MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 10.

5. A library comprising polynucleotides, wherein the polynucleotides in the library encode at least two, at least three, at least four, at least five, or at least ten antibodies, each having a unique masking peptide (MP), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), the MP being ligated to the N-terminus of the VH or VL of the antibody, the MP comprising a masking unit (MU) and a linkage unit (LU), the MU comprising an amino acid sequence according to formula (III): X1X2CX3X4X5X6X7X8CX9X10, where: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X4 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X6 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, R, S, T, V, and Y. X7 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X8 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X9 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X10 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine.

6. The above, X1 is an amino acid selected from the group consisting of A, F, H, N, P, S, and Y. X2 is an amino acid selected from the group consisting of A, L, P, S, and V. X3 is an amino acid selected from the group consisting of A, I, K, P, R, and V. X4 is an amino acid selected from the group consisting of A, G, L, P, V, and Y. X5 is an amino acid selected from the group consisting of F, I, L, P, R, and S. X6 is an amino acid selected from the group consisting of F, K, L, P, and Y. X7 is an amino acid selected from the group consisting of I, N, P, S, and V. X8 is an amino acid selected from the group consisting of A, F, L, and Y. X9 is an amino acid selected from the group consisting of G, K, Q, S, and V, and, X10 is an amino acid selected from the group consisting of G, Q, R, S, T, and V, and is the library according to claim 5.

7. The library according to claim 6, wherein the MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 19.

8. A library comprising polynucleotides, wherein the polynucleotides in the library encode at least two, at least three, at least four, at least five, or at least ten antibodies, each having a unique masking peptide (MP), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), the MP being linked to the N-terminus of the VH or VL of the antibody, the MP comprising a masking unit (MU) and a linkage unit (LU), the MP comprising a masking unit (MU) and a linkage unit (LU), the MU comprising an amino acid sequence according to formula (IV): X1X2CX3X4X5X6X7X8X9CX10X11, where: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X4 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X6 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X7 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X8 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X9 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X10 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X11 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine.

9. The above, X1 is an amino acid selected from the group consisting of A, H, L, P, S, and T. X2 is an amino acid selected from the group consisting of A, D, F, L, and Y. X3 is an amino acid selected from the group consisting of E, L, P, and R. X4 is an amino acid selected from the group consisting of A, E, K, P, and R. X5 is an amino acid selected from the group consisting of E, F, G, and L. X6 is an amino acid selected from the group consisting of A, F, P, T, and Y. X7 is an amino acid selected from the group consisting of A, P, S, T, and V. X8 is an amino acid selected from the group consisting of A, N, P, and S. X9 is an amino acid selected from the group consisting of V and Y. X10 is an amino acid selected from the group consisting of I, P, and R, and, X11 is an amino acid selected from the group consisting of E, G, I, P, and V, and is the library according to claim 8.

10. The library according to claim 9, wherein the MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 21 to 27.

11. A library comprising polynucleotides, wherein the polynucleotides in the library encode at least two, at least three, at least four, at least five, or at least ten antibodies, each having a unique masking peptide (MP), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), the MP being ligated to the N-terminus of the VH or VL of the antibody, the MP comprising a masking unit (MU) and a linkage unit (LU), the MU comprising an amino acid sequence according to formula (V): X1X2CX3X4X5X6X7X8X9X10CX11X12, where: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X4 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X6 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X7 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X8 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, R, S, T, V, and Y. X9 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X10 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X11 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X12 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine.

12. The above, X1 is an amino acid selected from the group consisting of A, F, P, S, and Y. X2 is an amino acid selected from the group consisting of H, L, P, S, and V. X3 is an amino acid selected from the group consisting of E, G, K, P, Q, and R. X4 is an amino acid selected from the group consisting of A, F, H, P, R, V, and Y. X5 is an amino acid selected from the group consisting of A, D, F, G, V, and Y. X6 is an amino acid selected from the group consisting of D, F, H, L, P, and V. X7 is an amino acid selected from the group consisting of H, K, P, R, and V. X8 is an amino acid selected from the group consisting of A, F, K, L, and P. X9 is an amino acid selected from the group consisting of A, D, F, L, and P. X10 is an amino acid selected from the group consisting of A, F, V, and Y. X11 is an amino acid selected from the group consisting of G, I, K, L, and R, and, X12 is an amino acid selected from the group consisting of A, E, K, P, R, and T, and is the library according to claim 11.

13. The library according to claim 12, wherein the MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-32 and 112-114.

14. The library according to any one of claims 1 to 13, wherein the MU does not contain the amino acid sequence of NG, DG, NXS, or NXT, where X is any amino acid.

15. The library according to any one of claims 1 to 14, wherein the MP further comprises an N-terminal unit (NU) connected to the N-terminus of the MU.

16. The library according to claim 15, wherein the N-terminal unit has a length of about 1 to 12 amino acid residues.

17. The library according to claim 16, wherein the N-terminal unit includes E, EVG, EVGSY (SEQ ID NO: 33), EVGVLDV (SEQ ID NO: 86), or EVGAESGVK (SEQ ID NO: 88).

18. The library according to any one of claims 1 to 17, wherein the LU does not include a cutting portion.

19. The LU is the library according to claim 18, wherein the LU includes a linker.

20. The linker comprises or consists of an amino acid sequence selected from the group consisting of GS, GGS, GGG, SGRG (SEQ ID NO: 104), GGGGS (SEQ ID NO: 105), GGGGT (SEQ ID NO: 36), SGGS (SEQ ID NO: 37), GGSG (SEQ ID NO: 106), GGSGG (SEQ ID NO: 107), GSGSG (SEQ ID NO: 108), GSGGG (SEQ ID NO: 109), GGGSG (SEQ ID NO: 110), GSSSG (SEQ ID NO: 111), GSGGS (SEQ ID NO: 77), SGGG (SEQ ID NO: 79), GGGS (SEQ ID NO: 80), and GGGGSGGGSGGGS (SEQ ID NO: 81), according to claim 19.

21. The library according to claim 18, wherein the LU comprises the amino acid sequence of sequence number 81.

22. The library according to any one of claims 1 to 17, wherein the LU includes a first cutting portion (C1).

23. The first cleavage site (C1) contains urokinase-type plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, tobacco etch virus (TEV) protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12. The library according to claim 22, wherein the protease cleavage sites are selected from the group consisting of ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE.

24. The library according to claim 23, wherein the first cleavage site (C1) comprises an amino acid sequence selected from the group consisting of SGRSA (SEQ ID NO: 34) and PLGLAG (SEQ ID NO: 35).

25. The library according to any one of claims 22 to 24, wherein the LU further includes a second cutting portion (C2).

26. The library according to claim 25, wherein the second cleavage site (C2) is a protease cleavage site of a protease selected from the group consisting of urokinase-type plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, tobacco etch virus (TEV) protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE.

27. The library according to claim 25, wherein the second cleavage site (C2) comprises an amino acid sequence selected from the group consisting of SGRSA (SEQ ID NO: 34) and PLGLAG (SEQ ID NO: 35).

28. The library according to any one of claims 25 to 27, wherein the first and second cutting portions are the same.

29. The library according to any one of claims 25 to 27, wherein the first and second cutting portions are different.

30. The library according to any one of claims 22 to 29, wherein the LU further includes a first linker (L1).

31. The Libraries of Claim 30, wherein the first linker (L1) comprises an amino acid sequence selected from the group consisting of GS, GGS, GGG, SGRG (SEQ ID NO: 104), GGGGS (SEQ ID NO: 105), GGGGT (SEQ ID NO: 36), SGGS (SEQ ID NO: 37), GGSG (SEQ ID NO: 106), GGSGG (SEQ ID NO: 107), GSGSG (SEQ ID NO: 108), GSGGG (SEQ ID NO: 109), GGGSG (SEQ ID NO: 110), GSSSG (SEQ ID NO: 111), GSGGS (SEQ ID NO: 77), SGGG (SEQ ID NO: 79), GGGS (SEQ ID NO: 80), and / GGGGGSGGGGGS (SEQ ID NO: 81).

32. The library according to claim 30 or 31, wherein the LU further includes a second linker (L2).

33. The Libraries of Claim 32, wherein the second linker (L2) comprises an amino acid sequence selected from the group consisting of GS, GGS, GGG, SGRG (SEQ ID NO: 104), GGGGS (SEQ ID NO: 105), GGGGT (SEQ ID NO: 36), SGGS (SEQ ID NO: 37), GGSG (SEQ ID NO: 106), GGSGG (SEQ ID NO: 107), GSGSG (SEQ ID NO: 108), GSGGG (SEQ ID NO: 109), GGGSG (SEQ ID NO: 110), GSSSG (SEQ ID NO: 111), GSGGS (SEQ ID NO: 77), SGGG (SEQ ID NO: 79), GGGS (SEQ ID NO: 80), and GGGGSGGGSGGGS (SEQ ID NO: 81).

34. The aforementioned LU extends from the N-terminus to the C-terminus: 1) First cutting site (C1) and first linker (L1); 2) First cut section (C1), first linker (L1), second cut section (C2), and second linker (L2); 3) First linker (L1), first cut section (C1), and second linker (L2); or 4) A library according to any one of claims 22 to 33, comprising a first linker (L1), a first cut portion (C1), a second linker (L2), and a second cut portion (C2).

35. The library according to any one of claims 22 to 34, wherein the LU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 38 to 42 and 119.

36. The library according to any one of claims 1 to 35, wherein the masking peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-65, 67-75, 83-85, 87, and 116-118.

37. The MP is connected to the N-terminus of the VL, and the library is as described in any one of claims 1 to 36.

38. The MP is ligated to the N-terminus of the VH, and the library is as described in any one of claims 1 to 36.

39. Each of the antibodies comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the MP is ligated to the N-terminus of the VH or VL of the antibody, the library according to any one of claims 1 to 36.

40. The library according to claim 39, wherein the antibody comprises scFv including VH and VL.

41. The library according to claim 39, wherein the antibody comprises Fab, which includes VH and VL.

42. The library according to claim 39, wherein the antibody or antibody comprises an antibody heavy chain and an antibody light chain.

43. The MP is ligated to the N-terminus of the VL of the antibody, and the library is as described in any one of claims 39 to 42.

44. Each of the antibodies comprises a heavy chain variable region (VH), and the MP is ligated to the N-terminus of the VH of the antibody, the library according to any one of claims 1 to 36.

45. The library according to claim 44, wherein the antibody comprises a VHH single-domain antibody or a VHH-Fc antibody containing VH, and the MP is ligated to the N-terminus of VH of the antibody.

46. The library according to any one of claims 1 to 45, wherein the polynucleotide encoding the polypeptide is present in the vector.

47. The library according to claim 46, wherein the vector is an expression vector or a presentation vector.

48. The library according to any one of claims 1 to 47, wherein the polynucleotide encoding the polypeptide is present in a host cell.

49. The library according to claim 48, wherein the cells are bacterial cells, yeast cells, insect cells, or mammalian cells.

50. The MU in the aforementioned library is 10 9 from 10 14 A library according to any one of claims 1 to 49, having diversity up to that point.

51. A library comprising antibodies encoded by polynucleotides in the library according to any one of claims 1 to 50.

52. The library according to claim 51, wherein each polypeptide is displayed on a cell surface or phage surface.

53. The library according to claim 52, wherein the cells are bacterial cells, yeast cells, insect cells, or mammalian cells.

54. A method for producing an antibody, comprising culturing host cells expressing a library containing the antibody described in any one of claims 51 to 52 under conditions suitable for the production of the antibody.

55. The method according to claim 54, further comprising recovering the antibody produced by the cells.

56. The method according to claim 54 or 55, further comprising testing the antibody for its ability to maintain a masked phenotype while remaining soluble.

57. A method for screening a masked antibody that binds to a target using a library according to any one of claims 1 to 53: a) Contacting the antibody expressed in the library with the target to determine a first binding affinity or the absence of detectable binding to the target; b) A step of contacting the target with the control antibody to determine a second binding affinity, and c) Select an expressed antibody that has a first binding affinity lower than the second binding affinity, or that does not bind to the target in a detectable manner. The first and second binding affinities are measured as KD, EC50, or IC50, according to the method.

58. The method according to claim 57, wherein the LU includes at least a first cutting portion (C1).

59. The method according to claim 58, wherein the control antibody is an antibody expressed in a library after the LU has been cleaved.

60. The method according to any one of claims 57 to 59, wherein the expressed antibody is selected if the binding affinity of the expressed antibody after the LU is cleaved is at least 2 times, at least 5 times, at least 10 times, at least 50 times, at least 100 times, at least 500 times, or at least 1000 times, the binding affinity of the expressed antibody before the LU was cleaved.

61. A method for screening a masked antibody that binds to a target using a library according to any one of claims 1 to 53, comprising the following steps: a) Determining the binding EC50 by contacting the antibody expressed in the library having the masking peptide with a first cell expressing the target antigen; b) A control antibody lacking the masking peptide is brought into contact with a first cell expressing the target antigen to determine its binding EC50; c) The antibody expressed in the library is brought into contact with a second cell expressing the target antigen to determine the binding EC50, wherein the second cell expresses a lower level of the target antigen than the first cell; d) A control antibody lacking the masking peptide is brought into contact with a second cell to determine the binding EC50; e) Determine the ratio of the EC50 in step a) to the EC50 in step b) as the first masking efficiency; f) The ratio of EC50 in step c) to EC50 in step d) is determined as the second masking efficiency; and, g) The method of selecting an expressed antibody having a second masking efficiency higher than the first masking efficiency.

62. The method according to claim 61, wherein step (g) comprises selecting an expressed antibody having a second masking efficiency that is at least 10%, at least 50%, at least 2 times, at least 3 times, at least 4 times, at least 5 times, or at least 10 times higher than a first masking efficiency.

63. The method according to claim 61 or 62, wherein the second masking efficiency is at least 50%, at least 2, at least 3, at least 4, at least 5, or at least 10 times higher than the first masking efficiency.

64. The method according to any one of claims 61 to 63, wherein the control antibody is an antibody having the same antigen-binding domain as the antibody expressed in the library.

65. The method according to any one of claims 61 to 64, wherein the control antibody is a parent antibody.

66. The method according to any one of claims 61 to 65, wherein the LU does not include the cut portion.

67. The method according to any one of claims 61 to 65, wherein the LU includes at least a first cutting portion (C1).

68. The method according to claim 67, wherein the control antibody is an antibody expressed in a library after the LU has been cleaved.

69. The method according to claim 68, further comprising cleaving the LU to produce the control antibody.

70. A method for identifying a masked antibody capable of concentration-dependent antigen binding, comprising the following steps: a) A step of determining the binding EC50 by contacting a masked antibody having the masking peptide with a first cell expressing a target antigen, wherein the masking peptide comprises a masking unit (MU) and a linkage unit (LU) from the N-terminus to the C-terminus; b) A control antibody lacking the masking peptide is brought into contact with a first cell expressing the target antigen to determine its binding EC50; c) The masked antibody is brought into contact with a second cell expressing the target antigen to determine the binding EC50, the second cell expressing a lower level of the target antigen than the first cell; d) A control antibody lacking the masking peptide is brought into contact with a second cell to determine the binding EC50; e) Determine the ratio of the EC50 in step a) to the EC50 in step b) as the first masking efficiency; f) The ratio of EC50 in step c) to EC50 in step d) is determined as the second masking efficiency; and, g) The method for identifying a concentration-dependent antigen-binding antibody when the second masking efficiency is higher than the first masking efficiency.

71. The method according to claim 70, wherein step (g) includes identifying a concentration-dependent antigen-binding antibody when the second masking efficiency is at least 10%, at least 50%, at least 2 times, at least 3 times, at least 4 times, at least 5 times, or at least 10 times higher than the first masking efficiency.

72. The method according to claim 70 or 71, wherein the control antibody is an antibody having the same antigen-binding domain as the masked antibody.

73. The method according to any one of claims 70 to 72, wherein the control antibody is a parent antibody.

74. The method according to any one of claims 70 to 73, wherein the LU does not include the cut portion.

75. The method according to any one of claims 70 to 73, wherein the LU includes at least a first cutting portion (C1).

76. The method according to claim 75, wherein the control antibody is a masked antibody after the LU has been cleaved.

77. The method according to claim 76, further comprising cleaving the LU to produce the control antibody.

78. The method according to any one of claims 61 to 77, wherein the second masking efficiency is at least 50%, at least 2 times, at least 3 times, at least 4 times, at least 5 times, or at least 10 times higher than the first masking efficiency.

79. An antibody encoded by one or more polynucleotides from the library according to any one of claims 1 to 50.

80. A kit comprising the library described in any one of claims 1 to 53.

81. A library comprising cells, wherein at least two, at least three, at least four, at least five, or at least ten cells in the library comprise the polynucleotides of the library according to any one of claims 1 to 50.

82. A masked antibody comprising a masking peptide (MP) and an antibody, wherein the antibody comprises an antibody light chain variable region (VL) and / or an antibody heavy chain variable region (VH), the MP being linked to the N-terminus of the VH or VL, and the MP comprising a masking unit (MU) and a linkage unit (LU) from the N-terminus to the C-terminus; the MU comprising an amino acid sequence according to formula (I): X1X2CX3(Xm)nX4X5CX6X7, where n is 2 to 8, where: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. Each Xm is independently an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X4 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X6 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X7 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. C is cysteine.

83. A masked antibody comprising a masking peptide (MP) and an antibody, wherein the antibody comprises an antibody light chain variable region (VL) and / or an antibody heavy chain variable region (VH), the MP being linked to the N-terminus of the VH or VL, and the MP comprising a masking unit (MU) and a linkage unit (LU) from the N-terminus to the C-terminus; the MU comprising an amino acid sequence according to formula (II): X1X2CX3X4X5X6X7CX8X9, where: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X4 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, E, F, G, H, K, L, P, Q, R, S, T, V, and Y. X6 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X7 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X8 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X9 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine.

84. The above, X1 is an amino acid selected from the group consisting of A, F, H, and V. X2 is an amino acid selected from the group consisting of A, L, and P. X3 is an amino acid selected from the group consisting of A, G, L, and R. X4 is an amino acid selected from the group consisting of E, G, K, and P. X5 is an amino acid selected from the group consisting of F, K, L, and V. X6 is an amino acid selected from the group consisting of F, L, P, and S. X7 is an amino acid selected from the group consisting of F, P, and Y. X8 is an amino acid selected from the group consisting of G, I, L, and P, and, X9 is an amino acid selected from the group consisting of E, Q, T, and V, the masked antibody according to claim 83.

85. The masked antibody according to claim 84, wherein the MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 10.

86. A masked antibody comprising a masking peptide (MP) and an antibody, wherein the antibody comprises an antibody light chain variable region (VL) and / or an antibody heavy chain variable region (VH), the MP being linked to the N-terminus of the VH or VL, and the MP comprising a masking unit (MU) and a linkage unit (LU) from the N-terminus to the C-terminus; the MU comprising an amino acid sequence according to formula (III): X1X2CX3X4X5X6X7X8CX9X10, where: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X4 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X6 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, R, S, T, V, and Y. X7 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X8 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X9 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X10 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine.

87. The above, X1 is an amino acid selected from the group consisting of A, F, H, N, P, S, and Y. X2 is an amino acid selected from the group consisting of A, L, P, S, and V. X3 is an amino acid selected from the group consisting of A, I, K, P, R, and V. X4 is an amino acid selected from the group consisting of A, G, L, P, V, and Y. X5 is an amino acid selected from the group consisting of F, I, L, P, R, and S. X6 is an amino acid selected from the group consisting of F, K, L, P, and Y. X7 is an amino acid selected from the group consisting of I, N, P, S, and V. X8 is an amino acid selected from the group consisting of A, F, L, and Y. X9 is an amino acid selected from the group consisting of G, K, Q, S, and V, and, X10 is an amino acid selected from the group consisting of G, Q, R, S, T, and V, and is the masked antibody according to claim 86.

88. The masked antibody according to claim 87, wherein the MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 19.

89. A masked antibody comprising a masking peptide (MP) and an antibody, wherein the antibody comprises an antibody light chain variable region (VL) and / or an antibody heavy chain variable region (VH), the MP being linked to the N-terminus of the VH or VL, and the MP comprising a masking unit (MU) and a linkage unit (LU) from the N-terminus to the C-terminus; the MU comprising an amino acid sequence according to formula (IV): X1X2CX3X4X5X6X7X8X9CX10X11, where: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X4 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X6 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X7 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, R, S, T, V, and Y. X8 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X9 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X10 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X11 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine.

90. The above, X1 is an amino acid selected from the group consisting of A, H, L, P, S, and T. X2 is an amino acid selected from the group consisting of A, D, F, L, and Y. X3 is an amino acid selected from the group consisting of E, L, P, and R. X4 is an amino acid selected from the group consisting of A, E, K, P, and R. X5 is an amino acid selected from the group consisting of E, F, G, and L. X6 is an amino acid selected from the group consisting of A, F, P, T, and Y. X7 is an amino acid selected from the group consisting of A, P, S, T, and V. X8 is an amino acid selected from the group consisting of A, N, P, and S. X9 is an amino acid selected from the group consisting of V and Y. X10 is an amino acid selected from the group consisting of I, P, and R, and, X11 is an amino acid selected from the group consisting of E, G, I, P, and V, the masked antibody according to claim 89.

91. The masked antibody according to claim 90, wherein the MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 21 to 27.

92. A masked antibody comprising a masking peptide (MP) and an antibody, wherein the antibody comprises an antibody light chain variable region (VL) and / or an antibody heavy chain variable region (VH), the MP being linked to the N-terminus of the VH or VL, and the MP comprising a masking unit (MU) and a linkage unit (LU) from the N-terminus to the C-terminus; the MU comprising an amino acid sequence according to formula (V): X1X2CX3X4X5X6X7X8X9X10CX11X12, where: X1 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X2 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X3 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X4 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X5 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X6 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X7 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. X8 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, P, R, S, T, V, and Y. X9 is an amino acid selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y. X10 is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y. X11 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X12 is an amino acid selected from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and, C is cysteine.

93. The above, X1 is an amino acid selected from the group consisting of A, F, P, S, and Y. X2 is an amino acid selected from the group consisting of H, L, P, S, and V. X3 is an amino acid selected from the group consisting of E, G, K, P, Q, and R. X4 is an amino acid selected from the group consisting of A, F, H, P, R, V, and Y. X5 is an amino acid selected from the group consisting of A, D, F, G, V, and Y. X6 is an amino acid selected from the group consisting of D, F, H, L, P, and V. X7 is an amino acid selected from the group consisting of H, K, P, R, and V. X8 is an amino acid selected from the group consisting of A, F, K, L, and P. X9 is an amino acid selected from the group consisting of A, D, F, L, and P. X10 is an amino acid selected from the group consisting of A, F, V, and Y. X11 is an amino acid selected from the group consisting of G, I, K, L, and R, and, X12 is an amino acid selected from the group consisting of A, E, K, P, R, and T, and is the masked antibody according to claim 92.

94. The masked antibody according to claim 93, wherein the MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-32 and 112-114.

95. The masked antibody according to any one of claims 82 to 94, wherein the MU does not contain the amino acid sequence of NG, DG, NXS, or NXT, and the X is any amino acid.

96. The masked antibody according to any one of claims 82 to 95, wherein the MP further comprises an N-terminal unit (NU) ligated to the N-terminus of the MU.

97. The masked antibody according to claim 96, wherein the N-terminal unit has a length of about 1 to 12 amino acid residues.

98. The masked antibody according to claim 97, wherein the N-terminal unit comprises E, EVG, EVGSY (SEQ ID NO: 33), EVGVLDV (SEQ ID NO: 86), or EVGAESGVK (SEQ ID NO: 88).

99. The masked antibody according to any one of claims 82 to 98, wherein the masked antibody is an activatable antibody.

100. The masked antibody according to any one of claims 82 to 99, wherein the LU does not contain a cleavage site.

101. The masked antibody according to claim 100, wherein the LU comprises a linker.

102. The masked antibody according to claim 101, wherein the linker comprises an amino acid sequence selected from the group consisting of GS, GGS, GGG, SGRG (SEQ ID NO: 104), GGGGS (SEQ ID NO: 105), GGGGT (SEQ ID NO: 36), SGGS (SEQ ID NO: 37), GGSG (SEQ ID NO: 106), GGSGG (SEQ ID NO: 107), GSGSG (SEQ ID NO: 108), GSGGG (SEQ ID NO: 109), GGGSG (SEQ ID NO: 110), GSSSG (SEQ ID NO: 111), GSGGS (SEQ ID NO: 77), SGGG (SEQ ID NO: 79), GGGS (SEQ ID NO: 80), and GGGGSGGSGGGS (SEQ ID NO: 81).

103. The masked antibody according to claim 101, wherein the linker comprises the amino acid sequence of SEQ ID NO:

81.

104. The masked antibody according to any one of claims 82 to 99, wherein the LU includes a first cleavage site (C1).

105. The first cleavage site (C1) contains urokinase-type plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, tobacco etch virus (TEV) protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, The masked antibody according to claim 104, wherein the masked antibody is a protease cleavage site of a protease selected from the group consisting of ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE.

106. The masked antibody according to claim 104, wherein the first cleavage site (C1) comprises an amino acid sequence selected from the group consisting of SGRSA (SEQ ID NO: 34) and PLGLAG (SEQ ID NO: 35).

107. The masked antibody according to any one of claims 104 to 106, wherein the LU further comprises a second cleavage site (C2).

108. The second cleavage site (C2) contains urokinase-type plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, tobacco etch virus (TEV) protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, The masked antibody according to claim 107, wherein the masked antibody is a protease cleavage site of a protease selected from the group consisting of ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE.

109. The masked antibody according to claim 107, wherein the second cleavage site (C2) comprises an amino acid sequence selected from the group consisting of SGRSA (SEQ ID NO: 34) and PLGLAG (SEQ ID NO: 35).

110. A masked antibody according to any one of claims 107 to 109, wherein the first and second cleavage sites are the same.

111. A masked antibody according to any one of claims 107 to 109, wherein the first and second cleavage sites are different.

112. The masked antibody according to any one of claims 104 to 111, wherein the LU further comprises a first linker (L1).

113. The masked antibody according to claim 112, wherein the first linker (L1) comprises an amino acid sequence selected from the group consisting of GS, GGS, GGG, SGRG (SEQ ID NO: 104), GGGGS (SEQ ID NO: 105), GGGGT (SEQ ID NO: 36), SGGS (SEQ ID NO: 37), GGSG (SEQ ID NO: 106), GGSGG (SEQ ID NO: 107), GSGSG (SEQ ID NO: 108), GSGGG (SEQ ID NO: 109), GGGSG (SEQ ID NO: 110), GSSSG (SEQ ID NO: 111), GSGGS (SEQ ID NO: 77), SGGG (SEQ ID NO: 79), GGGS (SEQ ID NO: 80), and GGGGSGGGSGGGS (SEQ ID NO: 81).

114. The masked antibody according to claim 112 or 113, wherein the LU further comprises a second linker (L2).

115. The masked antibody according to claim 114, wherein the second linker (L2) comprises an amino acid sequence selected from the group consisting of GS, GGS, GGG, SGRG (SEQ ID NO: 104), GGGGS (SEQ ID NO: 105), GGGGT (SEQ ID NO: 36), SGGS (SEQ ID NO: 37), GGSG (SEQ ID NO: 106), GGSGG (SEQ ID NO: 107), GSGSG (SEQ ID NO: 108), GSGGG (SEQ ID NO: 109), GGGSG (SEQ ID NO: 110), GSSSG (SEQ ID NO: 111), GSGGS (SEQ ID NO: 77), SGGG (SEQ ID NO: 79), GGGS (SEQ ID NO: 80), and GGGGSGGGSGGGS (SEQ ID NO: 81).

116. The aforementioned LU extends from the N-terminus to the C-terminus: 1) First cutting portion (C1) and first linker (L1); 2) First cut section (C1), first linker (L1), second cut section (C2), and second linker (L2); 3) First linker (L1), first cut section (C1), and second linker (L2); or 4) A masked antibody according to any one of claims 104 to 115, comprising a first linker (L1), a first cleavage site (C1), a second linker (L2), and a second cleavage site (C2).

117. The masked antibody according to any one of claims 104 to 116, wherein the LU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 38 to 42 and 119.

118. The masked antibody according to any one of claims 82 to 117, wherein the masking peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-65, 67-75, 83-85, 87, and 116-118.

119. The MP is ligated to the N-terminus of the VL, and the masked antibody is as described in any one of claims 82 to 118.

120. The MP is ligated to the N-terminus of VH, and the masked antibody is as described in any one of claims 82 to 118.

121. Each of the antibodies comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the MP is ligated to the N-terminus of the VH or VL of the antibody, wherein the masked antibody is as described in any one of claims 82 to 118.

122. The masked antibody according to claim 121, wherein the antibody comprises scFv including VH and VL.

123. The masked antibody according to claim 121, wherein the antibody comprises Fab containing VH and VL.

124. The masked antibody according to claim 121, wherein the antibody comprises an antibody heavy chain and an antibody light chain.

125. The MP is ligated to the N-terminus of the VL of the antibody, and the masked antibody is as described in any one of claims 121 to 123.

126. The antibody comprises a VHH single-domain antibody or a VHH-Fc antibody containing VH, wherein the MP is ligated to the N-terminus of VH of the antibody, and the masked antibody is as described in any one of claims 82 to 118.

127. A polynucleotide encoding a masked antibody according to any one of claims 82 to 126.

128. An expression vector comprising the polynucleotide described in claim 127 operably linked to a promoter.

129. A host cell comprising the expression vector according to claim 128.

130. The host cell according to claim 129, wherein the host cell is a bacterial cell, a yeast cell, an insect cell, or a mammalian cell.

131. A method for producing a masked antibody, comprising culturing the host cells described in claim 129 or 130 under conditions suitable for producing a masked antibody.

132. The method according to claim 131, further comprising recovering the antibody produced by the cells.

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