Masked antibodies, libraries, and methods of use thereof

Abstract

The present disclosure relates to polynucleotides and polynucleotide libraries useful for screening and / or identifying one or more masked binding polypeptides (e.g., masked antibodies), as well as polypeptides and polypeptide libraries useful for screening and / or identifying masked polypeptides (e.g., masked antibodies), cells, methods, and kits related thereto.

Description

[0001] Cross-references to related applications This application claims priority to International Patent Application No. PCT / CN2023 / 087999, filed on April 13, 2023, the entire contents of which are incorporated herein by reference.

[0002] Reference to electronic sequence listing The full text of the electronic sequence list (695402001741seqlist.xml; size: 99,142 bytes; creation date: April 4, 2024) is incorporated herein by reference. Technical Field

[0003] This disclosure relates to polynucleotides and polynucleotide libraries that can be used to screen and / or identify one or more masked binding peptides (e.g., masked antibodies), as well as peptides and peptide libraries that can be used to screen and / or identify masked peptides (e.g., masked antibodies), and related cells, methods, and kits. Background Technology

[0004] Masked-binding peptides, such as masked antibodies, can exhibit an "activatable" conformation, making the antigen-binding portion therein less likely to bind to its target. For example, the masking peptide is less likely to bind to its target when it is not cleaved compared to when it is cleaved in the presence of one or more specific proteases. Therefore, these masked-binding peptides provide antigen-specific binding proteins that can bind to their targets under certain conditions (e.g., in the presence of an antigen-rich and / or protease-rich tumor microenvironment (TME); antigens such as CTLA-4 are upregulated in TME lymph nodes, and MMP2 / 9 and other proteases are also upregulated in the TME).

[0005] Despite the development of numerous masked binding peptides, the development of such proteins is inefficient due to deficiencies in the design and screening processes for masked peptides. Masked antibodies are typically designed and screened to select masking peptides with high binding rates and low dissociation rates to bind to the antibody-binding site of the target antigen. This often results in the identified masking peptide remaining self-inhibitory to the lysed antibody due to its low dissociation rate viscosity, thus hindering its binding to the target antigen. This strong self-inhibitory binding of the masking peptide may reduce the antibody's potency in binding to its target in vivo. Furthermore, antibodies selected from these libraries often exhibit high levels of binding to antigens located in dissimilar locations or normal tissues via peripheral circulation, leading to significant non-target effects and impacting safety. Additionally, existing masked antibodies are often designed for rapid lysis in vivo, which may result in the circulation of unmasked antibodies in the bloodstream, thus contributing to toxicity associated with binding to antigens in non-target tissues.

[0006] Therefore, new concepts and improved methods and products are needed to identify dynamic rather than self-inhibiting peptides for use with masked binding peptides that have a variety of desirable properties, such as masked antibodies. Summary of the Invention

[0007] To meet the above and other needs, this document discloses polynucleotide libraries, for example, for screening and / or recognizing masked binding peptides (e.g., masked antibodies). The libraries described herein provide masked antibodies with ideal stability, structural and chemical diversity, and downstream developability.

[0008] In one aspect, this disclosure provides a polynucleotide library encoding masked-binding peptides (e.g., masked antibodies) that possess therapeutic efficacy, stability, and a desirable therapeutic index. As described herein, the disclosed libraries are designed to identify masked-binding peptides (e.g., masked antibodies) capable of binding to target antigens enriched or upregulated in the tumor microenvironment (TME), without being limited by the adhesion of the masking peptide to the antibody binding site. Therefore, the masked-binding peptides (e.g., masked antibodies) preferentially accumulate in regions with sufficiently high concentrations of target antigen and bind strongly to unmasked antibodies upon cleavage of the masking peptide. For example, in some embodiments, the dissociation rate constant (kc) of the masking peptide (e.g., the masking peptide of a masked antibody) from the antibody binding site is [not specified in the original text]. off The concentration of the masked binding peptide is high and it is not self-inhibited. In addition, the masked binding peptide (e.g., the masked antibody) does not accumulate in areas with low concentrations of the target antigen (e.g., the target in normal tissue), thereby reducing off-target effects and improving the therapeutic window of the masked binding peptide (e.g., the masked antibody) generated from the published library.

[0009] Therefore, masked antibodies generated from the library accumulate in regions of high antigen concentration. In these high-concentration antigen regions, the masking units cleave significantly, generating unmasked antibodies with high affinity for the target antigen. Conversely, in tissues with relatively low target antigen concentrations (e.g., blood), masked antibodies generated from the library do not accumulate. Furthermore, the high dissociation rate prevents masked antibodies from adhering to the antigen in these low-concentration microenvironments, thereby mitigating any potential nonspecific effects.

[0010] The polynucleotide libraries disclosed herein are designed to encode masked binding peptides (e.g., masked antibodies) having masked sequences (masking units) that enable the masked peptides to bind to target antigens without significantly adhering to the target antigens in normal tissues. As described herein, an optimal balance can be achieved through appropriate selection of the masking units encoded by the library. In particular, the structured peptide libraries disclosed herein aim to balance the composition of charged and polar amino acids with hydrophobic amino acids in the masking units, thereby achieving competitive binding between the antigen and the dynamically masking peptide through concentration-dependent antigen binding for transient activation.

[0011] In some implementations, these libraries are designed to enrich glycine and proline residues in the loop region of a peptide (e.g., an antibody). Antibodies encoded by such libraries can fold into structured loops to effectively mask the binding of the target antigen and the masked antibody without generating excessive self-inhibition or adhesion, thereby preventing competitive binding between the antigen and the masked antibody through the masking peptide.

[0012] In some implementations, these libraries were designed to avoid high-risk post-translational modification (PTM) sites, including free cysteine ​​residues, glycosylation sites, deamidation sites, aspartate isomerization sites, and oxidation sites. For example, in a particular implementation, NG, M, W, and NX[S / T] sites were removed from the identified masking units.

[0013] In some embodiments, the polynucleotide library encodes antibodies for masking. Specifically, in some aspects, this document provides a library containing 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 antibody having a unique masking peptide (MP), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), and the MP is linked to the N-terminus of the VH or VL of the antibody, wherein the MP comprises a masking unit (MU) and a linker unit (LU), wherein the MU comprises an amino acid sequence according to formula (I): X1X2CX3(X m ) n X4X5CX6X7, 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. 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 X mAmino acids 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 stands for cysteine.

[0014] In some implementations, the value of n ranges from 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 specific implementation, n is 2, 3, 4, 5, 6, 7, or 8.

[0015] In other respects, this document provides a library containing 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 antibody having a unique masking peptide (MP), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), and the MP is linked to the N-terminus of the VH or VL of the antibody, wherein the MP comprises a masking unit (MU) and a linker unit (LU), wherein 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 stands for cysteine.

[0016] In some implementations, 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.

[0017] In some embodiments, the polynucleotide in the library encodes a masking peptide (MP), wherein the MP comprises a masking unit (MU) and a linker unit (LU). In some embodiments, the MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-10.

[0018] In one aspect, this article provides a library containing 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 antibody having a unique masking peptide (MP), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), and the MP is linked to the N-terminus of the VH or VL of the antibody, wherein the MP comprises a masking unit (MU) and a linker unit (LU), wherein the MU comprises an amino acid sequence according to formula (III): X1X2CX3X4X5X6X7X8CX9X 10 ,in: 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. X 10 The amino acids selected are those from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and... C stands for cysteine.

[0019] In some implementations, 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 X 10 The amino acids are selected from the group consisting of G, Q, R, S, T, and V. In some embodiments, the polynucleotide in the library encodes a masking peptide (MP), wherein the MP includes a masking unit (MU) and a linker unit (LU). In some embodiments, the MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-19.

[0020] In one aspect, this article provides a library containing 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 antibody having a unique masking peptide (MP), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), and the MP is linked to the N-terminus of the VH or VL of the antibody, wherein the MP comprises a masking unit (MU) and a linker unit (LU), wherein the MU has an amino acid sequence according to formula (IV): X1X2CX3X4X5X6X7X8X9CX 10 X 11 ,in: 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. X 10 The amino acids selected are those from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X 11 The amino acids selected are those from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and... C stands for cysteine.

[0021] In some implementations, 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. X 10 The amino acids selected are those from the group composed of I, P, and R, and X 11 The amino acids selected are those composed of E, G, I, P, and V.

[0022] In some embodiments, this document provides a library containing polynucleotides, wherein the polynucleotides in the library encode a masking peptide (MP), the MP comprising a masking unit (MU) and a linker unit (LU). In some embodiments, the MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-27.

[0023] In one aspect, a library comprising polynucleotides is provided, 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 antibody having a unique masking peptide (MP), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), and the MP is linked to the N-terminus of the VH or VL of the antibody, wherein the MP comprises a masking unit (MU) and a linker unit (LU), wherein the MU comprises an amino acid sequence according to formula (V): X1X2CX3X4X5X6X7X8X9X 10 CX 11 X 12 ,in: 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. X 10 The amino acids selected are those from the group consisting of A, D, F, H, L, P, S, V, and Y. X 11 The amino acids selected are those from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X 12 The amino acids selected are those from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and... C stands for cysteine.

[0024] In some implementations, 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. X 10 The amino acids selected are from the group consisting of A, F, V, and Y. X 11 The amino acids selected are those derived from the group consisting of G, I, K, L, and R. X 12 The amino acids selected are those composed of groups A, E, K, P, R, and T.

[0025] In some embodiments, the polynucleotide in the library encodes a masking peptide (MP), wherein the MP comprises a masking unit (MU) and a linker 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.

[0026] In some embodiments of the foregoing aspects, the masking unit (MU) does not contain an amino acid sequence of NG, DG, NXS, or NXT, where X represents any amino acid. In some embodiments of the foregoing aspects, the masking peptide (MP) further comprises an N-terminal unit (NU) linked to the N-terminus of the MU. In some embodiments, the N-terminal unit is approximately 1-12 amino acid residues in length. In some embodiments, the N-terminal unit comprises E, EVG, EVGSY (SEQ ID NO: 33), EVGVLDV (SEQ ID NO: 86), or EVGAESGVK (SEQ ID NO: 88).

[0027] In some embodiments of the above aspects, LU does not contain a cleavage site. In some embodiments, LU contains a linker. In some embodiments, the linker comprises or 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), GGSSG (SEQ ID NO: 108), GGSGG (SEQ ID NO: 109), GGGSG (SEQ ID NO: 110), GSSSG (SEQ ID NO: 111), GGSGS (SEQ ID NO: 77), SGGG (SEQ ID NO: 79), GGGS (SEQ ID NO: 80), and GGGGSGGSGGGGS (SEQ ID NO: 81). In some embodiments, LU comprises the amino acid sequence of SEQ ID NO: 81.

[0028] In some implementations of the above aspects, LU includes a first cleavage site (C1). In some embodiments, the first cleavage site (C1) is a protease cleavage site selected from the group consisting of urokinase plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, tobacco mosaic virus (TEV) protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspases-1, caspases-2, caspases-3, caspases-4, caspases-5, caspases-6, caspases-7, caspases-8, caspases-9, caspases-10, caspases-11, caspases-12, caspases-13, caspases-14, and TACE. In some embodiments, the first cleavage site (C1) comprises an amino acid sequence selected from SGRSA (SEQ ID NO: 34) and PLGLAG (SEQ ID NO: 35). In some embodiments, LU also comprises a second cleavage site (C2). In some embodiments, the second cleavage site (C2) is a protease cleavage site selected from the group consisting of urokinase plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, tobacco mosaic virus (TEV) protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspases-1, caspases-2, caspases-3, caspases-4, caspases-5, caspases-6, caspases-7, caspases-8, caspases-9, caspases-10, caspases-11, caspases-12, caspases-13, caspases-14, and TACE. In some embodiments, the second cleavage site (C2) comprises an amino acid sequence selected from SGRSA (SEQ ID NO: 34) and PLGLAG (SEQ ID NO: 35). In some embodiments, the first and second cleavage sites are identical. In some embodiments, the first and second cleavage sites are different. In some embodiments, LU further comprises a first linker (L1).In some embodiments, the first linker (L1) comprises an amino acid sequence selected from the following: 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), GGSGG (SEQ ID NO: 109), GGGSG (SEQ ID NO: 110), GSSSG (SEQ ID NO: 111), GGSGS (SEQ ID NO: 77), SGGG (SEQ ID NO: 79), GGGS (SEQ ID NO: 80), and GGGGSGGSGGGGS (SEQ ID NO: 81). In some embodiments, L1 also comprises a second linker (L2). In some embodiments, the second linker (L2) comprises an amino acid sequence selected from the following: 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), GGSGG (SEQ ID NO: 109), GGGSG (SEQ ID NO: 110), GSSSG (SEQ ID NO: 111), GGSGS (SEQ ID NO: 77), SGGG (SEQ ID NO: 79), GGGS (SEQ ID NO: 80), and GGGGSGGSGGGGS (SEQ ID NO: 81). In some embodiments, LU comprises, 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 SEQ ID NOs: 38-42 and 119.

[0029] In some embodiments of the above aspects, 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 aspects, MP is linked to the N-terminus of VL. In some embodiments of the above aspects, MP is linked to the N-terminus of VH.

[0030] In some embodiments of the above aspects, each antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), and an MP is attached to the N-terminus of the antibody's VH or VL. In some embodiments, the antibody comprises an scFv consisting of VH and VL. In some embodiments, the antibody comprises a Fab consisting of VH and VL. In some embodiments, the antibody comprises an antibody heavy chain and an antibody light chain. In some embodiments, the MP is attached to the N-terminus of the antibody's VL.

[0031] In some embodiments of the above aspects, each antibody includes a heavy chain variable region (VH), with the MP linked to the N-terminus of the antibody's VH. In some embodiments, the antibody comprises a VHH single-domain antibody or a VHH-Fc antibody containing a VH, wherein the MP is linked to the N-terminus of the antibody's VH.

[0032] In some embodiments of the above aspects, the polynucleotide encoding the polypeptide is located in a vector. In some embodiments, these vectors are expression vectors or display vectors. In some embodiments, the polynucleotide encoding the polypeptide is located in a host cell. In some embodiments, these cells are bacterial cells, yeast cells, insect cells, or mammalian cells. In some embodiments of the above aspects, the MU in the library has 10 9 Up to 10 14 The diversity.

[0033] In other respects, this document provides a library containing antibodies encoded by polynucleotides from any of the libraries described above. In some embodiments, each polypeptide is displayed on the cell surface or the surface of a bacteriophage. In some embodiments, the cell may be a bacterial cell, yeast cell, insect cell, or mammalian cell.

[0034] In other respects, this document provides a method for producing antibodies, comprising culturing host cells expressing any of the antibody libraries described above under conditions suitable for antibody production. In some embodiments, the method for producing antibodies further includes recovering antibodies produced by the cells. In some embodiments, the method for producing antibodies further includes testing the ability of the antibody to retain the masked phenotype in a soluble state.

[0035] In some aspects, this document provides a method for screening masked antibodies bound to a target using any of the above-described libraries, comprising: a) contacting an antibody expressed in the library with the target to determine a first binding affinity for the target or lack of detectable binding; b) contacting a control antibody with the target to determine a second binding affinity; and c) selecting an expressed antibody with a first binding affinity lower than the second binding affinity or undetectable binding to the target, wherein the first and second binding affinity assays are KD, EC50, or IC50. In some embodiments, the LU contains at least one first cleavage site (C1). In some embodiments, the control antibody is an antibody expressed in the LU cleavage library. In some embodiments, the expressed antibody is selected if the binding affinity of the expressed antibody after LU cleavage is at least 2, at least 5, at least 10, at least 50, at least 100, at least 500, or at least 1000 times that of the expressed antibody before LU cleavage.

[0036] In other respects, this document provides a method for screening masked antibodies that bind to a target using any of the libraries described above, comprising: a) contacting an antibody expressed in a library containing a masking peptide with a first cell expressing a target antigen to determine the EC50 binding; b) contacting a control antibody lacking the masking peptide with the first cell expressing the target antigen to determine the EC50 binding; c) contacting an antibody expressed in the library with a second cell expressing the target antigen to determine the EC50 binding, wherein the target antigen expressed in the second cell is at a lower level than that in the first cell; d) contacting a control antibody lacking the masking peptide with the second cell to determine the EC50 binding; e) determining the ratio of the EC50 in step a) to the EC50 in step b) as a first masking efficiency; f) determining the ratio of the EC50 in step c) to the EC50 in step d) as a second masking efficiency; and g) selecting an expressed antibody whose second masking efficiency is higher than the first masking efficiency. In some embodiments, step (g) includes selecting an expression antibody with a second masking efficiency that is at least 10%, at least 50%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 10-fold higher than the first masking efficiency. In some embodiments, the second masking efficiency is at least 50%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 10-fold higher than the first masking efficiency. In some embodiments, the control antibody is an antibody having the same antigen-binding domain as the antibody expressed in the library. In some embodiments, the control antibody is a parent antibody. In some embodiments, the LU does not contain a cleavage site. In some embodiments, the LU contains at least one first cleavage site (C1). In some embodiments, the control antibody is an antibody expressed in the library after LU cleavage. In some embodiments, the method includes cleaving the LU to generate a control antibody.

[0037] In other aspects, the present invention provides a method for identifying a masked antibody capable of concentration-dependent antigen binding, comprising: a) contacting a masked antibody having a masking peptide with a first cell expressing a target antigen to determine EC50 binding, wherein the masking peptide comprises a masking unit (MU) and a linker unit (LU) from the N-terminus to the C-terminus; b) contacting a control antibody lacking the masking peptide with the first cell expressing the target antigen to determine EC50 binding; c) contacting a masked antibody with a second cell expressing the target antigen to determine EC50 binding, wherein the second cell expresses a lower level of the target antigen than the first cell; d) contacting a control antibody lacking the masking peptide with the second cell to determine EC50 binding; e) determining the ratio of EC50 in step a) to EC50 in step b) as a first masking efficiency; f) determining the ratio of EC50 in step c) to EC50 in step d) as a second masking efficiency; and g) if the second masking efficiency is higher than the first masking efficiency, then identifying an antibody capable of concentration-dependent antigen binding. In some embodiments, if the second masking efficiency is at least 10%, at least 50%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 10-fold higher than the first masking efficiency, then step (g) includes recognizing an antibody capable of concentration-dependent antigen binding. 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 LU does not contain a cleavage site. In some embodiments, the LU contains at least one first cleavage site (C1). In some embodiments, the control antibody is a masked antibody after LU cleavage. In some embodiments, the method includes cleaving the LU to generate a control antibody. In some embodiments, the second masking efficiency is at least 50%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 10-fold higher than the first masking efficiency.

[0038] In other respects, this article provides an antibody encoded by one or more polynucleotides from any of the above libraries.

[0039] In other respects, this article provides a kit containing any of the libraries described above.

[0040] In other respects, any of the above-mentioned libraries contains cells, wherein at least two, at least three, at least four, at least five, or at least ten cells contain the polynucleotides of the above-mentioned library.

[0041] In other aspects, the present invention provides 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), wherein the MP is linked to the N-terminus of the VH or VL, wherein the MP comprises a masking unit (MU) and a linking unit (LU) from the N-terminus to the C-terminus; wherein the MU comprises an amino acid sequence according to formula (I): X1X2CX3(X m ) n X4X5CX6X7, where n is 2-8, and: 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 X m Amino acids 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 stands for cysteine.

[0042] In some implementations, n ranges from 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 implementation, n is 2, 3, 4, 5, 6, 7, or 8.

[0043] In other aspects, the present invention provides 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), wherein the MP is linked to the N-terminus of the VH or VL, and the MP comprises a masking unit (MU) and a linking unit (LU) from the N-terminus to the C-terminus; wherein the MU comprises an amino acid sequence according to formula (II): X1X2CX3X4X5X6X7CX8X9, 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, 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 stands for cysteine.

[0044] In some implementations, MU comprises an amino acid sequence selected from the group of SEQ ID NOs: 7-10.

[0045] In other respects, this article provides 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), wherein the MP is linked to the N-terminus of the VH or VL, wherein the MP comprises a masking unit (MU) and a linking unit (LU) from the N-terminus to the C-terminus; wherein the MU comprises an amino acid sequence according to formula (III): X1X2CX3X4X5X6X7X8CX9X 10 ,in: 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. X 10 The amino acids selected are those from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and... C stands for cysteine.

[0046] In some implementations, 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 X 10 The amino acids selected are those composed of G, Q, R, S, T, and V.

[0047] In some embodiments, MU comprises an amino acid sequence consisting of the group consisting of SEQ ID NOs: 12-19.

[0048] In other respects, this article provides 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), wherein the MP is linked to the N-terminus of the VH or VL, wherein the MP comprises a masking unit (MU) and a linking unit (LU) from the N-terminus to the C-terminus; wherein the MU comprises an amino acid sequence according to formula (IV): X1X2CX3X4X5X6X7X8X9CX 10 X 11 ,in: 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. X 10 The amino acids selected are those from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X 11 The amino acids selected are those from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and... C stands for cysteine.

[0049] In some implementations, 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. X 10The amino acids selected are those from the group composed of I, P, and R, and X 11 The amino acids selected are those composed of E, G, I, P, and V.

[0050] In some implementations, MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-27.

[0051] In other respects, this article provides 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), wherein the MP is linked to the N-terminus of the VH or VL, wherein the MP comprises a masking unit (MU) and a linking unit (LU) from the N-terminus to the C-terminus; wherein the MU comprises an amino acid sequence according to formula (V): X1X2CX3X4X5X6X7X8X9X 10 CX 11 X 12 ,in: 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. X 10 The amino acids selected are those from the group consisting of A, D, F, H, L, P, S, V, and Y. X 11 The amino acids selected are those from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X 12The amino acids selected are those from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and... C stands for cysteine.

[0052] In some implementations, 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. X 10 The amino acids selected are from the group consisting of A, F, V, and Y. X 11 The amino acids selected are those derived from the group consisting of G, I, K, L, and R. X 12 The amino acids selected are those composed of groups A, E, K, P, R, and T.

[0053] In some embodiments, MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-32 and 112-114.

[0054] In some aspects, in any of the above-described masked antibodies, MU does not contain the amino acid sequence of NG, DG, NXS, or NXT, where X is any amino acid. In some embodiments, MP further includes an N-terminal unit (NU) linked to the N-terminus of MU. In some embodiments, the N-terminal unit is approximately 1-12 amino acid residues in length. In some embodiments, the N-terminal unit comprises 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 some embodiments, LU contains a linker. In some embodiments, 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), GGSSG (SEQ ID NO: 108), GGSGG (SEQ ID NO: 109), GGGSG (SEQ ID NO: 110), GSSSG (SEQ ID NO: 111), GGSGS (SEQ ID NO: 77), SGGG (SEQ ID NO: 79), GGGS (SEQ ID NO: 80), and GGGGSGGSGGGGS (SEQ ID NO: 81). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 81. In some embodiments, LU comprises a first cleavage site (C1). In some embodiments, 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 mosaic virus (TEV) protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspases-1, caspases-2, caspases-3, caspases-4, caspases-5, caspases-6, caspases-7, caspases-8, caspases-9, caspases-10, caspases-11, caspases-12, caspases-13, caspases-14, and a protease selected from the TACE group.In some embodiments, 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). In some embodiments, LU further comprises a second cleavage site (C2). In some embodiments, 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 mosaic virus (TEV) protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspases-1, caspases-2, caspases-3, caspases-4, caspases-5, caspases-6, caspases-7, caspases-8, caspases-9, caspases-10, caspases-11, caspases-12, caspases-13, caspases-14, and a protease cleavage site selected from the TACE group. In some embodiments, the second cleavage site (C2) comprises an amino acid sequence selected from the groups SGRSA (SEQ ID NO: 34) and PLGLAG (SEQ ID NO: 35). In some embodiments, the first and second cleavage sites are the same. In some embodiments, the first and second cleavage sites are different.

[0055] In some aspects, in any of the masked antibodies described above, LU further comprises a first adapter (L1). In some embodiments, the first adapter (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), GGSSG (SEQ ID NO: 108), GGSGG (SEQ ID NO: 109), GGGSG (SEQ ID NO: 110), GSSSG (SEQ ID NO: 111), GGSGS (SEQ ID NO: 77), SGGG (SEQ ID NO: 79), GGGS (SEQ ID NO: 80), and GGGGSGGSGGGGS (SEQ ID NO: 81). In some embodiments, the LU further comprises a second linker (L2). In some embodiments, 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), GGSSG (SEQ ID NO: 108), GGSGG (SEQ ID NO: 109), GGGSG (SEQ ID NO: 110), GSSSG (SEQ ID NO: 111), GGSGS (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 comprises, from the N-terminus to the C-terminus: 1) The first cleavage site (C1) and the first linker (L1); 2) First cleavage site (C1), first connector (L1), second cleavage site (C2), and second connector (L2); 3) The first linker (L1), the first cleavage site (C1), and the second linker (L2); or 4) First connector (L1), first cleavage site (C1), second connector (L2), and second cleavage site (C2).

[0056] In some embodiments, LU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 38-42 and 119.

[0057] In some embodiments, in any of the masked antibodies described above, 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, MP is linked to the N-terminus of VL. In some embodiments, MP is linked to the N-terminus of VH.

[0058] 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 linked to the N-terminus of the antibody's VH or VL. In some embodiments, the antibody comprises an scFv containing both VH and VL. In some embodiments, the antibody comprises a Fab containing both VH and VL. In some embodiments, the antibody comprises an antibody heavy chain and an antibody light chain. In some embodiments, the MP is linked to the N-terminus of the antibody's VL.

[0059] 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 MP is linked to the N-terminus of the antibody VH.

[0060] In some embodiments, in any of the aforementioned polynucleotides, the polynucleotide encodes the aforementioned masked antibody. In some embodiments, the expression vector containing the polynucleotide is operatively linked to a promoter. In some embodiments, the expression vector is contained in a host cell. In some embodiments, the host cell is a bacterial cell, yeast cell, insect cell, or mammalian cell. In some embodiments, a method of producing a masked antibody includes culturing a host cell under conditions suitable for producing a masked antibody. In some embodiments, the method of producing a masked antibody further includes recovering the antibody produced by the cell.

[0061] The masked binding peptides (e.g., masked antibodies) identified from the library of this invention possess superior properties. First, by using a masking peptide with a relatively high dissociation rate at the antibody binding site, post-activation self-inhibition (e.g., cleavage of the masking unit) is reduced, preventing effective binding of antigen and antibody after peptide cleavage. Second, the concentration-dependent binding of the masked peptide (e.g., masked antibody) to the antigen concentrated around the target region (e.g., tumor microenvironment) increases the local concentration of the masked antibody within the target region. In the case of masking peptides with cleavable linkers, the masked antibody subsequently undergoes time-dependent cleavage of the masking peptide to achieve permanent activation because the target region (e.g., tumor microenvironment) is rich in proteases capable of such cleavage. Third, the kinetic control of peptide cleavage by the enzyme-rich microenvironment within the target region (e.g., tumor microenvironment) is another important property. The kinetics of masking peptide cleavage in the tumor microenvironment can be modulated by selecting different protease cleavage sites in the masking peptide (e.g., sites where different levels of proteases exist in the tumor microenvironment) and by varying the number of protease cleavage sites in the linker unit of the masking peptide (e.g., one versus two protease cleavage sites). This modulation can lead to a “slow-release” effect of locally activated masked antibodies within the tumor microenvironment.

[0062] In some embodiments, after selecting the masked binding peptide (e.g., the masked antibody), additional sequences of the masking peptide can be modified to adjust and optimize folding, chemistry, manufacturing and control (CMC) solubility, thermal and cyclic stability, pharmacokinetics (PK), and anti-drug antibodies (ADAs) associated with the immunogenicity of the masking peptide. For example, the linker sequence and N-terminal peptide (e.g., amino acid residues linked to the N-terminus of the masking peptide) can be modulated to control these factors.

[0063] On the other hand, this disclosure provides masked antibodies generated from the libraries described herein. In some embodiments, the masked antibody has a masked portion (MP) comprising a masking unit (MU) and a linker unit (LU), as described herein. In other embodiments, the LU is cleaved after administration of the masked antibody. In other embodiments, the LU is not cleaved after administration of the masked antibody. It should be understood that the administered concentration of the cleavable antibody will be higher than that of the non-cleavable antibody.

[0064] On the other hand, the half-maximal effective concentration (EC50) can be compared in different antigen concentration environments (e.g., environments with high antigen concentration and environments with low antigen concentration). 50 The selection of masking peptides (e.g., masked antibodies) is based on their masking efficiency and / or masking effectiveness. In a particular embodiment, peptides with lower EC50 in high-concentration antigen regions than in low-concentration antigen regions are selected. 50(e.g., enhancing efficacy) masked peptides (e.g., masked antibodies). In other embodiments, masked antibodies are selected that have reduced masking efficiency in high-concentration antigen regions compared to low-concentration antigen regions. In some embodiments, this concentration-dependent selection of masked peptides (e.g., masked antibodies) is performed after pre-selecting two or more masking peptides (e.g., masked antibodies) from a library. In some such embodiments, the library is the library disclosed herein. In other such embodiments, the library is not the library disclosed herein.

[0065] In some embodiments, the masking efficiency of the masked peptide (e.g., the masked antibody) is about 2 to 50 times lower in an antigen-enriched environment (e.g., a tumor microenvironment) than in an environment with normal blood or antigen levels (e.g., healthy tissue). In some embodiments, the masking efficiency of the masked peptide (e.g., the masked antibody) is about 3 to 20 times lower in an antigen-enriched environment (e.g., a tumor microenvironment) than in an environment with normal blood or antigen levels (e.g., healthy tissue). In some embodiments, the masking efficiency of the masked peptide (e.g., the masked antibody) is about 4 to 10 times lower in an antigen-enriched environment (e.g., a tumor microenvironment) than in an environment with normal blood or antigen levels (e.g., healthy tissue).

[0066] It should be understood that one, some, or all of the features of the various embodiments described above and herein can be combined to form other embodiments of this disclosure. These and other aspects of this disclosure will be understood by those skilled in the art. These and other embodiments of this disclosure are further described in the following detailed description. Attached Figure Description

[0067] Figures 1A-1C show the concentration-dependent binding of the anti-HER2 masking antibody to cells expressing different concentrations of HER2 antigen. Figure 1A shows binding to SCOV3 cells, which have high levels of HER2 expression and high concentrations of HER2 antigen. Figure 1B shows binding to MCF7 cells, which have low levels of HER2 expression and low concentrations of HER2 antigen. Figure 1C shows binding to A549 cells, which have low levels of HER2 expression and low concentrations of HER2 antigen.

[0068] Figure 2 shows the time-dependent cleavage of isolated anti-CTLA4 masking antibodies TY24652 and TY26294 by MMP-9 under in vitro conditions.

[0069] Figures 3A-3DThis study demonstrates the time-dependent lysis of anti-CTLA4-masking antibodies TY24652 and TY26294 in a mouse H22 tumor model. Mice were administered 5 mg / kg of each antibody. Fresh tissues were isolated from mice at 24 and 96 hours post-administration, and the lysed and intact antibodies were analyzed by Western blot. Figure 3A Western blots of lysed and intact antibodies were shown in tissues collected 24 hours after administration. Figure 3B A bar chart showing the relative number of intact and lysed antibodies in tissues collected 24 hours after drug administration. Figure 3C Western blots of lysed and intact antibodies were shown in tissues collected 96 hours after administration. Figure 3D A bar chart showing the relative number of intact and lysed antibodies in tissues collected 96 hours after drug administration.

[0070] Figures 4A-4C This study demonstrates the in vivo time-dependent cleavage of the anti-CTLA4 masking antibody TY26294 in a mouse SHP-77 xenograft model. Mice were intravenously injected with a single dose of 10 mg / kg of TY26294, and plasma and tumor samples were collected at different time points to analyze the presence of cleaved and intact antibodies. Figure 4A The study showed the intact and fragmented forms of TY26294 in plasma samples over time. Figure 4B The intact and fragmented forms of TY26294 in SHP-77 tumor samples over time are shown. Figure 4C The study showed a comparison between TY26294 cleavage in tumors and plasma. Detailed Implementation

[0071] Overview This article describes an improved library for selecting masked antigen-binding peptides (e.g., masked antibodies) that offer improved safety in treatment. In the library described herein, the masking peptide linked to the antigen-binding peptide (e.g., masked antibody) is designed with a masked sequence (masking unit) having a relatively high dissociation rate, allowing concentration-dependent binding and reducing autoinhibition. Both cleavable and non-cleavable linker units, as well as the N-terminal peptide, are optimized, and the library exhibits good development potential. Several unique features are built into the improved masking unit, making the library described herein highly effective in identifying masking units of target antibodies with improved efficacy and safety profiles.

[0072] First, the masking unit was designed with enriched charged side-chain amino acids D / E / H / K / R. Together with polar side-chain amino acids S / T / N / Q / Y, these charged and polar amino acids constitute the majority of the masking unit residues in the library composition. Therefore, the identified masking unit provides a balanced ratio of charged and polar amino acid residues to hydrophobic amino acids in the masked antigen-binding domain. Second, the masking unit contains a loop region defined by two cysteine ​​residues forming an intramolecular disulfide bond. Importantly, glycine and proline residues are crucial to the peptide backbone structure and are intentionally enriched in the loop region to achieve optimal loop formation. Therefore, the identified masking unit can fold into a structured loop to effectively shield the binding of antigens and masked antibodies without generating unnecessary self-inhibition or stickiness, thus preventing competitive binding of the masked peptide between antigens and masked antibodies. Third, the library design avoids high-risk post-translational modification (PTM) sites, including free cysteine ​​residues (excluding the two cysteine ​​residues flanking the loop region), glycosylation sites, deamidation sites, aspartate isomerization sites, and oxidation sites. For example, NG, M, W, and NX[S / T] sites were removed from the identified masking units. These design considerations result in selected masked antibodies with less "stickiness" and less self-inhibitory masking units compared to previous masked antibodies. The selected antibodies are able to competitively bind in a concentration-dependent manner to the antigen-binding domain (ABD), such as in the tumor microenvironment (TME) where antigen concentrations are high, even without dependence on the removal of the masking peptide from the antibody by cleavage. Therefore, the selected masked antibodies can be enriched and temporarily activated in the TME, while masked antibodies present in other tissues (such as the bloodstream) remain masked by the masking units. In the case of masked antibodies with cleavable linkers, after enrichment and transient activation, time-dependent permanent activation occurs via proteases in the TME, which can be modulated by altering the N-terminal and cleavable linker sequences in the masked antibody. These properties enable the generation of masked antibodies with high binding affinity in the TME, thereby achieving high antitumor potency while minimizing the toxicity of antibodies bound outside the TME.

[0073] I. Definition Before describing this disclosure in detail, it should be understood that this disclosure is not limited to specific compositions or biological systems, which are of course subject to variation. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0074] Unless otherwise expressly stated, as used herein, the singular forms “a / an” and “the” include indicators of a plural number of species. Thus, for example, reference to “a molecule” may optionally include a combination of two or more of the stated molecules, and so on.

[0075] The term "about" in this document refers to the typical range of error for a corresponding value, as is well known to those skilled in the art. References to "about" a value or parameter herein include (and describe) embodiments relating to that value or parameter itself.

[0076] It should be understood that aspects and embodiments of this disclosure include aspects and embodiments that are “comprising,” “composed of,” and “substantially composed of.”

[0077] As used herein in phrases such as “A and / or B”, the term “and / or” is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, as used herein in phrases such as “A, B, and / or C”, the term “and / or” is intended to cover 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).

[0078] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. The term "amino acid analog" refers to a compound that has the same basic chemical structure as a naturally occurring amino acid, but whose C-terminal carboxyl group, N-terminal amino group, or side chain functional group has been chemically modified to another functional group. The term "amino acid mimic" refers to a chemical compound that has a structure different from the general chemical structure of an amino acid, but functions similarly to a naturally occurring amino acid.

[0079] As used in this article, the 20 common amino acids and their abbreviations follow their usual usage. See, for example, Immunology—ASynthesis (2nd edition, edited by E.S. Golub and D.R. Gren, Sinauer Associates, Sunderland, Mass. (1991)).

[0080] The terms “polypeptide,” “protein,” and “peptide” are used interchangeably in this document and may refer to a polymer composed of two or more amino acids.

[0081] As used interchangeably herein, “polynucleotide” or “nucleic acid” refers to a nucleotide polymer of any length and includes both DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogues, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides may contain modified nucleotides such as methylated nucleotides and their analogues. If present, modifications to the nucleotide structure may be conferred before or after polymer assembly. Non-nucleotide components may be intercalated in the sequence of the nucleotides. Polynucleotides may include one or more modifications performed post-synthesis, such as conjugation with a label. Other types of modifications include, for example, “caps”; substitution of one or more naturally occurring nucleotides with analogs; internucleotide modifications such as those with uncharged bonds (e.g., methylphosphonates, triphosphates, aminophosphates, carbamates, etc.) and those with charged bonds (e.g., thiophosphates, dithiophosphates, etc.); those containing side-attached moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.); those containing intercalating agents (e.g., acridine, psoralen, etc.); those containing chelating agents (e.g., metals, radioactive metals, boron, oxidizing metals, etc.); those containing alkylating agents; those with modified bonds (e.g., α-terminal isomeric nucleic acids, etc.); and unmodified forms of one or more polynucleotides. Furthermore, any hydroxyl group normally present in sugars can be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional bonds with additional nucleotides, or conjugated to solid or semi-solid carriers. The 5' and 3' terminal OH groups may be phosphorylated or substituted with an amine or an organic end-capping group having 1 to 20 carbon atoms. Other hydroxyl groups may also be derivatized to obtain a standard protecting group. Polynucleotides may also include similar forms of ribose or deoxyribose commonly known in the art, including, for example, 2'-O-methyl, 2'-O-allyl, 2'-fluoro or 2'-azidoribose, carbocyclic sugar analogs, α-terminal isomers, epimeric sugars such as arabinose, xylose or lythose, pyranose, furanose, heptacarbonose, acyclic analogs, and basic nucleoside analogs such as methylriboside. One or more phosphodiester bonds may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which the phosphate ester is replaced by P(O)S (“thioester”), P(S)S (“dithioester”), (O)NR2 (“amid”), P(O)R, P(O)OR', CO, or CH2 (“methylal”), wherein each R or R' is independently H or optionally a substituted or unsubstituted alkyl group (1-20 Cs) containing an ether (-O-) linker, aryl, alkenyl, cycloalkyl, cycloalkenyl, or aralkyl group. Not all links in polynucleotides need to be identical. The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA.

[0082] The term "isolated nucleic acid" refers to a genomic, cDNA, or synthetically derived nucleic acid molecule, or a combination thereof, that is separate from other nucleic acid molecules present in natural sources of nucleic acids. For example, with respect to genomic DNA, the term "isolated" includes nucleic acid molecules that are separate from chromosomes naturally associated with the genomic DNA. Preferably, the "isolated" nucleic acid does not contain sequences naturally side-attached to the nucleic acid (i.e., sequences located at the 5' and 3' ends of the target nucleic acid).

[0083] In this document, the term "library" refers to a collection of two or more entities that share a common category. For example, a library containing polynucleotides may refer to a group of two or more polynucleotides. The term "library" is used in the broadest sense herein and explicitly covers sub-libraries that may or may not be combined.

[0084] As used herein, "unique" means that a member of a set is different from the other members of that set. For example, a unique masking antibody in a library may refer to a masking antibody that has a specific masking peptide sequence that is not shared by other masking antibodies in the library. In practice, it should be understood that a "unique" member in the physical realization of a library can exist in more than one copy. For example, a library may contain multiple "unique" masking antibodies, with one or more of the "unique" masking antibody molecules existing in more than one copy.

[0085] As used herein, “diversity” refers to variety and / or heterogeneity. For example, the diversity of antibodies in a library may refer to the presence of multiple antibodies with unique sequences in the library, or to the presence of multiple masking antibodies with unique sequences in the masking peptide.

[0086] The term “antibody” is used in the broadest sense herein and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, masking antibodies (e.g., activatable or inactivatable antibodies), multispecific antibodies (e.g., bispecific antibodies), and antibody fragments or single-domain antibodies derived from human and natural sources (e.g., single-chain variable fragments or scFv or VHH single-domain fragments), provided they exhibit the desired biological activity.

[0087] In some implementations, the term "antibody" refers to an antigen-binding protein (i.e., an immunoglobulin) having a basic tetrapeptide chain structure consisting of two identical heavy (H) chains and two identical light (L) chains. Each L chain is linked to the H chain by a covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds, depending on the isotype of the H chains. Each heavy chain has a variable region (abbreviated herein as V) at its N-terminus. H ), followed by the constant region. The heavy chain constant region contains three structural domains, namely C H1 C H2and C H3 Each light chain has a variable region at the N-terminus (abbreviated as V in this paper). I ), followed by a constant region at its other end. The constant region of the light chain contains a structural domain, namely C L V L With V H Align, and C L Aligned with the first constant structural domain (CH1) of the heavy chain. V H and V L They pair together to form a single antigen-binding site. IgM antibodies consist of 5 basic heterotetrameric units and an additional polypeptide called the J chain, thus containing 10 antigen-binding sites, while secretory IgA antibodies can polymerize to form a multivalent aggregate containing 2-5 basic tetrameric units and the J chain.

[0088] The VH and VL regions can be further subdivided into highly variable regions, called hypervariable regions (HVRs), based on structural and sequence analysis. HVRs are interspersed with more conserved regions, called framework regions (FWs) (see, for example, Chen et al. (1999) J. Mol. Biol. (1999) 293, 865-881). Each VH and VL consists of three HVRs and four FWs, arranged in the following order from the amino terminus to the carboxyl terminus: 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.

[0089] Table 1 below provides exemplary CDR definitions based on various algorithms known in the art.

[0090] Table 1. CDR Definition

[0091] 1 Residue numbering follows Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., US Dept. of Health and Human Services, “Sequences of proteins of immune interest” (1991).

[0092] 2Residue numbering follows Chothia et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273: 927-948 (1997).

[0093] 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).

[0094] 4 Residue numbering follows the nomenclature of Lefranc MP et al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001).

[0095] 5 Residue numbering follows the nomenclature of Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001).

[0096] The variable regions of both the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Within both the light and heavy chains, the variable and constant regions are linked by “J” regions having about 12 or more amino acids, with the heavy chain also including “D” regions having about 10 or more amino acids (see, for example, Fundamental Immunology, Chapter 7 (Paul, W., ed., 2nd ed., Raven Press, NY) (1989)).

[0097] The L-chain from any vertebrate species can be designated as one of two distinct types (called κ and λ) based on the amino acid sequence of its constant domain. Antibodies can be designated as different species or isotypes depending on the amino acid sequence of their constant domain (CH) of the heavy chain. Five classes of antibodies exist: IgA, IgD, IgE, IgG, and IgM, which have heavy chains named α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu), respectively. IgG antibodies can be further classified into four subclasses—IgG1, IgG2, IgG3, and IgG4—based on the γ heavy chains Y1-Y4.

[0098] The terms “antigen-binding fragment,” “antigen-binding portion,” or “antigen-binding domain” of an antibody are used interchangeably herein and refer to one or more portions of an antibody that retain the ability to bind to an antigen. Examples of “antigen-binding fragments” of an antibody include: (i) Fab fragments, i.e., fragments composed of V... L V H C L and C H1 (ii) A monovalent segment composed of structural domains; (iii) A F(ab′)2 segment, i.e., a divalent segment containing two Fab segments connected by a disulfide bridge in the hinge region; (iv) A segment composed of V H and C H1 (iv) Fd fragments composed of structural domains; V-shaped segments formed by the single arm of the antibody. L and V H Fv fragments composed of structural domains; (v) scFv fragments, composed of V-arms of antibody fused via linkers. L and V H Domain composition; (vi) dAb fragment (Ward et al., Nature 341:544-546 (1989)), also known as VHH single variable domain, composed of V H Domain composition; (vii) VHH-Fc antibody, consisting of a VH domain fused to an Fc domain; and (viii) a separated complementarity-determining region (CDR).

[0099] The term "masked antibody" refers to an antibody or antigen-binding fragment thereof containing a masking peptide that interferes with, hinders, reduces, prevents, inhibits, or competes with the antigen-binding domain of the antibody for binding to its target. Masked antibodies can be generated by linking a masking peptide to the antigen-binding domain of an antibody. In some embodiments, the masked antibody or antigen-binding fragment thereof exhibits a first binding affinity to the target in an inactive state (e.g., inhibited or masked by the masking peptide) and a second binding affinity to the target in an activated state (e.g., not inhibited or masked by the masking peptide (e.g., a masking peptide cleaved from the antibody)), wherein the second binding affinity is greater than the first binding affinity. These embodiments may be referred to as "activatable antibodies." Activatable antibodies can be activated in a variety of ways. For example, an activateable antibody may contain a cleavable linker that, when cleaved, leads to antibody activation. Therefore, an activated masked antibody can be generated by linking a masking peptide containing an activatable portion (e.g., a cleavable site) to the antigen-binding domain of an antibody. In another example, the masked, activatable antibody may not be cleavable, but can be activated under concentration-dependent conditions, such as high concentrations of the target antigen. Masked and activatable antibodies have been described, for example, in U.S. Patent Publication Nos. 2019 / 0241886 and 2021 / 0207126, the contents of which are incorporated herein by reference in their entirety.

[0100] The term "masking peptide" refers to a peptide that inhibits the binding of an antigen-binding domain to its target antigen and typically comprises a masking unit (MU) and a linker unit (LU) from the N-terminus to the C-terminus. The C-terminus of the masking peptide is typically linked to the N-terminus of the VH or VL of the antigen-binding domain. In some embodiments, the masking peptide or a portion thereof (e.g., MU) interferes with or inhibits the binding of the antigen-binding domain to its target so effectively that the binding of the antigen-binding domain to its target is extremely low and / or below the detection limit (e.g., undetectable binding in ELISA or flow cytometry assays). In other embodiments, the masking peptide or a portion thereof (e.g., MU) binds weakly to the antigen-binding domain of an antibody, thereby allowing the binding of the antigen-binding domain to its target to be detected under certain conditions (e.g., detectable binding in ELISA or flow cytometry assays with high antigen concentrations). The masked antibodies or peptides described herein may contain one or more linkers, e.g., within the LU, between the MU and LU, between the LU and the VH or VL, or between the VH and the Fc hinge region. The masking peptide may further include an N-terminal unit having 1-12 amino acids at the N-terminus.

[0101] The LU of a masking peptide typically contains at least one linker and may or may not contain at least one cleavage site. The cleavage site generally contains a cleavable amino acid sequence, for example, serving as a substrate for enzymes and / or cysteine-cysteine ​​pairs capable of forming reducible disulfide bonds. Therefore, when the terms “cleavage,” “cleavable,” “cleaved,” etc., are used in conjunction with a cleavage site, these terms cover the disruption of the disulfide bond between cysteine-cysteine ​​pairs, for example, by enzymatic cleavage by a protease, and by disulfide bond reduction induced by exposure to a reducing agent. The amino acid sequence of the cleavage site may overlap with or be contained within the MU. A masked antibody or masked peptide may contain a cleavage site configured to mediate antibody or peptide activation. For example, when the cleavage site of an activated masking antibody having a cleavable masking peptide is intact (e.g., not cleaved by the corresponding enzyme, and / or contains unreduced cysteine-cysteine ​​disulfide bonds), the masking peptide or a portion thereof may interfere with or inhibit the binding of the antigen-binding domain to its target.

[0102] In some cases, the LU of a masking peptide may be non-cleavable and may not contain a cleavable site. Activatable masking antibodies with LUs lacking cleavable sites can be activated by high antigen concentrations, for example, in a tumor microenvironment (TME) if the tumor expresses high levels of the antigen. This can lead to transient antibody activation. For example, antibodies can be activated in a concentration-dependent manner in tumors or tissues that highly express the target antigen and may be deactivated once they move from the tumor or tissue to an environment with low antigen concentrations (e.g., once they enter the circulatory system).

[0103] 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 by the difference or ratio of a certain property (e.g., binding affinity to the target antigen) or an activity (e.g., prevention of target antigen binding to a ligand) of a masked antibody (e.g., an activatable antibody) relative to a corresponding unmasked antibody ("parent antibody"), wherein the masked antibody has both an antigen-binding domain and a masking peptide, while the unmasked antibody has the same antigen-binding domain but lacks the masking peptide. Masking efficiency can also be measured by the difference or ratio of the binding affinity of a masked antibody or masked peptide containing an antigen-binding domain to that of an unmasked antibody or unmasked peptide containing an antigen-binding domain (e.g., the masking peptide is cleaved from the antibody or temporarily activated through competitive binding between the antigen and the masked antibody). For example, masking efficiency can be measured by the EC50 or K0 of the masked antibody in its inactive state (e.g., inhibited, masked, and / or uncleaved) binding to the target antigen. D Divide the EC50 or K value of the activated state of the unmasked antibody (e.g., uninhibited, unmasked, and / or cleaved) binding to the target antigen. DOr, the EC50 or K-type protein of a parental antibody (e.g., an unlinked masking peptide) that binds to the target antigen. D The EC50 value can be measured using ELISA or Jurkat NFAT reporter assays, as described in, for example, U.S. Patent Publication No. US20210207126 A1. D The value can be measured, for example, using surface plasmon resonance.

[0104] The term "competitive binding" refers to the interaction between two antibodies when they bind to a target. If, in the presence of a second antibody, the binding of the first antibody to its homologous epitope is detectably reduced compared to binding in the absence of the second antibody, then the first antibody competes with the second antibody for binding. Alternatively, the binding of the second antibody to its epitope may also be detectably reduced in the presence of the first antibody, but this is possible but not always the case. That is, the first antibody may inhibit the binding of the second antibody to its epitope, while the second antibody may not inhibit the binding of the first antibody to its corresponding epitope. However, when each antibody detectably inhibits the binding of another antibody to its homologous epitope, whether to the same, greater, or lesser extent, the antibodies are said to "cross-compete" with each other for binding to one or more of their respective epitopes.

[0105] The term "epitope" refers to a portion of an antigen that binds to an antibody (or its antigen-binding fragment). Epitopes can be formed from consecutive amino acids or from discontinuous amino acids juxtaposed through the ternary folding of a protein. Epitopes formed from consecutive amino acids are generally retained upon exposure to denaturing solvents, while epitopes formed through ternary folding are generally lost upon treatment with denaturing solvents. Epitopes can include different numbers of amino acids in a unique spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, X-ray crystallography, two-dimensional nuclear magnetic resonance, deuterium exchange combined with mass spectrometry, or site-directed mutagenesis, or all methods combined with computer modeling of the antigen and its complex structure with the bound antibody and its variants (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris 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. The generation and characterization of the antibody can also elucidate information about the desired epitope. Based on this information, antibodies binding to the same epitope can be competitively screened. The method to achieve this is to conduct cross-competitive studies to identify antibodies that compete to bind to each other, i.e., antibodies compete to bind to antigens. A high-throughput method based on this cross-competitive “sorting” of antibodies is described in PCT Publication No. WO 03 / 48731.

[0106] The term "germline" refers to the nucleotide sequence of antibody genes and gene segments that are passed from parent to offspring via germ cells. Germline sequences differ from the nucleotide sequences encoding antibodies in mature B cells, which have been altered during B cell maturation through recombination and hypermutation events.

[0107] The term "glycosylation site" refers to an amino acid residue recognized by eukaryotic cells as a location for attaching sugar residues. The amino acids in which carbohydrates (e.g., oligosaccharides) are attached are typically asparagine (N-linked), serine (O-linked), and threonine (O-linked) residues. Specific attachment sites are typically indicated 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 except proline. The terms "N-linked" and "O-linked" refer to the chemical groups that serve as attachment sites between the sugar molecule and the amino acid residues. N-linked sugars are attached via an amino group; O-linked sugars are attached via a hydroxyl group. The term "glycan occupancy" refers to the presence of a carbohydrate moiety attached to the glycosylation site (i.e., the glycan site is occupied). In the presence of at least two potential glycosylation sites on a polypeptide, none of the sites (occupied by 0-glycan sites), one (occupied by 1-glycan sites), or both (occupied by 2-glycan sites) can be occupied by the carbohydrate moiety.

[0108] The term "host cell" refers to a cellular system that can be engineered 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. This term covers not only the specific subject cell but also its progeny. Because certain modifications may occur in successive generations due to mutations or environmental influences, the progeny may not be identical to the parent cell but are still included within the scope of the term "host cell." "Human antibody" refers to an antibody whose amino acid sequence corresponds to that of an antibody produced by humans or human cells, or is derived from a non-human source using sequences encoding human antibody libraries or other human antibodies. This definition of human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues.

[0109] The term "humanized antibody" refers to a chimeric antibody that contains amino acid residues derived from human antibody sequences. Humanized antibodies may contain some or all of the CDRs or HVRs derived from non-human animals or synthetic antibodies, while the frame and constant regions of the antibody contain amino acid residues derived from human antibody sequences.

[0110] The term "illustrative antibody" refers to any of the antibodies described herein. These antibodies may be in any class (e.g., IgA, IgD, IgE, IgG, and IgM). Therefore, each antibody identified above encompasses antibodies in all five classes that have the same amino acid sequences in both the VL and VH regions. Furthermore, antibodies in the IgG class may be in any subclass (e.g., IgG1, IgG2, IgG3, and IgG4). Therefore, each antibody identified above in the IgG subclass encompasses antibodies in all four subclasses that have the same amino acid sequences in both the VL and VH regions. The amino acid sequences of the heavy chain constant regions of human antibodies in the five classes and the four IgG subclasses are known in the art.

[0111] "Separated" antibodies or binding molecules are antibodies or binding molecules that have been separated from components of their native environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods used to assess antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0112] The term "k" a "" refers to the association rate constant of a specific antibody-antigen interaction, while the term "k" refers to the rate of association of that interaction. d "" refers to the dissociation rate constant of a specific antibody-antigen interaction.

[0113] Term "K" D "" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction. It is determined by k d With k a The ratio (i.e., k) d / k a K is obtained and expressed as molar concentration (M). D Used as a measure of the affinity of an antibody for its binding partner. K D The smaller the K value, the tighter the antibody binds, or the higher the affinity between the antibody and the antigen. For example, antibodies with a nanomolar (nM) dissociation constant bind more tightly to a specific antigen than antibodies with a micromolar (μM) dissociation constant. The K value of an antibody... D The value can be determined using methods well-established in the art. Determining antibody K. DOne method is to use ELISA. For example, using the ELISA testing procedure.

[0114] The term "mammal" refers to any animal species in the class Mammalia. 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, cattle, sheep, goats, horses, and pigs; and captive wild animals such as lions, tigers, and elephants.

[0115] The term “prevention” or “avoidance”, when referring to a disease condition in mammals, means preventing or delaying the onset of the disease or the manifestation of its clinical or subclinical symptoms.

[0116] As used herein, “sequence identity” between two polypeptide sequences indicates the percentage of identical amino acids between the sequences. Amino acid sequence identity of a polypeptide can be routinely determined using known computer programs such as Bestfit, FASTA, or BLAST (see, for example, Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132:185-219 (2000); Altschul et al., J. Mol. Biol. 215:403-410 (1990); Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). When using Bestfit or any other sequence alignment program to determine whether a particular sequence has, for example, 95% identity with a reference amino acid sequence, parameters are set such that the percentage of identity is calculated over the full length of the reference amino acid sequence, and the homology interval of the total number of amino acid residues in the reference sequence is allowed to be up to 5%. The above-described method for determining the percentage of identity between polypeptides is applicable to all proteins, fragments, or variants disclosed herein.

[0117] As used herein, the terms “binding,” “binding to,” “specific binding,” or “specific to” refer to a measurable and reproducible interaction between a target and an antibody, such as binding, which determines the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that binds to or specifically binds to a target (which may be an epitope) is an antibody that binds to the target with greater affinity, affinity, ease, and / or longer duration compared to its binding to other targets. In one embodiment, the degree to which the antibody binds to an irrelevant target is less than about 10% of the antibody's binding to the target, as measured, for example, by radioimmunoassay (RIA). In some embodiments, the antibody that specifically binds to the target has a dissociation constant (Kd) of ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, or ≤ 0.1 nM. In some embodiments, the antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In another embodiment, specific binding may include, but is not required to be, exclusive binding.

[0118] The term "treatment" (or "treat") in relation to a disease symptom in mammals refers to an effect that results in a desired or beneficial outcome in a mammal suffering from that disease symptom. A desired or beneficial outcome 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, etc.), or cessation or inhibition of further development of the disease, symptom, or condition. In the context of treating cancer in mammals, a desired or beneficial outcome may include inhibition of further growth or spread of cancer cells, death of cancer cells, inhibition of cancer recurrence, reduction of cancer-related pain, or increased survival rate in the mammal. The effect may be subjective or objective. For example, if the mammal is a human, then the human may record an improvement in energy or vitality or a reduction in pain as an improved subjective symptom or response to therapy. Alternatively, a clinician may notice a reduction in tumor size or tumor burden based on physical examination, laboratory parameters, tumor markers, or imaging findings. For a response to treatment, clinicians can observe several laboratory signs, including standardized tests such as white blood cell count, red blood cell count, platelet count, erythrocyte sedimentation rate, and various enzyme levels. Additionally, clinicians may observe a reduction in detectable tumor markers. Alternatively, other tests can be used to assess objective improvement, such as sonography, MRI, and positron emission tomography (PET).

[0119] The term "vector" refers to a nucleic acid molecule capable of transporting foreign nucleic acid molecules. Foreign nucleic acid molecules are attached to vector nucleic acid molecules via recombination techniques, such as ligation or recombination. This allows the foreign nucleic acid molecule to multiply, be selected, further manipulated, or expressed in a host cell or organism. Vectors can be plasmids, bacteriophages, transposons, granules, chromosomes, viruses, or virions. One class of vectors can integrate into the host cell's genome after introduction and replicate along with the host genome (e.g., non-attached mammalian vectors). Another class of vectors can replicate autonomously in the host cell in which they are introduced (e.g., bacterial vectors with bacterial origins of replication and attached mammalian vectors). Another specific type of vector capable of guiding the expression of an operatively linked, expressible foreign nucleic acid is often referred to as an "expression vector." Expression vectors typically have control sequences that drive the expression of the operatively expressed foreign nucleic acid. Simpler vectors known as "transcription vectors" can only transcribe, but not translate: they can replicate in target cells, but do not express. The term "vector" encompasses all types of vectors, regardless of their function. A vector that can guide the expression of an expressible nucleic acid that is operatively linked to it is generally referred to as an "expression vector". Other examples of "vectors" may include display vectors (e.g., vectors that guide the expression and display of encoded polypeptides on the surface of viruses or cells such as bacterial cells, yeast cells, insect cells and / or mammalian cells).

[0120] As used herein, “subject,” “patient,” or “individual” can refer to a human or a non-human animal. “Non-human animal” can refer to any animal not classified as human, such as domesticated animals, farm or zoo animals, sporting animals, pet animals (such as dogs, horses, cats, cows, etc.), and animals used in research. Research animals can refer without limitation 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 monkeys, cynomolgus monkeys, chimpanzees, etc.). In some implementations, the subject, patient, or individual is a human.

[0121] "Effective amount" means an amount that, at the necessary dose and time, is sufficient to achieve at least one or more desired or indicated effects, including therapeutic or preventative outcomes. An effective amount can be provided by one or more administrations. For the purposes of this disclosure, an effective amount of an antibody, drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve preventative or therapeutic treatment. As is understood in clinical settings, an effective amount 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 amount administered as a monotherapy or combination therapy). Therefore, an "effective amount" can be considered in the context of administering one or more therapeutic agents, and a single agent can be considered to have been administered in an effective amount if, in combination with one or more other agents, a desired outcome is achieved or realized.

[0122] II. Library and its Construction Certain aspects of this disclosure relate to libraries for screening antibodies (e.g., masked antibodies) containing a masking peptide. In some embodiments, the library contains multiple polynucleotides, each encoding an antibody linked to a masking peptide (e.g., a masked antibody). In some embodiments, the library contains multiple antibodies, each linked to a masking peptide (e.g., multiple masked antibodies). In some embodiments, the antibodies (e.g., masked antibodies) containing the masking peptide in the library are displayed on cell and phage surfaces. The masking peptide may include a masking unit (MU) and a linker unit (LU) from the N-terminus to the C-terminus. One or more masking units in the library may contain unique sequences as shown in formulas (I), (II), (III), (IV), or (V) as described in Example 1. In some embodiments, the masking peptide further includes an N-terminal unit linked to the N-terminus of the MU. In some embodiments, the LU contains one or more linkers. In some embodiments, the LU contains one or more cleavage sites, such as protease cleavage sites. In some embodiments, the LU contains 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 a cleavage site. In some embodiments, the antibody includes an antibody heavy chain variable region (VH) and / or an antibody light chain variable region (VL). In some embodiments, a masking peptide is attached to the N-terminus of the antibody's VH or VL. The libraries described herein can be used to screen and / or identify one or more masked antibodies. Libraries of host cells or phages displaying antibodies with masking peptides (e.g., masked antibodies) are also provided herein.

[0123] In one aspect, this document provides a library containing polynucleotides, wherein the polynucleotides encode at least two, at least three, at least four, at least five, or at least ten antibodies, each antibody having a unique masking peptide (MP), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), and the MP is linked to the N-terminus of the VH or VL of the antibody, wherein the MP comprises a masking unit (MU) and a linker unit (LU), wherein the MU comprises an amino acid sequence according to formula (I): X1X2CX3(X m ) n X4X5CX6X7, where: n is 2-8. 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; 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 X... m Independently, amino acids are 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 selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y.

[0124] 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. C stands for cysteine.

[0125] In some implementations, 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 some implementations, n is 2, 3, 4, 5, 6, 7, or 8.

[0126] On the other hand, this article provides a library containing 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 antibody having a unique masking peptide (MP), wherein each antibody contains a heavy chain variable region (VH) and / or a light chain variable region (VL), and the MP is linked to the N-terminus of the VH or VL of the antibody, wherein the MP contains a masking unit (MU) and a linker unit (LU), wherein the MU contains 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. The amino acids in the groups are: X8 is selected from the amino acids in the group composed of A, E, G, I, K, L, P, Q, R, S, T and V; X9 is selected from the amino acids in the group composed of A, E, G, I, K, L, P, Q, R, S, T and V.

[0127] C stands for cysteine.

[0128] 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.

[0129] In some embodiments, the 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 has approximately 10 amino acid sequences. 8 Up to 5x10 9 (For example, 10) 9 Up to 5x109 The diversity of ).

[0130] On the other hand, this article provides a library containing 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 antibody having a unique masking peptide (MP), wherein each antibody contains a heavy chain variable region (VH) and / or a light chain variable region (VL), and the MP is linked to the N-terminus of the VH or VL of the antibody, wherein the MP contains a masking unit (MU) and a linker unit (LU), wherein the MU contains an amino acid sequence according to formula (III): X1X2CX3X4X5X6X7X8CX9X 10 ,in: X1 is an amino acid group composed of A, D, F, H, I, L, N, P, S, T, V, and Y; X2 is an amino acid group composed of A, D, F, H, L, P, S, V, and Y; X3 is an amino acid group composed of A, E, G, I, K, L, P, Q, R, S, T, and V; X4 is an amino acid group composed of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y; X5 is an amino acid group composed of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y; X6 is an amino acid group composed of A, D, E, F, G, H, I, K, L, P, R, S, T, V, and Y; X7 is an amino acid group composed of A, D, F, H, I, L, N, P, S, T, V, and Y. The amino acids in the group composed of Y, X8 are amino acids selected from the group composed of A, D, F, H, L, P, S, V and Y, and X9 are amino acids selected from the group composed of A, E, G, I, K, L, P, Q, R, S, T and V. 10 It is an amino acid group selected from A, E, G, I, K, L, P, Q, R, S, T and V.

[0131] C stands for cysteine.

[0132] 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; X... 10 It is a group of amino acids selected from G, Q, R, S, T and V.

[0133] In some embodiments, the 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 has approximately 10 9 Up to 10 11 (For example, 10) 10 Up to 10 11 The diversity of ).

[0134] In another aspect, this article provides a library containing 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 antibody having a unique masking peptide (MP), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), and the MP is linked to the N-terminus of the VH or VL of the antibody, wherein the MP comprises a masking unit (MU) and a linker unit (LU), wherein the MU comprises an amino acid sequence according to formula (IV): X1X2CX3X4X5X6X7X8X9CX 10 X 11 ,in: 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. The amino acids in the groups X7, X8, and X9 are selected from the groups composed of A, D, E, F, G, H, I, K, L, P, R, S, T, V, and Y. 10 It is an amino acid group selected from A, E, G, I, K, L, P, Q, R, S, T, and V, X 11 It is a group of amino acids selected from A, E, G, I, K, L, P, Q, R, S, T and V.

[0135] C stands for cysteine.

[0136] 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; X 10 It is an amino acid group composed of I, P, and R, X 11 It is an amino acid group composed of E, G, I, P and V.

[0137] In some embodiments, the 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 has approximately 10 11 Up to 5x10 12 (For example, 10) 12 Up to 5x10 12The diversity of ).

[0138] On the other hand, this article provides a library containing 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 antibody having a unique masking peptide (MP), wherein each antibody contains a heavy chain variable region (VH) and / or a light chain variable region (VL), and the MP is linked to the N-terminus of the VH or VL of the antibody, wherein the MP contains a masking unit (MU) and a linker unit (LU), wherein the MU contains an amino acid sequence according to formula (V): X1X2CX3X4X5X6X7X8X9X 10 CX 11 X 12 ,in: 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. The amino acids in the groups X7, X8, and X9 are selected from the groups composed of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. 10 It is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y, X 11 It is an amino acid group composed of A, E, G, I, K, L, P, Q, R, S, T, and V, X 12 It is an amino acid group selected from A, E, G, I, K, L, P, Q, R, S, T and V.

[0139] C stands for cysteine.

[0140] In some implementations, 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; X... 10 It is an amino acid group composed of A, F, V, and Y, X 11 It is an amino acid selected from the group consisting of G, I, K, L, and R, X 12 It is a group of amino acids selected from A, E, K, P, R and T.

[0141] In some embodiments, the 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 has approximately 10 12 Up to 5x10 13 (For example, 10) 13 Up to 2x10 13 The diversity of ).

[0142] On the other hand, this article provides a library containing 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 antibody having a unique masking peptide (MP), wherein each antibody contains a heavy chain variable region (VH) and / or a light chain variable region (VL) that specifically binds to CTLA4 in the absence of MP, and the MP is linked to the N-terminus of the VH or VL of the antibody, wherein the MP contains a masking unit (MU) and a linker unit (LU), wherein the MU contains an amino acid sequence according to formula (V): X1X2CX3X4X5X6X7X8X9X 10 CX 11 X 12 ,in: 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. The amino acids in the groups X7, X8, and X9 are selected from the groups composed of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. 10 It is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y, X 11 It is an amino acid group composed of A, E, G, I, K, L, P, Q, R, S, T, and V, X 12 It is an amino acid group selected from A, E, G, I, K, L, P, Q, R, S, T and V.

[0143] C stands for cysteine.

[0144] In some implementation schemes, X1 is an amino acid selected from the group composed of P, S, and Y; X2 is an amino acid selected from the group composed of H, P, and S; X3 is an amino acid selected from the group composed of E, K, Q, and R; X4 is an amino acid selected from the group composed of P, R, V, and Y; X5 is an amino acid selected from the group composed of A, F, G, and Y; X6 is an amino acid selected from the group composed of L, P, and V; X7 is an amino acid selected from the group composed of H, K, P, and R; X8 is an amino acid selected from the group composed of A, K, L, and P; X9 is an amino acid selected from the group composed of D, F, L, and P. 10 It is an amino acid group selected from A, F, and Y, X 11 It is an amino acid selected from the group consisting of I, K, L, and R, X 12 It is an amino acid group selected from E, K and R.

[0145] In some embodiments, the MU comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 29-32. In some embodiments, the MU in the library has approximately 10 12 Up to 5x10 13 (For example, 10) 13 Up to 2x10 13 The diversity of ).

[0146] On the other hand, this article provides a library containing 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 antibody having a unique masking peptide (MP), wherein each antibody contains a heavy chain variable region (VH) and / or a light chain variable region (VL) that specifically binds to CD137 in the absence of the MP, and the MP is linked to the N-terminus of the VH or VL of the antibody, wherein the MP contains a masking unit (MU) and a linker unit (LU), wherein the MU contains an amino acid sequence according to formula (V): X1X2CX3X4X5X6X7X8X9X 10 CX 11 X 12 ,in: 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. The amino acids in the groups X7, X8, and X9 are selected from the groups composed of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. 10 It is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y, X 11 It is an amino acid group composed of A, E, G, I, K, L, P, Q, R, S, T, and V, X 12It is an amino acid group selected from A, E, G, I, K, L, P, Q, R, S, T and V.

[0147] C stands for cysteine.

[0148] 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; X 10 It is an amino acid group selected from F and V, X 11 It is an amino acid selected from the group consisting of G, I, and R, X 12 It is an amino acid group selected from A, P and T.

[0149] In some embodiments, the 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 has approximately 10 12 Up to 5x10 13 (For example, 10) 13 Up to 2x10 13 The diversity of ).

[0150] This article also provides an antibody library containing masking peptides.

[0151] In one aspect, this document provides a library containing at least two, at least three, at least four, at least five, or at least ten antibodies, each antibody having a unique masking peptide (MP), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), and the MP is linked to the N-terminus of the VH or VL of the antibody, wherein the MP comprises a masking unit (MU) and a linker unit (LU), wherein the MU comprises an amino acid sequence according to formula (I): X1X2CX3(X m ) n X4X5CX6X7, where: n is 2-8. 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; 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 X... mIndependently, amino acids are 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 selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y.

[0152] 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. C stands for cysteine.

[0153] In some implementations, 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 some implementations, n is 2, 3, 4, 5, 6, 7, or 8.

[0154] On the other hand, this document provides a library containing at least two, at least three, at least four, at least five, or at least ten antibodies, each antibody having a unique masking peptide (MP), wherein each antibody contains a heavy chain variable region (VH) and / or a light chain variable region (VL), and the MP is linked to the N-terminus of the VH or VL of the antibody, wherein the MP contains a masking unit (MU) and a linker unit (LU), wherein the MU contains 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. The amino acids in the groups are: X8 is selected from the amino acids in the group composed of A, E, G, I, K, L, P, Q, R, S, T and V; X9 is selected from the amino acids in the group composed of A, E, G, I, K, L, P, Q, R, S, T and V.

[0155] C stands for cysteine.

[0156] 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.

[0157] In some embodiments, the 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 has approximately 10 amino acid sequences. 8 Up to 5x10 9 (For example, 10) 9 Up to 5x10 9 The diversity of ).

[0158] On the other hand, this article provides a library containing at least two, at least three, at least four, at least five, or at least ten antibodies, each antibody having a unique masking peptide (MP), wherein each antibody contains a heavy chain variable region (VH) and / or a light chain variable region (VL), and the MP is linked to the N-terminus of the VH or VL of the antibody, wherein the MP contains a masking unit (MU) and a linker unit (LU), wherein the MU contains an amino acid sequence according to formula (III): X1X2CX3X4X5X6X7X8CX9X 10 ,in: X1 is an amino acid group composed of A, D, F, H, I, L, N, P, S, T, V, and Y; X2 is an amino acid group composed of A, D, F, H, L, P, S, V, and Y; X3 is an amino acid group composed of A, E, G, I, K, L, P, Q, R, S, T, and V; X4 is an amino acid group composed of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y; X5 is an amino acid group composed of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y; X6 is an amino acid group composed of A, D, E, F, G, H, I, K, L, P, R, S, T, V, and Y; X7 is an amino acid group composed of A, D, F, H, I, L, N, P, S, T, V, and Y. The amino acids in the group composed of Y, X8 are amino acids selected from the group composed of A, D, F, H, L, P, S, V and Y, and X9 are amino acids selected from the group composed of A, E, G, I, K, L, P, Q, R, S, T and V. 10 It is an amino acid group selected from A, E, G, I, K, L, P, Q, R, S, T and V.

[0159] C stands for cysteine.

[0160] 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; X... 10 It is a group of amino acids selected from G, Q, R, S, T and V.

[0161] In some embodiments, the 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 has approximately 10 9 Up to 10 11 (For example, 10) 10 Up to 10 11 The diversity of ).

[0162] On the other hand, this article provides a library containing at least two, at least three, at least four, at least five, or at least ten antibodies, each antibody having a unique masking peptide (MP), wherein each antibody contains a heavy chain variable region (VH) and / or a light chain variable region (VL), and the MP is linked to the N-terminus of the VH or VL of the antibody, wherein the MP contains a masking unit (MU) and a linker unit (LU), wherein the MU contains the amino acid sequence according to formula (IV): X1X2CX3X4X5X6X7X8X9CX 10 X 11 ,in: 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. The amino acids in the groups X7, X8, and X9 are selected from the groups composed of A, D, E, F, G, H, I, K, L, P, R, S, T, V, and Y. 10 It is an amino acid group selected from A, E, G, I, K, L, P, Q, R, S, T, and V, X 11 It is a group of amino acids selected from A, E, G, I, K, L, P, Q, R, S, T and V.

[0163] C stands for cysteine.

[0164] 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; X 10 It is an amino acid group composed of I, P, and R, X 11 It is an amino acid group composed of E, G, I, P and V.

[0165] In some embodiments, the 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 has approximately 10 11 Up to 5x10 12 (For example, 10) 12 Up to 5x10 12 The diversity of ).

[0166] On the other hand, this document provides a library containing at least two, at least three, at least four, at least five, or at least ten antibodies, each antibody having a unique masking peptide (MP), wherein each antibody contains a heavy chain variable region (VH) and / or a light chain variable region (VL), and the MP is linked to the N-terminus of the VH or VL of the antibody, wherein the MP contains a masking unit (MU) and a linker unit (LU), wherein the MU contains an amino acid sequence according to formula (V): X1X2CX3X4X5X6X7X8X9X 10 CX 11 X 12 ,in: 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. The amino acids in the groups X7, X8, and X9 are selected from the groups composed of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. 10 It is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y, X 11 It is an amino acid group composed of A, E, G, I, K, L, P, Q, R, S, T, and V, X 12 It is an amino acid group selected from A, E, G, I, K, L, P, Q, R, S, T and V.

[0167] C stands for cysteine.

[0168] In some implementations, 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; X... 10 It is an amino acid group composed of A, F, V, and Y, X 11 It is an amino acid selected from the group consisting of G, I, K, L, and R, X 12 It is a group of amino acids selected from A, E, K, P, R and T.

[0169] In some embodiments, the 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 has approximately 10 12 Up to 5x10 13 (For example, 10) 13 Up to 2x10 13 The diversity of ).

[0170] On the other hand, the present invention provides a library comprising at least two, at least three, at least four, at least five, or at least ten antibodies, each antibody having a unique masking peptide (MP), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), capable of specifically binding to CTLA4 in the absence of MP, and the MP is linked to the N-terminus of the VH or VL of the antibody, wherein the MP comprises a masking unit (MU) and a linker unit (LU), wherein the MU comprises an amino acid sequence according to formula (V): X1X2X3X4X5X6X7X8X9X 10 CX 11 X 12 ,in: 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. The amino acids in the groups X7, X8, and X9 are selected from the groups composed of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. 10 It is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y, X 11 It is an amino acid group composed of A, E, G, I, K, L, P, Q, R, S, T, and V, X 12It is an amino acid group selected from A, E, G, I, K, L, P, Q, R, S, T and V.

[0171] C stands for cysteine.

[0172] In some implementation schemes, X1 is an amino acid selected from the group composed of P, S, and Y; X2 is an amino acid selected from the group composed of H, P, and S; X3 is an amino acid selected from the group composed of E, K, Q, and R; X4 is an amino acid selected from the group composed of P, R, V, and Y; X5 is an amino acid selected from the group composed of A, F, G, and Y; X6 is an amino acid selected from the group composed of L, P, and V; X7 is an amino acid selected from the group composed of H, K, P, and R; X8 is an amino acid selected from the group composed of A, K, L, and P; X9 is an amino acid selected from the group composed of D, F, L, and P. 10 It is an amino acid group selected from A, F, and Y, X 11 It is an amino acid selected from the group consisting of I, K, L, and R, X 12 It is an amino acid group selected from E, K and R.

[0173] In some embodiments, the MU comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 29-32. In some embodiments, the MU in the library has approximately 10 12 Up to 5x10 13 (For example, 10) 13 Up to 2x10 13 The diversity of ).

[0174] On the other hand, the present invention provides a library comprising at least two, at least three, at least four, at least five, or at least ten antibodies, each antibody having a unique masking peptide (MP), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) capable of specifically binding to CD137 in the absence of MP, and the MP is linked to the N-terminus of the VH or VL of the antibody, wherein the MP comprises a masking unit (MU) and a linker unit (LU), wherein the MU comprises an amino acid sequence according to formula (V): X1X2X3X4X5X6X7X8X9X 10 CX 11 X 12 ,in: 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. The amino acids in the groups X7, X8, and X9 are selected from the groups composed of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. 10 It is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y, X 11 It is an amino acid group composed of A, E, G, I, K, L, P, Q, R, S, T, and V, X 12 It is an amino acid group selected from A, E, G, I, K, L, P, Q, R, S, T and V.

[0175] C stands for cysteine.

[0176] 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; X 10 It is an amino acid group selected from F and V, X 11 It is an amino acid selected from the group consisting of G, I, and R, X 12 It is an amino acid group selected from A, P and T.

[0177] In some embodiments, the 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 has approximately 10 12Up to 5x10 13 (For example, 10) 13 Up to 2x10 13 The diversity of ).

[0178] In some implementations of the library described herein, MU does not contain amino acid sequences of M, NG, DG, NXS, NXT, or any combination thereof.

[0179] In some embodiments of the library described herein, the masking peptide (MP) is linked to the N-terminus of the VL. In other embodiments of the library described herein, the MP is linked to the N-terminus of the VL. In some embodiments of the library described herein, each antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the MP is linked to the N-terminus of the VH or VL of the antibody. In some embodiments of the library described herein, each antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the MP is linked to the N-terminus of the VL of the antibody. In some embodiments of the library described herein, each antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the MP is linked to the N-terminus of the VH of the antibody.

[0180] In some embodiments of the libraries described herein, each antibody comprises an scFv consisting of a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the MP is linked to the VL of the scFv. In some embodiments, the MP is linked to the VH of the scFv.

[0181] In some embodiments of the library described herein, each antibody comprises a Fab consisting of a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the MP is linked to the VL of the Fab. In some embodiments, the MP is linked to the VH of the Fab.

[0182] In some embodiments of the library described herein, each antibody comprises a heavy chain variable region (VH) with an MP linked to the N-terminus of the VH. In some variants, the antibody does not comprise a light chain variable region (VL). In some embodiments, the antibody is a VHH single-domain antibody (also known as a nanobody) comprising a heavy chain variable region (VH) with an MP linked to the N-terminus of the VH. In some embodiments, the antibody is a VHH-Fc antibody comprising a heavy chain variable region (VH) with an MP linked to the N-terminus of the VH of the VH.

[0183] In some embodiments, the library disclosed herein contains multiple polynucleotides encoding at least one, 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, or 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 A unique masking unit. In some embodiments, the library disclosed herein contains a variety of antibodies having masking peptides, including at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least twenty, at least thirty, at least fourty, at least fifty, at least ten hundred, at least twenty ... 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 A unique type of shading unit.

[0184] In some embodiments, the libraries of this disclosure include one or more vectors (e.g., expression vectors and / or display vectors) containing one or more of the polynucleotides of this disclosure (e.g., synthetic polynucleotides). In some embodiments, each antibody with a masking peptide in the library is fused with all or part of a protein (e.g., viral capsid proteins, bacterial surface proteins, yeast surface proteins, insect cell surface proteins, mammalian cell surface proteins) (i.e., forming a fusion protein). In some embodiments, the fusion protein is displayed on the surface of a particle or a host cell. In some embodiments, the libraries of this disclosure include host cells and particles (e.g., bacteriophages) displaying the antibodies with masking peptides of this disclosure.

[0185] This document also provides a method for preparing a library, for example, by providing and assembling a library of the present disclosure with a polynucleotide sequence (e.g., one or more synthetic polynucleotides). A polynucleotide encoding an antibody having a masking peptide can be cloned into any suitable vector to express a portion or the entire polypeptide sequence. In some embodiments, cloning the polynucleotide into a vector allows for the production of all or a portion of a fusion protein (e.g., viral capsid proteins, bacterial surface proteins, yeast surface proteins, insect cell surface proteins, mammalian cell surface proteins) and the display of a portion or the entire polypeptide on the surface of a particle or cell. Several types of vectors are available and can be used to implement this disclosure, such as phageparticle vectors. Phageparticle vectors typically contain multiple components, including a promoter, signal sequence, phenotypic selection gene, origin of replication site, and other essential components as known to those skilled in the art. In some embodiments, a polynucleotide encoding a polypeptide region can be cloned into a vector for expression in bacterial cells to achieve bacterial display, or for expression in yeast cells to achieve yeast display. Exemplary vectors are described in U.S. Pre-Publication No. 20160145604. In some embodiments, the vector is a display vector comprising, from 5' to 3', a polynucleotide encoding an amino acid sequence to be displayed on a surface (e.g., the surface of a bacteriophage, bacterium, yeast, insect, or mammalian cell), a restriction site, a second polynucleotide encoding a surface peptide capable of being displayed on the surface, and a second restriction site. In some embodiments, the second polynucleotide encodes a bacteriophage coat protein, a yeast exowall protein (such as Aga2), a bacterial outer membrane protein, a cell surface tethering domain, or an adaptor, or a truncated form or derivative thereof. In some embodiments, the surface peptide is used for bacteriophage display, yeast display, bacterial display, insect display, or mammalian display, or shuttle display between them. In some embodiments, when expressed, the amino acid sequence and the surface peptide are displayed on the surface as a fusion protein. In some embodiments, the vector also comprises a fusion tag at the 5' of the first restriction site or the 3' of the second restriction site.

[0186] Certain aspects of this disclosure relate to a cell population containing the vector described herein. Antibodies encoding a masking peptide, produced by any of the techniques described herein or other suitable techniques, can be expressed and screened to identify masking antibodies having the desired structure and / or activity. Protein expression can be performed, 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 (such as Chinese hamster ovary (CHO) cells). Methods for transfecting bacterial, yeast, or mammalian cells are known in the art and described in the references cited herein. The expression of proteins in these cell types (e.g., from libraries of this disclosure) and the screening of targeted masking antibodies are described in more detail below.

[0187] Alternatively, the polynucleotide can be expressed in an *E. coli* expression system such as that described by Pluckthun and Skerra (Meth. Enzymol., 1989, 178: 476; Biotechnology, 1991, 9: 273). Mutant proteins can be expressed to achieve secretion in a culture medium and / or in the bacterial cytoplasm, as described by Better and Horwitz, *Meth. Enzymol., 1989, 178: 476*. In some embodiments, the polypeptide is attached to the 3′ end of a sequence encoding a signal sequence such as ompA, phoA, or pelB (Lei et al., *J. Bacteriol.*, 1987, 169: 4379). These gene fusions are assembled in bicistronic constructs, thus allowing them to be expressed from a single vector and secreted into the periplasmic space of *E. coli*, where they will fold back and be recovered in their active form (Skerra et al., Biotechnology, 1991, 9: 273).

[0188] In other embodiments, using the secretion signaling and lipidation portions described, for example, in US20040072740; US20030100023; and US20030036092, the polypeptide sequence of this disclosure is expressed on the membrane surface of a prokaryote, such as Escherichia coli.

[0189] Alternatively, the polypeptide sequences disclosed herein can be expressed and screened by anchored periplasmic expression (APEx two-hybrid surface display) as described, for example, Jeong et al., PNAS, 2007, 104: 8247, or by other anchoring methods as described, for example, Mazor et al., Nature Biotechnology, 2007, 25: 563.

[0190] Higher eukaryotic cells, such as mammalian cells, including myeloma cells (e.g., NS / O cells), hybridoma cells, Chinese hamster ovary (CHO) cells, and human embryonic kidney (HEK) cells, can also be used to express the polypeptides disclosed herein. Polypeptides expressed in mammalian cells (e.g., activatable binding polypeptides, such as masking antibodies) can be engineered to be secreted into the culture medium or expressed on the cell surface.

[0191] In other embodiments, peptides or antibodies (e.g., masking antibodies) may be selected using mammalian cell display (Ho et al., PNAS, 2006, 103: 9637). In some embodiments, as described above and illustrated below, selection of peptides or antibodies may be performed after, for example, phage display to generate all or part of a peptide or antibody fused to a viral capsid protein (i.e., generating a fusion protein) and displaying a portion or the entire peptide or antibody on the surface of a particle or cell.

[0192] Certain aspects of this disclosure relate to a non-human animal comprising the polynucleotides or polynucleotide libraries of this disclosure. For example, the non-human animal of this disclosure may be modified such that its genome includes polynucleotides encoding polypeptides or antibodies with masking peptides of this disclosure. In some embodiments, the transgenic animal (e.g., a mouse) expresses the polypeptide or antibody encoded by the polynucleotide. Techniques for modifying the genome of a non-human animal are known in the art (e.g., methods for generating Xenomouse™).

[0193] In some embodiments, the masking peptide in the library described herein further comprises an N-terminal unit. In some embodiments, the N-terminal unit comprises or consists of 1-12 amino acid residues. In some embodiments, the N-terminal unit is attached to the N-terminus of the masking unit. In some embodiments, the N-terminal unit comprises amino acid E, or peptides EVG, EVGSY (SEQ ID NO: 33), EVGVLDV (SEQ ID NO: 86), or EVGAESGVK (SEQ ID NO: 88). In some embodiments, the N-terminal unit is composed of amino acid E, or peptides EVG, EVGSY (SEQ ID NO: 33), EVGVLDV (SEQ ID NO: 86), or EVGAESGVK (SEQ ID NO: 88).

[0194] In some embodiments of the library described herein, the connection unit (LU) includes one or more connectors. Any suitable connector known in the art (e.g., a flexible connector) may be used, including, for example: glycine polymer (G)n, where 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.); glycine-serine polymer (GS)n, where 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 GS, GGS, GGG, SGRG, GGGGS, GGGGT, SGGS, GGSG, GGSGG, GGSGG, GGGSG, GGSSG, GGSGS, GGSGS, SGGG, GGGS and / or GGGGSGGSGGGS; glycine-alanine polymer; alanine-serine polymer; and the like. The linker sequence can have any length, such as about 1 amino acid (e.g., glycine or serine) to about 20 amino acids (e.g., a 20-amino acid glycine polymer or a 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 length is any of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

[0195] In some embodiments of the libraries described herein, the linker unit (LU) comprises one or more of the adapters described herein and does not contain any cleavage sites. In some embodiments, the linker unit comprises a non-cleavable adapter. In a particular embodiment, the non-cleavable adapter comprises the amino acid sequence of SEQ ID NO: 81. In a particular embodiment, the masking peptide comprises the amino acid sequence of SEQ ID NO: 82.

[0196] In some embodiments, the linker unit (LU) in the library described herein includes one or more cleavage sites. In some embodiments, the LU includes at least one 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 can be recognized and / or cleaved by any protease known in the art can be used, including, for example, protease cleavage sites recognized and / or cleaved by 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 etching virus (TEV) protease; plasmin; thrombin; PSA; PSMA; ADAMS / ADAMTS (e.g., ADAM 8, ADAM 9, ADAM 10 ... 9. ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMDEC1, ADAMTS1, ADAMTS4 and / or ADAMTS5; kasparases (e.g., kasparase-1, kasparase-2, kasparase-3, kasparase-4, kasparase-5, kasparase-6, kasparase-7, kasparase-8, kasparase-9, kasparase-10, kasparase-11, kasparase-12, kasparase-13 and... / or caspasmin-14); aspartic proteases (e.g., RACE and / or renin); aspartic 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 ​​proteases (e.g., Cruzipain, podin and / or Otubain-2); K LK (e.g., KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13 and / or KLK14); metalloproteinases (e.g., Meprin, enkephalin, PSMA and / or BMP-1); serine proteases (e.g., activated protein C, cathepsin A, cathepsin G, gastrin and / or coagulation factor proteases (e.g., FVIIa, FIXa, FXa, FXIa, FXIIa)); elastase; Granulase B; guanidinobenzoic acidase; HtrA1; human neutrophil elastase; lactoferrin; marapsin; NS3 / 4A; PACE4; tPA; trypsin; type II transmembrane serine proteases (TTSPs) (e.g., DESC1, DPP-4, FAP, Hepsin, Matriptase-2, MT-SP1 / Matriptase, TMPRSS2, TMPRSS3 and / or TMPRSS4); etc.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, caspases-1, caspases-2, caspases-3, caspases-4, caspases-5, caspases-6, caspases-7, caspases-8, caspases-9, caspases-10, caspases-11, caspases-12, caspases-13, caspases-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 protease. In some embodiments, the protease cleavage site comprises the amino acid sequence SGRSA (SEQ ID NO:34), the amino acid sequence PLGLAG (SEQ ID NO:35), or a combination thereof.

[0197] In some embodiments, the LU further comprises a first adapter (L1). In some embodiments, the first adapter (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 comprises a (CS1)-L1 structure from the N-terminus to the C-terminus. In some embodiments, the LU further comprises a second adapter (L2). In some embodiments, L2 is located at the C-terminus of a second cleavage site (CS2). In some embodiments, the LU comprises a (CS1)-L1-(CS2)-L2 structure from the N-terminus to the C-terminus. In some embodiments, the first adapter (L1) is located at the N-terminus of the first cleavage site (CS1). In some embodiments, the LU comprises an L1-(CS1)-L2 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.

[0198] In some embodiments, the masking peptide of this disclosure comprises the structure MU-(CS1)-L1 from the N-terminus to the C-terminus. In some embodiments, the masking peptide of this disclosure comprises the structure (MU)-(CS1)-L1-(CS2)-L2 from the N-terminus to the C-terminus. In some embodiments, the masking peptide of this disclosure comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 43-65 and 67.

[0199] In some embodiments, the masking peptide of this disclosure comprises the following structure from the N-terminus to the C-terminus: MU-L1-(CS1)-L2. In some embodiments, the masking peptide of this disclosure comprises the following structure from the N-terminus to the C-terminus: MU-L1-(CS1)-L2-(CS2). In some embodiments, the masking peptide of this disclosure comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 68-75, 83-85, 87, and 116-119.

[0200] In some embodiments, this disclosure relates to a polynucleotide library encoding one or more antibodies or antibody libraries, and each antibody in the library comprises an antigen-binding domain (ABD). In some embodiments, the ABD comprises a variable region of the antibody light chain and / or a variable region of the antibody heavy chain. In some embodiments, the ABD comprises both a variable region of the antibody light chain and a variable region of the antibody heavy chain. In some embodiments, the ABD comprises a variable region of the antibody heavy chain but not a variable region of the antibody light chain. In some embodiments, the ABD of this disclosure comprises a variable region of the antibody light chain and / or a variable region of the antibody heavy chain that is specific to any target of interest, said targets 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.

[0201] In some embodiments, the antibody comprises a full-length antibody light chain and / or a full-length antibody heavy chain. The antibody light chain may be a κ or λ light chain. The antibody heavy chain may belong to any class, such as IgG, IgM, IgE, IgA, or IgD. In some embodiments, the antibody heavy chain is in the IgG class, such as IgG1, IgG2, IgG3, or IgG4 subclasses. Using methods known in the art, the antibody heavy chain described herein can be changed from one class or subclass to another.

[0202] Any one or more antibodies described herein may comprise any HVR sequence (e.g., one, two, or three of the heavy chain variable region HVR sequences, and / or one, two, or three of the 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 (in whole by reference), PCT application number PCT / CN2017 / 098299 (in whole by reference), PCT application number PCT / CN2017 / 098332 (in whole by reference), and / or U.S. Patent Application Publication No. 2021 / 0206855 (in whole by reference).

[0203] Any one or more antibodies described herein may include any HVR sequence (e.g., one, two, or three of the heavy chain variable region HVR sequences, and / or one, two, or three of the light chain variable region HVR sequences), heavy chain variable region sequences, and / or light chain variable region sequences of any antibody described herein (e.g., anti-CTLA4 antibody, anti-CD137 antibody).

[0204] In some embodiments, the antibody is an anti-CTLA4 antibody comprising (a) an antibody light chain variable region comprising HVR-L1 containing the amino acid sequence RASQSVRGRFLA (SEQ ID NO: 95), HVR-L2 containing the amino acid sequence DASNRATGI (SEQ ID NO: 96), and / or HVR-L3 containing the amino acid sequence YCQQSSSWPPT (SEQ ID NO: 97); and (b) an antibody heavy chain variable region comprising HVR-H1 containing the amino acid sequence YSISSGYHWSWI (SEQ ID NO: X98), HVR-H2 containing the amino acid sequence LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 99), and / or HVR-H3 containing the amino acid sequence ARSYVYFDY (SEQ ID NO: 100). In some embodiments, the antibody comprises an antibody light chain variable region containing 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 containing 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 comprises an antibody light chain variable region containing 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 containing 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 comprises an antibody light chain variable region containing the amino acid sequence of SEQ ID NO: 93 and an antibody heavy chain variable region containing the amino acid sequence of SEQ ID NO: 94.

[0205] Exemplary anti-CTLA4 antibody light chain variable region (SEQ ID NO: 93): DIQLTQSPSSLSASVGDRVTITCRASQSVRGRFLAWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSSSWPPTFGQGTKVEIKR Exemplary anti-CTLA4 antibody heavy chain variable region (SEQ ID NO: 94): EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWSWIRQAPGKGLEWLARIDWDDDKYYSTSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARSYVYFDYWGQGTLVTVSS III. Methods for screening masked antibodies In some respects, this article provides methods for screening masked antibodies in the polynucleotide and peptide libraries described herein.

[0206] In some embodiments, this document provides a method for screening masked antibodies that bind to a target using a polynucleotide library or a peptide library, the method comprising: a) contacting an antibody containing a masking peptide expressed in the library with the target to determine a first binding affinity or no detection of binding to the target; b) contacting a control antibody lacking the masking peptide with the target to determine a second binding affinity; and c) selecting an expressed antibody having a first binding affinity lower than the second binding affinity or no detection of binding to the target, wherein the first and second binding affinities are measured as KD, EC50, or IC50. In some embodiments, the masking peptide comprises a masking unit (MU) and a linker unit from the N-terminus to the C-terminus. In some embodiments, the LU comprises at least one first cleavage site (C1). In some embodiments, wherein the LU of e comprises at least one first cleavage site (L1), and the control antibody is a library-expressed antibody after LU cleavage. In some embodiments, the expression antibody of the library is selected if the binding affinity of the expression antibody after LU lysis is at least 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 1000, 2000, 300, 400, 500, 1000, 2000, 3000, 4000, or 5000 times that before LU lysis. In some embodiments, step (c) includes selecting expression antibodies that are not detected to bind to the target, for example, those that are not detected to bind to the target under the same contact conditions used to determine the second binding affinity in step (b). In some embodiments, the method includes determining the ratio of the first binding affinity in step a) to the second binding affinity in step b) to calculate the masking efficiency. In some implementations, the expression antibody of the library is selected if the 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.

[0207] In some embodiments, this document provides a method for screening masked antibodies that bind to a target using a polynucleotide library or a peptide library described herein, the method comprising the steps of: a) contacting an antibody expressing the library containing the masking peptide with a first cell expressing the target antigen to determine binding affinity (e.g., EC50 binding); b) contacting a control antibody lacking the masking peptide with the first cell expressing the target antigen to determine binding affinity (e.g., EC50 binding); c) contacting an antibody expressing the library with a second cell expressing the target antigen to determine binding affinity (e.g., EC50 binding), wherein the second cell expresses a lower level of the target antigen than the first cell; d) contacting a control antibody lacking the masking peptide with the second cell to determine binding affinity (e.g., EC50 binding); e) calculating the ratio of the binding affinity in step a) to the binding affinity in step b) as a first masking efficiency; f) calculating the ratio of the binding affinity in step c) to the binding affinity in step d) as a second masking efficiency; and g) selecting an expression antibody having a second masking efficiency higher than the first masking efficiency. In some embodiments, the first and second binding affinities are measured in terms of KD, EC50, or IC50. In some embodiments, step (g) includes selecting an expression 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 the first masking efficiency. In some embodiments, the control antibody is an antibody having the same antigen-binding domain as the library-expressing antibody. In some embodiments, the control antibody is a parent antibody. In some embodiments, the masking peptide comprises a masking unit (MU) and a linker 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 one first cleavage site (C1). In some embodiments, the LU contains at least one first cleavage site, and the control antibody is an antibody expressing a library of cleaved LU. In some embodiments, the LU contains at least one first cleavage site, and the method includes cleaving the LU to generate a control antibody.In some implementations, the second shielding 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 shielding efficiency.

[0208] In some embodiments, this document provides a method for screening masked antibodies that bind to a target using a polynucleotide library or a peptide library, the method comprising the steps of: a) contacting an expression antibody containing a masking peptide in the library with a first cell expressing a target antigen to determine the EC50 of binding; b) contacting a control antibody lacking the masking peptide with the first cell expressing the target antigen to determine the EC50 of binding; c) contacting an expression antibody in the library with a second cell expressing the target antigen to determine the EC50 of binding, wherein the second cell expresses a lower level of the target antigen than the first cell; d) contacting a control antibody lacking the masking peptide with the second cell to determine the EC50 of binding; e) calculating the ratio of the EC50 in step a) to the EC50 in step b) as a first masking efficiency; f) calculating the ratio of the EC50 in step c) to the EC50 in step d) as a second masking efficiency; and g) selecting an expression antibody having a second masking efficiency higher than the first masking efficiency. In some embodiments, step (g) includes selecting an expression 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 the first masking efficiency. In some embodiments, the control antibody is an antibody having the same antigen-binding domain as the antibody expressed in the library. In some embodiments, the control antibody is a parent antibody. In some embodiments, the masking peptide comprises a masking unit (MU) and a linker 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 one first cleavage site (C1). In some embodiments, the LU contains at least one first cleavage site, and the control antibody is an expressed antibody in a library after the LU has been cleaved. In some embodiments, the method includes cleaving the LU to generate a 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.

[0209] In some embodiments, this document provides a method for identifying a masked antibody with concentration-dependent antigen-binding ability, the method comprising the steps of: a) contacting a masked antibody having a masking peptide with a first cell expressing a target antigen to determine binding affinity (e.g., EC50 binding); b) contacting a control antibody lacking the masking peptide with the first cell expressing the target antigen to determine binding affinity (e.g., EC50 binding); c) contacting a masked antibody with a second cell expressing the target antigen to determine binding affinity (e.g., EC50 binding), wherein the second cell expresses a lower level of the target antigen than the first cell; d) contacting a control antibody lacking the masking peptide with the second cell to determine binding affinity (e.g., EC50 binding); e) determining the ratio of the binding affinity in step a) to the binding affinity in step b) as a first masking efficiency; f) determining the ratio of the binding affinity in step c) to the binding affinity in step d) as a second masking efficiency; and g) if the second masking efficiency is higher than the first masking efficiency, then identifying an antibody with concentration-dependent antigen-binding ability. In some embodiments, binding affinity is measured in terms of KD, EC50, or IC50. In some embodiments, step (g) includes identifying an antibody with concentration-dependent antigen-binding capacity 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-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, or at least 1000-fold 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 linker 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 one first cleavage site (C1). In some embodiments, the LU contains at least one first cleavage site (C1), and the control antibody is a masked antibody obtained by cleaving the LU. In some embodiments, the LU contains at least one first cleavage site (C1), and the method includes cleaving the LU to generate a control antibody.In some implementations, the second shielding 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 shielding efficiency.

[0210] In some embodiments, this document provides a method for identifying a masked antibody capable of concentration-dependent antigen binding, the method comprising the steps of: a) contacting a masked antibody having a masking peptide with a first cell expressing a target antigen to determine the EC50 of binding; b) contacting a control antibody lacking the masking peptide with the first cell expressing the target antigen to determine the EC50 of binding; c) contacting a masked antibody with a second cell expressing the target antigen to determine the EC50 of binding, wherein the second cell expresses a lower level of the target antigen than the first cell; d) contacting a control antibody lacking the masking peptide with the second cell to determine the EC50 of binding; e) determining the ratio of the EC50 in step a) to the EC50 in step b) as a first masking efficiency; f) determining the ratio of the EC50 in step c) to the EC50 in step d) as a second masking efficiency; g) if the second masking efficiency is higher than the first masking efficiency, then identifying an antibody capable of concentration-dependent antigen binding. In some embodiments, step (g) includes identifying an antibody capable of concentration-dependent antigen binding when 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 comprises a masking unit (MU) and a linker 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 one first cleavage site (C1). In some embodiments, wherein the LU contains at least one first cleavage site (C1), the method comprising cleaving the LU to generate a 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.

[0211] In some embodiments, wherein the linker unit (LU) of the library contains at least one cleavage site, the method comprising: a) contacting an expressed antibody or protein of the library with a target prior to cleavage of the linker unit (LU); b) contacting an expressed antibody or protein of the library with a target after cleavage of the LU; and c) selecting one or more expressed antibodies or proteins that have a higher binding affinity to the target after cleavage of the LU compared to before cleavage of the cleavage site.

[0212] In other embodiments, wherein the linker unit (LU) of the library does not contain at least one cleavage site (e.g., non-cleavable), the method includes: a) contacting the expression antibody of the library with a first medium, cell, or tissue expressing the target; b) contacting the expression antibody of the library with a second medium, cell, or tissue, wherein the target concentration in the second medium, cell, or tissue is lower than that in the first medium, cell, or tissue; and c) selecting an expression antibody that has a higher binding capacity to the first medium, cell, or tissue compared to the second medium, cell, or tissue. In some embodiments, wherein the linker unit (LU) of the library does not contain at least one cleavage site (e.g., non-cleavable), the method includes: a) contacting an expression antibody of the library with a first medium, cell, or tissue expressing the target; b) contacting a parent antibody lacking the masking peptide with the first medium, cell, or tissue expressing the target; c) contacting an expression antibody of the library with a second medium, cell, or tissue, wherein the target concentration in the second medium, cell, or tissue is lower than that in the first medium, cell, or tissue; d) contacting a parent antibody lacking the masking peptide with a second medium, cell, or tissue expressing the target; and e) selecting an expression antibody having the following characteristics: (i) lower binding capacity to the first and second mediums, cells, or tissues compared to the parent antibody; and (ii) higher binding capacity to the first medium, cell, or tissue compared to the second medium, cell, or tissue.

[0213] In some embodiments, the masked antibodies or proteins of this disclosure are context-dependent (e.g., activated in certain contexts, such as in a protease-rich tumor microenvironment (TME) or a TME with high concentrations of target antigens, and are only able to bind to their targets). In some embodiments, the masked antibodies of this disclosure provide improved safety compared to conventional unmasked antibodies (e.g., exhibiting reduced toxicity, not inducing significant changes in the weight of many organs, and not altering liver histopathology, hematology, and / or blood biochemistry, etc.). In some embodiments, the masked antibodies of this disclosure have improved pharmacokinetic properties compared to conventional unmasked antibodies (e.g., having a longer in vivo half-life).

[0214] In some embodiments, the masked antibody or protein of this disclosure includes (e.g., from the N-terminus to the C-terminus) a) a masking peptide comprising a masking unit (MU) and a linker 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 this disclosure includes (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 linker 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 a corresponding antibody or protein lacking the MU to the target and / or compared to the binding of the ABD to the target. In some embodiments, the masking unit (MU) has a masking efficiency of at least about 2.0 before activation (e.g., removing the MU from the antibody by lysing the LU; or contacting the masked antibody with a medium, cells, tissue, or tumor having a high concentration of target antigen). (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, at least about 75, at least about 100, at least about 150, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 1500, at least about 2000, at least about 2500, at least about 3000, at least about 3500, at least about 4000, at least about 4500, or at least about 5000).

[0215] In some embodiments, masking efficiency is measured as the difference in affinity of a masked antibody containing a masking unit (MU) for the target antigen before activation (e.g., before the MU is removed from the antibody by cleaving the LU; or before contacting the masked antibody with a culture medium, cells, tissue, or tumor having a high concentration of the target antigen) relative to the affinity of an antibody lacking the MU for the target antigen, or the difference in affinity of a masked antibody containing the MU for the target antigen (before activation) relative to the affinity of the antibody for the target antigen after activation (e.g., after the MU is removed from the antibody by cleaving the LU; or after contacting the masked antibody with a culture medium, cells, tissue, or tumor having a high concentration of the target antigen). In some embodiments, masking efficiency is measured by the binding of the masked antibody containing the masking unit (MU) to the EC. 50 (Before activation, e.g., before removing MU from the antibody by lysing LU; or before contacting the masked antibody with a culture medium, cells, tissue, or tumor containing a high concentration of target antigen) divided by the EC of the parent antibody.50 To measure (e.g., an unmasked antibody has the same ABD as the masking peptide but lacks the masking unit). In some implementations, EC 50 Measured by ELISA. In some implementations, the masking unit (MU) binds to the ABD and prevents the masked peptide from binding to its target.

[0216] In some embodiments, when the antibody is activated (e.g., by removing the masking unit (MU) from the antibody via a cleavage linker (LU); or when the antibody comes into contact with a medium, cell, tissue, or tumor having a high concentration of the target antigen), if the antibody's binding affinity to its target increases by at least about 2-fold (e.g., at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold), 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, 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, the antibodies of this disclosure are generally considered to be masking antibodies or activatable antibodies. In some embodiments, if the EC of the peptide is activated (e.g., by removing the masking unit (MU) from the antibody through cleavage linker (LU); or by contacting the antibody with a medium, cell, tissue, or tumor having a high concentration of target antigen), 50Reduce by 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, at least 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 about 250 times, at least about 500 times, at least about 750 times, 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, then the polypeptides of this disclosure are generally considered "activatable". In some embodiments, if the EC of the polypeptide is activated (e.g., by removing the masking unit (MU) from the antibody through cleavage linking unit (LU); or by contacting the antibody with a medium, cell, tissue, or tumor having a high concentration of target antigen), then the polypeptide is considered "activatable". 50 If the reduction is at least approximately 2-fold, then the antibody disclosed herein is generally considered "activatable". 50 It can be measured by, for example, ELISA or FACS analysis.

[0217] In some implementations, when the masking unit binds to the antigen-binding domain of the masked antibody, the antibody targets its K... D The K value of the antibody when the masking unit does not bind to the antigen-binding domain is approximately equal to the K value of the antibody. D Twice the amount of the target antigen (e.g., about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 25, about 50, about 75, about 100, about 250, about 500, about 750, or about 1000 or more) (e.g., after removing the masking unit by cleaving the linker; or after contact with a medium, cell, tissue, or tumor having a high concentration of the target antigen). In some embodiments, when the masking unit binds to the antigen-binding domain of the masked antibody, the antibody targets its K... D Approximately K of maternal antibodies DTwice as much (e.g., about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 25, about 50, about 75, about 100, about 250, about 500, about 750, about 1000, about 2000, about 2500, about 3000, about 3500, about 4000, about 4500, about 5000, about 5500, about 6000, about 6500, or about 7000 or more) (e.g., an unmasked antibody having the same antigen-binding domain (ABD) as the masking peptide but lacking the masking unit).

[0218] In some embodiments, the masking unit spatially impedes the binding of the masked antibody to its target, and / or allosterically impedes 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.

[0219] In some implementations, the dissociation constant of the masking unit to the antigen-binding domain (ABD) is greater than the dissociation constant of the masked antibody to the target (when in the activated form, for example, after the MU is removed from the antibody by cleavage linker (LU); or after the antibody comes into contact with a medium, cell, tissue or tumor having a high concentration of the target antigen). In some implementations, the dissociation constant of the masking unit to the antigen-binding domain is approximately twice that of the dissociation constant of the masked antibody to the target (when in activated form) (e.g., about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 25, about 50, about 75, about 100, about 250, about 500, about 750, about 1000, about 2000, about 2500, about 3000, about 3500, about 4000, about 4500, about 5000, about 5500, about 6000, about 6500, or about 7000 or more). In some embodiments, the dissociation constant of the masking unit to the antigen-binding domain is approximately equal to the dissociation constant of the masked antibody to the target (when in activated form). In some embodiments, the masking unit (MU) binds to the antigen-binding domain and prevents the peptide from binding to its target only when the peptide has not been activated (e.g., the MU has not yet been removed from the antibody by cleavage of the LU; or the antibody has not yet come into contact with a medium, cell, tissue, or tumor having a high concentration of the target antigen). In some embodiments, activation induces peptide cleavage within the cleavage site of the linker unit. In some embodiments, activation induces a conformational change in the peptide (e.g., a translocation of the masking unit (MU)) such that the masking peptide no longer prevents the peptide from binding to its target.

[0220] The masked antibodies described herein may be further modified. In some embodiments, those masked antibodies are linked to other molecular entities. Examples of other molecular entities include pharmaceutical formulations, peptides or proteins, detection agents or tags, and antibodies.

[0221] In some embodiments, the masked antibody of this disclosure is linked to a pharmaceutical formulation. Examples of pharmaceutical formulations include cytotoxic agents or other cancer therapeutic agents, as well as radioactive isotopes. Specific examples of cytotoxic agents include paclitaxel, cytochalasin B, bacitracin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, anthraquinone, mitoxantrone, mirtamicin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as their analogues or homologs. Therapeutic agents also include, for example, antimetabolites (such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil azithromycin), alkylating agents (such as dichloromethyldiethylamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamineplatin(II) (DDP) cisplatin), anthracyclines (such as daunorubicin (formerly known as doxorubicin) and doxorubicin), antibiotics (such as dermatomycin (formerly known as actinomycin), bleomycin, mirtramycin and atrazomycin (AMC)), and antimitotic agents (such as vincristine and vinblastine). Examples of radioisotopes that can be conjugated to antibodies for diagnostic or therapeutic purposes include, but are not limited to, iodine-131, indium-111, yttrium-90 and lutetium-177. Methods for attaching peptides to pharmaceutical formulations are known in the art, such as the use of various linker techniques. Examples of linker types include hydrazone, thioether, ester, disulfide, and peptide-containing linkers. For further discussion on the linkers and methods for attaching therapeutic agents to antibodies, see, for example, Saito et al., Adv. Drug Deliv. Rev. 55:199-215 (2003); Trail et al., Cancer Immunol. Immunother. 52:328-337 (2003); Payne, Cancer Cell 3:207-212 (2003); Allen, Nat. Rev. Cancer 2:750-763 (2002); Pastan and Kreitman, Curr. Opin. Investig. Drugs 3:1089-1091 (2002); Senter and Springer (2001) Adv. Drug Deliv. Rev. 53:247-264.

[0222] IV. Masked Antibodies Other aspects of this disclosure relate to masked antibodies (e.g., activatable antibodies) and derivatives thereof selected from the libraries described herein. In some embodiments, the masked antibody comprises an antibody and a masking peptide (MP). The masking peptide may comprise a masking unit (MU) and a linker unit (LU) from the N-terminus to the C-terminus. The MU may comprise or consist of the amino acid sequence shown in formulas (I), (II), (III), (IV), or (V) as 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 comprises one or more linkers. In some embodiments, the LU comprises one or more cleavage sites, such as protease cleavage sites. In some embodiments, the LU comprises one or more linker sequences in addition to one or more cleavage sites. In some embodiments, the LU comprises one or more linkers without cleavage sites. In some embodiments, the antibody comprises an antibody heavy chain variable region (VH) and / or an antibody light chain variable region (VL). In some embodiments, the masking peptide is linked to the N-terminus of the antibody's VH or VL. 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-chain variable fragment (scFv). In other embodiments, the masked antibody comprises a VHH single-domain fragment. In some embodiments, the masked antibody comprises a VHH-Fc antibody. In some embodiments, the masked antibody is expressed on the cell surface (e.g., displayed in yeast or mammalian cells). In some embodiments, the masked antibody is an activatable antibody.

[0223] In one aspect, a masked antibody is provided, comprising an antibody and a masking peptide (MP), wherein the antibody includes a heavy chain variable region (VH) and / or a light chain variable region (VL), and the MP is linked to the N-terminus of the VH or VL of the antibody, wherein the MP includes a masking unit (MU) and a linker unit (LU), wherein the MU contains an amino acid sequence according to formula (I): X1X2CX3(X m ) n X4X5CX6X7, where: n is 2-8. 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; 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 X... mIndependently, amino acids are 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 selected from the group consisting of A, D, F, H, I, L, N, P, S, T, V, and Y.

[0224] 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. C stands for cysteine.

[0225] In some implementations, 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 some implementations, n is 2, 3, 4, 5, 6, 7, or 8.

[0226] In one aspect, this article provides a masked antibody 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), and the MP is linked to the N-terminus of the VH or VL of the antibody, wherein the MP comprises a masking unit (MU) and a linker unit (LU), wherein 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. The amino acids in the groups are: X8 is selected from the amino acids in the group composed of A, E, G, I, K, L, P, Q, R, S, T and V; X9 is selected from the amino acids in the group composed of A, E, G, I, K, L, P, Q, R, S, T and V.

[0227] C stands for cysteine.

[0228] 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.

[0229] In some implementations, MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-10.

[0230] In one aspect, this article provides a masked antibody 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), and the MP is linked to the N-terminus of the VH or VL of the antibody, wherein the MP comprises a masking unit (MU) and a linker unit (LU), wherein the MU comprises an amino acid sequence according to formula (III): X1X2CX3X4X5X6X7X8CX9X 10 ,in: X1 is an amino acid group composed of A, D, F, H, I, L, N, P, S, T, V, and Y; X2 is an amino acid group composed of A, D, F, H, L, P, S, V, and Y; X3 is an amino acid group composed of A, E, G, I, K, L, P, Q, R, S, T, and V; X4 is an amino acid group composed of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y; X5 is an amino acid group composed of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y; X6 is an amino acid group composed of A, D, E, F, G, H, I, K, L, P, R, S, T, V, and Y; X7 is an amino acid group composed of A, D, F, H, I, L, N, P, S, T, V, and Y. The amino acids in the group composed of Y, X8 are amino acids selected from the group composed of A, D, F, H, L, P, S, V and Y, and X9 are amino acids selected from the group composed of A, E, G, I, K, L, P, Q, R, S, T and V. 10 It is an amino acid group selected from A, E, G, I, K, L, P, Q, R, S, T and V.

[0231] C stands for cysteine.

[0232] 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; X... 10 It is a group of amino acids selected from G, Q, R, S, T and V.

[0233] In some implementations, MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-19.

[0234] In one aspect, a masked antibody is provided, comprising an antibody and a masking peptide (MP), wherein the antibody includes a heavy chain variable region (VH) and / or a light chain variable region (VL), and the MP is linked to the N-terminus of the VH or VL of the antibody, wherein the MP includes a masking unit (MU) and a linker unit (LU), wherein the MU comprises an amino acid sequence according to formula (IV): X1X2CX3X4X5X6X7X8X9CX 10 X 11 ,in: 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. The amino acids in the groups X7, X8, and X9 are selected from the groups composed of A, D, E, F, G, H, I, K, L, P, R, S, T, V, and Y. 10It is an amino acid group selected from A, E, G, I, K, L, P, Q, R, S, T, and V, X 11 It is a group of amino acids selected from A, E, G, I, K, L, P, Q, R, S, T and V.

[0235] C stands for cysteine.

[0236] 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; X 10 It is an amino acid group composed of I, P, and R, X 11 It is an amino acid group composed of E, G, I, P and V.

[0237] In some implementations, MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-27.

[0238] In one aspect, this article provides a masked antibody 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), and the MP is linked to the N-terminus of the VH or VL of the antibody, wherein the MP comprises a masking unit (MU) and a linker unit (LU), wherein the MU comprises an amino acid sequence according to formula (V): X1X2CX3X4X5X6X7X8X9X 10 CX 11 X 12 ,in: 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. The amino acids in the groups X7, X8, and X9 are selected from the groups composed of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. 10 It is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y, X 11 It is an amino acid group composed of A, E, G, I, K, L, P, Q, R, S, T, and V, X 12 It is an amino acid group selected from A, E, G, I, K, L, P, Q, R, S, T and V.

[0239] C stands for cysteine.

[0240] In some implementations, 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; X... 10 It is an amino acid group composed of A, F, V, and Y, X 11 It is an amino acid selected from the group consisting of G, I, K, L, and R, X 12 It is a group of amino acids selected from A, E, K, P, R and T.

[0241] In some embodiments, MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-32 and 112-114.

[0242] In one aspect, 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) capable of specifically binding to CTLA4 in the absence of MP, and the MP is linked to the N-terminus of the VH or VL of the antibody, wherein the MP comprises a masking unit (MU) and a linker unit (LU), wherein the MU comprises an amino acid sequence according to formula (V): X1X2CX3X4X5X6X7X8X9X 10 CX 11 X 12 ,in: 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. The amino acids in the groups X7, X8, and X9 are selected from the groups composed of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. 10 It is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y, X 11 It is an amino acid group composed of A, E, G, I, K, L, P, Q, R, S, T, and V, X 12 It is an amino acid group selected from A, E, G, I, K, L, P, Q, R, S, T and V.

[0243] C stands for cysteine.

[0244] In some implementation schemes, X1 is an amino acid selected from the group composed of P, S, and Y; X2 is an amino acid selected from the group composed of H, P, and S; X3 is an amino acid selected from the group composed of E, K, Q, and R; X4 is an amino acid selected from the group composed of P, R, V, and Y; X5 is an amino acid selected from the group composed of A, F, G, and Y; X6 is an amino acid selected from the group composed of L, P, and V; X7 is an amino acid selected from the group composed of H, K, P, and R; X8 is an amino acid selected from the group composed of A, K, L, and P; X9 is an amino acid selected from the group composed of D, F, L, and P. 10 It is an amino acid group selected from A, F, and Y, X 11 It is an amino acid selected from the group consisting of I, K, L, and R, X 12 It is an amino acid group selected from E, K and R.

[0245] In some implementations, MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-32.

[0246] In one aspect, 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) capable of specifically binding to CD137 in the absence of MP, and the MP is linked to the N-terminus of the VH or VL of the antibody, wherein the MP comprises a masking unit (MU) and a linker unit (LU), wherein the MU comprises an amino acid sequence according to formula (V): X1X2CX3X4X5X6X7X8X9X 10 CX 11 X 12 ,in: 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. The amino acids in the groups X7, X8, and X9 are selected from the groups composed of A, D, E, F, G, H, I, K, L, P, Q, R, S, T, V, and Y. 10 It is an amino acid selected from the group consisting of A, D, F, H, L, P, S, V, and Y, X 11 It is an amino acid group composed of A, E, G, I, K, L, P, Q, R, S, T, and V, X 12 It is an amino acid group selected from A, E, G, I, K, L, P, Q, R, S, T and V.

[0247] C stands for cysteine.

[0248] 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; X 10 It is an amino acid group selected from F and V, X 11 It is an amino acid selected from the group consisting of G, I, and R, X 12 It is an amino acid group selected from A, P and T.

[0249] In some embodiments, MU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 112-114.

[0250] In some embodiments described herein, the MU of the masking antibody does not contain the amino acid sequence of M, NG, DG, NXS, NXT, or any combination thereof.

[0251] In some embodiments of the masked antibody described herein, the masking peptide (MP) is linked to the N-terminus of the VL. In other embodiments of the masked antibody described herein, the MP is linked 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 linked 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 linked 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 linked to the N-terminus of the VH of the antibody.

[0252] In some embodiments of the masked antibody described herein, the antibody comprises an scFv containing a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the MP is linked to the VL of the scFv. In some embodiments, the MP is linked to the VH of the scFv.

[0253] In some embodiments of the masked antibody described herein, the antibody comprises a Fab fragment containing a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the MP is linked to the VL of the Fab. In some embodiments, the MP is linked to the VH of the Fab.

[0254] In some embodiments described herein, the masked antibody includes a heavy chain variable region (VH) with an MP linked to the N-terminus of the VH. In some variants, the antibody does not include a light chain variable region (VL). In some embodiments, the antibody is a VHH single-domain antibody (also known as a nanobody) containing a heavy chain variable region (VH) with an MP linked to the N-terminus of the VH. In some embodiments, the antibody is a VHH-Fc antibody containing a heavy chain variable region (VH) with an MP linked to the N-terminus of the VH.

[0255] In some aspects, this document provides polynucleotides encoding the masked antibodies disclosed herein. In some aspects, this document provides vectors (e.g., expression vectors and / or display vectors) comprising one or more polynucleotides (e.g., synthetic polynucleotides) encoding the masked antibodies disclosed herein. In some embodiments, the antibody is fused with all or part of a protein (e.g., viral capsid proteins, bacterial surface proteins, yeast surface proteins, insect cell surface proteins, mammalian cell surface proteins) (i.e., creating a fusion protein). In some embodiments, the fusion protein is displayed on the surface of a particle or a host cell. In some aspects, this document provides host cells and particles (e.g., bacteriophages) displaying the masked antibodies described herein.

[0256] This document also provides a method for providing and assembling polynucleotides (e.g., synthetic polynucleotides) encoding masked antibodies of the present disclosure. Polynucleotides encoding antibodies with masking peptides can be cloned into any suitable vector to express a portion or the entire polypeptide sequence of the masked antibody. In some embodiments, the polynucleotide is cloned into a vector, thereby allowing the generation of masked antibodies (i.e., the creation of fusion proteins) fused to all or part of a protein (e.g., a viral capsid protein, a bacterial surface protein, a yeast surface protein, an insect cell surface protein, or a mammalian cell surface protein) and displayed on the surface of particles or cells. Several types of vectors are available and can be used to implement the present disclosure, such as phageparticle vectors. Phageparticle vectors typically contain multiple components, including a promoter, a signal sequence, a phenotypic selection gene, an origin of replication site, and other essential components as known to those skilled in the art. In some embodiments, polynucleotides encoding masked antibodies can be cloned into a vector to achieve bacterial display by expression in bacterial cells or yeast display by expression in yeast cells. Exemplary vectors are as described above and are described in U.S. Pre-Publication No. US20160145604.

[0257] Certain aspects of this disclosure relate to a cell population containing one or more vectors encoding one or more polynucleotides, which in turn encode 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 the masked antibodies can be performed, 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 described in the references cited herein. The expression of proteins and screening for target masked antibodies in these cell types (e.g., from libraries of this disclosure) are described in more detail below.

[0258] Alternatively, the polynucleotide can be expressed in an *E. coli* expression system such as that 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 the bacterial cytoplasm, as described by Better and Horwitz, *Meth. Enzymol., 1989, 178: 476*. In some embodiments, the masking antibody is attached to the 3′ end of a sequence encoding a signal sequence, such as the ompA, phoA, or pelB signal sequence (Lei et al., *J. Bacteriol.*, 1987, 169: 4379). These gene fusions are assembled in a bicistronic construct, thus allowing them to be expressed from a single vector and secreted into the periplasmic space of *E. coli*, where they will refold and can be recovered in their active form. (Skerra et al., Biotechnology, 1991, 9: 273).

[0259] In other embodiments, using the secretion signaling and lipidation portions described, for example, in US20040072740; US20030100023; and US20030036092, the masking antibody of this disclosure is expressed on the membrane surface of prokaryotes such as Escherichia coli.

[0260] Alternatively, the masking antibodies of this disclosure can be expressed and screened by anchoring periplasmic expression (APEx two-hybrid surface display), as described in Jeong et al., PNAS, 2007, 104: 8247, or by other anchoring methods, as described in Mazor et al., Nature Biotechnology, 2007, 25: 563.

[0261] Higher eukaryotic cells, such as mammalian cells, including myeloma cells (e.g., NS / O cells), hybridoma cells, Chinese hamster ovary (CHO) cells, and human embryonic kidney (HEK) cells, can also be used to express the masking antibodies of this disclosure. Masking antibodies expressed in mammalian cells can be engineered to be secreted into a culture medium or expressed on the cell surface.

[0262] Certain aspects of this disclosure relate to a non-human animal comprising the polynucleotides or polynucleotide libraries of this disclosure. For example, the non-human animal of this disclosure may be modified such that its genome includes polynucleotides encoding the masked antibodies of this disclosure. In some embodiments, the transgenic animal (e.g., a mouse) expresses the masked antibodies described herein. Techniques for modifying the genome of a non-human animal are known in the art (e.g., methods for generating Xenomouse™).

[0263] In some embodiments described herein, the masking peptide of the masking antibody further comprises an N-terminal unit. In some embodiments, the N-terminal unit comprises or consists of 1-12 amino acid residues. In some embodiments, the N-terminal unit is attached to the N-terminus of the masking unit. In some embodiments, the N-terminal unit comprises amino acid E, or peptides EVG, EVGSY (SEQ ID NO: 33), EVGVLDV (SEQ ID NO: 86), or EVGAESGVK (SEQ ID NO: 88). In some embodiments, the N-terminal unit is composed of amino acid E, or peptides EVG, EVGSY (SEQ ID NO: 33), EVGVLDV (SEQ ID NO: 86), or EVGAESGVK (SEQ ID NO: 88).

[0264] In some embodiments described herein, the linker unit (LU) of the masked antibody comprises one or more linkers. Any suitable linker known in the art (e.g., flexible linkers) can be used, including, for example: glycine polymer (G)n, where 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.); glycine-serine polymer (GS)n, where 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 GS, GGS, GGG, SGRG, GGGGS, GGGGT, SGGS, GGSG, GGSGG, GGSGG, GGGSG, GGSSG, GGSGS, GGSGS, SGGG, GGGS and / or GGGGSGGSGGGS; glycine-alanine polymer; alanine-serine polymer; and the like. The linker sequence can be of any length, for example, from about 1 amino acid (e.g., glycine or serine) to about 20 amino acids (e.g., a 20-amino acid glycine polymer or a 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 length is any of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

[0265] In some embodiments of the masked antibody described herein, the linker unit (LU) comprises one or more adapters described herein and does not contain any cleavage site. In some embodiments, the linker unit comprises a non-cleavable adapter. In some embodiments, the non-cleavable adapter comprises the amino acid sequence of SEQ ID NO: 81.

[0266] In some embodiments described herein, the linker unit (LU) of the masked antibody includes one or more cleavage sites. In some embodiments, the LU includes at least one 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 (e.g., a protease known to co-localize with a target containing a polypeptide) known to be recognized and / or cleaved by any protease known in the art can be used, including, for example, protease cleavage sites recognized and / or cleaved by: 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 etching virus (TEV) protease; plasmin; thrombin; PSA; PSMA; ADAMS / ADAMTS (e.g., ADAM 8, ADAM 9, ADAM 10 ... 9. ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMDEC1, ADAMTS1, ADAMTS4 and / or ADAMTS5; Casparases (e.g., Casparase-1, Casparase-2, Casparase-3, Casparase-4, Casparase-5, Casparase-6, Casparase-7, Casparase-8, Casparase-9, Casparase-10, Casparase-11, Casparase-12, Casparase-13 and / or Casparase-14); Aspartic proteases (e.g., RACE and / or renin); Aspartic 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 ​​proteases (e.g., Cruzipain, podin and / or Otubain-2); KLKs (e.g., KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13 and / or KLK14); metalloproteinases (e.g., methyldopa, enkephalin, PSMA and / or BMP-1);Serine proteases (e.g., activated protein C, cathepsin A, cathepsin G, gastrinase and / or coagulation factor proteases (e.g., FVIIa, FIXa, FXa, FXIa, FXIIa)); elastase; granzyme B; guanidinobenzoic acidase; HtrA1; human neutrophil elastase; lactoferrin; marapsin; NS3 / 4A; PACE4; tPA; trypsin-like proteins; type II transmembrane serine proteases (TTSPs) (e.g., DESC1, DPP-4, FAP, hepsin, Matriptase-2, MT-SP1 / Matriptase, TMPRSS2, TMPRSS3 and / or TMPRSS4); etc. In some embodiments, the first protease cleavage site is a cleavage site selected from the following proteases: 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, caspases-1, caspases-2, caspases-3, caspases-4, caspases-5, caspases-6, caspases-7, caspases-8, caspases-9, caspases-10, caspases-11, caspases-12, caspases-13, caspases-14, and TACE. In some embodiments, the first protease cleavage site is a cleavage site selected from the following proteases: uPA, MMP-2, MMP-9, and / or TEV protease. In some embodiments, the protease cleavage site comprises the amino acid sequence SGRSA (SEQ ID NO: 34), the amino acid sequence PLGLAG (SEQ ID NO: 35), or a combination thereof.

[0267] In some embodiments of the masked antibody described herein, the LU further comprises a first adapter (L1). In some embodiments, the first adapter (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 comprises the structure (CS1)-L1 from the N-terminus to the C-terminus. In some embodiments, the LU further comprises a second adapter (L2). In some embodiments, L2 is located at the C-terminus of a second cleavage site (CS2). In some embodiments, the LU comprises the structure (CS1)-L1-(CS2)-L2 from the N-terminus to the C-terminus. In some embodiments, the first adapter (L1) is located at the N-terminus of the first cleavage site (CS1). In some embodiments, the LU comprises the structure L1-(CS1)-L2-(CS2) from the N-terminus to the C-terminus. In some embodiments, the LU comprises the structure L1-(CS1)-L2-(CS2) 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.

[0268] In some embodiments of the masked antibody described herein, the masking peptide comprises the structure MU-(CS1)-L1 from the N-terminus to the C-terminus. In some embodiments of this disclosure, the masking peptide comprises the structure (MU)-(CS1)-L1-(CS2)-L2 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.

[0269] In some embodiments, the masking peptide comprises the following structure from the N-terminus to the C-terminus: MU-L1-(CS1)-L2. In some embodiments, the masking peptide comprises the following structure from the N-terminus to the C-terminus: MU-L1-(CS1)-L2-(CS2). In some embodiments, the masking peptide of this disclosure comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 68-75, 83-85, 87, and 116-118.

[0270] In some embodiments, the masked antibody comprises an antigen-binding domain (ABD). In some embodiments, the ABD comprises a variable region of the antibody light chain and / or a variable region of the antibody heavy chain. In some embodiments, the ABD comprises both a variable region of the antibody light chain and a variable region of the antibody heavy chain. In some embodiments, the ABD comprises a variable region of the antibody heavy chain and does not comprise a variable region of the antibody light chain. In some embodiments, the ABD of this disclosure comprises a variable region of the antibody light chain and / or a variable region of the antibody heavy chain that is specific to 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.

[0271] In some embodiments, the masked antibody comprises a full-length antibody light chain and / or a full-length antibody heavy chain. The antibody light chain may be a κ light chain or a λ light chain. The antibody heavy chain may be of any class, such as IgG, IgM, IgE, IgA, or IgD. In some embodiments, the antibody heavy chain belongs to an IgG class, such as a subclass of IgG1, IgG2, IgG3, or IgG4. Using methods known in the art, the antibody heavy chain described herein can be changed from one class or subclass to another.

[0272] The masking antibodies described herein may include any HVR sequence (e.g., one, two, or three of the heavy chain variable region HVR sequences, and / or one, two, or three of the 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 numbers PCT / CN2017 / 098333 (in whole or in part, by reference), PCT application numbers PCT / CN2017 / 098299 (in whole or in part, by reference), PCT application numbers PCT / CN2017 / 098332 (in whole or in part, by reference), and / or U.S. Patent Application Publication No. 2021 / 0206855 (in whole or in part, by reference).

[0273] The masking antibodies described herein may incorporate any HVR sequence (e.g., one, two, or three of the heavy chain variable region HVR sequences, and / or one, two, or three of the light chain variable region HVR sequences), heavy chain variable region sequences, and / or light chain variable region sequences of any antibody described herein (e.g., anti-CTLA4 antibody, anti-CD137 antibody).

[0274] In some embodiments, the masked antibody is an anti-CTLA4 antibody comprising (a) an antibody light chain variable region comprising HVR-L1 containing the amino acid sequence RASQSVRGRFLA (SEQ ID NO: 95), HVR-L2 containing the amino acid sequence DASNRATGI (SEQ ID NO: 96), and / or HVR-L3 containing the amino acid sequence YCQQSSSWPPT (SEQ ID NO: 97); and (b) an antibody heavy chain variable region comprising HVR-H1 containing the amino acid sequence YSISSGYHWSWI (SEQ ID NO: X98), HVR-H2 containing the amino acid sequence LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 99), and / or HVR-H3 containing the amino acid sequence ARSYVYFDY (SEQ ID NO: 100). In some embodiments, the masked antibody comprises an antibody light chain variable region containing 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 containing 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 comprises an antibody light chain variable region containing 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 containing 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 comprises an antibody light chain variable region containing the amino acid sequence of SEQ ID NO: 93, and an antibody heavy chain variable region containing the amino acid sequence of SEQ ID NO: 94.

[0275] The masked antibodies disclosed herein can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In some embodiments, an isolated nucleic acid encoding any masked antibody is provided. The nucleic acid may encode a V containing the antibody. L and / or V containing antibodies H The amino acid sequence (e.g., the light and / or heavy chains of an antibody). In some embodiments, one or more vectors (e.g., expression vectors) containing such nucleic acids are provided herein. In some embodiments, a host cell containing said nucleic acid is provided. In one embodiment, the host cell contains (e.g., transformed with the following) one or two vectors encoding the masked antibody described herein. In some embodiments, the host cell is a eukaryotic cell, such as yeast cells, insect cells, Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NSO, Sp20 cells), plant cells, or bacterial cells. In some embodiments, a method for preparing a masked antibody is provided, wherein the method includes culturing the host cell provided above containing the nucleic acid encoding said masked antibody under conditions suitable for expressing said masked antibody, and optionally recovering said masked antibody from said host cell (or host cell culture medium).

[0276] In any of the above embodiments, 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.

[0277] In some implementations, the masking peptide does not have one of the following sequences:

[0278] V. Composition In other aspects, this disclosure provides a composition comprising one or more masked antibodies as described herein. In some embodiments, the composition is a pharmaceutical composition comprising a polypeptide (e.g., a masked binding polypeptide, such as a masked antibody) and a pharmaceutically acceptable carrier. The composition can be prepared by conventional methods known in the art.

[0279] The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in formulations for delivering peptides (e.g., masked antibodies). Carriers can be anti-adhesives, binding agents, coating agents, disintegrants, fillers or diluents, preservatives (such as antioxidants, antibacterial agents, or antifungal agents), sweeteners, absorption delay agents, wetting agents, emulsifiers, buffers, etc. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), dextrose, vegetable oils (such as olive oil), saline, buffer solutions, buffered saline, and isotonic agents such as sugars, polyols, sorbitol, and sodium chloride.

[0280] Compositions can be in any suitable form, such as liquids, semi-solids, and solid dosage forms. Examples of liquid dosage forms include solutions (e.g., injectable and infusionable 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 peptides (e.g., masked-binding peptides, such as masked antibodies) is a sterile liquid, such as a solution, suspension, or dispersion for injection or infusion. Sterile solutions can be prepared by incorporating a peptide (e.g., a masked-binding peptide, such as a masked antibody) in a desired amount into a suitable carrier, followed by sterile microfiltration. Dispersions can be prepared by incorporating a peptide (e.g., a masked-binding peptide, such as a masked antibody) into a sterile medium containing a basic dispersion medium and other carriers. For sterile powders used to prepare sterile liquids, preparation methods include vacuum drying and freeze-drying (lyophilization) to produce a powder of the active ingredient plus any additional desired components from its previously sterile filtered solution. Various dosage forms of the composition can be prepared using conventional techniques known in the art.

[0281] The relative amount of peptides (e.g., masked antibodies) included in a composition will vary depending on many factors, such as the specific peptide and carrier used, the dosage form, and the desired release and pharmacokinetic characteristics. The amount of peptide (e.g., masked binding peptides, such as masked antibodies) in a single dosage form will typically be the amount that produces a therapeutic effect, but may also be a smaller amount. Generally, this amount will range from about 0.01% to about 99%, from about 0.1% to about 70%, or from about 1% to about 30% relative to the total weight of the dosage form.

[0282] In addition to peptides (e.g., masking antibodies), the composition may also include one or more additional therapeutic agents. Examples of additional therapeutic agents are described below. The appropriate amount of additional therapeutic agent to be included in the composition can be readily selected by those skilled in the art and will vary depending on many factors, such as the specific pharmaceutical agent and carrier used, the dosage form, and the desired release and pharmacokinetic characteristics. The amount of additional therapeutic agent included in a single dosage form is typically the amount of the pharmaceutical agent that produces the therapeutic effect, but can also be a smaller amount.

[0283] Any peptides (e.g., masked antibodies) and / or compositions (e.g., pharmaceutical compositions) described herein may be used to prepare agents (e.g., agents for treating cancer in subjects in need or for delaying its progression).

[0284] VI. Reagent Kit On the other hand, this document provides a kit comprising polynucleotides, peptides, antibodies with masking peptides, or cells or phages displaying peptides or antibodies with masking peptides of the present disclosure. In some embodiments, the kit further includes a packaging insert containing instructions on expressing, modifying, screening, or otherwise using a library, for example, to identify a target masking antibody. In some embodiments, the kit further includes one or more buffers, for example, for storing, transferring, transfecting, or otherwise using one or more polynucleotides (e.g., synthetic polynucleotides). In some embodiments, the kit further includes one or more containers for storing one or more polynucleotides. In some embodiments, the kit further includes one or more vectors, for example, for transfecting host cells with one or more polynucleotides.

[0285] On the other hand, this document provides a kit comprising the peptides and / or compositions described herein. In some embodiments, the kit further includes a packaging insert containing instructions for using selected masked binding peptides (e.g., masked antibodies), antibodies, and / or compositions. In some embodiments, the kit further includes one or more buffers, such as those for storing, transferring, administering, or otherwise using the masked binding peptides (e.g., masked antibodies) and / or compositions. In some embodiments, the kit further includes one or more containers for storing or administering (e.g., using a syringe, etc.) the masked binding peptides, antibodies, and / or compositions.

[0286] The foregoing written description is considered sufficient to enable those skilled in the art to implement this disclosure. The following examples are provided for illustrative purposes only and are not intended to limit the scope of this disclosure in any way. In fact, various modifications to this disclosure, other than those shown and described herein, will become apparent to those skilled in the art based on the foregoing description and fall within the scope of the appended claims.

[0287] Example Example 1: A method for identifying masking peptides of masked antibodies As mentioned above, there is a need for improved methods and products for identifying masking peptides (MPs) of masked antibodies. Based on data analysis of antibody structural motifs and an accumulated SAFEbody set, a novel masking unit (MU) library was designed. The library design considered antibody stability, structural and chemical diversity, and downstream developability. Therefore, this paper describes an improved masking peptide library designed and implemented using highly developable masking units. The improved masking unit library possesses several unique features that make it highly effective in identifying masking units of target antibodies with high developability: 1) This library is designed to balance the chemical diversity of the MU region. Amino acids with charged side chains, such as E / D / H / K / R, are preferred (enriched) in the MU library. Together with amino acids with polar side chains, S / T / N / Q / Y, these charged and polar amino acids constitute the bulk of the library composition.

[0288] 2) The design of the peptide library included the identification of stable motifs that showed high frequency in data analysis. Key residues for early chain compression during folding, such as glycine and proline, were intentionally retained in the loop region to accelerate loop formation.

[0289] 3) The library was designed to avoid high-risk post-translational modification (PTM) sites, including free cysteine ​​residues, glycosylation sites, deamidation sites, aspartate isomerization sites, and oxidation sites. For example, the new MU library excludes NG, DG, and NX[S / T] sites.

[0290] This article provides a multinucleotide library (SAFEbody masking unit library) containing a polypeptide encoding a peptide, wherein the polypeptide includes a masking peptide (MP) and an antigen-binding domain (ABD); wherein the MP includes a masking unit (MU) and a linker unit (LU) from the N-terminus to the C-terminus; wherein the MP is linked to the N-terminus of the ABD; wherein the MU contains an amino acid sequence according to formula (I).

[0291] X1X2CX3(X m ) n X4, X5, X6, X7 Where n is between 2 and 8, and 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 X mAmino acids 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 represents the amino acid cysteine.

[0292] Four exemplary constrained peptide libraries (CPLs) were designed to identify potential masking units (MUs) (Table 2A). The diversity of the libraries (possible peptide combinations) ranged from approximately 5 x 10^6. 9 Up to 2x10 13 Their composition is listed in Table 2B below.

[0293] Table 2A: Diversity of Masking Units in Library Design

[0294] 1 X represents an amino acid; the subscript indicates the number of amino acids at each position. 2 C represents the cysteine ​​amino acid residue. Table 2B: Amino acid composition at each position in the masking unit (MU) library

[0295] 1 X represents an amino acid; the subscript indicates the number of amino acids at each position.

[0296] 2 C represents the cysteine ​​amino acid residue.

[0297] For optimization purposes, two cleavage peptide sequences were introduced into the linker unit of the construct, immediately following these masking unit sequences. The protease recognition sites introduced during library construction were: SGRSA (SEQ ID NO: 34) for urokinase-type plasminogen activator (uPA) protease and PLGLAG (SEQ ID NO: 35) for matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) proteases. These recognition sites have been used by numerous research groups for in vivo tumor cell-specific activation of the target (see, for example, Ke et al. (1997) J Biol Chem 272(33):20456-62; Gerspach et al. (2006) Cancer Immunol Immunother 55(12):1590-600; and Jiang et al. (2004) Proc Natl Acad Sci USA 101(51):17867-72).

[0298] The masking peptide also includes an N-terminal unit fused to the C-terminus of the masking unit. In this embodiment, EVGSY (SEQ ID: 33) is used as the N-terminal unit.

[0299] To identify masking peptide sequences that can effectively mask parental antibodies, the N-terminus of the variable region of the light chain of the anti-CTLA4 scFv antigen-binding domain of the parental antibody was fused to the C-terminus of the masking peptide. In this embodiment, the masking units in the improved peptide library were directly fused to the N-terminus of the light chain of the parental antigen-binding domain, and a yeast library displaying the fusion protein on the surface of yeast cells was constructed. The yeast library then underwent multiple rounds of FACS-based screening: first, yeast clones with low antigen-binding affinity were enriched; then, the enriched yeast clones were treated with protease to remove the masking units, and clones with high antigen-binding affinity were selected. After 4-5 rounds of screening, plasmids were extracted from these clones, and the masking unit sequences were confirmed by DNA sequencing. Table 3 lists the enrichment of certain amino acids when optimizing the masking units in the four libraries. Tables 4A-D list exemplary masking units generated by library screening. Table 5 lists exemplary masked peptide sequences after library selection, where the N-terminal unit is fused with both the masking unit and the linker unit from the N-terminus to the C-terminus.

[0300] Table 3: Enrichment of specific amino acids after screening in each masking unit library

[0301]

[0302]

[0303]

[0304]

[0305]

[0306] Table 4D: Example sequences of occlusion units in Library 4 Table 5: Exemplary masking peptides

[0307]

[0308] 1 The underlined N-terminal unit EVGSY (SEQ ID NO:33) is attached to the N end of each shielding unit.

[0309] 2 Bold text indicates invariant cleavage sites, such as SGRSA (SEQ ID NO:34) or PLGLAG (SEQ ID NO:35); italic text indicates variable connection sequences, such as... GGGGT (SEQ ID NO:36) or SGGS (SEQ ID NO:37).

[0310] 3 The connecting unit sequence SGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 38) is attached to the C end of each shielding unit.

[0311] Masking efficiency can be further improved by optimizing the N-terminal unit sequence and linker unit. Tables 6A-6C identify the masking unit sequence (BC3855) in combination 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 linker units: SGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 38), SGRGPLGLAGGS (SEQ ID NO: 39); SGGGPLGLAGGS (SEQ ID NO: 40); GGGPLGLAGGS (SEQ ID NO: 41); GGGPLGLAGGGS (SEQ ID NO: 42); and GGGGSGGSGGGS (SEQ ID NO: 81). The resulting properties vary among the different constructs. Exemplary masking peptides with different linker units are shown in Tables 6A-6C.

[0312] Table 6a. N-terminal unit, masking unit, linker, and cleavage site sequences of antibodies with C1-L1-C2-L2 configuration linker units.

[0313]

[0314]

[0315]

[0316] Masking peptides against anti-CTLA4 were screened using the above library. Recombinant human CTLA4-Fc was diluted to 1 μg / mL in PBS and coated onto Maxisorp plates overnight at 4°C. The plates were blocked with PBS containing 3% skim milk at 37°C for 1 hour. After washing, 100 μL of a three-fold serial dilution of the antibody was added to each well. After incubation at 37°C for 1 hour, the plates were washed four times, and 100 μL of HRP-labeled anti-human IgG (Fab-specific) (1:6000 dilution) (1:6000 dilution) was added to each well. The plates were incubated at 37°C for 1 hour, washed four times, and then 50 μL of TMB substrate solution was added to each well. The plates were incubated at room temperature. After stopping the reaction with 50 μL of H2SO4 in each well, the absorbance was measured at 450 nm. The ELISA data were fitted using an asymmetric sigmoid (five-parameter logistic equation) model in GraphPad Prism 6 software to evaluate EC50. 50 Experiments were conducted once for the activatable antibodies TY24463, TY24464, TY24649, and TY24652, and twice for TY24148, TY24466, and TY24465, thus obtaining two calculated masking efficiencies for each activatable antibody. The masking efficiency for each activatable antibody was determined by estimating the EC50 of the activatable antibody. 50 EC with parental antibody (TY21580) 50 The results were calculated by division. As shown in Table 7, compared to the parental antibody, all activatable antibodies exhibited significantly reduced binding to their antigens, with calculated masking efficiencies ranging from 773 to 6925. The differences in masking efficiency may be due to EC... 50 The masking efficiency of each activatable antibody may be within the calculated range due to variations in measurement values ​​and data fitting (e.g., the masking efficiency of the activatable antibody TY22465 ranges from 3749 to 5097). These results indicate that the various masking peptides identified from CPLs maintain their masking efficiency when expressed in mammalian cells and as part of the intact IgG molecule. Furthermore, masking efficiency will vary depending on the density of the immobilized antigen or the density in the cell line used. Higher antigen concentrations result in lower masking efficiency.

[0317] Table 7. Exemplary optimized masking peptides derived by altering the N-terminal and linker units surrounding the BC3855 masking unit.

[0318]

[0319] 1 N-terminal units (E or EVGSY) are indicated by an underscore. 2 Bold text indicates unchanging cleavage sites, such as SGRSA (SEQ ID NO: 34) or PLGLAG (SEQ ID NO: 35). 3 Italics indicate variant connection sequences, such as GGGGT (SEQ ID NO: 36) or SGGS (SEQ ID NO: 37) HMW: High molecular weight LMW: Low molecular weight ME: Occlusion efficiency The purified, activatable antibody was treated with a protease that recognizes the cleavage sequence, and then the removal of the masking peptide was tested to determine if its activity was restored. 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. SDS-PAGE analysis confirmed that the masking peptide had been removed from the light chain. The masking efficiency was then measured by ELISA following the same procedure.

[0320] Example 2: Effect of N-terminal peptide on the masking efficiency of masking peptides Of the three units of a masking peptide (MP), while the masking unit (MU) plays a decisive role in the ability to mask the parental antibody, the N-terminal unit and the linker unit (LU) also influence its masking efficiency. In particular, the N-terminal unit can act as an effective multiplier to broaden the masking efficiency range of a given masking unit. Selecting a set of short peptides as universal N-terminal units can be applied to any masking peptide to adjust its masking efficiency. Table 8 shows the N-terminal units tested using a TY24652, and their masking efficiencies measured and benchmarked. The selection of various N-terminal units can also be used to fine-tune the properties of the masked antibody, such as its isoelectric point (PI) and charge distribution.

[0321] Table 8. Other masking peptide sequences with different N-terminal peptides.

[0322]

[0323] Example 3: Concentration-dependent binding activity - binding with HER2+ tumor cell lines The concentration-dependent binding activity of parental and masked anti-HER2 antibodies TY23477 and TY24925 in HER2+ tumor cell lines with different antigen expression levels was measured by flow cytometry. Although TY23477 and TY24925 antibodies possess cleavable linker units, these units were not cleaved in in vitro assays. Therefore, these results indicate concentration-dependent binding of the antibodies without requiring cleavage to remove the masking peptide.

[0324] In short, SKOV3, MCF7, or A549 cells are grown at a density of 1.0 × 10⁶ cells per well. 5 Cells were seeded at a density of [insert density here] in 96-well plates, and serially diluted test antibodies were added. Cells were incubated in 1% FBS / 1640 buffer at 4°C for 30 minutes. Cells were then washed twice with DPBS and incubated again at 4°C for 30 minutes, followed by the addition of secondary antibody APC-anti-human IgG Fc antibody. Finally, cells were washed twice more with DPBS and resuspended in FACS buffer for flow cytometry analysis. FlowJo analysis was then used to plot the relationship between MFI values ​​and concentration, and four-parameter nonlinear regression fitting was performed using GraphPad Prism software to obtain EC50 values.

[0325] like Figure 1A-1C As shown in Table 9A, compared with the parental antibody trastuzumab, the EC50 of both masked antibodies TY23477 and TY24925 for binding to tumor cell lines was reduced. Comparing the EC50 of the masked antibodies with the parental antibody, the masking efficiency was lower in SKOV3 cells with high antigen expression levels, but higher in MCF7 and A549 cells. The masking peptides of the anti-HER2 masked antibodies are shown in Table 9B.

[0326] Table 9A. Concentration-dependent binding of masked anti-Her2 antibody in cell lines with different antigen expression levels.

[0327]

[0328]

[0329] The concentration-dependent binding between antigens and masked antibodies indicates that competitive binding between masked antibodies and antigens serves as a mechanism for enriching masked antibodies around antigen-rich regions, such as tumor-induced upregulated CTLA-4. These data demonstrate the design of dynamic rather than viscous masking peptides with relatively high dissociation rates in this invention's innovative masking technique. This is significantly different from other self-inhibiting masking techniques. The sequence composition and structural characterization of such masking peptide libraries are discussed in detail herein.

[0330] Example 4: Time-dependent fragmentation Time-dependent cleavage of purified enzymes with masked CTLA4 antibody The time-dependent cleavage of TY24652 and TY26294 was evaluated. Recombinant human MMP-9 (prepared internally) was first activated by APMA (100 μg / mL MMP-9 and 1 mM APMA incubated at 37 °C for 24 h). TY24652 or TY26294 was mixed with activated MMP-9 and reacted at 37 °C for 900 s, 1800 s, 3600 s, 7200 s, and 14400 s, respectively, in a reaction buffer of 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij35 (w / v), pH 7.5. After cleavage, the percentage of cleaved light chains to total light chains was analyzed by reducing SDS-PAGE. The masking peptides of the anti-CTLA4 antibody used in this study are shown in Table 10.

[0331] Table 10. Masking peptides of masked anti-CTLA4 antibodies

[0332] like Figure 2 As shown, both TY24652 and TY26294 were fragmented in a time-dependent manner. Complete fragmentation occurred between 2 and 4 hours.

[0333] Quantitative analysis of lysed antibodies in a mouse H22 tumor model was performed using the traditional Western blotting method. In this study, the time-dependent in vivo lysis of masked antibodies TY22404 and TY24652 in a mouse H22 tumor model or in plasma and liver was measured using the conventional Western blotting (WB) method.

[0334] Fresh tissues obtained from mice treated with TY22404 and TY24652 (5 mg / kg) were homogenized into powder on ice using a handheld electric homogenizer (Woxin, Wuxi). The homogenized tissues were then resuspended in RIPA lysis buffer (CST) containing the protease inhibitor mixture, following the instructions for use of the protease inhibitor mixture. AmMag was used to further homogenize the tissues. TM Protein A magnetic beads (GenScript) were used to capture antibodies from tissue homogenates by incubation at room temperature for 3 hours via rotation. The purified antibodies were separated on a 12% SDS-PAGE gel (Beyotime) under reducing denaturing conditions and then transferred to a PVDF membrane (Millipore). Masked and lysed antibody signals were detected using horseradish peroxidase-labeled purified mouse anti-goat IgG (H+L) antibody (Jackson ImmunoResearch). The blot was scanned using an Amersham Imager 600 (GE Healthcare Life Sciences).

[0335] like Figures 3A-3D As shown, these results indicate that the masked antibodies TY22404 and TY24652 can be 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 was higher at 96 hours post-treatment than at 24 hours. Additionally, TY22404 was more readily cleaved than TY24652, suggesting that the number of cleavage sites (one in TY24652 and two in TY22404) can be used to modulate the kinetics of the masked antibody based on cleavage activation.

[0336] Plasma and tumor pharmacokinetic data of TY26294 in the SHP-77 xenograft model.

[0337] TY26294 is an anti-CD47 antibody with a masked peptide sequence LTVDYFCDIDPLYCNAGGGPLGLAGSGGS (SEQ ID NO: 92). CB17 SCID mice burdened with SHP-77 tumors received a single intravenous injection of TY26294 at a dose of 10 mg / kg. Plasma and tumor samples were collected at different time points. The concentrations of intact and fragmented forms of TY26294 were analyzed using an established ELISA method.

[0338] like Figures 4A-4C As shown, these results indicate that the masked antibody TY26294 can be selectively cleaved over time in mouse SHP-77 tumor sites and plasma.

[0339] Example 5: Recognition of masking peptides against human CD137 antibodies The restricted peptide library described in Example 1 was used to screen for masking units against VHH antibodies targeting human CD137. A cleavage peptide sequence was introduced into the linker unit of the construct, following the masking unit sequence. Specifically, the protease recognition site PLGLAG (SEQ ID NO: 35) of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) was used. This recognition site has been used by many research groups in in vivo tumor cell-specific activation targets (see, for example, Ke et al. (1997) J Biol Chem 272(33):20456-62; Gerspach et al. (2006) Cancer Immunol Immunother 55(12):1590-600; and Jiang et al. (2004) Proc Natl Acad Sci USA 101(51):17867-72).

[0340] 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. Masking units from the modified peptide library 4 (Table 2B) were directly fused to the N-terminus of the VHH antibody, and a yeast library was constructed to display the fusion protein on the surface of yeast cells for screening of masking peptides. The yeast library then underwent multiple rounds of FACS-based screening: first, yeast clones with low antigen-binding capacity were enriched; then, the enriched yeast clones were treated with protease to remove the masking units; finally, clones with high antigen-binding capacity were selected.

[0341] Yeast cells (1x10) 8Yeast cells were harvested after induction in glucose-free medium, washed once with PBSA buffer, and then incubated for 1 hour at room temperature with different concentrations of biotinylated antigen. Following antigen incubation, yeast cells were washed with PBSA buffer and incubated at 4°C for 30 minutes with either streptavidin fluorescent dye (phycoerythrin (PE)-streptavidin; 1:500 dilution, eBioscience #2-4317-87) or goat anti-human Fc Alexa Fluor® 647 conjugate (Jackson #109-606-098). Cells were then washed twice with PBSA buffer and sorted at 2–3 OD / mL (MoFlo XDP). Cells with low antigen binding were collected at a biotinylated CD137-HisFc concentration of 2 nM. After the first sorting, the collected yeast cells were treated with TEV protease (2 μg / OD cells, Genscript) at 30°C for 30 minutes to confirm protease cleavage-mediated activation of the target antibody. After multiple rounds of selection, single clones were sorted on selective media and cultured individually to confirm cleavage-mediated activation of antigen binding. 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.

[0342] Table 11A: Exemplary masking peptides for masking anti-CD137 VHH antibodies

[0343] 1 N-terminal unit marked with an underline EVGSY (SEQ ID NO:33) is attached to the N end of each shielding unit.

[0344] 2 bolded part The cleavage site is PLGLAG (SEQ ID NO: 35), which remains unchanged. italics Some have variable connector sequences, such as GGG or SGGS (SEQ ID NO: 37).

[0345] 3 The connecting unit sequence GGGPLGLAGSGGS (SEQ ID NO: 119) is attached to the C end of each masking unit.

[0346] Table 11B. Sequences of the N-terminal unit, masking unit, linker, and cleavage site of anti-CD137 VHH antibody.

[0347] To test the masking efficiency of the identified masking units, selected activatable anti-CD137 VHH single-domain antibodies were fused to the human Fc domain. The sequences of VHH single-domain antibodies fused to the unmasked peptide in the Fc domain are shown in Table 12. The masking efficiency of the exemplary activatable antibodies in Table 11A was measured using an ELISA-based assay. Recombinant human CD137-His was diluted to 2 μg / mL in PBS and coated onto Maxisorp plates overnight at 4°C. The wells were blocked with PBS containing 3% skim milk at 37°C for 1 hour. After washing, 100 μL of a three-fold serially diluted antibody was added to each well. After incubation at 37°C for 1 hour, the wells were washed four times, and 100 μL of HRP-labeled anti-human IgG (Fc specific) (1033 ng / mL) was added to each well. The plate was incubated at 37°C for 1 hour, washed four times, and then 50 μL of TMB substrate solution was added to each well, followed by incubation at room temperature. The reaction was terminated with 50 μL of H₂SO₄ per well, and the absorbance at 450 nm was measured. EC50 was evaluated by fitting the ELISA data to a sigmoidal (four-parameter logistic equation) model using GraphPad Prism software.

[0348] Table 12. Anti-CD137 VHH-Fc antibodies

[0349] The masking efficiency of the activated antibody TY28851 was calculated by dividing the EC50 of TY28851 bound to CD137 by the EC50 of the parental VHH-Fc antibody (TY28974, SEQ ID 115) bound to CD137. TY28851 showed a masking efficiency of 43. These results demonstrate the significant efficacy of the library described herein in selecting masked antibodies in multi-target and antibody forms with high masking efficiency.

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 having unique masking peptides (MPs), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), and the MP is linked to the N-terminus of the antibody VH or VL, wherein the MP comprises a masking unit (MU) and a linking unit (LU), wherein the MU comprises as shown in formula (I): X1X2CX3(X m ) n The amino acid sequence X4X5CX6X7, where n is 2 to 8, 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. Each X m Amino acids 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 stands for 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 having unique masking peptides (MPs), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), and the MP is linked to the N-terminus of the antibody VH or VL, wherein the MP comprises a masking unit (MU) and a linking unit (LU), wherein the MU comprises an amino acid sequence of formula (II): X1X2CX3X4X5X6X7CX8X9, 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, 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 stands for cysteine.

3. The library of claim 2, wherein: 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.

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

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 having unique masking peptides (MPs), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), and the MP is linked to the N-terminus of the antibody VH or VL, wherein the MP comprises a masking unit (MU) and a linking unit (LU), wherein the MU comprises as shown in formula (III): X1X2CX3X4X5X6X7X8CX9X 10 The amino acid sequence, in which: 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. X 10 The amino acids selected are those from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and... C stands for cysteine.

6. The library of claim 5, wherein: 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 X 10 The amino acids selected are those composed of G, Q, R, S, T, and V.

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

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 having unique masking peptides (MPs), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), and the MP is linked to the N-terminus of the antibody VH or VL, wherein the MP comprises a masking unit (MU) and a linker unit (LU); wherein the MU comprises as shown in formula (IV): X1X2CX3X4X5X6X7X8X9CX 10 X 11 The amino acid sequence, in which: 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. X 10 The amino acids selected are those from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X 11 The amino acids selected are those from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and... C stands for cysteine.

9. The library of claim 8, wherein: 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. X 10 The amino acids selected are those from the group composed of I, P, and R, and X 11 The amino acids selected are those composed of E, G, I, P, and V.

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

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 having unique masking peptides (MPs), wherein each antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), and the MP is linked to the N-terminus of the antibody VH or VL, wherein the MP comprises a masking unit (MU) and a linking unit (LU), wherein the MU comprises the following formula (V): X1X2X3X4X5X6X7X8X9X 10 CX 11 X 12 The amino acid sequence, in which: 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. X 10 The amino acids selected are those from the group consisting of A, D, F, H, L, P, S, V, and Y. X 11 The amino acids selected are those from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X 12 The amino acids selected are those from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and... C stands for cysteine.

12. The library of claim 11, wherein: 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. X 10 The amino acids selected are from the group consisting of A, F, V, and Y. X 11 The amino acids selected are those derived from the group consisting of G, I, K, L, and R. X 12 The amino acids selected are those composed of groups A, E, K, P, R, and T.

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

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

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

16. The library of claim 15, wherein the N-terminal unit is about 1-12 amino acid residues long.

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

18. The library of any one of claims 1 to 17, wherein the LU does not contain cleavage sites.

19. The library of claim 18, wherein the LU comprises a connector.

20. The library of claim 19, wherein the linker comprises or is composed 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), GGSGG (SEQ ID NO: 109), GGGSG (SEQ ID NO: 110), GSSSG (SEQ ID NO: 111), GGSGS (SEQ ID NO: 77), SGGG (SEQ ID NO: 79), GGGS (SEQ ID NO: 80), and GGGGSGGSGGGGS (SEQ ID NO: 81).

21. The library of claim 18, wherein LU comprises the amino acid sequence of SEQ ID NO:

81.

22. The library of any one of claims 1 to 17, wherein LU comprises a first cleavage site (C1).

23. The library of claim 22, wherein 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, cytoplasm, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspases-1, caspases-2, caspases-3, caspases-4, caspases-5, caspases-6, caspases-7, caspases-8, caspases-9, caspases-10, caspases-11, caspases-12, caspases-13, caspases-14, and TACE.

24. The library of 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 of any one of claims 22 to 24, wherein LU further comprises a second cleavage site (C2).

26. The library of 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, cytoplasm, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspases-1, caspases-2, caspases-3, caspases-4, caspases-5, caspases-6, caspases-7, caspases-8, caspases-9, caspases-10, caspases-11, caspases-12, caspases-13, caspases-14, and TACE.

27. The library of 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 of any one of claims 25 to 27, wherein the first and second cleavage sites are identical.

29. The library of any one of claims 25 to 27, wherein the first and second cleavage sites are different.

30. The library of any one of claims 22 to 29, wherein LU further comprises a first connector (L1).

31. The library 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), GGSSG (SEQ ID NO: 108), GGSGG (SEQ ID NO: 109), GGGSG (SEQ ID NO: 110), GSSSG (SEQ ID NO: 111), GGSGS (SEQ ID NO: 77), SGGG (SEQ ID NO: 79), GGGS (SEQ ID NO: 80) and / GGGGSGGSGGGS (SEQ ID NO: 81).

32. The library of claim 30 or 31, wherein LU further comprises a second connector (L2).

33. The library 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), GGSSG (SEQ ID NO: 108), GGSGG (SEQ ID NO: 109), GGGSG (SEQ ID NO: 110), GSSSG (SEQ ID NO: 111), GGSGS (SEQ ID NO: 77), SGGG (SEQ ID NO: 79), GGGS (SEQ ID NO: 80), and GGGGSGGSGGGGS (SEQ ID NO: 81).

34. The library of any one of claims 22 to 33, wherein the LU comprises, from the N-end to the C-end: 1) First cleavage site (C1) and first connector (L1); 2) First cleavage site (C1), first connector (L1), second cleavage site (C2), and second connector (L2); 3) First connector (L1), first cleavage site (C1), and second connector (L2); or 4) First connector (L1), first cleavage site (C1), second connector (L2) and second cleavage site (C2).

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

36. The library of 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 library of any one of claims 1 to 36, wherein MP is connected to the N-terminus of VL.

38. The library of any one of claims 1 to 36, wherein MP is connected to the N-terminus of VH.

39. The library of any one of claims 1 to 36, wherein each antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), and MP is linked to the N-terminus of the antibody VH or VL.

40. The library of claim 39, wherein the antibody comprises scFv containing VH and VL.

41. The library of claim 39, wherein the antibody comprises Fab containing VH and VL.

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

43. The library of any one of claims 39 to 42, wherein MP is linked to the N-terminus of antibody VL.

44. The library of any one of claims 1 to 36, wherein each antibody comprises a heavy chain variable region (VH), and MP is linked to the N-terminus of the antibody VH.

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

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

47. The library of claim 46, wherein the carrier is an expression carrier or a display carrier.

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

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

50. The library of any one of claims 1 to 49, wherein the MU in the library has 10 9 Up to 10 14 The diversity.

51. A library comprising an antibody encoded by a polynucleotide of any one of claims 1 to 50.

52. The library of claim 51, wherein each polypeptide is displayed on the cell surface or the phage surface.

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

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

55. The method of claim 54, further comprising recovering antibodies produced by cells.

56. The method of claim 54 or 55 further comprises testing the ability of the antibody to retain the masked phenotype when soluble.

57. A method for screening masked antibodies bound to a target using a library as described in any one of claims 1 to 53, the method comprising: The antibody expressed in the library is contacted with the target to determine the first binding affinity of the target or the lack of detectable binding. The control antibody was contacted with the target to determine the second binding affinity; and Select expression antibodies with a first binding affinity lower than the second binding affinity or undetectable binding to the target. The first and second binding affinity measurements are KD, EC50, or IC50.

58. The method of claim 57, wherein LU comprises at least one first cleavage site (C1).

59. The method of claim 58, wherein the control antibody is an antibody expressed in a LU lysed library.

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

61. A method for screening masked antibodies bound to a target using a library as described in any one of claims 1 to 53, the method comprising the following steps: Antibodies expressed from a library containing a masking peptide were contacted with first cells expressing the target antigen to determine EC50 binding. A control antibody lacking the masking peptide was contacted with first cells expressing the target antigen to determine EC50 binding. The antibody expressed in the library was contacted with a second cell expressing the target antigen to determine the binding EC50, wherein the second cell expressed a lower level of the target antigen than the first cell; d) Contact the control antibody lacking the masking peptide with the second cell to determine EC50 binding; e) Determine the ratio of EC50 in step a) to EC50 in step b) as the first shading efficiency; f) Determine the ratio of EC50 in step c) to EC50 in step d) as the second shading efficiency; and g) Select expression antibodies whose second masking efficiency is higher than that of the first masking efficiency.

62. The method of claim 61, wherein step (g) comprises selecting an expression antibody whose second masking efficiency is at least 10%, 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.

63. The method of any one of claims 61 or 62, wherein the second occlusion 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 occlusion efficiency.

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

66. The method of any one of claims 61 to 65, wherein the LU does not contain a cleavage site.

67. The method of any one of claims 61 to 65, wherein LU comprises at least one first cleavage site (C1).

68. The method of claim 67, wherein the control antibody is an antibody expressed in a LU lysed library.

69. The method of claim 68, wherein the method comprises cleaving LU to produce a control antibody.

70. A method for identifying a masked antibody capable of concentration-dependent antigen binding, the method comprising the steps of: a) Contact a masked antibody with a masking peptide with a first cell expressing a target antigen to determine the binding of EC50, wherein the masking peptide comprises a masking unit (MU) and a linker unit (LU) from the N-terminus to the C-terminus. b) Contact the control antibody lacking the masking peptide with the first cell expressing the target antigen to determine EC50 binding; c) The masked antibody is contacted 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) Contact the control antibody lacking the masking peptide with the second cell to determine EC50 binding; e) Determine the ratio of EC50 in step a) to EC50 in step b) as the first shading efficiency; f) Determine the ratio of EC50 in step c) to EC50 in step d) as the second shading efficiency; and g) If the second masking efficiency is higher than the first masking efficiency, then an antibody capable of concentration-dependent antigen binding is identified.

71. The method of claim 70, wherein 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, then step (g) comprises identifying an antibody capable of performing concentration-dependent antigen binding.

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

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

74. The method of any one of claims 70 to 73, wherein the LU does not contain a cleavage site.

75. The method of any one of claims 70 to 73, wherein LU comprises at least one first cleavage site (C1).

76. The method of claim 75, wherein the control antibody is a masked antibody after LU lysis.

77. The method of claim 76, wherein the method comprises cleaving LU to produce a control antibody.

78. The method of any one of claims 61 to 77, wherein the second occlusion 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 occlusion efficiency.

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

80. A kit comprising a library of 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 as claimed in 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), wherein the MP is linked to the N-terminus of the VH or VL, wherein the MP comprises a masking unit (MU) and a linking unit (LU) from the N-terminus to the C-terminus; wherein the MU comprises as shown in formula (I): X1X2CX3(X m ) n The amino acid sequence X4X5CX6X7, where n is 2 to 8, 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. Each X m Amino acids 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 stands for 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), wherein the MP is linked to the N-terminus of the VH or VL, wherein the MP comprises a masking unit (MU) and a linking unit (LU) from the N-terminus to the C-terminus; wherein the MU comprises an amino acid sequence of formula (II): X1X2CX3X4X5X6X7CX8X9, 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, 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 stands for cysteine.

84. The masked antibody of claim 83, wherein: 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.

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

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), wherein the MP is linked to the N-terminus of the VH or VL, wherein the MP comprises a masking unit (MU) and a linking unit (LU) from the N-terminus to the C-terminus; wherein the MU comprises as shown in formula (III): X1X2CX3X4X5X6X7X8CX9X 10 The amino acid sequence, in which: 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. X 10 The amino acids selected are those from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and... C stands for cysteine.

87. The masked antibody of claim 86, wherein: 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 X 10 The amino acids selected are those composed of G, Q, R, S, T, and V.

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

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), wherein the MP is linked to the N-terminus of the VH or VL, wherein the MP comprises a masking unit (MU) and a linking unit (LU) from the N-terminus to the C-terminus; wherein the MU comprises as shown in formula (IV): X1X2CX3X4X5X6X7X8X9CX 10 X 11 The amino acid sequence, in which: 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. X 10 The amino acids selected are those from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X 11 The amino acids selected are those from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and... C stands for cysteine.

90. The masked antibody of claim 89, wherein: 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. X 10 The amino acids selected are those from the group composed of I, P, and R, and X 11 The amino acids selected are those composed of E, G, I, P, and V.

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

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), wherein the MP is linked to the N-terminus of the VH or VL, wherein the MP comprises a masking unit (MU) and a linking unit (LU) from the N-terminus to the C-terminus; wherein the MU comprises the following formula (V): X1X2X3X4X5X6X7X8X9X 10 CX 11 X 12 The amino acid sequence, in which: 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. X 10 The amino acids selected are those from the group consisting of A, D, F, H, L, P, S, V, and Y. X 11 The amino acids selected are those from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V. X 12 The amino acids selected are those from the group consisting of A, E, G, I, K, L, P, Q, R, S, T, and V, and... C stands for cysteine.

93. The masked antibody of claim 92, wherein: 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. X 10 The amino acids selected are from the group consisting of A, F, V, and Y. X 11 The amino acids selected are those derived from the group consisting of G, I, K, L, and R. X 12 The amino acids selected are those composed of groups A, E, K, P, R, and T.

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

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

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

97. The masked antibody of claim 96, wherein the N-terminal unit is about 1-12 amino acid residues long.

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

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

101. The masked antibody of claim 100, wherein LU comprises a linker.

102. The masked antibody of 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), GGSSG (SEQ ID NO: 108), GGSGG (SEQ ID NO: 109), GGGSG (SEQ ID NO: 110), GSSSG (SEQ ID NO: 111), GGSGS (SEQ ID NO: 77), SGGG (SEQ ID NO: 79), GGGS (SEQ ID NO: 80), and GGGGSGGSGGGGS (SEQ ID NO: 81).

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

81.

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

105. The masked antibody of claim 104, wherein 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, cytoplasm, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, and caspasmin-1. Kasparase-2, Kasparase-3, Kasparase-4, Kasparase-5, Kasparase-6, Kasparase-7, Kasparase-8, Kasparase-9, Kasparase-10, Kasparase-11, Kasparase-12, Kasparase-13, Kasparase-14 and TACE.

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

108. The masked antibody of claim 107, 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, cytoplasm, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspases-1, caspases-2, caspases-3, caspases-4, caspases-5, caspases-6, caspases-7, caspases-8, caspases-9, caspases-10, caspases-11, caspases-12, caspases-13, caspases-14, and TACE.

109. The masked antibody of 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. The masked antibody of any one of claims 107 to 109, wherein the first and second cleavage sites are identical.

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

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

113. The masked antibody of claim 112, wherein the first adapter (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), GGSSG (SEQ ID NO: 108), GGSGG (SEQ ID NO: 109), GGGSG (SEQ ID NO: 110), GSSSG (SEQ ID NO: 111), GGSGS (SEQ ID NO: 77), SGGG (SEQ ID NO: 79), GGGS (SEQ ID NO: 80), and GGGGSGGSGGGS (SEQ ID NO: 81).

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

115. The masked antibody of claim 114, wherein the second adapter (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), GGSSG (SEQ ID NO: 108), GGSGG (SEQ ID NO: 109), GGGSG (SEQ ID NO: 110), GSSSG (SEQ ID NO: 111), GGSGS (SEQ ID NO: 77), SGGG (SEQ ID NO: 79), GGGS (SEQ ID NO: 80), and GGGGSGGSGGGGS (SEQ ID NO: 81).

116. The masked antibody of any one of claims 104 to 115, wherein LU comprises, from the N-terminus to the C-terminus: 1) First cleavage site (C1) and first connector (L1); 2) First cleavage site (C1), first connector (L1), second cleavage site (C2), and second connector (L2); 3) First connector (L1), first cleavage site (C1), and second connector (L2); or 4) First connector (L1), first cleavage site (C1), second connector (L2) and second cleavage site (C2).

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

118. The masked antibody of 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 masked antibody of any one of claims 82 to 118, wherein MP is linked to the N-terminus of VL.

120. The masked antibody of any one of claims 82 to 118, wherein MP is linked to the N-terminus of VH.

121. The masked antibody of any one of claims 82 to 118, wherein each antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), and MP is linked to the N-terminus of the antibody VH or VL.

122. The masked antibody of claim 1, wherein the antibody comprises scFv containing VH and VL.

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

124. Any masked antibody as claimed in claim 121, wherein the antibody may comprise an antibody heavy chain and an antibody light chain.

125. The masked antibody of any one of claims 121 to 123, wherein MP is linked to the N-terminus of antibody VL.

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

127. A polynucleotide encoding a masked antibody as described in any one of claims 82 to 126.

128. An expression vector comprising the polynucleotide of claim 127 operatively linked to a promoter.

129. A host cell comprising the expression vector as described in claim 128.

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

131. A method for generating masked antibodies, the method comprising culturing host cells as described in claim 129 or 130 under conditions suitable for generating masked antibodies.

132. The method of claim 131, further comprising recovering antibodies produced by cells.

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