Anti-CTLA4 antibodies and methods of preparing and using the same

Abstract

Provided herein are cross-reactive antibodies (or antigen-binding fragments thereof) that bind to human CTLA4, activatable antibodies that bind to human CTLA4, nucleic acid molecules encoding the antibodies, their pharmaceutical compositions, and methods of therapeutic use thereof (e.g., for treating cancer).

Description

[0001] Cross-references to related applications

[0002] This application claims priority to International Application No. PCT / CN2018 / 075064, filed on February 2, 2018, which is incorporated herein by reference in its entirety.

[0003] Sequence list submission using ASCII text files

[0004] The following submission, made in ASCII text file, is incorporated herein by reference in its entirety: Sequence List in Computer-Readable Form (CRF) (file name: 695402000541SEQLIST.TXT, record date: February 1, 2019, size: 102KB). Technical Field

[0005] This disclosure relates to cross-reactive antibodies that bind to human cytotoxic T-lymphocyte protein 4 (CTLA4), precision / situation-dependent activatable antibodies that bind to human CTLA4, nucleic acids encoding said antibodies, pharmaceutical compositions thereof, and therapeutic uses thereof. Background Technology

[0006] CTLA4 is a member of the immunoglobulin (Ig) protein superfamily that plays a role in downregulating T cell activation and maintaining immunogenic homeostasis. Antibody-mediated CTLA4 blockade has been shown to enhance anti-cancer immune responses in an allogeneic mouse prostate cancer model (Kwon et al. (1997) Proc Natl Acad Sci USA, 94(15): 8099-103). Furthermore, CTLA4 function blockade has been shown to enhance anti-tumor T cell responses in tumor-carrying mice at various tumor growth stages (Yang et al. (1997) Cancer Res 57(18): 4036-41; Hurwitz et al. (1998) Proc Natl Acad Sci USA 95(17): 10067-7). However, the development of antibody-based therapeutics suitable for human use remains challenging due to the often poor safety profiles from preclinical animal models to humans. Therefore, there is a need for anti-CTLA4 antibodies that exhibit cross-reactivity across different species, such as humans and laboratory animals (e.g., mice, monkeys, rats, etc.), to enable concurrent animal model studies and the provision of therapeutic candidates suitable for humans. Furthermore, there is a need to develop safer anti-CTLA4 antibodies that are active only in certain conditions, such as in protease-rich tumor microenvironments.

[0007] All references cited in this document, including patent applications, patent publications, non-patent literature, and UniProtKB / Swiss-Prot / GenBank accession numbers, are incorporated herein by reference in their entirety as if each individual reference had been specifically and individually indicated to be incorporated by reference. Summary of the Invention

[0008] To meet the above and other needs, this document discloses antibodies (e.g., cross-reactive antibodies) that bind to human CTLA4 and their antigen-binding fragments. The disclosed anti-CTLA4 antibody or its antigen-binding fragment has at least one (e.g., one, some, or all) of the following functional properties: (a) at 500 nM or less K D (a) binds to human, cynomolgus monkey, mouse, rat and / or dog CTLA4; (b) exhibits antagonistic activity against human CTLA4; (c) does not bind to human PD-1, PD-L1, PD-L2, LAG3, TIM3, B7-H3, CD95, CD120a, OX40, CD40, BTLA, VISTA, ICOS and / or B7-H4 at concentrations up to 100 nM; (d) exhibits cross-reactivity with monkey, mouse, rat and / or dog CTLA4; (e) induces ADCC effects (e.g., against Treg); (f) activates human PBMCs (e.g., stimulates the secretion of IL-2 and / or IFNγ); (g) inhibits tumor cell growth and establishes immune memory against tumor cells; (h) has therapeutic effects on cancer; and (i) blocks the binding of human CTLA4 to human CD80 and / or human CD86 (see Examples 1-5 below).

[0009] This document discloses a precise / situation-dependent reactivatable antibody that binds to human CTLA4 when in its active form rather than its inactive form; that is, the reactivatable antibody binds to CTLA4 (active) only after cleaving the cleavable moiety (CM) to remove the masking moiety (MM). In some embodiments, the masking moiety (MM) described herein efficiently masks antibody activity and / or reduces or completely inhibits antigen binding, while in some embodiments, it contains no chemically unstable methionine and / or tryptophan residues. Furthermore, the reactivatable antibodies identified and described herein are as potent as their parent antibodies in treating a variety of cancer types, while exhibiting significantly reduced cytotoxicity in susceptible animals (NOD mice).

[0010] Therefore, in one aspect, this document provides an anti-CTLA4 antibody (e.g., a human antibody) that binds to human CTLA4 and is cross-reactive with CTLA4 peptides from at least one non-human animal selected from the group consisting of cynomolgus monkeys, mice, rats, and dogs. In some embodiments, the antibody binds to both cynomolgus monkey CTLA4 and mouse CTLA4. In some embodiments that can be combined with any prior embodiment, the antibody has a dissociation constant (K0) of about 350 nM or less (e.g., about 300 nM or less, about 200 nM or less, about 100 nM or less, about 50 nM or less, or about 10 nM or less). D ) Combines with human CTLA4, cynomolgus monkey CTLA4, mouse CTLA4, rat CTLA4 and / or dog CTLA4. In some implementations, K D Measured by surface plasmon resonance (SPR). In some embodiments, antibody binding to CTLA4 induces antibody-dependent cytotoxicity (ADCC) against CTLA4-expressing cells. In some embodiments, antibody binding to CTLA4 induces ADCC against Treg cells. In some embodiments, binding of the anti-CTLA4 antibody described herein induces antibody-dependent cytotoxicity (ADCC) against CTLA4-expressing human cells or human Treg cells, wherein, in vitro, the ADCC activity of the anti-CTLA4 antibody is higher than that of ipilimumab, and wherein both antibodies contain the wild-type human IgG1 Fc region. In some embodiments, binding of the anti-CTLA4 antibody described herein induces antibody-dependent cytotoxicity (ADCC) against CTLA4-expressing human cells or human Treg cells, wherein, in vitro, the ADCC activity of the anti-CTLA4 antibody is twice or more than that of ipilimumab, and wherein both antibodies contain the wild-type human IgG1 Fc region. In some embodiments, in vitro, the EC50 of the anti-CTL4 antibody ADCC activity is 50% or less of the EC50 of the iprimarab ADCC activity. Assays for measuring ADCC activity are described in Examples 3 and 15. In some embodiments, in mouse cancer models, the anti-CTLA4 antibody selectively reduces Treg cells in the tumor microenvironment (e.g., reduces the percentage of Treg cells among tumor-infiltrating lymphocytes) compared to PBMCs or spleen. See, for example, Example 18.

[0011] In some embodiments that can be combined with any prior embodiment, the antibody specifically binds to epitopes containing amino acid residues at ligand-binding sites of human CTLA4, such as the CD80 and / or CD86 binding sites of human CTLA4. In some embodiments, the antibody specifically binds to epitopes similar to ligand-binding sites of human CTLA4, such as the CD80 and / or CD86 binding sites of human CTLA4. In some embodiments, the antibody specifically binds to epitopes containing amino acid residues Y105 and L106 of human CTLA4, wherein the amino acid residues are numbered according to SEQ ID NO: 207. In some embodiments, the antibody does not bind to residue I108 of human CTLA4, wherein the amino acid residues are numbered according to SEQ ID NO: 207. In some embodiments, the anti-CTLA4 antibody blocks the binding of CD80 and / or CD86 to human CTLA4. In some embodiments, the anti-CTLA4 antibody has a higher IC50 than the Ipalimab for blocking the binding of CD80 and / or CD86 to human CTLA4. In some embodiments, in assays where CTLA4 is in solution or displayed on the cell surface and plate-bound with CD86 or CD80, the anti-CTLA4 antibody has an IC50 that is 3.5 times or higher (including 3.9 times or higher) than that of the iprimarab for blocking the binding of CD80 and / or CD86 to human CTLA4. See Example 13, Table 23. Figures 57A-57D and Figure 58 The determination of the blocking activity (ligand competition) and IC50 of the antibody is described in Examples 3 and 13.

[0012] In some embodiments that can be combined with any prior embodiment, the antibody comprises a heavy chain variable region and a light chain variable region, a) wherein the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, wherein the HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of: Formula (I): X1TFSX2YX3IHWV (SEQ ID NO: 1), wherein X1 is F or Y, X2 is D or G, and X3 is A, G, or W; Formula (II): YSIX1SGX2X3WX4WI (SEQ ID NO: 2), wherein X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, or S; and Formula (III): FSLSTGGVAVX1WI (SEQ ID NO: 3), wherein X1 is G or S; wherein the HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of: Formula (IV): IGX1IX2HSGSTYYSX3SLKSRV (SEQ ID NO: 3). NO: 4), wherein X1 is D or E, X2 is S or Y, and X3 is P or Q; Formula (V): IGX1ISPSX2GX3TX4YAQKFQGRV (SEQ ID NO: 5), wherein X1 is I or W, X2 is G or S, X3 is G or S, and X4 is K or N; and Formula (VI): VSX1ISGX2GX3X4TYYADSVKGRF (SEQ ID NO: 6), wherein X1 is A, G or S, X2 is S or Y, X3 is G or S, and X4 is S or T; and wherein the HVR-H3 comprises an amino acid sequence selected from the group consisting of: Formula (VII): ARX1X2X3X4FDX5 (SEQ ID NO: 4) NO: 7), where X1 is G, R or S, X2 is A, I or Y, X3 is D, V or Y, X4 is A, E or Y, and X5 is I or Y; Equation (VIII): ARX1GX2GYFDX3 (SEQ ID NO: 8), where X1 is D or L, X2 is F or Y, and X3 is V or Y; Equation (IX): ARX1X2X3X4AX5X6FDY (SEQ ID NO: 9), where X1 is L or R, X2 is I or P, X3 is A or Y, X4 is S or T, X5 is T or Y, and X6 is A or Y; Equation (X): ARDX1X2X3GSSGYYX4GFDX5 (SEQ ID NO: 10), where X1 is I or V, X2 is A or H, X3 is P or S, X4 is D or Y, and X5 is F or V;And b) wherein the light chain variable region comprises HVR-L1, HVR-L2 and HVR-L3, wherein the HVR-L1 comprises an amino acid sequence according to a group consisting of: Formula (XI): RASQX1X2X3SX4LX5 (SEQ ID NO: 11), wherein X1 is G or S, X2 is I or V, X3 is G or S, X4 is S or Y, and X5 is A or N; Formula (XII): RASQX1VX2X3RX4LA (SEQ ID NO: 12), wherein X1 is S or T, X2 is F, R or S, X3 is G or S, and X4 is F or Y; and Formula (XIII): RASX1SVDFX2GX3SFLX4 (SEQ ID NO: 11). NO: 13), wherein X1 is E or Q, X2 is D, F, H or Y, X3 is F, I or K, and X4 is A, D or H; wherein the HVR-L2 comprises an amino acid sequence according to formula (XIV): X1ASX2X3X4X5GX6 (SEQ ID NO: 14), wherein X1 is A or D, X2 is N, S or T, X3 is L or R, X4 is A, E or Q, X5 is S or T, and X6 is I or V; and wherein the HVR-L3 comprises an amino acid sequence according to a formula selected from the group consisting of: formula (XV): YCX1X2X3X4X5X6PX7T (SEQ ID NO: 14). NO: 15), where X1 is E, Q or V, X2 is H or Q, X3 is A, G, H, R or S, X4 is D, L, S or Y, X5 is E, G, P, Q or S, X6 is L, T, V or W, and X7 is F, L, P, W or Y; Formula (XVI): YCQQX1X2X3WPPWT (SEQ ID NO: 16), where X1 is S or Y, X2 is D or Y, and X3 is Q or Y;Formula (XVII): YCQX1YX2SSPPX3YT (SEQ ID NO: 17), where X1 is H or Q, X2 is T or V, and X3 is E or V. In some embodiments, HVR-H1 contains an amino acid sequence selected from the group consisting of SEQ ID NO: 18-29, HVR-H2 contains an amino acid sequence selected from the group consisting of SEQ ID NO: 30-39, HVR-H3 contains an amino acid sequence selected from the group consisting of SEQ ID NO: 40-52, HVR-L1 contains an amino acid sequence selected from the group consisting of SEQ ID NO: 53-65, HVR-L2 contains an amino acid sequence selected from the group consisting of SEQ ID NO: 66-69, and HVR-L3 contains an amino acid sequence selected from the group consisting of SEQ ID NO: 70-81. In some embodiments, the antibody comprises: a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 18, HVR-H2 containing the amino acid sequence of SEQ ID NO: 30, HVR-H3 containing the amino acid sequence of SEQ ID NO: 40, HVR-L1 containing the amino acid sequence of SEQ ID NO: 53, HVR-L2 containing the amino acid sequence of SEQ ID NO: 66, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 70; b) HVR-H1 containing the amino acid sequence of SEQ ID NO: 19, HVR-H2 containing the amino acid sequence of SEQ ID NO: 31, HVR-H3 containing the amino acid sequence of SEQ ID NO: 41, HVR-L1 containing the amino acid sequence of SEQ ID NO: 54, HVR-L2 containing the amino acid sequence of SEQ ID NO: 67, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 71; c) HVR-H1 containing the amino acid sequence of SEQ ID NO: 20, HVR-L2 containing the amino acid sequence of SEQ ID NO: 66, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 70. HVR-H2 containing the amino acid sequence of SEQ ID NO: 32, HVR-H3 containing the amino acid sequence of SEQ ID NO: 42, HVR-L1 containing the amino acid sequence of SEQ ID NO: 55, HVR-L2 containing the amino acid sequence of SEQ ID NO: 66, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 72; d) HVR-H1 containing the amino acid sequence of SEQ ID NO: 21, HVR-H2 containing the amino acid sequence of SEQ ID NO: 33, HVR-H3 containing the amino acid sequence of SEQ ID NO: 43, HVR-L1 containing the amino acid sequence of SEQ ID NO: 56, HVR-L2 containing the amino acid sequence of SEQ ID NO: 68, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 73;e) HVR-H1 containing the amino acid sequence of SEQ ID NO: 22, HVR-H2 containing the amino acid sequence of SEQ ID NO: 34, HVR-H3 containing the amino acid sequence of SEQ ID NO: 44, HVR-L1 containing the amino acid sequence of SEQ ID NO: 57, HVR-L2 containing the amino acid sequence of SEQ ID NO: 66, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 74; f) HVR-H1 containing the amino acid sequence of SEQ ID NO: 23, HVR-H2 containing the amino acid sequence of SEQ ID NO: 35, HVR-H3 containing the amino acid sequence of SEQ ID NO: 45, HVR-L1 containing the amino acid sequence of SEQ ID NO: 58, HVR-L2 containing the amino acid sequence of SEQ ID NO: 66, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 75; g) HVR-H1 containing the amino acid sequence of SEQ ID NO: 24, HVR-H2 containing the amino acid sequence of SEQ ID NO: 32, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 23. HVR-H3 containing the amino acid sequence of SEQ ID NO: 46, HVR-L1 containing the amino acid sequence of SEQ ID NO: 59, HVR-L2 containing the amino acid sequence of SEQ ID NO: 66, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 76; h) HVR-H1 containing the amino acid sequence of SEQ ID NO: 25, HVR-H2 containing the amino acid sequence of SEQ ID NO: 36, HVR-H3 containing the amino acid sequence of SEQ ID NO: 47, HVR-L1 containing the amino acid sequence of SEQ ID NO: 60, HVR-L2 containing the amino acid sequence of SEQ ID NO: 69, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 77; i) HVR-H1 containing the amino acid sequence of SEQ ID NO: 26, HVR-H2 containing the amino acid sequence of SEQ ID NO: 37, HVR-H3 containing the amino acid sequence of SEQ ID NO: 48, HVR-L1 containing the amino acid sequence of SEQ ID NO: 61, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 76. HVR-L2 containing the amino acid sequence NO: 66 and HVR-L3 containing the amino acid sequence SEQ ID NO: 78;j) HVR-H1 containing the amino acid sequence of SEQ ID NO: 27, HVR-H2 containing the amino acid sequence of SEQ ID NO: 32, HVR-H3 containing the amino acid sequence of SEQ ID NO: 49, HVR-L1 containing the amino acid sequence of SEQ ID NO: 62, HVR-L2 containing the amino acid sequence of SEQ ID NO: 67, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 79; k) HVR-H1 containing the amino acid sequence of SEQ ID NO: 28, HVR-H2 containing the amino acid sequence of SEQ ID NO: 37, HVR-H3 containing the amino acid sequence of SEQ ID NO: 50, HVR-L1 containing the amino acid sequence of SEQ ID NO: 63, HVR-L2 containing the amino acid sequence of SEQ ID NO: 67, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 80; l) HVR-H1 containing the amino acid sequence of SEQ ID NO: 18, HVR-H2 containing the amino acid sequence of SEQ ID NO: 38, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 49. HVR-L3 containing the amino acid sequence of SEQ ID NO: 51, HVR-L1 containing the amino acid sequence of SEQ ID NO: 64, HVR-L2 containing the amino acid sequence of SEQ ID NO: 67, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 81; or m) HVR-L1 containing the amino acid sequence of SEQ ID NO: 29, HVR-L2 containing the amino acid sequence of SEQ ID NO: 39, HVR-L3 containing the amino acid sequence of SEQ ID NO: 52, HVR-L1 containing the amino acid sequence of SEQ ID NO: 65, HVR-L2 containing the amino acid sequence of SEQ ID NO: 68, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 77. In some embodiments that can be combined with any prior embodiment, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 82-94, and / or the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 95-107. In some embodiments that can be combined with any prior embodiment, the antibody comprises: a) a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 82 or a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or greater) sequence identity with the amino acid sequence of SEQ ID NO: 82, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 95 or a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or greater) sequence identity with the amino acid sequence of SEQ ID NO: 95;b) A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 83 or a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or greater) sequence identity with the amino acid sequence of SEQ ID NO: 83, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 96 or a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or greater) sequence identity with the amino acid sequence of SEQ ID NO: 96; c) A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 84 or a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or greater) sequence identity with the amino acid sequence of SEQ ID NO: 84, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 97 or a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or greater) sequence identity with the amino acid sequence of SEQ ID NO: 97; d) A heavy chain variable region containing SEQ ID NO: 84 or a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or greater) sequence identity with the amino acid sequence of SEQ ID NO: 97; The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 85 or a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or greater) sequence identity with the amino acid sequence of SEQ ID NO: 85, and the light chain variable region containing the amino acid sequence of SEQ ID NO: 98 or a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or greater) sequence identity with the amino acid sequence of SEQ ID NO: 98; e) the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 86 or a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or greater) sequence identity with the amino acid sequence of SEQ ID NO: 86, and the light chain variable region containing the amino acid sequence of SEQ ID NO: 99 or a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or greater) sequence identity with the amino acid sequence of SEQ ID NO: 99; f) the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 99. The heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 87 or a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or greater) sequence identity with the amino acid sequence of SEQ ID NO: 87, and the light chain variable region comprising the amino acid sequence of SEQ ID NO: 100 or a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or greater) sequence identity with the amino acid sequence of SEQ ID NO: 100;g) A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 88 or a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or greater) sequence identity with the amino acid sequence of SEQ ID NO: 88, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 101 or a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or greater) sequence identity with the amino acid sequence of SEQ ID NO: 101; h) A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 89 or a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or greater) sequence identity with the amino acid sequence of SEQ ID NO: 89, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 102 ... The light chain variable region containing the amino acid sequence of SEQ ID NO: 102 having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or greater) sequence identity; i) a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 90 or a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or greater) sequence identity with the amino acid sequence of SEQ ID NO: 90, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 103 or a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or greater) sequence identity with the amino acid sequence of SEQ ID NO: 103; j) a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 91 or a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or greater) sequence identity with the amino acid sequence of SEQ ID NO: 91, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 104 or a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or greater) sequence identity with the amino acid sequence of SEQ ID NO: 104, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 104, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 102, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 103, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 103, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 102, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 103, and a heavy chain variable region k) a light chain variable region containing a variant of SEQ ID NO: 104 having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or greater) sequence identity; k) a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 92 or a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or greater) sequence identity; and k) a light chain variable region containing the amino acid sequence of SEQ ID NO: 105 or a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or greater) sequence identity.1) A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 93 or a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or greater) sequence identity with the amino acid sequence of SEQ ID NO: 93, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 106 or a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or greater) sequence identity with the amino acid sequence of SEQ ID NO: 106; or m) a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 94 or a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or greater) sequence identity with the amino acid sequence of SEQ ID NO: 94, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 107 ..., and a light chain variable region containing the amino acid sequence of SEQ ID NO: NO: 107 is a variant of the light chain variable region having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or greater) sequence identity.

[0013] In some embodiments, the anti-CTLA4 antibody described herein comprises a heavy chain variable region and a light chain variable region, wherein one, two, three, four, five, or six HVRs of the antibody comprise the HVR sequences shown in Table A. In some embodiments, the anti-CTLA4 antibody comprises a heavy chain variable region containing HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 comprises the amino acid sequence of SEQ ID NO: 23, or HVR-H2 comprises the amino acid sequence of SEQ ID NO: 35, or HVR-H3 comprises the amino acid sequence of SEQ ID NO: 45. In some embodiments, the anti-CTLA4 antibody comprises a light chain variable region containing HVR-L1, HVR-L2, and HVR-L3, wherein HVR-L1 comprises the amino acid sequence of SEQ ID NO: 58, or HVR-L2 comprises the amino acid sequence of SEQ ID NO: 66, or HVR-L3 comprises the amino acid sequence of SEQ ID NO: 75. In some embodiments, the HVR-H2 of the antibody comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the anti-CTLA4 antibody comprises (a) a heavy chain variable region comprising HVR-H1 containing the amino acid sequence of SEQ ID NO: 23, HVR-H2 containing the amino acid sequence of SEQ ID NO: 35, and HVR-H3 containing the amino acid sequence of SEQ ID NO: 45, and / or a light chain variable region comprising HVR-L1 containing the amino acid sequence of SEQ ID NO: 58, HVR-L2 containing the amino acid sequence of SEQ ID NO: 66, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 75. In some embodiments, one, two, three, four, five, or six of the HVRs of the antibody may contain one, two, or three conserved amino acid substitutions in the HVR. In some embodiments, the anti-CTLA4 antibody comprises (b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 87 or an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence of SEQ ID NO: 87, and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 100 or an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence of SEQ ID NO: 100.

[0014] In some embodiments that can be combined with any prior embodiment, the antibody is an antibody fragment. In some embodiments, the fragment is a Fab, Fab', Fab'-SH, F(ab')2, Fv, or scFv fragment. In some embodiments that can be combined with any prior embodiment, the antibody comprises an IgG1, IgG2, IgG3, or IgG4 Fc region (such as a human IgG1, IgG2, IgG3, or IgG4 Fc region). In some embodiments, the antibody comprises human IgG1 or a variant having enhanced ADCC activity. In some embodiments, the antibody comprises human IgG1 with reduced trehalose (or non-trehalose). In some embodiments, the antibody is a human antibody.

[0015] Other aspects of this disclosure relate to an antibody that competes with or cross-competes with any antibody described herein for binding to human CTLA4. Antibodies that bind to the same and / or substantially the same epitopes as any antibody described herein are also provided herein.

[0016] Other aspects of this disclosure relate to an activatable antibody comprising: a) a first polypeptide comprising, from its N-terminus to its C-terminus, a masking portion (MM), a cleavable portion (CM), and a target-binding portion (TBM), wherein the MM comprises according to formula (XVIII): X m CX n CZ o The amino acid sequence of (SEQ ID NO: 134), wherein m is 2-10, n is 3-10, and o is 1-10, wherein each X is independently selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and wherein each Z is independently selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P; wherein when the CM is not cleaved, the MM inhibits the binding of the activatable antibody to human CTLA4; wherein the CM contains at least a first cleavage site; and wherein the TBM contains an antibody heavy chain variable region (VH); and b) a second polypeptide containing an antibody light chain variable region (VL); and wherein when the CM is cleaved, the activatable antibody binds to human CTLA4 via the VH and the VL. In some embodiments, m is 3-10.

[0017] Other aspects of this disclosure relate to an activatable antibody comprising: a) a polypeptide comprising, from its N-terminus to its C-terminus, a masking portion (MM), a cleavable portion (CM), and a target-binding portion (TBM), wherein the MM comprises according to formula (XVIII): X m CX n CZ oThe amino acid sequence of (SEQ ID NO: 134), wherein m is 2-10, n is 3-10, and o is 1-10, wherein each X is independently selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and wherein each Z is independently selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P; wherein when the CM is not cleaved, the MM inhibits the binding of the activatable antibody to human CTLA4; wherein the CM contains at least a first cleavage site; and wherein the TBM contains an antibody light chain variable region (VL); and b) a second polypeptide containing an antibody heavy chain variable region (VH); and wherein when the CM is cleaved, the activatable antibody binds to human CTLA4 via the VH and the VL. In some embodiments, m is 3-10.

[0018] Other aspects of this disclosure relate to an activatable antibody comprising: a polypeptide comprising, from its N-terminus to its C-terminus, a masking portion (MM), a cleavable portion (CM), and a target-binding portion (TBM), wherein the MM comprises according to formula (XVIII): X m CX n CZ o The amino acid sequence of (SEQ ID NO: 134), wherein m is 2-10, n is 3-10, and o is 1-10, wherein each X is independently selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and wherein each Z is independently selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P; wherein when the CM is not cleaved, the MM inhibits the binding of the activatable antibody to human CTLA4; wherein the CM contains at least a first cleavage site; wherein the TBM contains an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH) from the N-terminus to the C-terminus; and wherein when the CM is cleaved, the activatable antibody binds to human CTLA4 via the VH and the VL. In some embodiments, m is 3-10.

[0019] Other aspects of this disclosure relate to an activatable antibody comprising: a polypeptide comprising, from its N-terminus to its C-terminus, a masking portion (MM), a cleavable portion (CM), and a target-binding portion (TBM), wherein the MM comprises according to formula (XVIII): X m CX n CZ oThe amino acid sequence of (SEQ ID NO: 134), wherein m is 2-10, n is 3-10, and o is 1-10, wherein each X is independently selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and wherein each Z is independently selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P; wherein when the CM is not cleaved, the MM inhibits the binding of the activatable antibody to human CTLA4; wherein the CM contains at least a first cleavage site; wherein the TBM contains an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) from the N-terminus to the C-terminus; and wherein when the CM is cleaved, the activatable antibody binds to human CTLA4 through the VH and the VL.

[0020] In some embodiments of any of the activatable antibodies described above, m is 2, 3, 4, 5, or 6. In some embodiments, m is 6. In some embodiments, n is 6-8. In some embodiments, n is 6. In some embodiments, o is 1-2. In some embodiments, o is 2. In some embodiments that can be combined with any prior embodiment, each X is not M, W, or C. In some embodiments that can be combined with any prior embodiment, X of formula (XVIII) m Each X in the formula (XVIII) is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P. In some embodiments that can be combined with any prior embodiment, X in formula (XVIII) n Each X in MM is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P. In some embodiments, MM comprises amino acids selected from X. m CPDHPYPCXX (SEQ IDNO: 181), X m CDAFYPYCXX (SEQ ID NO: 182), X m CDSHYPYCXX (SEQ ID NO: 183) and X mThe amino acid sequence of the group consisting of CVPYYYACXX (SEQ ID NO: 184), wherein m is 2-10, and wherein each X is independently selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y. In some embodiments, each X is not M, W, or C. In some embodiments, each X is independently selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P. In some embodiments that can be combined with any prior embodiment, the masking portion (MM) comprises an amino acid sequence selected from SEQ ID NO: 141-147. In some embodiments that can be combined with any prior embodiment, MM also comprises an additional amino acid sequence at its N-terminus. In some embodiments, the additional amino acid sequence includes the amino acid sequence of SEQ ID NO: 148.

[0021] In some embodiments that can be combined with any prior embodiment, the first cleavage site 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, and Tobacco Etch virus. Virus (TEV) protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, Caspase-1, Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10, Caspase-11, Caspase-12, Caspase-13, Caspase-14, and TACE. In some embodiments that can be combined with any prior embodiment, the CM further includes a first linker (L1) at the C-terminus of the first cleavage site. In some embodiments, L1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 156-163. In some embodiments that can be combined with any prior embodiment, the CM further includes a second cleavage site. In some embodiments, the second cleavage site is located at the C-terminus of L1. In some embodiments, the second cleavage site is a protease cleavage site 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 etching 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 and the second cleavage site are different. In some embodiments that can be combined with any of the prior embodiments, CM also includes a second linker (L2) at the C-terminus of the second cleavage site. In some embodiments, L2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 156-163.In some embodiments that can be combined with any prior embodiment, the CM also includes a third connector (L3) at the N-terminus of the first cleavage site. In some embodiments that can be combined with any prior embodiment, the CM includes at least a first protease cleavage site and is cleaved by one or more proteases 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 etching 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.

[0022] In some embodiments that can be combined with any prior embodiment, the activatable antibody comprises the following formula (XXIX): EVGSYX1X2X3X4X5X6CX7X8X9X10X11X12CX13X14SGRSAGGGGTENLYFQGSGGS (SEQ The masked portion (MM) and cleavable portion (CM) of the amino acid sequence of SEQ ID NO: 164, wherein X1 is A, D, I, N, P, or Y; X2 is A, F, N, S, or V; X3 is A, H, L, P, S, V, or Y; X4 is A, H, S, or Y; X5 is A, D, P, S, V, or Y; X6 is A, D, L, S, or Y; X7 is D, P, or V; X8 is A, D, H, P, S, or T; X9 is A, D, F, H, P, or Y; X10 is L, P, or Y; X11 is F, P, or Y; X12 is A, P, S, or Y; X13 is A, D, N, S, T, or Y; and X14 is A, S, or Y. In some embodiments that can be combined with any prior embodiment, the activatable antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 165-179.

[0023] In some embodiments that can be combined with any prior embodiment, VL comprises HVR-L1 containing the amino acid sequence of SEQ ID NO: 58, HVR-L2 containing the amino acid sequence of SEQ ID NO: 66, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 75. In some embodiments that can be combined with any prior embodiment, VL comprises the amino acid sequence of SEQ ID NO: 100, or a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99%, or greater) sequence identity with the amino acid sequence of SEQ ID NO: 100. In some embodiments that can be combined with any prior embodiment, VH comprises HVR-H1 containing the amino acid sequence of SEQ ID NO: 23, HVR-H2 containing the amino acid sequence of SEQ ID NO: 35, and HVR-H3 containing the amino acid sequence of SEQ ID NO: 45. In some embodiments that can be combined with any prior embodiment, VH comprises the amino acid sequence of SEQ ID NO: 87, or a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or greater) sequence identity with the amino acid sequence of SEQ ID NO: 87.

[0024] Other aspects of this disclosure relate to a pharmaceutical composition comprising any antibody and / or activatable antibody described herein and a pharmaceutically acceptable carrier.

[0025] Other aspects of this disclosure relate to polynucleotides encoding any antibodies and / or activatable antibodies described herein. In some embodiments, the polynucleotide comprises a sequence selected from SEQ ID NO: 108-133.

[0026] Other aspects of this disclosure relate to a vector comprising any of the polynucleotides described herein. In some embodiments, the vector is an expression vector and / or a display vector.

[0027] Other aspects of this disclosure relate to a host cell comprising any of the polynucleotides and / or vectors described herein. In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is a Chinese hamster ovary (CHO) cell.

[0028] Other aspects of this disclosure relate to a method for preparing antibodies or activatable antibodies, the method comprising culturing any of the host cells described herein under conditions suitable for producing said antibodies or activatable antibodies. In some embodiments, the method further includes recovering the antibodies or activatable antibodies produced by the cells.

[0029] Other aspects of this disclosure relate to a method of treating a subject in need of cancer or delaying the progression of said cancer, the method comprising administering to said subject an effective amount of any antibody, activatable antibody, and / or pharmaceutical composition described herein. In some embodiments, the cancer is liver cancer, digestive system cancer (e.g., colon cancer, colorectal cancer), lung cancer, bone cancer, heart cancer, brain cancer, kidney cancer, bladder cancer, blood cancer (e.g., leukemia), skin cancer, breast cancer, thyroid cancer, pancreatic cancer, head and / or neck cancer, eye-related cancer, male reproductive system cancer (e.g., prostate cancer, testicular cancer), or female reproductive system cancer (e.g., uterine cancer, cervical cancer). Other aspects of this disclosure relate to a method of reducing the size of a solid tumor in a subject in need, said solid tumor having a size of approximately 400-1000 mm. 3 The method comprises administering to the subject an effective amount of any antibody, activatable antibody, and / or pharmaceutical composition described herein. In some embodiments, the solid tumor has a size of approximately 400-800 mm. 3The size. In some embodiments, the method further includes administering an effective amount of at least one additional therapeutic agent to the subject. In some embodiments, the at least one additional therapeutic agent is selected from the group consisting of viral gene therapy, immune checkpoint inhibitors, targeted therapy, radiotherapy, vaccination therapy, and chemotherapy. In some embodiments, the at least one additional therapeutic agent is selected from the group consisting of: pomalyst, revlimid, lenalidomide, pomalidomide, thalidomide, DNA alkylation platinum-containing derivative cisplatin, 5-fluorouracil, cyclophosphamide, anti-CD137 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-CD20 antibody, anti-CD40 antibody. Antibody, anti-DR5 antibody, anti-CD1d antibody, anti-TIM3 antibody, anti-SLAMF7 antibody, anti-KIR receptor antibody, anti-OX40 antibody, anti-HER2 antibody, anti-ErbB-2 antibody, anti-EGFR antibody, cetuximab, rituximab, trastuzumab, pembrolizumab, radiotherapy, single-dose radiation, fractionated radiation, focal radiation, whole-organ radiation, IL-12, IFNα, GM-CSF, chimeric antigen receptor, inherited metastatic T cells, anticancer vaccines, and oncolytic viruses. In some embodiments, the method includes administering an effective amount of the anti-CTLA4 antibody, activating antibody, or pharmaceutical composition described herein to the subject before or after surgery used to remove a tumor in the subject. In some embodiments, the anti-CD137 antibody includes an antibody heavy chain variable region comprising HVR-H1 containing the amino acid sequence FSLSTGGVGVGWI (SEQ ID NO: 223), HVR-H2 containing the amino acid sequence LALIDWADDKYYSPSLKSRL (SEQ ID NO: 224), and HVR-H3 containing the amino acid sequence ARGGSDTVIGDWFAY (SEQ ID NO: 225), and an antibody light chain variable region comprising HVR-L1 containing the amino acid sequence RASQSIGSYLA (SEQ ID NO: 226), HVR-L2 containing the amino acid sequence DASNLETGV (SEQ ID NO: 227), and HVR-L3 containing the amino acid sequence YCQQGYYLWT (SEQ ID NO: 228).In some embodiments, the anti-CD137 antibody comprises an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 229 or a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the sequence of SEQ ID NO: 229; and / or an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 230 or a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the sequence of SEQ ID NO: 230.

[0030]

[0031] It should be understood that one, some, or all of the properties 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 become apparent to those skilled in the art. These and other embodiments of this disclosure are further described in the following detailed description. Attached Figure Description

[0032] Figure 1A-Figure 1B This demonstrates the binding of the antibody to CTLA4, as measured by ELISA. Figure 1A The image shows the binding of the indicated antibody to human CTLA4. Figure 1B The image shows the binding of the indicated antibody to canine CTLA4.

[0033] Figure 2 The species cross-reactivity of the indicated antibody, isotype control, or mediator (PBSA) with HEK293F cells that transiently overexpress empty vector (pIRES) or mouse or human CTLA4 is shown, as determined by flow cytometry.

[0034] Figure 3 The binding of antibody TY21580 or its isotype control to activated human, monkey, and mouse T cells is shown, as determined by flow cytometry.

[0035] Figures 4A-4C The antibody's specificity for CTLA4 was demonstrated, as measured by flow cytometry. Figure 4A This demonstrates the binding of the indicated antibody or isotype control to HEK293F cells that transiently overexpress human PD-1, CTLA4, LAG3, TIM3, B7-H3, or the empty vector (293F). Figure 4BThis demonstrates the binding of the indicated antibody, isotype control, or mediator (PBSA) to HEK293F cells that transiently overexpress human CD95, CD120a, OX40, CD40, CTLA4, or an empty vector (pIRES). Figure 4C This demonstrates the binding of the indicated antibody, isotype control, or mediator (PBSA) to HEK293F cells that transiently overexpress human TIM3, CTLA4, PD-L1, LAG3, BTLA, VISTA, PD-L2, ICOS, B7-H4, PD-1, B7-H3, or empty vector (pIRES).

[0036] Figures 5A-5D The blocking ability of antibodies can be demonstrated, as measured by ELISA. Figure 5A The antibodies TY21687, TY21689, TY21680, and TY21691 were shown to block the binding of human CD80 to human CTLA4. Figure 5B The antibodies TAC2114, TY21585, TY21587, TY21588, TY21589, TY21580 and TY21591 were shown to block the binding of human CD80 to human CTLA4. Figure 5C The antibodies TY21687, TY21689, TY21680, and TY21691 were shown to block the binding of human CD86 to human CTLA4. Figure 5D The antibodies TAC2114, TY21585, TY21587, TY21588, TY21589, TY21580 and TY21591 were shown to block the binding of human CD86 to human CTLA4.

[0037] Figures 6A-6B The blocking ability of the antibody is demonstrated, as measured by FACS. Figure 6A This demonstrates the ability of the indicated antibody, isotype control, or mediator (PBSA) to block the binding of human CD80 to HEK293F cells that transiently overexpress human CTLA4. Figure 6B This demonstrates the ability of the indicated antibody, isotype control, or mediator (PBSA) to block the binding of human CD86 to HEK293F cells that transiently overexpress human CTLA4.

[0038] Figure 7 The ability of the indicated antibody to bind to FcRn is shown, as determined by surface plasmon resonance (SPR).

[0039] Figures 8A-8B This demonstrates activation of human peripheral blood mononuclear cells (PBMCs) achieved by antibody TY21580 or an isotype control, as measured by ELISA. Figure 8AThis study demonstrates the effect on IL-2 secretion from CD3-stimulated human PBMCs treated with antibody TY21580 or isotype control. Figure 8B This study demonstrates the effect on IFNγ secretion from CD3-stimulated human PBMCs treated with antibody TY21580 or an isotype control.

[0040] Figure 9 The effect of the presence or absence of anti-CD3 antibody on IL-2 secretion from human PBMCs treated with antibody TY21580 was shown, as measured by ELISA.

[0041] Figure 10 The results showed that, under treatment with antibody TY21580, allotype control, or anti-PD-1 antibody, human dendritic cells (DCs) and allogeneic CD4+... + The effect of IFNγ secretion obtained from T cell co-culture, as measured by ELISA.

[0042] Figures 11A-11B This demonstrates the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of an exemplary antibody against HEK293F cells that transiently overexpress human CTLA4, as measured by a lactate dehydrogenase (LDH) release assay. Figure 11A This demonstrates the ADCC activity of HEK293F cells that transiently overexpress human CTLA4 and are co-incubated with human natural killer (NK) cells with antibody TY21580 or an isotype control. Figure 11B This study demonstrated the ADCC activity of HEK293F cells that transiently overexpressed human CTLA4 and were co-incubated with human NK cells against antibodies TY21580, TAC2114, or the isotype control.

[0043] Figures 12A-12B This demonstrates the ADCC activity of an exemplary antibody against human Tregs isolated from two donors, as determined by a calcein-AM release assay. Figure 12A The antibody TY21580, TAC2114, or the isotype control were shown to exhibit ADCC activity in human Treg cells (from donor #96) incubated with human NK cells. Figure 12B This demonstrates the ADCC activity of antibodies TY21580, TAC2114, or the isotype control on human Treg cells (from donor #12) incubated with human NK cells.

[0044] Figure 13 The antibody TY21580 or the isotype control demonstrated complement-dependent cytotoxic (CDC) activity against HEK293F cells transiently overexpressing human CTLA4, as determined by the calcein-AM release assay.

[0045] Figure 14 The antibody TY21580 or the isotype control showed activity against activated human CD4. + T cell CDC activity, as measured by calcein-AM release assay.

[0046] Figures 15A-15C This demonstrates the in vivo antitumor efficacy of antibody TY21580 or its isotype control in the MC38 isogenous mouse colorectal tumor model. Figure 15A Tumor growth curves are shown for different treatment groups of female C57BL / 6 mice carrying tumors established with MC38. Data points represent group means; error bars represent SEM. Figure 15B The individual tumor growth curves for each test group are displayed. Figure 15C This study demonstrates the reactivation of persistent memory indicating immune responses against MC38 tumor cells.

[0047] Figure 16 This demonstrates the in vivo antitumor efficacy of antibody TY21580 or its isotype control in a CT26 allogeneic mouse colorectal tumor model. Tumor growth curves of different treatment groups in female C57BL / 6 mice carrying tumors established by CT26 are shown. Data points represent group means; error bars represent SEM images.

[0048] Figure 17 This diagram illustrates the in vivo antitumor efficacy of antibodies TY21586, TY21580, or the isotype control in an H22 allogeneic mouse liver tumor model. Tumor growth curves are shown for different treatment groups of female C57BL / 6 mice carrying tumors established via H22. Data points represent group means; error bars represent SEM images.

[0049] Figure 18 This study demonstrates the in vivo antitumor efficacy of antibodies TY21580, TY21687, TY21687, TY21691, and TY21580, or the isotype control, in a Lewis genotype mouse lung tumor model. Tumor growth curves are shown for different treatment groups of female C57BL / 6 mice carrying tumors established via Lewis cells. Data points represent group means; error bars represent SEM images.

[0050] Figure 19 This demonstrates the in vivo antitumor efficacy of antibody TY21580 or the isotype control in a PAN02 genotype-based mouse pancreatic tumor model. Tumor growth curves are shown for different treatment groups of female C57BL / 6 mice carrying tumors established with PAN02. Data points represent group means; error bars represent SEM images.

[0051] Figures 20A-20BThis study demonstrates the in vivo antitumor efficacy of monotherapy with antibody TY21580, anti-CD137 antibody or isotype control, and combination therapy with TY21580 + anti-CD137 antibody in a 3LL allogeneic mouse lung tumor model. Figure 20A Tumor growth curves are shown for different treatment groups of female C57BL / 6 mice carrying tumors established via 3LL. Data points represent group means; error bars represent SEM. Figure 20B The individual tumor growth curves for each test group are displayed.

[0052] Figure 21 This study demonstrates the re-activation of persistent memory indicating immunity against H22 mouse liver tumor cells. On day 59, mice in the TY21580 treatment group that had a complete response were subcutaneously re-activated with H22 tumor cells at the opposite flank. Primitive mice were also inoculated with H22 tumor cells.

[0053] Figure 22 The time course of blood concentration of the indicated antibody, as determined by ELISA, is shown when administered intravenously to female BALB / c mice at a concentration of 10 mg / kg.

[0054] Figure 23 The time course of blood concentration of the indicated antibody, as determined by ELISA, when administered intravenously in cynomolgus monkeys at a concentration of 10 mg / kg.

[0055] Figure 24 The time course of blood concentrations of the indicated antibody administered intravenously in cynomolgus monkeys at a concentration of 10 mg / kg is compared with the presence of anti-drug antibodies (ADA) in these monkeys, as determined by ELISA.

[0056] Figures 25A-25B The mean spleen weight of male and female BALB / c mice was shown after repeated intraperitoneal administration of antibody TY21580 or the mediator control. Figure 25A The mean spleen weight of male BALB / c mice was shown after repeated intraperitoneal administration of antibody TY21580 or the mediator control on days 1, 4, 7, and 11. Figure 25B The mean spleen weight of female BALB / c mice was shown after repeated intraperitoneal administration of antibody TY21580 or the mediator control on days 1, 4, 7, and 11.

[0057] Figure 26 The histopathology of BALB / c mice is shown after repeated intraperitoneal administration of antibody TY21580 or the mediator control on days 1, 4, 7 and 11.

[0058] Figures 27A-27B The stability of the exemplary antibody is shown after storage at high concentrations. Figure 27A Size exclusion chromatography (SEC) profile of antibody TY21586 after storage at >100 mg / mL. Figure 27B The SEC profile of antibody TY21580 is shown after storage at >100 mg / mL.

[0059] Figure 28 The SEC profile of an exemplary antibody is shown under accelerated stress conditions.

[0060] Figure 29 This diagram illustrates a self-blocking peptide selection process performed on Fab fragments displayed on the surface of yeast using an anti-CTLA4 antibody.

[0061] Figure 30 This diagram illustrates the self-blocking peptide selection process performed on scFv fragments displayed on the surface of yeast using an anti-CTLA4 antibody.

[0062] Figures 31A-31B Demonstrating the functionality of Fab and scFv targeting CTLA4 in yeast, as determined by flow cytometry. Figure 31A This demonstrates the functionality of Fab targeting CTLA4 on the surface of yeast. Figure 31B Demonstrates the functionality of scFv targeting CTLA4 on the surface of yeast.

[0063] Figure 32 This demonstrates an exemplary selection process for activatable antibodies targeting human CTLA4. A yeast library displaying the fusion protein is subjected to several rounds of FACS-based screening.

[0064] Figures 33A-33B The CTLA4 binding affinity of an exemplary CTLA4-activated antibody clone is shown, as determined by flow cytometry. Figure 33A The binding affinity of CTLA4-activated antibody clones in scFv form compared to scFv fragments of the target antibody without the masking peptide is shown, including cases where the masking peptide of CTLA4-activated antibody clone B13287 remains intact, or cases where the masking peptide is cleaved by the TEV protease. Figure 33B This demonstrates the CTLA4 binding affinity of CTLA4-activated antibody clones in Fab form compared to the Fab fragment of the target antibody without the masking peptide, including cases where the masking peptide of the CTLA4-activated antibody clone B13189 remains intact, or cases where the masking peptide is cleaved by the TEV protease.

[0065] Figures 34A-34B The exemplary CTLA4 demonstrates the masking efficiency of activating antibodies TY22401, TY22403, TY22402, and TY22404 compared to the parental antibody TY21580. Figure 34A The association and dissociation curves of the indicated activatable antibody are shown relative to the parent antibody TY21580, as determined by the ForteBio system. Figure 34B A graph showing the relative ratio of bound, activatable antibodies compared to the parent antibody TY21580.

[0066] Figures 35A-35B This demonstrates the masking efficiency of an exemplary CTLA4-activated antibody against recombinant human CTLA4-Fc, as determined by ELISA. Figure 35A This shows the first batch of ELISA data indicating the binding of CTLA4-activated antibodies TY22401, TY22402, TY22403, and TY22404 to recombinant human CTLA4-Fc, compared to the parental antibody TY21580. Figure 35B The results showed that, compared to the parental antibody TY21580, CTLA4 could activate the binding of antibodies TY22563, TY22564, TY22565, and TY22566 to recombinant human CTLA4-Fc.

[0067] Figures 36A-36B The results showed that CTLA4 could activate the activity of antibody TY22404 after the masking peptide was removed. Figure 36A The SDS-PAGE results show the activated antibody TY22404 without treatment, with protease uPA, or with 5 or 10 units of protease MMP-9. Figure 36B The binding of the activated antibody TY22404 to the parent antibody TY21580, whether untreated, treated with protease uPA, or treated with protease MMP-9, was measured by ELISA.

[0068] Figures 37A-37C Showing a size exclusion chromatography (SEC) profile of an exemplary activatable antibody under accelerated stress conditions. Figure 37A This shows the SEC profile of the activated antibody TY22402 after six freeze-thaw cycles compared to control conditions. Figure 37B This shows the SEC profile of the activated antibody TY22402 after seven days at 50°C, compared to the control conditions. Figure 37C Shows the percentage of the SEC main peak area of ​​an exemplary activated antibody after seven days at 50°C, up to 28 days after storage at 40°C, or after six freeze-thaw cycles, compared to control conditions.

[0069] Figure 38 Shows the percentage of the SEC main peak area of ​​the activated antibodies TY22401 and TY22402 after storage at approximately 8 mg / mL or >150 mg / mL.

[0070] Figure 39 Masking efficiencies of untreated, activatable antibodies TY21580, TY22401, TY22402, and TY22566, incubated at pH 3.7 for 30 minutes or 1 hour, as determined by the ForteBio system, are shown.

[0071] Figures 40A-40B This demonstrates activation of human peripheral blood mononuclear cells (PBMCs) achieved by isotype control antibody, parental antibody TY21580, or exemplary CTLA4-activating antibodies TY22401, TY22402, or TY22404, as measured by ELISA. Figure 40A This demonstrates the effect on IL-2 secretion from CD3 cells via sensitized human PBMCs stimulated with an isotype control antibody, parental antibody TY21580, and exemplary CTLA4-activating antibodies TY22401, TY22402, or TY22404. Figure 40B This demonstrates the effect on IFNγ secretion from CD3 cells via sensitized human PBMCs stimulated with an isotype control antibody, parental antibody TY21580, and exemplary CTLA4-activating antibodies TY22401, TY22402, or TY22404.

[0072] Figure 41 This demonstrates antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the isotype control antibody, parental antibody TY21580, or exemplary activatable antibodies TY22401, TY21580, or TY22404 against HEK293F cells that transiently overexpress human CTLA4, as determined by ADCC reporter assay.

[0073] Figures 42A-42B This demonstrates the in vivo antitumor efficacy of the parental antibody TY21580, the isotype control antibody, or the exemplary CTLA4-activated antibodies TY22401, TY22402, or TY22566 in an MC38 isogenous mouse colorectal tumor model. Figure 42A Tumor growth curves are shown for different treatment groups of female C57BL / 6 mice carrying tumors established with MC38. Data points represent group means; error bars represent SEM. Figure 42B The figures show the growth curves of individual tumors in the groups treated with TY21580, TY22401, TY22402, and TY22566.

[0074] Figure 43This diagram illustrates the in vivo antitumor efficacy of an allotype control antibody, parental antibody TY21580, or one of the three activatable antibodies in a CT26 allogeneic mouse colorectal tumor model. Tumor growth curves are shown for different treatment groups of female C57BL / 6 mice carrying tumors established by CT26. Data points represent group means; error bars represent SEM images.

[0075] Figure 44 This diagram illustrates the in vivo antitumor efficacy of an allotype control antibody, parental antibody TY21580, or one of the three activatable antibodies in an H22 allogeneic mouse liver tumor model. Tumor growth curves are shown for different treatment groups of female C57BL / 6 mice carrying H22-established tumors. Data points represent group means; error bars represent SEM images.

[0076] Figures 45A-45B This demonstrates the in vivo antitumor efficacy of the parental antibody TY21580, the isotype control antibody, and the exemplary activatable antibodies TY22401, TY22402, or TY22566 in a 3LL isogenous mouse lung tumor model. Figure 45A Tumor growth curves are shown for different treatment groups of female C57BL / 6 mice carrying tumors established via 3LL. Data points represent group means; error bars represent SEM. Figure 45B The figures show the growth curves of individual tumors in the groups treated with TY21580, TY22401, TY22402, and TY22566.

[0077] Figures 46A-46C The time course of blood concentration of the test item (TA) administered intravenously to female BALB / c mice at a concentration of 10 mg / kg, as determined by ELISA. Figure 46A This shows the time course of blood concentrations of the activated antibody TY22401 administered intravenously to female BALB / c mice at a concentration of 10 mg / kg, compared to the parental antibody TY21580. Figure 46B This shows the time course of blood concentrations of the activated antibody TY22402 administered intravenously to female BALB / c mice at a concentration of 10 mg / kg, compared to the parental antibody TY21580. Figure 46C This shows the time course of blood concentrations of the activated antibody TY22404 administered intravenously to female BALB / c mice at a concentration of 10 mg / kg, compared to the parental antibody TY21580.

[0078] Figure 47 This diagram shows the repeated-dose toxicity of the isotype control antibody, parental antibody TY21580, and exemplary activatable antibodies TY22566, TY22401, and TY22402 using a NOD mouse model. The percentage of survival over 20 days is shown for each treatment group.

[0079] Figures 48A-48C The average spleen weight of BALB / c mice is shown after repeated intraperitoneal administration of the indicated activatable antibody. Figure 48A The mean spleen weight of BALB / c mice was shown after repeated intraperitoneal administration of the activating antibody TY22402, the parental antibody TY21580, or the isotype control on days 1, 4, 7, and 11. Figure 48B The mean spleen weight of BALB / c mice was shown after repeated intraperitoneal administration of the activating antibody TY22566, the parental antibody TY21580, or the isotype control on days 1, 4, 7, and 11. Figure 48C The mean spleen weight of BALB / c mice was shown after repeated intraperitoneal administration of the activating antibody TY22401, the parental antibody TY21580, or the isotype control on days 1, 4, 7, and 11.

[0080] Figure 49 This shows the size exclusion chromatography (SEC) profile of the indicated activatable antibody after seven days at 50°C, compared to control conditions.

[0081] Figure 50 This displays the size exclusion chromatography (SEC) profile of the indicated activatable antibody after storage at 40°C for 7, 14, 21, or 28 days, compared to control conditions.

[0082] Figure 51 This shows the size exclusion chromatography (SEC) profile of the indicated activatable antibody after six freeze-thaw cycles, compared to control conditions.

[0083] Figure 52 Shows the percentage of the SEC main peak ratio of the indicated activated antibody after storage at >115 mg / mL.

[0084] Figure 53 This displays an overview of the stability data.

[0085] Figure 54 Multiple sequence alignments depicting a portion of human and mouse CTLA4 were performed, locating contact residues between human CTLA4 and one of CD80, CD86, or ipelimab (based on two crystal structures). Contact amino acids are shaded in gray, key contact amino acids are boxed, dimer interface amino acids are indicated with dots, and amino acids that differ between mouse and human CTLA4 are underlined and indicated in bold. From top to bottom, the sequences shown are represented by SEQ ID NO: 203-208.

[0086] Figure 55A Describe the interaction between human CTLA4 and its ligand CD80. Figure 55BDescribe the interaction between human CTLA4 and its ligand CD86. Figure 55C Structural comparisons between human CTLA4 and mouse CTLA4 are depicted. Human CTLA4 is stained black, and mouse CTLA4 is stained white.

[0087] Figures 56A-56E The results from the epitope localization experiment are depicted, showing TY21580 based on flow cytometry. Figure 56A ), Ipalimab ( Figure 56B ), human CD80 ( Figure 56C ), human CD86 ( Figure 56D ) and mouse CD86 ( Figure 56E The binding ability of CTLA4 with human CTLA4, mouse CTLA4 and CTLA4 mutants.

[0088] Figures 57A-57D To depict the effects of TY21580 and iprimab on receptor-ligand binding blockade between human CTLA4 and CD80 or CD86. Figure 57A and Figure 57B The results showed that in the presence of serially diluted TY21580, iprimarab, or isotype control antibody, human CD80 ( Figure 57A ) or CD86 ( Figure 57B The binding curve of recombinant human CTLA4 protein to the plate, as measured by ELISA. Figures 57C-57D The results showed that, in the presence of serially diluted TY21580, iprimab, or allotype control antibody, human recombinant CTLA4 protein bound to the plate with human CD80 (…). Figure 57C ) or CD86 ( Figure 57D The binding curve, as measured by ELISA.

[0089] Figure 58 This study depicted the activation of reporter signaling mediated by CTLA4 blockade of the CD28 pathway achieved by anti-CTLA4 antibody. Jurkat / CTLA4 and aAPC / Raji cells were co-cultured in the presence of serially diluted anti-CTLA4 antibody, with human IgG1 anti-HEL antibody serving as an allotype control. After overnight incubation, luminescence signals were measured using a Bio-luciferase substrate, and relative luciferase units (RLUs) were normalized relative to a blank control. Results are expressed as mean RLU ± SEM. Experiments were performed in triplicate. Note: Data points at the highest concentration of TY21580 (500 μg / mL) were excluded from the analysis when fitting curves, as a significant hook effect was observed at this concentration.

[0090] Figure 59The activation of ADCC reporter signaling mediated by anti-CTLA4 antibody was depicted. Jurkat / NFAT-Luc / CD16 cells and HEK293F / hCTLA4 cells were co-cultured in the presence of serially diluted anti-CTLA4 antibody, with human IgG1 anti-HEL antibody serving as an isotype control. After 6 hours of incubation, luminescence signal was measured using ONE-Glo luciferase substrate. Relative luciferase units (RLUs) were normalized relative to the blank control, and results are expressed as mean RLU ± SEM. Experiments were performed in triplicate.

[0091] Figure 60A and Figure 60B The tumor growth curves of mice carrying MC38 tumors treated with anti-CTLA4 antibody were plotted. Figure 60A This study depicts the group-mean tumor growth over time in mice carrying MC38 tumors treated with an allotype control antibody (1 mg / kg, BIW), TY21580 (1 mg / kg or 0.2 mg / kg, BIW), or iprimab (1 mg / kg or 0.2 mg / kg, BIW). Data points represent the mean; error bars represent the standard error of the mean (SEM). Figure 60B The tumor growth over time in a single mouse carrying an MC38 tumor was depicted using an allotype control antibody (Group-1), TY21580 (Group-2 and Group-3), or iprimab (Group-4 and Group-5).

[0092] Figure 61A and Figure 61B To depict the effects of TY21580 and iprimab on the level of intratumoral regulatory T (Treg) cells in subcutaneous MC38 tumors from mice treated with TY21580 or iprimab. Figure 61A This shows the percentage of T regulatory (Treg) cells (CD4+CD25+) among CD4+ T cells isolated from the tumor. Figure 61B The ratio of cytotoxic T lymphocytes (CD8+ T cells) to Treg cells (i.e., CD8+ / Treg ratio) was plotted in a subpopulation of CD4+ T cells isolated from tumors. Each data point represents data from one mouse. Statistical analysis was performed using Prism 7 (GraphPad Software). P-values ​​were calculated using multiple t-tests. ns: P > 0.05; **: 0.001 < P < 0.01, ***: P < 0.001.

[0093] Figure 62A and Figure 62B To depict the effects of TY21580 and iprimab on the level of intratumoral regulatory T (Treg) cells in subcutaneous CT26 tumors from mice treated with TY21580 or iprimab. Figure 62AThis shows the percentage of T regulatory (Treg) cells (CD4+CD25+) among CD4+ T cells isolated from the tumor. Figure 62B The ratio of cytotoxic T lymphocytes (CD8+ T cells) to Treg cells (i.e., CD8+ / Treg ratio) was plotted in a subpopulation of CD4+ T cells isolated from tumors. Each data point represents data from one mouse. Statistical analysis was performed using Prism 7 (GraphPad Software). P-values ​​were calculated using multiple t-tests. ns: P > 0.05; **: 0.001 < P < 0.01, ***: P < 0.001.

[0094] Figure 63 Depicting FOXP3 in mice carrying CT26 tumors treated with an isotype control antibody or TY21580. + CD4 + CTLA4 expression levels on Treg cells, measured by mean fluorescence intensity (MFI). Each data point represents data from one mouse. Statistical analysis was performed using Prism 7 (GraphPad Software). P-values ​​were calculated using multiple t-tests. ns: P > 0.05; **: 0.001 < P < 0.01, ***: P < 0.001.

[0095] Figures 64A-64D The tumor growth curves of mice carrying H22 hepatocellular carcinoma were plotted after treatment with TY21580 or an isotype control antibody. Figure 64A Describing when the tumor reaches 500mm 3 Or 800mm 3 TY21580 treatment can begin at this time, or when the tumor reaches 500mm. 3 The mean tumor growth in the group that started treatment with the same type of control antibody at the time of initiation. Data points represent the mean of tumors from 8 mice / groups, and error bars represent the standard error of the mean (SEM). Figures 64B-64D Depict the growth of a single tumor in each mouse. Figure 64B The growth of tumors in mice treated with an allotype control antibody was depicted, where tumors reached 500 mm. 3 Treatment should begin at that time; Figure 64C The growth of tumors in mice treated with TY21580 was depicted, including when the tumor reached 500 mm. 3 Treatment should begin at that time; Figure 64D The growth of tumors in mice treated with TY21580 was depicted, including when the tumor reached 800 mm. 3 Treatment should begin at that time.

[0096] Figure 65A and Figure 65BThe masking efficiency of exemplary activatable antibodies containing a masking peptide of variable length, compared to the parent antibody TY21580, is depicted. Masking efficiency was determined using an ELISA-based method. Figure 65A and Figure 65B This represents two experimental setups that use the same experimental methods to test various activatable anti-CTLA4 antibodies.

[0097] Figure 66 The masking efficiency of exemplary activatable antibodies containing cleavage peptides of varying lengths compared to the parental antibody TY21580 is depicted. Masking efficiency was determined using an ELISA-based method. Detailed Implementation

[0098] I. General Techniques

[0099] The techniques and procedures described or referenced herein are generally well understood by those skilled in the art and are typically employed by those skilled in the art using conventional methods, such as those widely used as described below: Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd Edition (2001), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (F.M. Usubel et al., eds., (2003)); Methods in Enzymology (Academic Press, Inc.); PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames, and G.G. Taylor, eds., (1995)); Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual and Animal Cell Culture (R.I. Freshney, ed., (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (edited by JECellis, 1998) Academic Press; Animal Cell Culture (edited by RIFreshney, 1987); Introduction to Cell and Tissue Culture (JPMather and PERoberts, 1998) PlenumPress; Cell and Tissue Culture: Laboratory Procedures (edited by A.Doyle, JBGriffiths and DG Newell, 1993-8) J.Wiley and Sons; Handbook of Experimental Immunology (eds. DMWeir and CC Blackwell); Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos, 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., 1994); Current Protocols in Immunology (JEColigan et al., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (edited by D. Catty., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (edited by P. Shepherd and C. Dean, Oxford University Press, 2000); Using Antibodies: ALaboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (edited by M. Zanetti and JD. Capra, Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (edited by V.T. DeVita et al., J.L. Bippincott Company, 1993).

[0100] II. Definition

[0101] Before describing this disclosure in detail, it should be understood that this disclosure is not limited to any particular composition or biological system that may vary without doubt. 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.

[0102] As used herein, unless the context explicitly states otherwise, the singular forms “a / an” and “the” include plural indicators. Thus, for example, reference to “a molecule” optionally includes a combination of two or more of the molecules described, and so on.

[0103] As used herein, the term "about" refers to a typical range of error for a given value that is readily known to those skilled in the art. References to "about" a value or parameter herein include (and describe) embodiments of that value or parameter itself.

[0104] It should be understood that the aspects and embodiments of this disclosure described herein include aspects and embodiments that are “comprising,” “composed of,” and “substantially composed of.”

[0105] 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).

[0106] The term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, and 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 compound whose structure differs from the general chemical structure of an amino acid, but functions in a manner similar to that of a naturally occurring amino acid.

[0107] As used in this article, the twenty common amino acids and their abbreviations follow conventional usage. See, for example, Immunology-A Synthesis (2nd edition, edited by ESGolob and DRGren, Sinauer Associates, Sunderland, Mass. (1991)).

[0108] The terms “polypeptide,” “protein,” and “peptide” are used interchangeably in this document and may refer to a polymer of two or more amino acids.

[0109] 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 with 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 supports. 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. The polynucleotide may also contain similar forms of ribose or deoxyribose commonly known in the art, including, for example, 2'-O-methyl-ribose; 2'-O-allyl-ribose; 2'-fluoro-ribose or 2'-azido-ribose; carbocyclic sugar analogs; α-terminal isomers; epimeric sugars such as arabinose, xylose, or lythose; pyranose; furanose; sedoheptulose; acyclic analogs; and non-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 (“amide”), P(O)R, P(O)OR', CO, or CH2 (“methylal”), wherein each R or R' is independently H or optionally contains an ether (-O-) linker, an aryl, alkenyl, cycloalkyl, cycloalkenyl, or aralkyl substituted or unsubstituted alkyl group (1-20 Cs). Not all links in polynucleotides need to be identical.The previous description applies to all polynucleotides mentioned herein, including RNA and DNA.

[0110] The term "isolated nucleic acid" refers to a genomic, cDNA, or synthetically derived nucleic acid molecule, or a combination thereof, isolated from other nucleic acid molecules present in the natural source of said nucleic acid. For example, with respect to genomic DNA, the term "isolated" includes nucleic acid molecules naturally isolated from the chromosomes to which the genomic DNA is naturally associated. Preferably, the "isolated" nucleic acid does not contain sequences naturally side-attached to said nucleic acid, i.e., sequences located at the 5′ and 3′ ends of the target nucleic acid.

[0111] The term “antibody” is used in the broadest sense herein and explicitly covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies), and antibody fragments (e.g., Fab, Fab', Fab'-SH, F(ab')2, Fv, and / or single-chain variable fragments or scFv) as long as they exhibit the desired biological activity.

[0112] 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 H2 and 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.

[0113] V H District and V LThe region can be further subdivided into highly variable regions, known as high-variability regions (HVRs), based on structural and sequence analysis. HVRs are interspersed with more conserved regions known as frame regions (FWs) (see, for example, Chen et al. (1999) J. Mol. Biol. (1999) 293, 865-881). Each V H and V L It 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.

[0114] The variable regions of 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 the light and heavy chains, the variable and constant regions are joined 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)).

[0115] L-chains from any vertebrate species can be designated as one of two distinct types, called κ and λ, based on the amino acid sequence of their constant domain. Antibodies can be designated as different classes or isotypes depending on the amino acid sequence of the constant domain (CH) of the antibody heavy chain. There are five antibody classes: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated as α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu), respectively. The IgG antibody class can be further subdivided into four subclasses based on the γ heavy chain Y1-Y4: IgG1, IgG2, IgG3, and IgG4.

[0116] The term "antibody derivative" or "derivative of an antibody" refers to a molecule capable of binding to the same antigen (e.g., CTLA4) that the antibody binds to, and which contains the amino acid sequence of the antibody linked to an additional molecular entity. The amino acid sequence contained in the antibody derivative can be the full-length heavy chain, the full-length light chain, any one or more portions of the full-length heavy chain, any one or more portions of the full-length light chain, any other one or more fragments of the antibody, or the complete antibody. The additional molecular entity can be a chemical or biological molecule. Examples of additional molecular entities include chemical groups, amino acids, peptides, proteins (such as enzymes, antibodies), and compounds. The additional molecular entity can have any utility, such as being used as a detection agent, label, biomarker, drug, or therapeutic agent. The amino acid sequence of the antibody can be attached to or linked to the additional molecular entity by chemical conjugation, genetic fusion, non-covalent association, or other means. The term "antibody derivative" also encompasses chimeric antibodies, humanized antibodies, and molecules obtained by modifications to the amino acid sequence of the CTLA4 antibody, such as conserved amino acid substitution, addition, and insertion.

[0117] The term "antigen-binding fragment" or "antigen-binding portion" of an antibody refers to one or more portions of the antibody that retain the ability to bind to the antigen (e.g., CTLA4) that the antibody is bound to. Examples of "antigen-binding fragments" of antibodies 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 bivalent segment consisting of two Fab segments connected by a disulfide bridge at the hinge region; and (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 The Fv fragment, composed of structural domains, and the (v)dAb fragment (Ward et al., Nature 341: 544-546 (1989)), are composed of V H Structural domain composition; and (vi) separated complementarity determination regions (CDRs).

[0118] The term “binding molecule” encompasses (1) antibodies, (2) antigen-binding fragments of antibodies, and (3) antibody derivatives, each as defined herein.

[0119] The term "CTLA4" is used in this application and includes human CTLA4 (e.g., UniProt accession number P16410) and its variants, subtypes, and species homologs (e.g., mouse CTLA4 (UniProt accession number P09793), rat CTLA4 (UniProt accession number Q9Z1A7), dog CTLA4 (UniProt accession number Q9XSI1), cynomolgus monkey CTLA4 (UniProt accession number G7PL88), etc.). Therefore, binding molecules (e.g., antibodies or activatable antibodies) as defined and disclosed herein may also bind CTLA4 from species other than humans. In other cases, binding molecules may be completely specific to human CTLA4 and may not exhibit species cross-reactivity or other types of cross-reactivity.

[0120] The term "CTLA4 antibody" refers to an antibody as defined herein that can bind to human CTLA4.

[0121] The term "chimeric antibody" refers to an antibody that contains amino acid sequences derived from different animal species, such as those with variable regions derived from human antibodies and constant regions derived from mouse immunoglobulins.

[0122] The term "competitive binding" refers to the interaction between two antibodies when they bind to their target. If, in the presence of a second antibody, the binding of the first antibody to its homologous epitope is detectably reduced compared to the binding of the first antibody in the absence of the second antibody, then the first and second antibodies compete for binding. An alternative where the binding of the second antibody to its epitope is also detectably reduced in the presence of the first antibody is possible, but not required. That is, the first antibody may inhibit the binding of the second antibody to its epitope without the second antibody inhibiting 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" for binding to one or more of their respective epitopes.

[0123] The term "epitope" refers to the portion of an antigen to which an antibody (or its antigen-binding fragment) binds. Epitopes can be formed from either adjacent amino acids or non-adjacent amino acids adjacent through the ternary folding of a protein. Epitopes formed from consecutive amino acids are generally preserved upon exposure to denaturing solvents, while epitopes formed through ternary folding are generally lost upon treatment with denaturing solvents. Epitopes can comprise a variety of numbers of amino acids with a unique spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, X-ray crystallography, 2D nuclear magnetic resonance, a combination of deuterium and hydrogen exchange with mass spectrometry, or site-directed mutagenesis, or all of these methods combined with computational modeling of the complex structure of the antigen and its binding antibody and its variants (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, ed. GEMorris (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, it becomes possible to competitively screen antibodies that bind to the same epitope. One method to achieve this is to conduct cross-competition studies to discover antibodies that competitively bind to each other, i.e., antibodies competitively bind to antigens. A high-throughput method for “classifying” antibodies based on their cross-competition is described in PCT Publication No. WO 03 / 48731.

[0124] The term "germline" refers to the nucleotide sequence of antibody genes and gene segments that are passed from parent to offspring through 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 high mutation rates.

[0125] The term "glycosylation site" refers to an amino acid residue that is recognized by eukaryotic cells as a linking site for sugar residues. The amino acids linking carbohydrates such as oligosaccharides are typically asparagine (N-linked), serine (O-linked), and threonine (O-linked) residues. Specific linking sites are usually indicated by an amino acid sequence referred to herein as the "glycosylation site sequence." The glycosylation site sequence used 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 act as linking sites between sugar molecules and amino acid residues. N-linked sugars are linked by an amino group; O-linked sugars are linked by a hydroxyl group. The term "glycan occupancy" refers to the presence of a carbohydrate moiety linked to the glycosylation site (i.e., the glycan site is occupied). When at least two potential glycosylation sites are present on a polypeptide, no (0 glycan site occupied), one (1 glycan site occupied), or two (2 glycan site occupied) sites can be occupied by the carbohydrate moiety.

[0126] 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, such as HEK293F cells and HEK293T cells; or human tissue or hybridoma cells, yeast cells, insect cells (e.g., S2 cells), bacterial cells (e.g., E. coli cells), and cells contained within transgenic animals or cultured tissues. The term covers not only the specific subject cell but also its progeny. Because certain modifications can occur in successive generations due to mutations or environmental influences, the progeny may not be identical to the parent cells but are still included within the scope of the term "host cell."

[0127] "Human antibody" is an antibody having an amino acid sequence corresponding to an amino acid sequence produced by humans or human cells or derived from a non-human source utilizing a human antibody lineage or other human antibody coding sequence. This definition of human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues.

[0128] The term "humanized antibody" refers to a chimeric antibody containing amino acid residues derived from human antibody sequences. Humanized antibodies may contain some or all of the CDR or HVR derived from non-human animals or synthetic antibodies, while the frame region and constant region of the antibody contain amino acid residues derived from human antibody sequences.

[0129] The term "illustrative antibody" refers to any of the antibodies described and designated in this disclosure as those listed in Tables A and B, and any antibody containing the six HVRs and / or VHs and VLs of the antibodies listed in Tables A and B. These antibodies may be in any class (e.g., IgA, IgD, IgE, IgG, and IgM). Therefore, each antibody identified above covers antibodies in all five classes with the same V... L District and V H Antibodies with specific amino acid sequences. Furthermore, antibodies in the IgG class can be in any subclass (e.g., IgG1, IgG2, IgG3, and IgG4). Therefore, each of the antibodies identified above in the IgG subclass covers antibodies in all four subclasses with the same V... L District and V H Antibodies with specific amino acid sequences. 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.

[0130] "Separated" antibodies or binding molecules (e.g., activatable antibodies) 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).

[0131] The term "K" a "k" refers to the association rate constant of a specific binding molecule-antigen interaction, where the term "k" is... d "" refers to the dissociation rate constant of the interaction between a specific binding molecule and an antigen.

[0132] The 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 nanovolume molar concentration (nM) dissociation constant bind more tightly to specific antigens than antibodies with a microvolume molar concentration (μM) dissociation constant. The K value of an antibody... D The value can be determined using methods well-established in the art. A K value for determining antibodies... D The method involves using surface plasmon resonance (SPR), typically utilizing biosensor systems such as System. For example, using BIACORE. TM The system's measurement procedure (BIAcore measurement) is described in at least Example 3 of this disclosure.

[0133] 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.

[0134] The term "preventing" in relation to a disease condition in mammals refers to preventing or delaying the onset of a disease, or preventing the appearance of clinical or subclinical symptoms of a disease.

[0135] As used herein, “sequence identity” between two polypeptide sequences indicates the percentage of identical amino acids between the sequences. The 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 is, for example, 95% identical to a reference amino acid sequence, parameters are set to calculate the percentage of identity across the full length of the reference amino acid sequence and to allow for vacancies in the homology to account for up to 5% of the total number of amino acid residues in the reference sequence. The method mentioned above in determining the percentage of identity between polypeptides is applicable to all proteins, fragments or variants disclosed herein.

[0136] As used herein, the terms “binding,” “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 that target with greater affinity, strength, ease, and / or duration of binding compared 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.

[0137] The term "treat / treating / treatment" in relation to a disease condition in mammals refers to an effect that results in a desired or beneficial outcome in the mammal having the disease condition. 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 the cessation or suppression of further development of the disease, ailment, or condition. In the case of treating cancer in mammals, a desired or beneficial outcome may include inhibiting further growth or spread of cancer cells, causing cancer cell death, suppressing cancer recurrence, reducing cancer-related pain, or improving the survival of the mammal. The outcome may be subjective or objective. For example, if the mammal is a human, then the person may notice an improvement in energy or vitality or a reduction in pain as a subjective sign of improvement or response to therapy. Alternatively, a clinician may perceive a reduction in tumor size or tumor burden based on physical examination, laboratory parameters, tumor markers, or radiographic studies. Some laboratory signs of response to treatment that clinicians can observe include normalization of 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 decrease in detectable tumor markers. Alternatively, other tests may be used to assess objective improvement, such as ultrasound scans, MRI, and positron emission tomography (PET).

[0138] The term "vector" refers to a nucleic acid molecule capable of delivering foreign nucleic acid molecules. Foreign nucleic acid molecules are linked 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 viral particles. One type of vector integrates into the host cell's genome after introduction and replicates along with the host genome (e.g., non-free mammalian vectors). Another type of vector can replicate autonomously in the host cell it is introduced into (e.g., bacterial vectors with bacterial origins of replication and free mammalian vectors). Another specific type of vector capable of guiding the expression of the 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, called "transcription vectors," can only be transcribed, not translated: they replicate, but are not expressed, in target cells. The term "vector" encompasses all types of vectors, regardless of their function. Vectors that can guide the expression of expressible nucleic acids that are operatively linked are generally referred to as “expression vectors”. 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).

[0139] As used herein, “subject,” “patient,” or “individual” can refer to a human or a non-human animal. “Non-human animal” can mean 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.

[0140] "Effective amount" means at least the amount that, at the necessary dose and for the necessary duration, effectively achieves one or more desired or indicated effects, including therapeutic or preventative outcomes. An effective amount can be provided by a single or multiple 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 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 be administered in an effective amount if a desired outcome is achieved or realized in combination with one or more other agents.

[0141] III. Binding molecules of human CTLA4

[0142] This disclosure relates in part to isolated binding molecules that bind human CTLA4, including CTLA4 antibodies, antigen-binding fragments of CTLA4 antibodies, and derivatives of CTLA4 antibodies. In some embodiments, the binding molecule is any antibody described herein, including antibodies described with respect to specific amino acid sequences of HVR, variable regions (VL, VH), and light and heavy chains (e.g., IgG1, IgG2, IgG4). In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a humanized antibody and / or a chimeric antibody. In some embodiments, this disclosure relates to binding molecules that bind human CTLA4 and have at least one (e.g., at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or all nine) of the following functional properties: (a) at 500 nM or less K D(a) binds to human, cynomolgus monkey, mouse, rat and / or dog CTLA4; (b) exhibits antagonistic activity against human CTLA4; (c) does not bind to human PD-1, PD-L1, PD-L2, LAG3, TIM3, B7-H3, CD95, CD120a, OX40, CD40, BTLA, VISTA, ICOS and / or B7-H4 at concentrations up to 100 nM; (d) exhibits cross-reactivity with monkey, mouse, rat and / or dog CTLA4; (e) induces ADCC effects (e.g., against Treg); (f) activates human PBMCs (e.g., stimulates the secretion of IL-2 and / or IFNγ); (g) inhibits tumor cell growth; (h) has therapeutic effects on cancer; and (i) blocks the binding of human CTLA4 to human CD80 and / or human CD86. In some embodiments, in assays where human CD80 and / or CD86 are immobilized (or plate-bound) or when human CTLA4 protein is present on the cell surface, the anti-CTLA4 antibody described herein exhibits lower activity in blocking the binding of CD80 and / or CD86 to human CTLA4 compared to iprimarab. See also Figure 57C and Figure 57D and Figure 58 In some implementations, the anti-CTLA4 antibody described herein selectively reduces Treg cells in the tumor microenvironment compared to Treg reduction in PBMCs or the spleen. In some implementations, the anti-CTLA4 antibody described herein exhibits higher Treg reduction activity in the tumor microenvironment compared to ipelamalbrine. See also Figures 61A-61B , Figures 62A-62B and Figure 63 This article also provides one or more anti-CTLA4 antibodies or antigen-binding fragments that cross-compete with one or more of the antibody or antigen-binding fragments described herein for binding to human CTLA4.

[0143] In some embodiments, the antibody or antigen-binding fragment is at a Kc of about 500 nM or less (e.g., about 500 nM or less, about 450 nM or less, about 400 nM or less, about 350 nM or less, about 300 nM or less, about 250 nM or less, about 200 nM or less, about 150 nM or less, about 100 nM or less, about 90 nM or less, about 80 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 25 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, about 0.1 nM or less, etc.). D This formulation binds to CTLA4 in humans, cynomolgus monkeys, mice, rats, and / or dogs. In some embodiments, the antibody or antigen-binding fragment is at approximately 350 nM or less. DThis formulation binds to CTLA4 in humans, cynomolgus monkeys, mice, rats, and / or dogs. In some embodiments, the antibody or antigen-binding fragment is at approximately 100 nM or less. D Binds to human CTLA4. In some embodiments, the antibody or antigen-binding fragment is at approximately 50 nM or less K. D Binds to human CTLA4. In some embodiments, the antibody or antigen-binding fragment is at approximately 10 nM or less. D Binding to human CTLA4. Measuring the K-value of the antibody or antigen-binding fragment. D The method can be performed using any method known in the art, including, for example, surface plasmon resonance, ELISA, isothermal titration calorimetry, membrane binding assay, EMSA, etc. In some embodiments, K D Measurements were taken by surface plasmon resonance or ELISA (see, for example, Example 3 below).

[0144] In some embodiments, the antibody or antigen-binding fragment described herein has antagonistic activity against human CTLA4. In some embodiments, when cells expressing human CTLA4 (e.g., human cells) are contacted with the antibody or antigen-binding fragment, the antibody or antigen-binding fragment inhibits one or more activities of human CTLA4 (e.g., CTLA4 blocking, as measured by an increase in reporter gene signal, such as using a CLA4 blocking reporter gene assay).

[0145] In some embodiments, the antibody or antigen-binding fragment is cross-reactive with CTLA4 in monkeys (e.g., cynomolgus monkeys), mice, rats, and / or dogs. In some embodiments, the antibody or antigen-binding fragment is cross-reactive with monkey CTLA4. In some embodiments, the antibody or antigen-binding fragment is cross-reactive with mouse CTLA4. In some embodiments, the antibody or antigen-binding fragment is cross-reactive with rat CTLA4. In some embodiments, the antibody or antigen-binding fragment is cross-reactive with dog CTLA4. In some embodiments, the antibody or antigen-binding fragment is cross-reactive with CTLA4 in monkeys and mice; CTLA4 in monkeys and rats; CTLA4 in monkeys and dogs; CTLA4 in mice and rats; CTLA4 in mice and dogs; CTLA4 in rats and dogs; CTLA4 in monkeys, mice, and rats; CTLA4 in monkeys, mice, and dogs; CTLA4 in monkeys, rats, and dogs; CTLA4 in mice, rats, and dogs; or CTLA4 in monkeys, mice, rats, and dogs. In some implementations, if the antibody or antigen-binding fragment has a Kc of less than about 500 nM (e.g., less than about 1 nM, less than about 10 nM, less than about 25 nM, less than about 50 nM, less than about 75 nM, less than about 100 nM, less than about 150 nM, less than about 200 nM, less than about 250 nM, less than about 300 nM, less than about 350 nM, etc.), DWhen a non-human CTLA4 molecule is bound, the antibody or antigen-binding fragment exhibits cross-reactivity. Methods for measuring antibody cross-reactivity are known in the art, including but not limited to surface plasmon resonance, ELISA, isothermal titration calorimetry, membrane binding assay, EMSA, etc. In some embodiments, cross-reactivity is measured by ELISA (see, for example, Example 3 below).

[0146] In some embodiments, after the antibody binds to CTLA4 expressed on cells, the antibody induces an ADCC effect against CTLA4-expressing cells (e.g., against CTLA4-expressing human cells such as Tregs). Methods for measuring the ADCC effect (e.g., in vitro methods) are known in the art, including but not limited to the method described in Example 3 below. In some embodiments, the antibody induces an ADCC effect of more than about 10% relative to a control (e.g., an allotype control or iprimarab), such that it induces an ADCC effect of more than about 10% (e.g., more than about 15%, more than about 20%, more than about 25%, more than about 30%, more than about 35%, more than about 40%, etc.).

[0147] In some embodiments, the antibody or antigen-binding fragment is capable of inhibiting tumor cell growth and / or proliferation. In some embodiments, when contacted with the antibody or antigen-binding fragment, tumor cell growth and / or proliferation is inhibited by at least about 5% (e.g., at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 99%), relative to corresponding tumor cells not contacted with the antibody or antigen-binding fragment (or relative to corresponding tumor cells contacted with an isotype control antibody). In some embodiments, when the antibody or antigen-binding fragment is administered to a subject, the antibody or antigen-binding fragment is capable of reducing tumor volume in the subject. In some embodiments, the antibody or antigen-binding fragment is capable of reducing the tumor volume in a subject by at least about 5% (e.g., at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 99%) relative to the initial tumor volume in the subject (e.g., before administration of the antibody or antigen-binding fragment; compared to the corresponding tumor in a subject administered with an isotype control antibody). Methods for monitoring tumor cell growth / proliferation, tumor volume, and / or tumor suppression are known in the art, including, for example, the methods described in Example 4 below.

[0148] In some implementations, the antibody or antigen-binding fragment has a therapeutic effect on cancer. In some implementations, the antibody or antigen-binding fragment alleviates one or more signs or symptoms of cancer. In some implementations, when the antibody or antigen-binding fragment is administered, a subject with cancer experiences partial or complete remission.

[0149] On the other hand, this disclosure provides isolated antibodies that compete or cross-competitively bind to human CTLA4 with any of the illustrative antibodies of this disclosure, such as TY21585, TY21586, TY21587, TY21588, TY21589, TY21580, TY21591, TY21686, TY21687, TY21689, TY21680, TY21691 and / or TY21692. In a particular embodiment, this disclosure provides isolated antibodies that compete or cross-competitively bind to the same epitope on human CTLA4 with any of the illustrative antibodies of this disclosure. The ability of an antibody to compete or cross-competitively bind with another antibody can be determined using standard binding assays known in the art, such as BIAcore analysis, ELISA assays, or flow cytometry. For example, an illustrative antibody of this disclosure can be made to bind to human CTLA4 under saturation conditions, and then the ability of a test antibody to bind to CTLA4 can be measured. If the test antibody can bind to CTLA4 simultaneously with the illustrative antibody, then the test antibody binds to a different epitope than the illustrative antibody. However, if the test antibody cannot bind to CTLA4 simultaneously, then the test antibody will bind to the same epitope, overlapping epitope, or epitope closely adjacent to the epitope bound by the descriptive antibody. This assay can be performed using various methods such as ELISA, RIA, FACS, or surface plasmon resonance.

[0150] In some embodiments, the antibody or antigen-binding fragment blocks the binding between CTLA4 and one or more of its binding partners (e.g., human CTLA4 with human CD80, human CTLA4 with human CD86). In some embodiments, the antibody or antigen-binding fragment blocks the binding between CTLA4 and its ligand in vitro. In some embodiments, for blocking the binding of CTLA4 to CD80 and / or CD86, the antibody or antigen-binding fragment has a half-maximum inhibitory concentration (IC50) of about 500 nM or less (e.g., about 500 nM or less, about 400 nM or less, about 300 nM or less, about 200 nM or less, about 100 nM or less, about 50 nM or less, about 25 nM or less, about 10 nM or less, about 1 nM or less, etc.). 50 In some implementations, for blocking the binding of CTLA4 to CD80 and / or CD86, the antibody or antigen-binding fragment has a half-maximum inhibitory concentration (IC50) of about 100 nM or less. 50In some embodiments, when provided at concentrations of about 100 nM or greater (e.g., about 100 nM or greater, about 500 nM or greater, about 1 μM or greater, about 10 μM or greater, etc.), the antibody or antigen-binding fragment completely blocks the binding of human CTLA4 to CD80 and / or CD86. As used herein, the term "complete blocking" means that the antibody or antigen-binding fragment reduces the binding between the first protein and the second protein by at least about 80% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, etc.). Methods for measuring the ability of an antibody or antigen-binding fragment to block the binding of the first protein (e.g., human CTLA4) and the second protein (e.g., human CD80 or human CD86) are known in the art, including but not limited to BIAcore analysis, ELISA assays, and flow cytometry (see, for example, Example 3 below). In some implementations, the anti-CTLA4 antibody described herein has lower activity in blocking ligand binding compared to iprimab.

[0151] CTLA4 antibody

[0152] In some aspects, this disclosure provides a segregated antibody that binds to human CTLA4. In some embodiments, the antibody is in K+ at 1000 nM or less (e.g., 50 nM or less, 10 nM or less). D The antibody binds to human CTLA4, as measured by surface plasmon resonance. In some embodiments, the antibody exhibits cross-reactivity with at least one non-human species selected from cynomolgus monkeys, mice, rats, and dogs.

[0153] In some aspects, this disclosure provides an isolated antibody that specifically binds to an epitope similar to the ligand-binding site of human CTLA4. In some embodiments, the antibody specifically binds to an epitope similar to the CD80 binding site of human CTLA4. In some embodiments, the antibody specifically binds to an epitope similar to the CD86 binding site of human CTLA4. In some embodiments, the antibody specifically binds to an epitope containing one or more amino acid residues in a ligand-binding site of human CTLA4 (e.g., CD80 and / or CD86 binding sites). In some embodiments, the antibody specifically binds to an epitope on human CTLA4 that is different from the epitope of the iprimarone. In some embodiments, the epitope does not contain amino acid residues in the CC' ring motif of human CTLA4. In some embodiments, the epitope does not contain amino acid residues L106 or I108 of human CTLA4. In some embodiments, the antibody specifically binds to an epitope containing amino acid residues Y105 and L106 of human CTLA4, but not I108, wherein the amino acid residues are numbered according to SEQ ID NO: 207.

[0154]

[0155] In one aspect, this disclosure provides a separated antibody comprising a heavy chain variable region and a light chain variable region, a) wherein the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, wherein the HVR-H1 comprises an amino acid sequence selected from the formulas: Formula (I): X1TFSX2YX3IHWV (SEQ ID NO: 1), wherein X1 is F or Y, X2 is D or G, and X3 is A, G, or W; Formula (II): YSIX1SGX2X3WX4WI (SEQ ID NO: 2), wherein X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, or S; and Formula (III): FSLSTGGVAVX1WI (SEQ ID NO: 3), wherein X1 is G or S; the HVR-H2 comprises an amino acid sequence selected from the formulas: Formula (IV): IGX1IX2HSGSTYYSX3SLKSRV (SEQ ID NO: 3). NO: 4), where X1 is D or E, X2 is S or Y, and X3 is P or Q; Formula (V): IGX1ISPSX2GX3TX4YAQKFQGRV (SEQ ID NO: 5), where X1 is I or W, X2 is G or S, X3 is G or S, and X4 is K or N; and Formula (VI): VSX1ISGX2GX3X4TYYADSVKGRF (SEQ ID NO: 6), where X1 is A, G, or S, X2 is S or Y, X3 is G or S, and X4 is S or T; and the HVR-H3 comprises an amino acid sequence selected from the following formula: Formula (VII): ARX1X2X3X4FDX5 (SEQ ID NO: 4). NO: 7), where X1 is G, R, or S, X2 is A, I, or Y, X3 is D, V, or Y, X4 is A, E, or Y, and X5 is I or Y; Equation (VIII): ARX1GX2GYFDX3 (SEQ ID NO: 8), where X1 is D or L, X2 is F or Y, and X3 is V or Y; Equation (IX): ARX1X2X3X4AX5X6FDY (SEQ ID NO: 9), where X1 is L or R, X2 is I or P, X3 is A or Y, X4 is S or T, X5 is T or Y, and X6 is A or Y; and Equation (X): ARDX1X2X3GSSGYYX4GFDX5 (SEQ ID NO: 8). NO: 10), wherein X1 is I or V, X2 is A or H, X3 is P or S, X4 is D or Y, and X5 is F or V; and / or b) wherein the light chain variable region comprises HVR-L1, HVR-L2 and HVR-L3, wherein the HVR-L1 comprises an amino acid sequence selected from the following formula: Formula (XI): RASQX1X2X3SX4LX5 (SEQ ID NO: 11), wherein X1 is G or S, X2 is I or V, X3 is G or S, X4 is S or Y, and X5 is A or N;Formula (XII): RASQX1VX2X3RX4LA (SEQ ID NO: 12), where X1 is S or T, X2 is F, R or S, X3 is G or S, and X4 is F or Y; and Formula (XIII): RASX1SVDFX2GX3SFLX4 (SEQ ID NO: 13), where X1 is E or Q, X2 is D, F, H or Y, X3 is F, I or K, and X4 is A, D or H; the HVR-L2 comprises according to Formula (XIV): X1ASX2X3X4X5GX6 (SEQ ID NO: 12). The amino acid sequence of (SEQ ID NO: 14), wherein X1 is A or D, X2 is N, S or T, X3 is L or R, X4 is A, E or Q, X5 is S or T, and X6 is I or V; and the HVR-L3 comprises an amino acid sequence selected from the following formulas: Formula (XV): YCX1X2X3X4X5X6PX7T (SEQ ID NO: 15), wherein X1 is E, Q or V, X2 is H or Q, X3 is A, G, H, R or S, X4 is D, L, S or Y, X5 is E, G, P, Q or S, X6 is L, T, V or W, and X7 is F, L, P, W or Y; Formula (XVI): YCQQX1X2X3WPPWT (SEQ ID NO: 15). NO: 16), where X1 is S or Y, X2 is D or Y, and X3 is Q or Y; and formula (XVII): YCQX1YX2SSPPX3YT (SEQ ID NO: 17), where X1 is H or Q, X2 is T or V, and X3 is E or V.

[0156] In some embodiments, the antibody comprises: a) HVR-H1 containing an amino acid sequence selected from SEQ ID NO: 18-29; HVR-H2 containing an amino acid sequence selected from SEQ ID NO: 30-39; and HVR-H3 containing an amino acid sequence selected from SEQ ID NO: 40-52; and / or b) HVR-L1 containing an amino acid sequence selected from SEQ ID NO: 53-65; HVR-L2 containing an amino acid sequence selected from SEQ ID NO: 66-69; and HVR-L3 containing an amino acid sequence selected from SEQ ID NO: 70-81. In some embodiments, the antibody comprises one, two, three, four, five, or all six of the HVRs shown for any of the exemplary antibodies described in Table A below.

[0157] Table A: Anti-CTLA4 HVR sequences

[0158]

[0159]

[0160]

[0161] In some embodiments, the antibody comprises HVR-H1 containing the amino acid sequence of SEQ ID NO: 18, HVR-H2 containing the amino acid sequence of SEQ ID NO: 30, HVR-H3 containing the amino acid sequence of SEQ ID NO: 40, HVR-L1 containing the amino acid sequence of SEQ ID NO: 53, HVR-L2 containing the amino acid sequence of SEQ ID NO: 66, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 70. In some embodiments, the antibody comprises HVR-H1 containing the amino acid sequence of SEQ ID NO: 19, HVR-H2 containing the amino acid sequence of SEQ ID NO: 31, HVR-H3 containing the amino acid sequence of SEQ ID NO: 41, HVR-L1 containing the amino acid sequence of SEQ ID NO: 54, HVR-L2 containing the amino acid sequence of SEQ ID NO: 67, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 71. In some embodiments, the antibody comprises HVR-H1 containing the amino acid sequence of SEQ ID NO: 20, HVR-H2 containing the amino acid sequence of SEQ ID NO: 32, HVR-H3 containing the amino acid sequence of SEQ ID NO: 42, HVR-L1 containing the amino acid sequence of SEQ ID NO: 55, HVR-L2 containing the amino acid sequence of SEQ ID NO: 66, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 72. In some embodiments, the antibody comprises HVR-H1 containing the amino acid sequence of SEQ ID NO: 21, HVR-H2 containing the amino acid sequence of SEQ ID NO: 33, HVR-H3 containing the amino acid sequence of SEQ ID NO: 43, HVR-L1 containing the amino acid sequence of SEQ ID NO: 56, HVR-L2 containing the amino acid sequence of SEQ ID NO: 68, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 73. In some embodiments, the antibody comprises HVR-H1 containing the amino acid sequence of SEQ ID NO: 22, HVR-H2 containing the amino acid sequence of SEQ ID NO: 34, HVR-H3 containing the amino acid sequence of SEQ ID NO: 44, HVR-L1 containing the amino acid sequence of SEQ ID NO: 57, HVR-L2 containing the amino acid sequence of SEQ ID NO: 66, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 74.In some embodiments, the antibody comprises HVR-H1 containing the amino acid sequence of SEQ ID NO: 23, HVR-H2 containing the amino acid sequence of SEQ ID NO: 35, HVR-H3 containing the amino acid sequence of SEQ ID NO: 45, HVR-L1 containing the amino acid sequence of SEQ ID NO: 58, HVR-L2 containing the amino acid sequence of SEQ ID NO: 66, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 75. In some embodiments, the antibody comprises HVR-H1 containing the amino acid sequence of SEQ ID NO: 24, HVR-H2 containing the amino acid sequence of SEQ ID NO: 32, HVR-H3 containing the amino acid sequence of SEQ ID NO: 46, HVR-L1 containing the amino acid sequence of SEQ ID NO: 59, HVR-L2 containing the amino acid sequence of SEQ ID NO: 66, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 76. In some embodiments, the antibody comprises HVR-H1 containing the amino acid sequence of SEQ ID NO: 25, HVR-H2 containing the amino acid sequence of SEQ ID NO: 36, HVR-H3 containing the amino acid sequence of SEQ ID NO: 47, HVR-L1 containing the amino acid sequence of SEQ ID NO: 60, HVR-L2 containing the amino acid sequence of SEQ ID NO: 69, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 77. In some embodiments, the antibody comprises HVR-H1 containing the amino acid sequence of SEQ ID NO: 26, HVR-H2 containing the amino acid sequence of SEQ ID NO: 37, HVR-H3 containing the amino acid sequence of SEQ ID NO: 48, HVR-L1 containing the amino acid sequence of SEQ ID NO: 61, HVR-L2 containing the amino acid sequence of SEQ ID NO: 66, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 78. In some embodiments, the antibody comprises HVR-H1 containing the amino acid sequence of SEQ ID NO: 27, HVR-H2 containing the amino acid sequence of SEQ ID NO: 32, HVR-H3 containing the amino acid sequence of SEQ ID NO: 49, HVR-L1 containing the amino acid sequence of SEQ ID NO: 62, HVR-L2 containing the amino acid sequence of SEQ ID NO: 67, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 79.In some embodiments, the antibody comprises HVR-H1 containing the amino acid sequence of SEQ ID NO: 28, HVR-H2 containing the amino acid sequence of SEQ ID NO: 37, HVR-H3 containing the amino acid sequence of SEQ ID NO: 50, HVR-L1 containing the amino acid sequence of SEQ ID NO: 63, HVR-L2 containing the amino acid sequence of SEQ ID NO: 67, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 80. In some embodiments, the antibody comprises HVR-H1 containing the amino acid sequence of SEQ ID NO: 18, HVR-H2 containing the amino acid sequence of SEQ ID NO: 38, HVR-H3 containing the amino acid sequence of SEQ ID NO: 51, HVR-L1 containing the amino acid sequence of SEQ ID NO: 64, HVR-L2 containing the amino acid sequence of SEQ ID NO: 67, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 81. In some embodiments, the antibody comprises HVR-H1 containing the amino acid sequence of SEQ ID NO: 29, HVR-H2 containing the amino acid sequence of SEQ ID NO: 39, HVR-H3 containing the amino acid sequence of SEQ ID NO: 52, HVR-L1 containing the amino acid sequence of SEQ ID NO: 65, HVR-L2 containing the amino acid sequence of SEQ ID NO: 68, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 77.

[0162] In some embodiments, the antibody comprises: a) a heavy chain variable region containing an amino acid sequence selected from SEQ ID NO: 82-94; and / or b) a light chain variable region containing an amino acid sequence selected from SEQ ID NO: 95-107. In some embodiments, the antibody comprises a heavy chain variable region containing 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 a sequence selected from SEQ ID NO: 82-94, and / or a light chain variable region containing 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 a sequence selected from SEQ ID NO: 95-107. In some embodiments, the antibody comprises the heavy chain variable region and light chain variable region of any of the exemplary antibodies described in Table B below. In some implementations, the antibody comprises one, two, or all three HVRs of the heavy chain variable region and / or one, two, or all three HVRs of the light chain variable region as shown for any of the exemplary antibodies described in Table B below.

[0163] Table B: Amino acid sequence of the anti-CTLA4 variable region

[0164]

[0165]

[0166]

[0167] In some embodiments, the antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 82 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 95. In some embodiments, the antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 83 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 96. In some embodiments, the antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 84 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 97. In some embodiments, the antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 85 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 98. In some embodiments, the antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 86 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 99. In some embodiments, the antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 87 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 100. In some embodiments, the antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 88 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 101. In some embodiments, the antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 89 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 102. In some embodiments, the antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 90 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 103. In some embodiments, the antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 91 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 104. In some embodiments, the antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 92 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 105. In some embodiments, the antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 93 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 106. In some embodiments, the antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 94 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 107.

[0168] In some embodiments, the antibody of this disclosure cross-competes with antibodies comprising: a) HVR-H1 containing an amino acid sequence selected from SEQ ID NO: 18-29; HVR-H2 containing an amino acid sequence selected from SEQ ID NO: 30-39; and HVR-H3 containing an amino acid sequence selected from SEQ ID NO: 40-52; and / or b) HVR-L1 containing an amino acid sequence selected from SEQ ID NO: 53-65; HVR-L2 containing an amino acid sequence selected from SEQ ID NO: 66-69; and HVR-L3 containing an amino acid sequence selected from SEQ ID NO: 70-81. In some embodiments, the antibody of this disclosure cross-competes with antibodies comprising one, two, three, four, five, or all six of the HVRs shown for any of the exemplary antibodies described in Table A. In some embodiments, the antibodies of this disclosure cross-competitively bind to human CTLA4 with antibodies comprising: a) a heavy chain variable region containing an amino acid sequence selected from SEQ ID NO: 82-94; and / or b) a light chain variable region containing an amino acid sequence selected from SEQ ID NO: 95-107. In some embodiments, the antibodies of this disclosure cross-competitively bind to human CTLA4 with antibodies comprising VH and / or VL as shown for any of the exemplary antibodies described in Table B.

[0169] The CTLA4 antibody described herein can be in any class, such as IgG, IgM, IgE, IgA, or IgD. In some embodiments, the CTLA4 antibody is in the IgG class, such as IgG1, IgG2, IgG3, or IgG4 subclasses. Using methods known in the art, the CTLA4 antibody can be converted from one class or subclass to another. An exemplary method for producing an antibody in the desired class or subclass includes the steps of: separating the nucleic acid of the heavy chain encoding the CTLA4 antibody and the nucleic acid of the light chain encoding the CTLA4 antibody, separating the nucleic acid of the V... H The sequence of regions makes V H The sequence is linked to a heavy chain constant region encoding the desired class or subclass, expressing the light chain gene and heavy chain construct in cells, and collecting CTLA4 antibodies. The antibodies disclosed herein can be monoclonal or polyclonal antibodies. The antibodies disclosed herein can be monospecific or multispecific (e.g., bispecific, trispecific, etc.) antibodies. In some embodiments, the CTLA4 antibodies described herein may include one or more Fc mutations (e.g., Fc mutations that regulate (increase or decrease) ADCC or CDC activity). Any suitable Fc mutation known in the art can be used in the CTLA4 antibodies of this disclosure.

[0170] In some embodiments, the antibody of this disclosure is a bispecific antibody that binds to a first target and a second target, wherein the first target is human CTLA4. In some embodiments, the bispecific antibody binds to a first target and a second target, wherein the first target is human CTLA4, and wherein the bispecific antibody comprises a) HVR-H1 containing an amino acid sequence selected from SEQ ID NO: 18-29; HVR-H2 containing an amino acid sequence selected from SEQ ID NO: 30-39; and HVR-H3 containing an amino acid sequence selected from SEQ ID NO: 40-52; and / or b) HVR-L1 containing an amino acid sequence selected from SEQ ID NO: 53-65; HVR-L2 containing an amino acid sequence selected from SEQ ID NO: 66-69; and HVR-L3 containing an amino acid sequence selected from SEQ ID NO: 70-81. In some embodiments, the bispecific antibody binds to a first target and a second target, wherein the first target is human CTLA4, and wherein the bispecific antibody comprises one, two, three, four, five, or all six of the HVRs shown for any of the exemplary antibodies described in Table A. In some embodiments, the bispecific antibody binds to a first target and a second target, wherein the first target is human CTLA4, and wherein the bispecific antibody comprises: a) a heavy chain variable region containing an amino acid sequence selected from SEQ ID NO: 82-94; and / or b) a light chain variable region containing an amino acid sequence selected from SEQ ID NO: 95-107. In some embodiments, the bispecific antibody binds to a first target and a second target, wherein the first target is human CTLA4, and wherein the bispecific antibody comprises VH and / or VL shown for any of the exemplary antibodies described in Table B. In some implementations, the second target is PD-1, PD-L1, PD-L2, LAG3, TIM3, B7-H3, CD95, CD120a, OX40, CD40, BTLA, VISTA, ICOS, Her1, Her2, Her3, or B7-H4.

[0171] The antibodies disclosed herein can be produced by any technique known in the art, including conventional monoclonal antibody methods such as standard somatic cell hybridization (see, for example, Kohler and Milstein, Nature 256:495 (1975)), viral or oncogenic transformation of B lymphocytes, or recombinant antibody techniques as detailed herein (see, for example, Examples 1 and 2). In some embodiments, the antibodies of this disclosure are produced using any libraries and / or methods described in PCT application number PCT / CN2017 / 098333 (into the entirety by reference) and / or PCT application number PCT / CN2017 / 098299 (into the entirety by reference).

[0172] Hybridoma generation is a highly established procedure. The mouse system is a common animal system used for hybridoma preparation. Immunization protocols and techniques for isolating immunized spleen cells for fusion are known in the art. Fusion couples (e.g., mouse myeloma cells) and fusion procedures are also known. A well-known method for preparing human CTLA4 antibodies provided in this disclosure involves using XenoMouse. TM Animal system. XenoMouse TMMice are engineered strains of mice that contain large segments of the heavy and light chain loci of human immunoglobulins and are defective in antibody production (see, for example, Green et al., (1994) Nature Genetics 7: 13-21; WO2003 / 040170). Animals are immunized with the CTLA4 antigen. The CTLA4 antigen is isolated and / or purified CTLA4. It may be a fragment of CTLA4, such as the extracellular domain of CTLA4. Immunization of animals can be performed by any method known in the art (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Press, 1990). Methods for immunizing non-human animals such as mice, rats, sheep, goats, pigs, cattle, and horses are well known in the art (see, for example, Harlow and Lane (above) and U.S. Patent No. 5,994,619). The CTLA4 antigen may be administered with an adjuvant to stimulate an immune response. Exemplary adjuvants include full or incomplete Freund's adjuvant, RIBI (muramicyl dipeptide), or ISCOM (immunostimulatory complex). After immunization of an animal with the CTLA4 antigen, antibody-producing immortalized cell lines are prepared from cells isolated from the immunized animal. Following immunization, the animal is sacrificed, and lymph node and / or spleen B cells are immortalized. Methods for immortalizing cells include, but are not limited to, transferring them with oncogenes, infecting them with oncogenes, culturing them under conditions suitable for selecting immortalized cells, subjecting them to oncogenetic or mutagenic compounds, fusing them with immortalized cells such as myeloma cells, and inactivating tumor suppressor genes (see, for example, Harlow and Lane (above)). If fusion with myeloma cells is used, the myeloma cells preferably do not secrete immunoglobulin peptides (non-secreting cell lines). Immortalized cells are screened using CTLA4, a portion thereof, or cells expressing CTLA4. Select, clone, and further screen CTLA4 antibody-producing cells, such as hybridomas, for desired characteristics, including robust growth, high antibody yield, and desired antibody characteristics, as discussed further below. Hybridomas can be expanded in vivo in syngeneic animals, in animals lacking an immune system, such as nude mice, or in vitro in cell culture. Methods for selecting, cloning, and expanding hybridomas are well known to those skilled in the art.

[0173] The antibodies disclosed herein can also be prepared using phage display or yeast display methods. Display methods for isolating human antibodies have been established in the art (see, for example, Knappik et al. (2000) J. Mol. Biol. 296, 57-86; Feldhaus et al. (2003) Nat Biotechnol 21: 163-170; also see the methods of Examples 1 and 2 below).

[0174] antigen-binding fragments

[0175] In some other respects, this disclosure provides antigen-binding fragments of any CTLA4 antibody described herein.

[0176] The antigen-binding fragment may contain any sequence of any antibody described herein. In some embodiments, the antigen-binding fragment comprises (1) the light chain of the CTLA4 antibody; (2) the heavy chain of the CTLA4 antibody; (3) a variable region from the light chain of the CTLA4 antibody; (4) a variable region from the heavy chain of the CTLA4 antibody; (5) one or more HVRs (e.g., one, two, three, four, five, or six HVRs) of the CTLA4 antibody; or (6) the amino acid sequences of three HVRs from the light chain of the CTLA4 antibody and three HVRs from the heavy chain of the CTLA4 antibody.

[0177] In some embodiments, this disclosure provides antigen-binding fragments of antibodies selected from those listed in Tables A and B.

[0178] In some implementations, the antigen-binding fragment of the CTLA4 antibody includes: (i) a Fab fragment, which is composed of V L V H C L and C H (ii) A monovalent segment composed of a structural domain; (iii) A divalent segment consisting of two Fab segments connected at the hinge region by a disulfide bridge; (iv) A segment composed of V H and C H (iv) The Fd fragment composed of a single-arm antibody; L and V H The Fv segment composed of structural domains; the (v)dAb segment (Ward et al., (1989) Nature 341: 544-546), which is composed of V H The structural domains consist of (vi) isolated CDR and (vii) a single-chain antibody (scFv), which is a V containing the antibody. L The region is linked to the V of the antibody. HPolypeptides in the region (see, for example, Bird et al. (1988) Science 242: 423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883).

[0179] Antibody derivatives

[0180] In some other respects, this disclosure provides derivatives of any CTLA4 antibody described herein.

[0181] In some embodiments, the antibody derivative is obtained by modifying the amino acid sequence of the illustrative antibody (e.g., "parent antibody") of this disclosure while maintaining the overall molecular structure of the parent antibody's amino acid sequence. The amino acid sequence of any region of the parent antibody chain can be modified, such as a frame region, HVR region, or constant region. Types of modification include substitution, insertion, deletion, or combinations thereof of one or more amino acids of the parent antibody.

[0182] In some embodiments, the antibody derivative comprises at least 65%, at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the amino acid sequence as set forth in any of SEQ ID NO: 82-107. L or V HIn some embodiments, the antibody derivative comprises an HVR-H1 amino acid sequence region that is at least 65%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in any of SEQ ID NOs: 18-29. In some embodiments, the antibody derivative comprises an HVR-H2 amino acid sequence region that is at least 65%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in any of SEQ ID NOs: 30-39. In some embodiments, the antibody derivative comprises an HVR-H3 amino acid sequence region that is at least 65%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in any of SEQ ID NOs: 40-52. In some embodiments, the antibody derivative comprises an HVR-L1 amino acid sequence region that is at least 65%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in any of SEQ ID NOs: 53-65. In some embodiments, the antibody derivative comprises an HVR-L2 amino acid sequence region that is at least 65%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in any of SEQ ID NOs: 66-69. In some embodiments, the antibody derivative comprises an HVR-L3 amino acid sequence region that is at least 65%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in any of SEQ ID NOs: 70-81.

[0183] In some specific embodiments, the derivative comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 conserved or non-conserved substitutions for the amino acid sequence as set forth in any of SEQ ID NO: 18-107, and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 additions and / or deletions.

[0184] Amino acid substitution encompasses both conservative and non-conservative substitution. The term "conservative amino acid substitution" refers to the replacement of one amino acid with another, where the two amino acids are similar in certain physicochemical properties, such as the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the residues involved. For example, substitution can generally occur in each of the following groups: (a) nonpolar (hydrophobic) amino acids, such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; (b) polar neutral amino acids, such as glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; (c) positively charged (basic) amino acids, such as arginine, lysine, and histidine; and (d) negatively charged (acidic) amino acids, such as aspartic acid and glutamic acid.

[0185] Modifications can be made at any position in the amino acid sequence of the antibody, including HVR, frame regions, or constant regions. In one embodiment, this disclosure provides a V containing the illustrative antibody of this disclosure. H and V LHVR sequences, but antibody derivatives containing frame sequences that differ from those of the illustrative antibodies described herein. These frame sequences are available from public DNA databases or publicly available references that include germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes are available in the Genbank database or the “VBase” human germline sequence database (Kaba et al., Sequences of Proteins of Immunological Interest, 5th ed., USDapartment of Health and Human Services, NIH Publication No. 91-3242 (1991); Tomlinson et al., J. Mol. Biol. 227: 776-798 (1992); and Cox et al., Eur. J. Immunol. 24: 827-836 (1994)). Frame sequences that can be used to construct antibody derivatives include those structurally similar to those used by the illustrative antibodies of this disclosure. For example, the HVR-H1, HVR-H2, and HVR-H3 sequences of the explanatory antibody, as well as the HVR-L1, HVR-L2, and HVR-L3 sequences, can be transplanted onto a frame region that has the same sequence as the frame region in the germline immunoglobulin gene from which the frame sequence originates, or the HVR sequence can be transplanted onto a frame region that contains one or more mutations compared to the germline sequence.

[0186] In some embodiments, the antibody derivative is a chimeric antibody comprising the amino acid sequence of the illustrative antibody of this disclosure. In one example, one or more HVRs derived from one or more illustrative antibodies are combined with HVRs derived from an antibody from a non-human animal such as a mouse or rat. In another example, all HVRs of the chimeric antibody are derived from one or more illustrative antibodies. In some specific embodiments, the chimeric antibody comprises one, two, or three HVRs derived from the heavy chain variable region of the illustrative antibody and / or one, two, or three HVRs derived from the light chain variable region of the illustrative antibody. The chimeric antibody can be produced using conventional methods known in the art.

[0187] Another type of modification is to make V H and / or V LAmino acid residue mutations within the HVR region of the chain. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce one or more mutations, and the effects on antibody binding or other target functional properties can be assessed in in vitro or in vivo assays known in the art. Typically, conserved substitutions are introduced. Mutations can be amino acid additions and / or deletions. Furthermore, at most one, two, three, four, or five residues within the HVR region are typically altered. In some embodiments, the antibody derivative contains 1, 2, 3, or 4 amino acid substitutions in the heavy chain HVR and / or the light chain HVR. In another embodiment, the amino acid substitution is replacing one or more cysteine ​​residues in the antibody with another residue, such as, but not limited to, alanine or serine. The cysteine ​​can be typical or atypical cysteine. In one embodiment, the antibody derivative has 1, 2, 3, or 4 conserved amino acid substitutions in the heavy chain HVR region relative to the amino acid sequence of the illustrative antibody.

[0188] It can also be used for V H and / or V L The framework residues within the region are modified. Typically, these framework variants are prepared to reduce the immunogenicity of the antibody. One approach is to “revert” one or more framework residues to the corresponding germline sequence. Antibodies that have been mutated in recipient cells may contain framework residues different from the germline sequence from which the antibody originated. These residues can be identified by comparing the antibody framework sequence with the germline sequence from which the antibody originated. To revert framework region sequences to their germline configuration, somatic mutations can be “reverted” to germline sequences via, for example, site-directed mutagenesis or PCR-mediated mutagenesis.

[0189] Furthermore, modifications can be made to the Fc region of the illustrative antibody, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. In one example, the hinge region of CH1 is modified to alter the number of cysteine ​​residues in the hinge region, for example, by increasing or decreasing it. This method is further described in U.S. Patent No. 5,677,425. Changing the number of cysteine ​​residues in the hinge region of CH1 can, for example, facilitate the assembly of light and heavy chains or increase or decrease the stability of the antibody. In another case, the Fc hinge region of the antibody is mutated to reduce the biological half-life of the antibody.

[0190] Furthermore, the antibodies of this disclosure can be modified according to conventional experiments known in the art to alter their underlying glycosylation sites or patterns. In another aspect, this disclosure provides derivatives of CTLA4 antibodies containing at least one mutation in a variable region of the light or heavy chain that alters the glycosylation pattern in said variable region. Such antibody derivatives may have increased affinity and / or altered specificity in relation to binding antigens. Mutations may add new glycosylation sites in the V region, alter the position of one or more V region glycosylation sites, or remove pre-existing V region glycosylation sites. In one embodiment, this disclosure provides a derivative of a CTLA4 antibody having a potentially N-linked glycosylation site at asparagine in a heavy chain variable region, wherein said potentially N-linked glycosylation site in one heavy chain variable region is removed. In another embodiment, this disclosure provides a derivative of a CTLA4 antibody having a potentially N-linked glycosylation site at asparagine in a heavy chain variable region, wherein said potentially N-linked glycosylation sites in two heavy chain variable regions are removed. Methods for altering the glycosylation pattern of antibodies are known in the art, such as those described in U.S. Patent No. 6,933,368, the disclosure of which is incorporated herein by reference.

[0191] In another aspect, this disclosure provides an antibody derivative comprising an antibody as described herein, or an antigen-binding fragment thereof, linked to an additional molecular entity. Examples of the additional molecular entity include pharmaceutical formulations, peptides or proteins, detection agents or labels, and antibodies.

[0192] In some embodiments, the antibody derivative comprises an antibody of the present disclosure linked to a pharmaceutical formulation. Examples of pharmaceutical formulations include cytotoxic agents or other cancer therapeutic agents and radioactive isotopes. Specific examples of cytotoxic agents include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, and actinomycin D. 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, and decarbazine), alkylating agents (such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, and mitomycin C). C) and cis-dichlorodiamineplatin(II) (DDP), anthracyclines (e.g., daunorubicin (formerly known as doxorubicin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly known as actinomycin), bleomycin, mithramycin, and antramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine). Examples of radioactive isotopes that can be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine. 131 ,indium 111 ,yttrium 90 Helu 177Methods for attaching antibodies to pharmaceutical formulations are known in the art, such as the use of various linker technologies. Examples of linker types include hydrazone, thioether, ester, dithioether, and peptide-containing linkers. For further discussion of the linkers and methods used to attach 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.

[0193] In some embodiments, the antibody derivative is a CTLA4 antibody multimer, such as an antibody dimer, trimer, or a higher-order oligomer of a monomeric antibody, which is a polymeric form of the CTLA4 antibody. The individual monomers within the antibody multimer can be the same or different. Furthermore, the individual antibodies within the multimer can have the same or different binding specificities. Antibody multimerization can be achieved through the natural aggregation of antibodies. For example, a certain percentage of purified antibody formulations (e.g., purified IgG4 molecules) spontaneously form protein aggregates containing antibody homodimers and other higher-order antibody multimers. Alternatively, antibody homodimers can be formed using chemical linking techniques known in the art, such as by using a cross-linking agent. Suitable crosslinking agents include those with heterobifunctionality, i.e., those having two distinct reactive groups separated by a suitable spacer (such as m-cis-butenylimide-benzoyl-N-hydroxysuccinimide, 4-(cis-butenylimide-methyl)cyclohexane-1-carboxylic acid succinimide, and S-acetylthio-acetic acid N-succinimide), or those with homobifunctionality (such as disuccinimide octanoate). The linkers are commercially available, for example, from Pierce Chemical Company, Rockford, IL. Antibody polymerization can also be achieved using recombinant DNA techniques known in the art.

[0194] Examples of other antibody derivatives provided by this disclosure include single-chain antibodies, miniature bifunctional antibodies, domain antibodies, nanobodies, and monoclonal antibodies. A "single-chain antibody" (scFv) consists of a V... L The structural domain is connected to V H The domain is composed of a single polypeptide chain, in which V L Domain and VH Domain pairing forms a monovalent molecule. Single-chain antibodies can be prepared according to methods known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). “Miniature bifunctional antibodies” consist of two chains, each containing a heavy chain variable region linked to a light chain variable region on the same polypeptide chain, connected by a short peptide linker, wherein the two regions on the same chain do not pair with each other, but rather with complementary domains on the other chain to form a bispecific molecule. Methods for preparing miniature bifunctional antibodies are known in the art (see, for example, Holliger P. et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448 and Poljak RJ et al., (1994) Structure 2:1121-1123). Domain-specific antibodies (dAbs) are small, functional binding units of antibodies, corresponding to the variable regions of the antibody's heavy or light chain. Domain-specific antibodies are well expressed in bacterial, yeast, and mammalian cell systems. Further details regarding domain-specific antibodies and methods of their production are known in the art (see, for example, U.S. Patents 6,291,158; 6,582,915; 6,593,081; 6,172,197; 6,696,245; European Patents 0368684 and 0616640; WO05 / 035572, WO04 / 101790, WO04 / 081026, WO04 / 058821, WO04 / 003019, and WO03 / 002609). Nanobodies are derived from the heavy chain of antibodies. Nanobodies typically contain a single variable domain and two constant domains (CH2 and CH3) and retain the antigen-binding capacity of the original antibody. Nanobodies can be prepared by methods known in the art (see, for example, U.S. Patent Nos. 6,765,087, 6,838,254, and WO 06 / 079372). Monoclonal antibodies consist of a light chain and a heavy chain of an IgG4 antibody. Monoclonal antibodies can be prepared by removing the hinge region of an IgG4 antibody. Further details regarding monoclonal antibodies and methods for their preparation can be found in WO2007 / 059782.

[0195] IV. Activated binding peptides targeting CTLA4

[0196] This disclosure also relates in part to precise / situation-dependent activatable binding peptides (i.e., activatable antibodies) that bind to human CTLA4, including activatable antibodies comprising any anti-CTLA4 antibody described herein (e.g., anti-CTLA4 antibody, anti-CTLA4 antibody binding fragment, and / or anti-CTLA4 antibody derivative); antigen-binding fragments of activatable anti-CTLA4 antibodies; and / or derivatives of activatable anti-CTLA4 antibodies. In some embodiments, the activatable anti-CTLA4 antibodies described herein may have an improved safety profile. For example, the anti-CTLA4 antibodies described herein may have better safety limits, as assessed by changes in spleen weight. Changes in spleen size with increasing dose of the administered drug are used as a benchmark to assess the safety limits of the drug candidate used. Figures 48A-48B As shown, the activatable anti-CTLA4 antibody described herein has a better safety margin compared to the parent antibody (the antibody without the masking portion).

[0197] In some embodiments, the activatable antibody of this disclosure comprises: (a) a masking portion (MM); (b) a cleavable portion (CM); and (c) a target-binding portion (TBM). In some embodiments, the MM is any masking portion described herein. In some embodiments, the CM is any cleavable portion described herein. In some embodiments, the TBM is any target-binding portion described herein (e.g., a target-binding portion (TBM) comprising a variable region of the antibody light chain and / or a variable region of the antibody heavy chain, such as the VH and / or VL of any anti-CTLA4 antibody described herein). In some embodiments, when the CM is not cleaved, the MM interferes with and / or inhibits the binding of the activatable antibody to its target (e.g., human CTLA4 or human CD137). In some embodiments, when the CM is cleaved, the activatable antibody is able to bind to its target (e.g., human CTLA4 or human CD137).

[0198] In some embodiments, the activatable antibody comprises: (a) a polypeptide comprising a masking portion (MM), a cleavable portion (CM), and a target-binding portion (TBM) from its N-terminus to its C-terminus, wherein the MM is any masking portion described herein, the CM is any cleavable portion described herein, and wherein the TBM comprises an antibody light chain variable region (VL); and (b) an antibody heavy chain variable region (VH).

[0199] In some embodiments, the activatable antibody comprises: (a) a polypeptide comprising a masking portion (MM), a cleavable portion (CM), and a target-binding portion (TBM) from its N-terminus to its C-terminus, wherein the MM is any masking portion described herein, the CM is any cleavable portion described herein, and wherein the TBM comprises an antibody heavy chain variable region (VH); and (b) an antibody light chain variable region (VL).

[0200] In some embodiments, the activatable antibody comprises: a polypeptide comprising a masking portion (MM), a cleavable portion (CM), and a target-binding portion (TBM) from its N-terminus to its C-terminus, wherein the MM is any masking portion described herein, the CM is any cleavable portion described herein, and wherein the TBM comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL).

[0201] The terms "activatable binding peptide," "ABP," or "activatable antibody" include peptides comprising a target-binding moiety (TBM), a cleavable moiety (CM), and a masking moiety (MM). In some embodiments, the TBM comprises an amino acid sequence that binds to a target. In some embodiments, the TBM comprises an antigen-binding domain (ABD) of an antibody or an antibody fragment thereof (e.g., any antibody or antigen-binding fragment described herein). In some embodiments, the antigen-binding domain comprises a heavy chain variable region containing one, two, or three of the heavy chain variable regions (HVRs) described herein, and a light chain variable region containing one, two, or three of the light chain variable regions (HVRs) described herein (e.g., one, two, or three of the heavy chain variable region (HVR) sequences shown in Table A, and / or one, two, or three of the light chain variable region (HVR) sequences, including all six HVRs of any exemplary antibody shown in Table A). In some embodiments, the antigen-binding domain comprises a heavy chain variable region containing any heavy chain variable region sequence described herein, and a light chain variable region containing any light chain variable region sequence described herein (e.g., heavy chain variable region sequences and / or light chain variable region sequences as shown in Table B). In some embodiments, the TBM (e.g., containing an ABD) comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), wherein the VH and the VL form a binding domain for binding a target in the absence of a MM. In some embodiments, the VH and VL are covalently linked, for example in an scFv. In some embodiments, the VH and VL are not covalently linked. In some embodiments, the VH and VL form a Fab fragment. In some embodiments, the VH is linked to an antibody heavy chain constant region, and the VL is linked to an antibody light chain constant region.

[0202] In some embodiments, the activatable antibody comprises a polypeptide comprising, from its N-terminus to its C-terminus, the following structure: masking moiety (MM)-cleavable moiety (CM)-VL, and the activatable antibody further comprises a second polypeptide comprising VH (e.g., a Fab fragment). In some embodiments, the activatable antibody comprises a polypeptide comprising, from its N-terminus to its C-terminus, the following structure: masking moiety (MM)-cleavable moiety (CM)-VL-VH (e.g., scFv). In some embodiments, the activatable antibody comprises a polypeptide comprising, from its N-terminus to its C-terminus, the following structure: masking moiety (MM)-cleavable moiety (CM)-VH, and the activatable antibody further comprises a second polypeptide comprising VL (e.g., a Fab fragment). In some embodiments, the activatable antibody comprises a polypeptide comprising, from its N-terminus to its C-terminus, the following structure: masking moiety (MM)-cleavable moiety (CM)-VH-VL (e.g., scFv).

[0203] CMs typically include cleavable amino acid sequences, such as those that act as substrates for enzymes, and / or cysteine-cysteine ​​pairs capable of forming reducible disulfide bonds. Therefore, when the terms “cleavage,” “cleavable,” “cleaved,” etc., are used in association with CMs, the terms encompass enzymatic cleavage, for example, achieved by a protease, and the disruption of disulfide bonds between cysteine-cysteine ​​pairs by disulfide bond reduction induced by exposure to a reducing agent.

[0204] MM refers to the amino acid sequence in which the CM of an activatable antibody interferes with or inhibits the binding of TBM to its target when the MM is intact (e.g., not cleaved by the corresponding enzyme, and / or contains unreduced cysteine-cysteine ​​disulfide bonds). In some embodiments, the MM interferes with or inhibits the binding of TBM to its target so efficiently that the binding of TBM to its target is extremely low and / or below the detection limit (e.g., the binding cannot be detected in ELISA or flow cytometry assays). The amino acid sequence of the CM may overlap with or be included within the MM. It should be noted that, for convenience, “ABP” or “activatable antibody” is used herein to refer to ABP or activatable antibodies in their uncleaved (or “native”) state and in their cleaved state. It will be apparent to those skilled in the art that in some embodiments, a cleaved ABP may lack MM due to, for example, the cleavage of the CM by a protease, which results in the release of at least the MM (e.g., when the MM is not covalently bonded to the ABP by a disulfide bond between cysteine ​​residues). An exemplary ABP is described in more detail below.

[0205] In some embodiments, the masking portion (MM) interferes with, hinders, reduces, prevents, or inhibits the target-binding portion from binding to its target, or competes with the target-binding portion for binding to its target (e.g., an "inactive" activatable antibody). In some embodiments, the masking portion (MM) interferes with, hinders, reduces, prevents, or inhibits the target-binding portion from binding to its target, or competes with the target-binding portion for binding to its target, only when the peptide has not yet been activated (e.g., activated by a change in pH (increase or decrease), activated by a change in temperature (increase or decrease), activated after contact with a second molecule (such as a small molecule or protein ligand), etc.). In some embodiments, activation induces cleavage of the peptide within the cleavage portion. In some embodiments, activation induces a conformational change in the peptide (e.g., a shift of the masking portion (MM)) such that the masking portion no longer prevents the activatable antibody from binding to its target. In some embodiments, the masking portion (MM) interferes with or hinders the target-binding portion from binding to its target only when the cleavable portion (CM) has not yet been cleaved by one or more proteases that cleave within the cleavable portion (CM), reduces the ability of the target-binding portion to bind to its target, prevents or inhibits the target-binding portion from binding to its target, or competes with the target-binding portion for binding to its target. In some embodiments, prior to activation, the masking portion (MM) has a masking efficiency of at least about 2.0 (e.g., at least about 2.0, at least about 3.0, at least about 4.0, at least about 5.0, at least about 6.0, at least about 7.0, at least about 8.0, at least about 9.0, at least about 10, at least about 25, at least about 50, 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, etc.). In some embodiments, masking efficiency is measured as the difference in affinity (before activation) of an activatable antibody containing a masking portion (MM) to its target relative to the affinity of a peptide lacking a masking portion to its target (e.g., the difference in affinity of an activatable antibody containing a masking portion (MM) (before activation) relative to a parent antibody lacking a masking portion (MM) for the target antigen (such as CTLA4), or the difference in affinity (before activation) of an activatable antibody containing a masking portion (MM) for the target antigen (such as CTLA4) relative to the affinity of the activatable antibody for the target antigen after activation). In some embodiments, masking efficiency is measured by the binding of an activatable antibody containing a masking portion (MM) to the EC. 50 (Before activation) Divide by the EC of the parent antibody 50 To measure (e.g., by measuring EC with ELISA) 50(See, for example, the method of Example 8). In some embodiments, masking efficiency is measured as the difference in affinity of the activatable antibody containing the masking portion (MM) for its target before activation relative to the difference in affinity of the activatable antibody containing the masking portion (MM) for its target after activation (e.g., the difference in affinity of the activatable antibody for the target antigen (such as CTLA4) before activation relative to the difference after activation). In some embodiments, the masking portion (MM) binds to the target-binding portion (TBM) and prevents the activatable antibody from binding to its target (e.g., an "inactive" activatable antibody). In some embodiments, the masking portion (MM) has a dissociation constant with respect to binding to the target-binding portion (TBM) that is greater than the dissociation constant of the target-binding portion (TBM) with respect to its target.

[0206] In some embodiments, after the activatable antibody has been activated (e.g., by treatment with one or more proteases that cleave within the cleavable moiety (CM), by pH change (increase or decrease), by temperature change (increase or decrease), by contact with a second molecule (such as an enzyme or protein ligand), etc.), the masking moiety (MM) does not interfere with or hinder the target-binding moiety (TBM) from binding to its target, reduces the ability of the target-binding moiety (TBM) to bind to its target, prevents or inhibits the target-binding moiety (TBM) from binding to its target, or competes with the target-binding moiety (TBM) for binding to its target. In some embodiments, after the cleavable moiety (CM) has been cleaved by one or more proteases that cleave within the cleavable moiety (CM), the masking moiety (MM) does not interfere with or hinder the target-binding moiety (TBM) from binding to its target, reduces the ability of the target-binding moiety (TBM) to bind to its target, prevents or inhibits the target-binding moiety (TBM) from binding to its target, or competes with the target-binding moiety (TBM) for binding to its target. In some implementations, after activation, the masking portion (MM) has a masking efficiency of up to about 1.75 (e.g., up to about 1.75, up to about 1.5, up to about 1.4, up to about 1.3, up to about 1.2, up to about 1.1, up to about 1.0, up to about 0.9, up to about 0.8, up to about 0.7, up to about 0.6, or up to about 0.5, etc.) (e.g., the relative affinity of the activated antibody after activation compared to the affinity of the parent antibody).

[0207] In some embodiments, the activatable antibody of this disclosure contains a pair of cysteine ​​residues at a fixed position to ensure a constrained conformation of the activatable antibody, and / or a masking moiety (MM) with few or no chemically unstable residues (such as methionine or tryptophan). Advantageously, including a pair of cysteine ​​residues at the fixed position ensures a constrained conformation of the activatable antibody, thereby tending to exhibit increased binding affinity and / or specificity. Furthermore, the activatable antibody of this disclosure includes a masking moiety with few or no residues such as methionine or tryptophan that are detrimental to the manufacturing process.

[0208] In some embodiments, the activated antibodies of this disclosure are situation-dependent (e.g., activated in certain conditions (such as in a tumor microenvironment rich in proteases) and are only able to bind their targets). In some embodiments, the activated antibodies of this disclosure provide improved safety compared to more conventional non-activated antibodies (e.g., exhibiting reduced toxicity, not inducing significant changes in the weight of many organs, not altering liver histopathology, hematology, and / or blood biochemistry, etc.). In some embodiments, the activated antibodies of this disclosure have improved pharmacokinetic properties (e.g., a longer in vivo half-life) compared to more conventional non-activated antibodies.

[0209] Anti-CTLA4 can activate antibody activity

[0210] In some embodiments, this disclosure relates to an activated antibody that binds to human CTLA4 when in its active form (e.g., the activated antibody is active after cleavage in the cleavable portion (e.g., by one or more proteases), but inactive before cleavage in the cleavable portion (e.g., by one or more proteases). In some embodiments, when in its active form, the activated antibody has at least one of the following functional properties (e.g., at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or all nine): (a) K at 500 nM or less, for example, about 10 nM or less. D(a) binds to human, cynomolgus monkey, mouse, rat and / or dog CTLA4; (b) exhibits antagonistic activity against human CTLA4; (c) does not bind to human PD-1, PD-L1, PD-L2, LAG3, TIM3, B7-H3, CD95, CD120a, OX40, CD40, BTLA, VISTA, ICOS and / or B7-H4 at concentrations up to 100 nM; (d) exhibits cross-reactivity with monkey, mouse, rat and / or dog CTLA4; (e) induces ADCC effects (e.g., against Treg); (f) activates human PBMCs (e.g., stimulates the secretion of IL-2 and / or IFNγ); (g) inhibits tumor cell growth; (h) has therapeutic effects on cancer; and (i) inhibits the binding of human CTLA4 to human CD80 and / or human CD86. This article also provides one or more activatable antibodies that compete or cross-competitively bind to human CTLA4 with one or more of the CTLA4-targeting and / or anti-CTLA4 antibodies described herein.

[0211] In some implementations, when in an inactive form, the antibody can be activated at approximately 500 nM or greater K. D This formulation binds to CTLA4 in humans, cynomolgus monkeys, mice, rats, and / or dogs. In some embodiments, when in active form, the antibody can be activated at approximately 500 nM or less (e.g., approximately 500 nM or less, approximately 450 nM or less, approximately 400 nM or less, approximately 350 nM or less, approximately 300 nM or less, approximately 250 nM or less, approximately 200 nM or less, approximately 150 nM or less, approximately 100 nM or less, approximately 90 nM or less, approximately 80 nM or less, approximately 70 nM or less, approximately 60 nM or less, approximately 50 nM or less, approximately 40 nM or less, approximately 30 nM or less, approximately 25 nM or less, approximately 20 nM or less, approximately 10 nM or less, approximately 1 nM or less, approximately 0.1 nM or less, etc.). D This formulation binds to CTLA4 in humans, cynomolgus monkeys, mice, rats, and / or dogs. In some embodiments, when in active form, the antibody can be activated at approximately 350 nM or less K. D This formulation binds to CTLA4 in humans, cynomolgus monkeys, mice, rats, and / or dogs. In some embodiments, when in active form, the antibody can be activated at approximately 100 nM or less K+. D Binds to human CTLA4. In some embodiments, when in active form, the antibody can be activated at approximately 50 nM or less K. D Binds to human CTLA4. In some embodiments, when in active form, the antibody can be activated at approximately 10 nM or less K. D Binding to human CTLA4. Measuring the K of activatable antibodies. DThe method can be performed using any method known in the art, including, for example, surface plasmon resonance, ELISA, isothermal titration calorimetry, membrane binding assay, EMSA, etc. In some embodiments, K D Measured by ELISA (see, for example, the following examples).

[0212] In some embodiments, when in an inactive form, the activated antibody does not have antagonistic activity against human CTLA4. In some embodiments, when in an active form, the activated antibody has antagonistic activity against human CTLA4 (e.g., inducing ADCC effects (e.g., against Tregs), activating PBMCs (e.g., by activating, inducing, and / or stimulating IL-2 and / or IFNγ secretion), blocking the binding of human CTLA4 to human CD80 and / or human CD86, etc.). In some embodiments, when in an active form, the activated antibody represses one or more activities of human CTLA4 (e.g., repressing one or more activities of human CTLA4 when cells expressing human CTLA4 (e.g., human cells) are contacted by the activated antibody).

[0213] In some embodiments, when in an inactive form, the activated antibody does not cross-react with CTLA4 in monkeys (e.g., cynomolgus monkeys), mice, rats, and / or dogs. In some embodiments, when in an active form, the activated antibody cross-reacts with CTLA4 in monkeys (e.g., cynomolgus monkeys), mice, rats, and / or dogs. In some embodiments, when in an active form, the activated antibody cross-reacts with monkey CTLA4. In some embodiments, when in an active form, the activated antibody cross-reacts with mouse CTLA4. In some embodiments, when in an active form, the activated antibody cross-reacts with rat CTLA4. In some embodiments, when in an active form, the activated antibody cross-reacts with dog CTLA4. In some embodiments, when in active form, the activated antibody exhibits cross-reactivity with CTLA4 in monkeys and mice; CTLA4 in monkeys and rats; CTLA4 in monkeys and dogs; CTLA4 in mice and rats; CTLA4 in mice and dogs; CTLA4 in rats and dogs; CTLA4 in monkeys, mice, and rats; CTLA4 in monkeys, mice, and dogs; CTLA4 in monkeys, rats, and dogs; CTLA4 in mice, rats, and dogs; or CTLA4 in monkeys, mice, rats, and dogs. In some embodiments, when in active form, the activated binding peptide exhibits cross-reactivity at about 350 nM (e.g., at about 1 nM, at about 10 nM, at about 25 nM, at about 50 nM, at about 75 nM, at about 100 nM, at about 150 nM, at about 200 nM, at about 250 nM, at about 300 nM, at about 350 nM). Methods for measuring cross-reactivity are known in the art, including but not limited to surface plasmon resonance, ELISA, isothermal titration calorimetry, membrane binding assay, EMSA, etc.

[0214] In some embodiments, when in an inactive form, the activated antibody does not induce an ADCC effect (e.g., against CTLA4-expressing human cells such as Tregs). In some embodiments, when in an inactive form, the activated antibody has a reduced ADCC effect compared to a control binding peptide (e.g., a parental antibody) (e.g., against CTLA4-expressing human cells such as Tregs). In some embodiments, when in an active form, the activated antibody induces an ADCC effect (e.g., against CTLA4-expressing cells such as Tregs). Methods for measuring ADCC effects (e.g., in vitro methods) are known in the art, including but not limited to the methods described in the following examples. In some embodiments, when in an inactive form, the activated antibody induces an ADCC effect of less than about 10% (e.g., induces less than about 10%, less than about 5%, less than about 1%, etc.) compared to a control (e.g., a parental antibody). In some implementations, when in active form, the activated antibody induces an ADCC effect of more than about 10% relative to a control (e.g., an isotype control) (e.g., induces ADCC of more than about 10%, more than about 15%, more than about 20%, more than about 25%, more than about 30%, more than about 35%, more than about 40%, etc.).

[0215] In some embodiments, the activatable antibody is capable of inhibiting tumor cell growth and / or proliferation. In some embodiments, when exposed to the activatable antibody, tumor cell growth and / or proliferation are inhibited by at least about 5% (e.g., at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 99%), relative to corresponding tumor cells not exposed to the activatable antibody (or relative to corresponding tumor cells exposed to an isotype control antibody). In some embodiments, when the activatable antibody is administered to a subject, the activatable antibody is capable of reducing tumor volume in the subject. In some embodiments, the activated antibody is capable of reducing the tumor volume in a subject by at least about 5% (e.g., at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 99%) relative to the initial tumor volume in the subject (e.g., before administration of the activated antibody; compared to the corresponding tumor in a subject administered with an isotype control antibody). Methods for monitoring tumor cell growth / proliferation, tumor volume, and / or tumor suppression are known in the art, including, for example, methods described in the following examples.

[0216] In some implementations, the activatable antibody has a therapeutic effect on cancer. In some implementations, the activatable antibody alleviates one or more signs or symptoms of cancer. In some implementations, when the activatable antibody is administered, a subject with cancer experiences partial or complete remission.

[0217] In some embodiments, this disclosure provides, when in active form, isolated and reactivatable antibodies that compete or cross-competitively bind to human CTLA4 with antibodies comprising: a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 23; HVR-H2 containing the amino acid sequence of SEQ ID NO: 35; and HVR-H3 containing the amino acid sequence of SEQ ID NO: 45; and / or b) HVR-L1 containing the amino acid sequence of SEQ ID NO: 58; HVR-L2 containing the amino acid sequence of SEQ ID NO: 66; and HVR-L3 containing the amino acid sequence of SEQ ID NO: 75. In some embodiments, this disclosure provides, when in active form, isolated and reactivatable antibodies that compete or cross-competitively bind to human CTLA4 with antibodies comprising: a) a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 87; and / or b) a light chain variable region containing the amino acid sequence of SEQ ID NO: 100. The ability of an activatable antibody to competitively or cross-competitively bind to an antibody can be determined using standard binding assays known in the art, such as BIAcore analysis, ELISA assays, or flow cytometry. For example, an antibody (e.g., as described above) can be saturated to bind to human CTLA4, and then the ability of the activatable antibody (in its active form) to bind to CTLA4 can be measured. If the activatable antibody can bind to CTLA4 simultaneously with the antibody, then the activatable antibody binds to a different epitope. However, if the activatable antibody cannot bind to CTLA4 simultaneously, then the activatable antibody binds to the same epitope, overlapping epitopes, or epitopes closely adjacent to the epitope bound by the antibody. This assay can be performed using various methods such as ELISA, RIA, FACS, or surface plasmon resonance.

[0218] In some embodiments, the activated antibody (in its inactive form) does not inhibit the binding of CTLA4 to one or more of its binding partners (e.g., human CTLA4 with human CD80, human CTLA4 with human CD86). In some embodiments, the activated antibody (in its active form) inhibits the binding of CTLA4 to one or more of its binding partners (e.g., human CTLA4 with human CD80, human CTLA4 with human CD86). In some embodiments, the activated antibody inhibits the binding of CTLA4 to its ligand in vitro. In some implementations, for inhibiting the binding of CTLA4 to CD80 and / or CD86, the activating antibody has a half-maximum inhibitory concentration (IC50) of about 500 nM or less (e.g., about 500 nM or less, about 400 nM or less, about 300 nM or less, about 200 nM or less, about 100 nM or less, about 50 nM or less, about 25 nM or less, about 10 nM or less, about 1 nM or less, etc.). 50 In some implementations, the activatable antibody has a half-maximum inhibitory concentration (IC50) of about 100 nM or less for inhibiting the binding of CTLA4 to CD80 and / or CD86. 50 In some embodiments, when provided at concentrations of about 100 nM or greater (e.g., about 100 nM or greater, about 500 nM or greater, about 1 μM or greater, about 10 μM or greater, etc.), the activatable antibody completely inhibits the binding of human CTLA4 to CD80 and / or CD86. As used herein, the term "completely inhibiting" means that the activatable antibody is able to reduce the binding between the first protein and the second protein by at least about 80% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, etc.). Methods for measuring the ability of a peptide to inhibit the binding of the first protein (e.g., human CTLA4) and the second protein (e.g., human CD80 or human CD86) are known in the art, including but not limited to BIAcore analysis, ELISA assays, and flow cytometry.

[0219] Covered area (MM)

[0220] In some embodiments, this disclosure relates to an activatable antibody comprising a masking portion (MM). In some embodiments, the masking portion (MM) comprises according to formula (XVIII): X m CX n CZ oThe amino acid sequence of (SEQ ID NO: 134), wherein m is 2-10, n is 3-10, and o is 1-10, wherein each X is independently selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and wherein each Z is independently selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P. In some embodiments, X is not W, M, and / or C. In some embodiments, X of formula (XVIII) m Each X in the formula is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P, and / or X of formula (XVIII). n Each X in the formula is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P. In some embodiments, MM comprises amino acids according to formula (XX): (NNK) m TGY(NNK) n TGY(NHC) o The polypeptide encoded by the polynucleotide sequence (SEQ ID NO: 136), wherein each N is independently A, G, T or C, each K is independently T or G, each Y is independently T or C, and each H is independently A, T or C.

[0221] In some implementations, the masking portion (MM) comprises according to formula (XIX): Z m CZ n CZ o The amino acid sequence of (SEQ ID NO: 135), wherein m is 2-10, n is 3-10, and o is 1-10, and each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P.

[0222] In some implementations, m is 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10. In some implementations, m is 6-8. In some implementations, m is 3, 4, 5, 6, 7, 8, 9, or 10. In some implementations, m is 6.

[0223] In some implementations, n is 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10. In some implementations, n is 6-8. In some implementations, n is 3, 4, 5, 6, 7, 8, 9, or 10. In some implementations, n is 6. In some implementations, n is 8.

[0224] In some implementations, o is 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10. In some implementations, o is 1-2. In some implementations, o is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some implementations, o is 2.

[0225] In some embodiments, the masking portion (MM) comprises an amino acid sequence according to formula (XXI): Z6CX6CZ2 (SEQ ID NO: 137), wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P.

[0226] In some embodiments, the masking portion (MM) comprises an amino acid sequence according to formula (XXII): Z6CX8CZ2 (SEQ ID NO: 138), wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P.

[0227] In some embodiments, the first peptide (FP) comprises an amino acid sequence according to formula (XXIII): (Z6)C(Z6)C(Z2) (SEQ ID NO: 139), wherein each Z is an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P.

[0228] In some embodiments, the masking portion (MM) comprises an amino acid sequence according to formula (XXIV): (Z6)C(Z8)C(Z2) (SEQ ID NO: 140), wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P. In some embodiments, the activatable antibody comprises an amino acid selected from X. m CPDHPYPCXX (SEQ ID NO: 181), X m CDAFYPYCXX (SEQ ID NO: 182), X m CDSHYPYCXX (SEQ ID NO: 183) and X m The masked portion (MM) of the sequence of the group consisting of CVPYYYACXX (SEQ ID NO: 184), where m is 2-10, and each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y. In some embodiments, the activatable antibody comprises a masking portion (MM) containing the sequence EVGSYNFVADSCPDHPYPCSA (SEQ ID NO: 189), EVGSYIVHHSDCDAFYPYCDS (SEQ ID NO: 190), EVGSYYSAYPACDSHYPYCNS (SEQ ID NO: 191), EVGSYPNPSSDCVPYYYACAY (SEQ ID NO: 192), EVGSYYSAYPACDSHYPYCQS (SEQ ID NO: 193), EVGSYPQPSSDCVPYYYACAY (SEQ ID NO: 195), or EVGSYPNPASDCVPYYYACAY (SEQ ID NO: 196). In some implementations, MM includes the sequences EDCVPYYYACAY (SEQ ID NO: 213), EVGSSDCVPYYYACAY (SEQ ID NO: 214), EDCDAFYPYCDS (SEQ ID NO: 215), or EVGHSDCDAFYPYCDS (SEQ ID NO: 216).

[0229] In some embodiments, the masking portion (MM) comprises an amino acid sequence selected from NFVADSCPDHPYPCSA (SEQ ID NO: 141), IVHHSDCDAFYPYCDS (SEQ ID NO: 142), YSAYPACDSHYPYCNS (SEQ ID NO: 143), PNPSSSDCVPYYYACAY (SEQ ID NO: 144), YSAYPACDSHYPYCQS (SEQ ID NO: 145), PQPSSDCVPYYYACAY (SEQ ID NO: 146), and PNPSSSDCVPYYYACAY (SEQ ID NO: 147).

[0230] In some embodiments, any masking portion (MM) described herein may further include one or more additional amino acid sequences (e.g., one or more polypeptide tags). Examples of suitable additional amino acid sequences may include, but are not limited to, purification tags (such as his tags, flag tags, maltose-binding proteins, and glutathione S-transferase tags), detection tags (such as tags detectable by photometric methods (e.g., red or green fluorescent proteins), tags with detectable enzyme activity (e.g., alkaline phosphatase), tags containing secretion sequences, leader sequences, and / or stabilization sequences, protease cleavage sites (e.g., furin cleavage sites, TEV cleavage sites, thrombin cleavage sites), etc. In some embodiments, one or more additional amino acid sequences are located at the N-terminus of the masking portion (MM). In some embodiments, the additional amino acid sequence comprises or consists of the sequence EVGSY (SEQ ID NO: 148).

[0231] In some embodiments, prior to activation (e.g., prior to treatment with one or more proteases that cleave within the cleavable portion (CM), prior to exposure to (local) pH changes (increases or decreases), prior to temperature changes (increases or decreases), prior to contact with a second molecule (such as a small molecule or protein ligand), etc.), the masking portion binds to the target-binding portion (TBM) and inhibits the binding of the activated antibody to its target, but after activation (e.g., after treatment with one or more proteases that cleave within the cleavable portion (CM), after exposure to (local) pH changes (increases or decreases), after temperature changes (increases or decreases), after contact with a second molecule (such as a small molecule or protein ligand), etc.), the TBM does not bind and / or inhibits the binding of the activated antibody to its target. In some embodiments, when the CM is not cleaved, the masking portion (MM) inhibits the binding of the activated antibody to its target, but when the CM is cleaved, it does not inhibit the binding of the activated antibody to its target. In some implementations, the masking portion (MM) has a dissociation constant with respect to binding TBM that is greater than the dissociation constant of the activated antibody with respect to its target (when in active form) (e.g., at least about 1.5 times larger, at least about 2 times larger, at least about 2.5 times larger, at least about 3 times larger, at least about 3.5 times larger, at least about 4 times larger, at least about 4.5 times larger, at least about 5 times larger, at least about 10 times larger, at least about 100 times larger, at least about 500 times larger, etc.).

[0232] Decomposable component (CM)

[0233] In some embodiments, this disclosure relates to an activatable antibody comprising a cleavable moiety (CM). In some embodiments, the cleavable moiety (CM) is cleaved and / or destroyed by treatment with one or more proteases that cleave within the cleavable moiety (CM), by pH changes (increases or decreases), by temperature changes (increases or decreases), and / or by contact with a second molecule (such as a small molecule or protein ligand).

[0234] In some embodiments, the cleavable portion (CM) includes at least a first cleavage site (CS1) (e.g., a first protease cleavage site). In some embodiments, the first cleavage site is a first protease cleavage site. Any suitable protease cleavage site that is recognized and / or cleaved by any protease known in the art (e.g., a protease known to co-localize with a target of an activatable antibody containing CM) can be used, including protease cleavage sites recognized and / or cleaved by, for example, urokinase-type plasminogen activator (uPA); matrix metalloproteinases (e.g., MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-19, MMP-20, MMP-2...). 3. MMP-24, MMP-26 and / or MMP-27); Tobacco Etching Virus (TEV) protease; Fibrinolytic enzyme; Thrombin; PSA; PSMA; ADAMS / ADAMTS (e.g., ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMDEC1, ADAMTS1, ADAMTS4 and / or ADAMTS5); Kaspar enzymes (e.g., Kaspar enzyme-1, Kaspar enzyme-2, Kaspar enzyme-3, Kaspar enzyme-4, Kaspar enzyme-5, Kaspar enzyme-6, Kaspar enzyme-7, Kaspar enzyme-8, Kaspar enzyme-9 ...1, Kaspar enzyme-2, Kaspar enzyme-3, Kaspar enzyme-4, Kaspar enzyme-5, Kaspar enzyme-6, Kaspar enzyme-7, Kaspar enzyme-8, Kaspar enzyme-9, Kaspar enzyme-1, Kaspar enzyme-2, Kaspar enzyme-2, Kaspar enzyme-2, Kaspar enzyme-2, Kaspar enzyme-2, Kaspar enzyme-2, Kaspar enzyme-2, Kaspar enzyme-2, Kaspar enzyme-2, Kaspar enzyme-2, Kaspar enzyme-2, Kaspar enzyme-2, Kaspar enzyme-2, Kaspar enzyme-2, Kaspar enzyme-2 Kasparase-10, Kasparase-11, Kasparase-12, Kasparase-13 and / or Kasparase-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, Legumain and / or Otubain-2); KLK (e.g., KLK4, ... KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13 and / or KLK14); metalloproteinases (e.g., meprin, neprilysin, PSMA and / or BMP-1); serine proteases (e.g., activated protein C, cathepsin A, cathepsin G, chomase and / or coagulation factor proteases (such as FVIIa, FIXa, FXa, FXIa, FXIIa)); elastase; granzyme B; guanidinylbenzoic acidase; HtrA1; human neutrophil elastase;Lactoferrin; marapsin; NS3 / 4A; PACE4; tPA; tryptase; type II transmembrane serine proteases (TTSPs) (e.g., DESC1, DPP-4, FAP, hepsin, matrix protease-2, MT-SP1 / matrix protease, 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 an amino acid sequence selected from SGRSA (SEQ ID NO: 149), PLGLAG (SEQ ID NO: 150), and ENLYFQG (SEQ ID NO: 151).

[0235] In some embodiments, the activatable antibody comprises a masked portion (MM) and a cleavable portion (CM) containing an amino acid sequence according to formula (XXV): EVGSY(Z6)C(Z6)C(Z2)SGRSA (SEQ ID NO: 152), wherein each Z is independently an amino acid selected from D, A, Y, S, T, N, I, L, F, V, H and P.

[0236] In some embodiments, the activatable antibody comprises a masked portion (MM) and a cleavable portion (CM) containing an amino acid sequence according to formula (XXVI): EVGSY(Z6)C(X6)C(Z2)SGRSA (SEQ ID NO: 153), wherein each X is independently selected from amino acids A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and wherein each Z is independently selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P.

[0237] In some embodiments, the activatable antibody comprises a masked portion (MM) and a cleavable portion (CM) containing an amino acid sequence according to formula (XXVII): EVGSY(Z6)C(Z8)C(Z2)SGRSA (SEQ ID NO: 154), wherein each Z is independently an amino acid selected from D, A, Y, S, T, N, I, L, F, V, H and P.

[0238] In some embodiments, the activatable antibody comprises a masked portion (MM) and a cleavable portion (CM) containing an amino acid sequence according to formula (XXVIII): EVGSY(Z6)C(X8)C(Z2)SGRSA (SEQ ID NO: 155), wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P.

[0239] In some embodiments, the cleavable portion (CM) further includes a first linker (L1). In some embodiments, the first linker (L1) is located at the C-terminus of the first cleavage site (CS1) (e.g., the first protease cleavage site). In some embodiments, the cleavable portion (CM) comprises the following structure from the N-terminus to the C-terminus: (CS1)-L1.

[0240] Any suitable connector (e.g., flexible connector) known in the art can be used, including, for example: glycine polymer (G)n, where n is an integer of at least 1 (e.g., 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, etc.); glycine-serine polymer (GS)n, where n is an integer of at least 1 (e.g., 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, etc.), such as GGGGS (SEQ ID NO: 156), SGGS (SEQ ID NO: 157), GGSG (SEQ ID NO: 158), GGSGG (SEQ ID NO: 159), GGSSG (SEQ ID NO: 160), GGSGG (SEQ ID NO: 161), GGGSG (SEQ ID NO: 162) and / or GSSSG (SEQ ID NO: 163); glycine-alanine polymer; alanine-serine polymer; etc. 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. In some embodiments, the linker comprises an amino acid sequence selected from SEQ ID NO: 159-163. In some embodiments, the linker comprises an amino acid sequence of SEQ ID NO: 156 or 157.

[0241] In some embodiments, the cleavable portion (CM) further includes at least a second cleavage site (e.g., at least a second, at least a third, at least a fourth, at least a fifth, etc.). In some embodiments, the cleavable portion (CM) further includes a second cleavage site (CS2). In some embodiments, the second cleavage site is a second protease cleavage site. The second protease cleavage site may be any suitable protease cleavage site recognized and / or cleaved by any of the proteases described above. In some embodiments, the first (CS1) and second (CS2) cleavage sites are protease cleavage sites recognized and / or cleaved by the same protease. In some embodiments, the first (CS1) and second (CS2) cleavage sites are protease cleavage sites recognized and / or cleaved by different proteases (e.g., the first protease cleavage site is recognized and / or cleaved by uPA, while the second protease cleavage site is recognized and / or cleaved by MMP-2; the first protease cleavage site is recognized and / or cleaved by uPA, while the second protease cleavage site is recognized and / or cleaved by MMP-9; the first protease cleavage site is recognized and / or cleaved by uPA, while the second protease cleavage site is recognized and / or cleaved by TEV protease; etc.). In some embodiments, at least the second cleavage site (CS2) is located at the C-terminus of the first connector (L1). In some embodiments, the cleavable portion (CM) comprises the following structure from the N-terminus to the C-terminus: (CS1)-L1-(CS2).

[0242] In some embodiments, the cleavable portion (CM) further comprises at least a second adapter (e.g., at least a second, at least a third, at least a fourth, at least a fifth, etc.). In some embodiments, the cleavable portion (CM) further comprises a second adapter (L2). The second adapter (L2) may be any suitable adapter described above. In some embodiments, the second adapter comprises an amino acid sequence selected from SEQ ID NO: 156-163. In some embodiments, the first (L1) and second (L2) adapters are identical (e.g., both adapters comprise the sequence of SEQ ID NO: 156 or 157). In some embodiments, the first (L1) and second (L2) adapters are different (e.g., the first adapter (L1) comprises the amino acid sequence of SEQ ID NO: 156, while the second adapter (L2) comprises the amino acid sequence of SEQ ID NO: 157, etc.). In some embodiments, at least the second adapter (L2) is located at the C-terminus of the second cleavage site (CS2). In some embodiments, the cleavable portion (CM) comprises the following structure from the N-terminus to the C-terminus: (CS1)-L1-(CS2)-L2.

[0243] Exemplary MM-CM sequence

[0244] In some embodiments, the activatable antibody of this disclosure comprises the following structure from the N-terminus to the C-terminus: (FP)-(PCS1)-L1-(PCS2)-L2. In some embodiments, the activatable antibody comprises according to formula (XXIX)EVGSYX1X2X3X4X5X6CX7X8X9X 10 X 11 X 12 CX 13 X 14The amino acid sequence of SGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 164), wherein X1 is A, D, I, N, P or Y, X2 is A, F, N, S or V, X3 is A, H, L, P, S, V or Y, X4 is A, H, S or Y, X5 is A, D, P, S, V or Y, X6 is A, D, L, S or Y, X7 is D, P or V, X8 is A, D, H, P, S or T, X9 is A, D, F, H, P or Y, X10 is L, P or Y, X11 is F, P or Y, X12 is A, P, S or Y, X13 is A, D, N, S, T or Y, and X14 is A, S or Y.In some embodiments, the activatable antibodies of this disclosure comprise the following amino acid sequences: EVGSYDALHYACPPDYYACYYSGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 165); EVGSYNSYHAYCPHPLYPCTASGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 166); EVGSYASSAVLCVTAYFSCNSSGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 167); EVGSYNFVADSCPDHPYPCSASGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 168); EVGSYNFVADSCPDHPYPCSASGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 169); EVGSYIVHHSDCDAFYPYCDSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 169). NO: 170); EVGSYIVHHSDCDAFYPYCDSSSGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 171); EVGSYYSAYPACDSHYPYCNSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 172); EVGSYYSAYPACDSHYPYCNSSGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 173); EVGSYPNPSSDCVPYYYACAYSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 174); EVGSYPNPSSDCVPYYYACAYSGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 175); EVGSYYSAYPACDSHYPYCQSSGRSAGGGGSPLGLAGSGGS (SEQ ID SEQ ID NO: 176); EVGSYYSAYPACDSHYPYCNSAGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 177); EVGSYPQPSSDCVPYYYACAYSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 178); and / or EVGSYPNPASDCVPYYYACAYSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 179). In some embodiments, the polypeptides of this disclosure comprise the following structure from the N-terminus to the C-terminus: (FP)-(PCS1)-L1-(PCS2)-L2-(TBM).

[0245] In some embodiments, the activatable antibody comprises the amino acid sequence SGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 220), SGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 221), or SGRSAPLGLA (SEQ ID NO: 222). In some embodiments, the activatable antibody comprises the sequence EV(Zn)C(X8)C(Z2)SGRSA (SEQ ID NO: 217), EDC(Z6)C(Z2)SGRSA (SEQ ID NO: 218), or EDC(Z6)C(Z2)PLGLA (SEQ ID NO: 219), wherein each X is independently selected from the group consisting of amino acids selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, wherein n is 1-11, and wherein each Z is independently selected from the group consisting of amino acids selected from D, A, Y, S, T, N, I, L, F, V, H, and P.

[0246] Target-binding membrane (TBM)

[0247] In some embodiments, this disclosure relates to an activatable antibody comprising a target-binding moiety (TBM). In some embodiments, the target-binding moiety (TBM) comprises a variable region of the antibody light chain and / or a variable region of the antibody heavy chain. In some embodiments, the target-binding moiety (TBM) comprises a variable region of the antibody light chain. In some embodiments, the target-binding moiety (TBM) comprises both a variable region of the antibody light chain and a variable region of the antibody heavy chain.

[0248] In some embodiments, the target-binding moiety (TBM) 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 be in 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.

[0249] Any one or more of the target-binding regions (TBMs) described herein may include: any HVR sequence described herein (e.g., one, two, or three of the heavy chain variable region HVR sequences shown in Table A above, and / or one, two, or three of the light chain variable region HVR sequences); any heavy chain variable region sequence and / or light chain variable region sequence described herein (e.g., heavy chain variable region sequence and / or light chain variable region sequence shown in Table B above); and / or any antibody described herein.

[0250] In some embodiments, the target-binding portion (TBM) comprises the sequence of one or more of the anti-CTLA4 antibodies described herein, including antibodies comprising specific amino acid sequences relating to HVR, variable regions (VL, VH), and / or light and heavy chains (e.g., IgG1, IgG2, IgG4). In some embodiments, the target-binding portion (TBM) comprises an antibody light chain variable region comprising HVR-L1 containing the amino acid sequence RASQSVRGRFLA (SEQ ID NO: 58), HVR-L2 containing the amino acid sequence DASNRATGI (SEQ ID NO: 66), and / or HVR-L3 containing the amino acid sequence YCQQSSSWPPT (SEQ ID NO: 75). In some embodiments, the target-binding portion (TBM) comprises an antibody light chain variable region containing the amino acid sequence of SEQ ID NO: 100 or a sequence having at least 90% (e.g., 95%, 96%, 97%, 98%, or 99%) sequence identity with the sequence of SEQ ID NO: 100. In some embodiments, the target-binding portion (TBM) comprises an antibody heavy chain variable region containing HVR-H1 with the amino acid sequence YSISSGYHWSWI (SEQ ID NO: 23), HVR-H2 with the amino acid sequence LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 35), and / or HVR-H3 with the amino acid sequence ARSYVYFDY (SEQ ID NO: 45). In some embodiments, the target-binding portion (TBM) includes an antibody heavy chain variable region containing the amino acid sequence of SEQ ID NO: 87 or a sequence having at least 90% (e.g., 95%, 96%, 97%, 98%, or 99%) sequence identity with the sequence of SEQ ID NO: 87. In some embodiments, the target binding portion (TBM) comprises: a) an antibody light chain variable region comprising HVR-L1 containing the amino acid sequence RASQSVRGRFLA (SEQ ID NO: 58), HVR-L2 containing the amino acid sequence DASNRATGI (SEQ ID NO: 66), and / or HVR-L3 containing the amino acid sequence YCQQSSSWPPT (SEQ ID NO: 75); and b) an antibody heavy chain variable region comprising HVR-H1 containing the amino acid sequence YSISSGYHWSWI (SEQ ID NO: 23), HVR-H2 containing the amino acid sequence LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 35), and / or HVR-H3 containing the amino acid sequence ARSYVYFDY (SEQ ID NO: 45).In some embodiments, the target binding region (TBM) includes an antibody light chain variable region containing the amino acid sequence of SEQ ID NO: 100 and an antibody heavy chain variable region containing the amino acid sequence of SEQ ID NO: 87.

[0251] Properties of activated binding peptides

[0252] In some embodiments, the activatable binding polypeptide (i.e., the activatable antibody) of this disclosure comprises: (a) a masking portion (MM), (b) a cleavable portion, and (c) a target-binding portion. In some embodiments, the masking portion (MM) binds to the target-binding portion (TBM) of the activatable antibody and reduces or inhibits the binding of the activatable binding portion to CTLA4 (e.g., human CTLA4) compared to the binding of a corresponding binding polypeptide lacking the masking portion to CTLA4 (e.g., human CTLA4), and / or compared to the binding of a parent antibody to CTLA4 (e.g., human CTLA4).

[0253] In some embodiments, an "activatable" binding peptide refers to a binding peptide that exhibits a first binding level to CTLA4 when in an inhibited, masked, and / or uncleaved state, and a second binding level to CTLA4 when in an uninhibited, unmasked, and / or cleaved state, wherein the second CTLA4 binding level is greater than the first CTLA4 binding level. In some embodiments, the opportunity for the activated binding peptide to approach CTLA4 increases after cleavage within the cleavable portion (e.g., by one or more proteases).

[0254] In some embodiments, the binding affinity of the peptide to CTLA4 (e.g., human CTLA4) increases by at least about 2-fold (e.g., at least about 2.5-fold, at least about 2.5-fold, at least about 2.5-fold) compared to before activation of the activatable antibody, after activation of the activatable antibody (e.g., after activation by treatment with one or more proteases that cleave within the cleavable moiety (CM), after activation by pH change (increase or decrease), after activation by temperature change (increase or decrease), after activation by contact with a second molecule (such as a small molecule), etc.) 3. When the concentration is at least about 3.5 times, at least about 4 times, at least about 4.5 times, at least about 5 times, at least about 5.5 times, at least about 6 times, at least about 6.5 times, at least about 7 times, at least about 7.5 times, at least about 8 times, at least about 8.5 times, at least about 9 times, at least about 9.5 times, at least about 10 times, at least about 25 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 250 times, at least about 500 times, at least about 750 times, or at least about 1000 times or more, the activatable antibody of this disclosure is generally considered to be an "activatable" binding peptide. In some embodiments, if, after "activation," the EC50 of the activatable antibody... 50If the concentration is reduced to at least about 1 / 2 (e.g., at least about 1 / 2, at least about 1 / 2.5, at least about 1 / 3, at least about 1 / 3.5, at least about 1 / 4, at least about 1 / 4.5, at least about 1 / 5, at least about 1 / 5.5, at least about 1 / 6, at least about 1 / 6.5, at least about 1 / 7, at least about 1 / 7.5, at least about 1 / 8, at least about 1 / 8.5, at least about 1 / 9, at least about 1 / 9.5, at least about 1 / 10, at least about 1 / 25, at least about 1 / 50, at least about 1 / 75, at least about 1 / 100, at least about 1 / 250, at least about 1 / 500, at least about 1 / 750 or at least about 1 / 1000, or reduced to less) (e.g., as measured by ELISA or FACS assay; see the examples below), then the activatable antibody of this disclosure is generally considered "activatable". In some embodiments, if the peptide's EC is reduced after treatment with a protease used for cleavage within the cleavable portion (CM), 50 If the concentration is reduced to at least about 1 / 2 (e.g., as measured by ELISA or FACS assay; see the examples below), then the activatable antibody of this disclosure is generally considered "activatable".

[0255] In some implementations, when the masking portion (MM) binds to the target-binding portion (TBM) of the activatable antibody, the activatable antibody reacts with respect to the K-type receptor of CTLA4. D This is when the masking portion (MM) does not bind to the target-binding portion (TBM) (e.g., after the “activation” of an activatable antibody (such as after protease treatment to cleave within the cleavable portion (CM))) and / or when the parental antibody has K with respect to CTLA4. D Approximately 2 times (e.g., approximately 2, approximately 2.5, approximately 3, approximately 3.5, approximately 4, approximately 4.5, approximately 5, approximately 5.5, approximately 6, approximately 6.5, approximately 7, approximately 7.5, approximately 8, approximately 8.5, approximately 9, approximately 9.5, approximately 10, approximately 25, approximately 50, approximately 75, approximately 100, approximately 250, approximately 500, approximately 750, or approximately 1000 or more). Methods for measuring affinity are known in the art, including, for example, methods described in the following examples.

[0256] In some implementations, relative to when the masking portion does not bind to the target-binding portion (e.g., after “activation” of the activatable antibody (such as after protease treatment to cleave within the cleavable portion (CM))), and / or relative to the parent antibody’s K with respect to CTLA4 D When the masking portion binds to the target-binding portion of the activatable antibody, the activatable antibody's K-axis with respect to CTLA4... DThe reduction is at least about 25% (e.g., at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%). Methods for measuring affinity are known in the art, including, for example, methods described in the following examples.

[0257] In some embodiments, the masking portion spatially prevents the binding of the activatable antibody to CTLA4, and / or allosterically prevents the binding of the activatable antibody to CTLA4. In some embodiments, the masking portion does not contain the amino acid sequence of the natural binding partner of the activatable antibody and / or the parent antibody.

[0258] In some embodiments, the dissociation constant of the masking portion with respect to the target-binding portion is greater than the dissociation constant of the activated antibody with respect to CTLA4 (when activated). In some embodiments, the dissociation constant of the masking portion with respect to the target-binding portion is approximately 2 (e.g., approximately 2, approximately 2.5, approximately 3, approximately 3.5, approximately 4, approximately 4.5, approximately 5, approximately 5.5, approximately 6, approximately 6.5, approximately 7, approximately 7.5, approximately 8, approximately 8.5, approximately 9, approximately 9.5, approximately 10, approximately 25, approximately 50, approximately 75, approximately 100, approximately 250, approximately 500, approximately 750, or approximately 1000 or more) times greater than the dissociation constant of the activated antibody with respect to CTLA4 (when activated). In some embodiments, the dissociation constant of the masking portion with respect to the target-binding portion is approximately equal to the dissociation constant of the activated antibody with respect to CTLA4 (when activated).

[0259] The activatable antibodies described herein can be further modified. In some embodiments, the activatable antibody is linked to an additional molecular entity. Examples of additional molecular entities include pharmaceutical formulations, peptides or proteins, detection agents or labels, and antibodies.

[0260] In some embodiments, the activatable antibody of this disclosure is linked to a pharmaceutical formulation. Examples of pharmaceutical formulations include cytotoxic agents or other cancer therapeutic agents and radioisotopes. 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 (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine), alkylating agents (e.g., dichloromethyldiethylamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamineplatin(II)(DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly known as doxorubicin) and doxorubicin), antibiotics (e.g., dermatomycin (formerly known as actinomycin), bleomycin, mirtamicin and atrazomycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine). Examples of radioactive isotopes that can be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine. 131 ,indium 111 ,yttrium 90 Helu 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, dithioether, and peptide-containing linkers. For further discussion of the linkers and methods used to attach 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.

[0261] V. Nucleic acid, vector, host cell, and recombinant methods for generating CTLA4 antibodies and / or precision / situation-dependent activatable antibodies.

[0262] Another aspect of this disclosure provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence of a binding molecule (e.g., an antibody or an activatable antibody) provided herein. The amino acid sequence encoded by the nucleotide sequence may be any portion of an antibody, such as an HVR, a sequence containing one, two, or three HVRs, a variable region of the heavy chain, a variable region of the light chain, or may be a full-length heavy chain or a full-length light chain. The nucleic acid of this disclosure may be, for example, DNA or RNA, and may or may not contain intron sequences. Typically, the nucleic acid is a cDNA molecule.

[0263] In some embodiments, this disclosure provides a nucleotide sequence comprising an amino acid sequence selected from the group consisting of: (1) the amino acid sequence of HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and / or HVR-L3 of the illustrative antibody described herein; (2) the variable region of the heavy chain and / or the variable region of the light chain of the illustrative antibody described herein; or (3) the full-length heavy chain or the full-length light chain of the illustrative antibody.

[0264] In some embodiments, the nucleic acid molecule comprises or consists of a nucleotide sequence encoding an amino acid sequence as described in any of SEQ ID NO: 18-107.

[0265] In some implementations, the nucleic acid molecule comprises or is composed of the nucleotide sequences described in Table C below.

[0266] Table C: Anti-CTLA4 variable region polynucleotide sequence

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273] The nucleic acids disclosed herein can be obtained using any suitable molecular biology technique. For antibodies expressed by hybridomas, the cDNA encoding the light and heavy chains of the antibody prepared via hybridoma can be obtained by PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), the nucleic acid encoding the antibody can be recovered from said library.

[0274] Encoding VH The isolated DNA from the region can be used to make V H The encoding DNA is operatively linked to another DNA molecule encoding the heavy chain constant regions (CH1, CH2, and CH3) to transform it into a full-length heavy chain gene. The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat et al. (1991) NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. Heavy chain constant regions can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant regions. For Fab fragment heavy chain genes, V H The encoding DNA can be operatively linked to another DNA molecule that encodes only the CH1 constant region of the heavy chain.

[0275] Encoding V L The isolated DNA from the region can be used to make V L The encoding DNA is operatively linked to another DNA molecule encoding the light chain constant region CL to transform it into a full-length light chain gene (and a Fab light chain gene). The sequences of human light chain constant region genes are known in the art (see, for example, Kabat et al. (1991) NIH Publication No. 91-3242), and DNA fragments covering these regions can be obtained by standard PCR amplification. The light chain constant region can be either a κ or λ constant region.

[0276] To create the scFv gene, so that V H Encoding DNA fragments and V L The encoding DNA fragment is operatively linked to another fragment encoding a flexible linker, such as the amino acid sequence (Gly4-Ser)3, to enable V H and V L The sequence can be expressed as a continuous single-chain protein, where V L District and V H The area is joined by the flexible joint (see, for example, Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and McCafferty et al., Nature 348: 552-554 (1990)).

[0277] This disclosure also provides a vector comprising the nucleic acid molecule described herein. In some embodiments, the vector is an expression vector or a display vector (e.g., a viral display vector, a bacterial display vector, a yeast display vector, an insect display vector, a mammalian display vector, etc.). The nucleic acid molecule may encode an amino acid sequence of a portion of a light chain or heavy chain (such as CDR or HVR; a light chain variable region or a heavy chain variable region), a full-length light chain or heavy chain, a polypeptide comprising a portion or full length of a heavy chain or light chain, or an antibody derivative or an antigen-binding fragment. In some embodiments, the vector is an expression vector that can be used to express a binding molecule such as an antibody or an antigen-binding fragment thereof. In some embodiments, a vector is provided herein in which a first vector comprises a polynucleotide sequence encoding a heavy chain variable region as described herein, and a second vector comprises a polynucleotide sequence encoding a light chain variable region as described herein. In some embodiments, a single vector comprises polynucleotides encoding both a heavy chain variable region as described herein and a light chain variable region as described herein.

[0278] To express the binding molecules of this disclosure, DNA encoding partial or full-length light and heavy chains is inserted into an expression vector such that the DNA molecule is operatively linked to transcriptional and translational control sequences. In this context, the term "operatively linked" means linking the antibody gene to the vector so that the transcriptional and translational control sequences within the vector perform their predetermined functions of regulating the transcription and translation of the DNA molecule. The expression vector and expression control sequences are selected to be compatible with the host cell used for expression. The antibody light chain gene and antibody heavy chain gene can be inserted into separate vectors, or both genes can be inserted into the same expression vector. The antibody gene is inserted into the expression vector by any suitable method (e.g., ligation of the antibody gene fragment and complementary restriction sites on the vector, or DNA ligation based on homologous recombination). The light chain variable regions and heavy chain variable regions of the antibodies described herein can be used to create full-length antibody genes of any antibody isotype and subclass by inserting them into expression vectors that encode the heavy chain constant regions and light chain constant regions of the desired isotype and subclass, so that V H Segments are operatively connected to one or more C segments within the carrier. H Section, and V L The C segment is operatively connected within the carrier. L The segment. Alternatively, the recombinant expression vector may encode a signal peptide that facilitates the secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into the vector so that the signal peptide is attached in-frame to the N-terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin).

[0279] In addition to the antibody sequence, the expression vectors of this disclosure typically carry regulatory sequences that control the expression of the antibody sequence in host cells. The term “regulatory sequence” is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain gene. Such regulatory sequences are described, for example, in Goeddel (GeneExpression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Those skilled in the art will understand that the design of expression vectors, including the selection of regulatory sequences, can depend on factors such as the selection of host cells to be transformed, the desired protein expression level, etc. Examples of regulatory sequences for mammalian host cell expression include viral elements that guide high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenoviruses (e.g., the adenovirus major late promoter (AdMLP)), and polyomaviruses. Alternatively, non-viral regulatory sequences, such as ubiquitin promoters or β-globulin promoters, may be used. In addition, regulatory elements consist of sequences from different sources, such as the SR promoter system, which contains sequences from the SV40 early promoter and long terminal repeat sequences from human T-cell leukemia virus type 1 (Takebe, Y. et al. (1988) Mol. Cell. Biol. 8: 466-472).

[0280] In addition to antibody chain genes and regulatory sequences, expression vectors may carry additional sequences, such as sequences regulating vector replication in host cells (e.g., origin of replication) and selectable marker genes. Selectable marker genes help select host cells into which the vector has been introduced (see, for example, U.S. Patents 4,399,216, 4,634,665, and 5,179,017, all by Axel et al.). For example, selectable marker genes typically confer resistance to drugs such as G418, hygromycin, or methotrexate in host cells into which the vector has been introduced. Selectable marker genes include the dihydrofolate reductase (DHFR) gene (used in conjunction with methotrexate selection / amplification in DHFR-host cells) and the neo gene (used for G418 selection).

[0281] For expression of the light and heavy chains, one or more expression vectors encoding the heavy and light chains are transfected into host cells using any suitable technique. The various forms of the term "transfection" are intended to encompass a wide range of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. Although it is possible to express the antibodies disclosed herein in either prokaryotic or eukaryotic host cells, expression of the antibodies in eukaryotic cells, and typically in mammalian host cells, is most typical.

[0282] This disclosure also provides a host cell containing the nucleic acid molecule provided by this disclosure. The host cell can be virtually any cell with an expression vector available. It can be, for example, higher eukaryotic host cells such as mammalian cells, lower eukaryotic host cells such as yeast cells, and prokaryotic cells such as bacterial cells. Methods for introducing recombinant nucleic acids into the host cell are known in the art, including, for example, transfection by calcium phosphate, DEAE-mediated transfection, electroporation, or phage infection.

[0283] Suitable prokaryotic hosts for transformation include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus.

[0284] Suitable eukaryotic hosts for transformation include yeast, insects (e.g., S2 cells), and mammalian cells. Mammalian host cells used for expressing the binding molecules of this disclosure include, for example, Chinese hamster ovary (CHO) cells (including dhfr-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220 (1980); Sharp, J. Mol. Biol. 159:601-621 (1982)), NSO myeloma cells, COS cells, HEK293F cells, HEK293T cells, and Sp2 cells. In particular, for use with NSO myeloma or CHO cells, another expression system is the GS (glutamine synthase) gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP338,841. In some embodiments, the antibodies of this disclosure are produced in CHO cells. In some embodiments, the antibodies of this disclosure are modified and do not include C-terminal lysine residues (e.g., the C-terminal lysine residues of the antibody heavy chain described herein are removed (e.g., before or during antibody production)). When an expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period sufficient to allow antibody expression in the host cell or secretion into the culture medium in which the host cell is grown. The antibody can be recovered from the culture medium using any suitable protein purification method known in the art (e.g., protein A chromatography and / or ion exchange chromatography).

[0285] VI. Composition

[0286] In other aspects, this disclosure provides a composition comprising a binding molecule (e.g., an antibody or an activatable antibody) provided by this disclosure. In one aspect, the composition is a pharmaceutical composition comprising a binding molecule (e.g., an antibody or an activatable antibody) and a pharmaceutically acceptable carrier. The composition can be prepared by conventional methods known in the art.

[0287] In some embodiments, this disclosure provides a composition comprising a binding molecule (e.g., an antibody or an activatable antibody) provided by this disclosure and a pharmaceutically acceptable carrier, wherein the binding molecule comprises a variable domain containing the HVR amino acid sequence disclosed herein, and wherein, relative to the total amount of binding molecules (e.g., antibodies or activatable antibodies) present in the composition, the composition comprises up to about 11%, 10%, 8%, 5%, 3%, or 2% of the binding molecules (e.g., antibodies or activatable antibodies) glycosylated at the asparagine site of the amino acid sequence. In another embodiment, the composition comprises at least about 2% of the binding molecules (e.g., antibodies or activatable antibodies) glycosylated at the asparagine site of the amino acid sequence, relative to the total amount of binding molecules (e.g., antibodies or activatable antibodies) present in the composition.

[0288] The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in formulations for delivering bound molecules. Carriers can be anti-adhesives, adhesives, coating agents, disintegrants, fillers or diluents, preservatives (such as antioxidants, antibacterial agents, or antifungal agents), sweeteners, absorption delay agents, humectants, 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, buffers, buffered saline, and isotonic agents such as sugars, polyols, sorbitol, and sodium chloride.

[0289] The composition may be in any suitable form, such as liquid, semi-solid, 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 a binding molecule (e.g., an antibody or an activatable antibody) is a sterile liquid, such as a solution, suspension, or dispersion for injection or infusion. Sterile solutions can be prepared by incorporating an antibody in a desired amount into a suitable carrier, followed by sterile microfiltration. Typically, dispersions are prepared by incorporating the binding molecule (e.g., an antibody or an activatable antibody) into a sterile medium containing a basic dispersion medium and other carriers. In the case of 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.

[0290] The relative amount of binding molecules (e.g., antibodies or activatable antibodies) included in the composition will vary depending on many factors, such as the specific binding molecules and carriers used, the dosage form, and the desired release and pharmacokinetic characteristics. The amount of binding molecules (e.g., antibodies or activatable antibodies) in a single dosage form will typically be the amount that produces a therapeutic effect, but can also be a smaller amount. Typically, 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.

[0291] In addition to binding molecules (such as antibodies or activatable antibodies), one or more additional therapeutic agents may be included in the composition. Examples of additional therapeutic agents are described below. The suitable 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 will typically be the amount of the pharmaceutical agent that produces a therapeutic effect, but may also be a smaller amount.

[0292] Any binding molecule (e.g., antibody or activatable antibody) and / or composition (e.g., pharmaceutical composition) described herein may be used to prepare a pharmaceutical agent (e.g., a pharmaceutical agent for treating cancer in a subject in need or for delaying the progression of said cancer).

[0293] VII. Use of combined molecules and pharmaceutical compositions

[0294] The conjugating molecules (e.g., antibodies or activating antibodies) and pharmaceutical compositions provided by this disclosure can be used for therapeutic, diagnostic, or other purposes, such as modulating immune responses, treating cancer, enhancing the efficacy of other cancer therapies, enhancing vaccine efficacy, or treating autoimmune diseases. Therefore, in other aspects, this disclosure provides methods of using conjugating molecules (e.g., antibodies or activating antibodies) or pharmaceutical compositions. In one aspect, this disclosure provides a method of treating a condition in a mammal, the method comprising administering an effective amount of a conjugating molecule (e.g., antibody or activating antibody) or composition provided by this disclosure to the mammal requiring treatment. The conjugating molecule (e.g., antibody or activating antibody) may be a CTLA4 antibody (e.g., a human anti-human CTLA4 antibody) or a CTLA4 activating antibody. In some embodiments, the mammal is a human.

[0295] In some implementations, the condition is cancer. Various cancers can be treated or prevented using the methods, uses, compositions, or agents provided in this disclosure.Examples of the cancers mentioned include lung cancers such as bronchial carcinomas (e.g., squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma), alveolar cell carcinomas, bronchial adenomas, chondromatous hamartomas (non-cancerous), and sarcomas (cancerous); cardiac cancers such as myxomas, fibromas, and rhabdomyosarcomas; and bone cancers such as osteochondroma, chondroma, chondroblastoma, chondromycinoid fibroma, osteoid osteoma, giant cell tumors, chondrosarcomas, multiple myeloma, osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, and Ewing's tumor (Ewing's sarcoma). Sarcoma and reticulum cell sarcoma; brain cancers such as gliomas (e.g., glioblastoma multiforme), anaplastic astrocytoma, astrocytoma, oligodendroglioma, medulloblastoma, chordoma, schwannoma, ependymoma, meningioma, pituitary adenoma, pineal tumor, osteoma, hemangioblastoma, craniopharyngioma, chordoma, germ cell tumor, teratoma, dermoid cyst, and hemangioma; cancers of the digestive system such as leiomyomas, epidermoid carcinomas, adenocarcinomas, leiomyosarcomas, gastric adenocarcinomas, and intestinal lipomas. Neurofibroma of the intestine, intestinal fibroma, colorectal polyps and colorectal cancer; liver cancer such as hepatocellular adenoma, hemangioma, hepatocellular carcinoma, fibrolamellar carcinoma, cholangiocarcinoma, hepatoblastoma and angiosarcoma; kidney cancer such as renal adenocarcinoma, renal cell carcinoma, adrenal adenoid tumor and transitional cell carcinoma of the renal pelvis; bladder cancer; hematologic malignancies such as acute lymphoblastic (lymphoblastic) leukemia, acute myeloid (myeloid, bone marrow-derived, medulloblastic, myelomonocytic) leukemia, chronic lymphocytic leukemia (e.g., Sezary syndrome). Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, mycosis fungoides, and myeloproliferative disorders (including myeloproliferative disorders such as polycythemia vera, myelofibrosis, thrombocytosis, and chronic myeloid leukemia); skin cancers such as basal cell carcinoma, squamous cell carcinoma, melanoma, Kaposi's sarcoma, and Paget's disease. Cancers include: head and neck cancers; eye-related cancers such as retinoblastoma and intraocular melanoma; male reproductive system cancers such as benign prostatic hyperplasia, prostate cancer, and testicular cancer (e.g., seminoma, teratoma, embryonal carcinoma, and choriocarcinoma); breast cancer; female reproductive system cancers such as uterine cancer (endometrial cancer), cervical cancer (cervical cancer), ovarian cancer (ovarian tumor), vulvar cancer, vaginal cancer, fallopian tube cancer, and hydatidiform mole; thyroid cancers (including papillary, follicular, anaplastic, or medullary carcinoma); pheochromocytoma (adrenal gland); non-cancerous growths of the parathyroid gland; pancreatic cancer; and blood cancers such as leukemia, myeloma, non-Hodgkin's lymphoma, and Hodgkin's lymphoma.

[0296] In another aspect, this disclosure provides a method for enhancing an immune response in a mammal, the method comprising administering to the mammal an effective amount of a binding molecule (e.g., an antibody or an activatable antibody) or composition provided by this disclosure. In some embodiments, the binding molecule is a CTLA4 antibody or an antigen-binding fragment thereof, and the mammal is a human. In some embodiments, the binding molecule is a CTLA4 activatable antibody, and the mammal is a human. The term “enhancing an immune response” or its grammatical variations mean any response that stimulates, arouses, increases, improves, or strengthens the immune system of a mammal. An immune response may be a cellular response (i.e., cell-mediated, such as mediated by cytotoxic T lymphocytes) or a humoral response (i.e., antibody-mediated), and may be a primary or secondary immune response. Examples of enhancing an immune response include activating PBMCs and / or T cells (including increasing the secretion of one or more cytokines such as IL-2 and / or IFNγ). Enhancement of the immune response can be assessed using a number of in vitro or in vivo measurements known to those skilled in the art, including but not limited to cytotoxic T lymphocyte assays, cytokine release, tumor regression, survival of tumor-carrying animals, antibody production, immune cell proliferation, expression of cell surface markers, and cytotoxicity. Generally, the methods of this disclosure enhance the immune response achieved by mammals when compared to the immune response achieved by untreated mammals or mammals not treated using the described methods.

[0297] In implementing treatment methods, the binding molecule (e.g., an antibody or an activating antibody) may be administered alone as a monotherapy or in combination with one or more additional therapeutic agents or therapies. Therefore, in another aspect, this disclosure provides a combination therapy comprising a binding molecule (e.g., an antibody or an activating antibody) combined with one or more additional therapeutic agents or therapies for separate, sequential, or simultaneous administration. The term "additional therapeutic agent" may refer to any therapeutic agent other than the binding molecule (e.g., an antibody or an activating antibody) provided by this disclosure. In one particular aspect, this disclosure provides a combination therapy for treating cancers in mammals, the combination therapy comprising administering to the mammal an effective amount of the binding molecule (e.g., an antibody or an activating antibody) provided herein combined with one or more additional therapeutic agents. In another embodiment, the mammal is a human.

[0298] A wide variety of cancer therapeutic agents can be used in combination with binding molecules (e.g., antibodies or activating antibodies) provided by this disclosure. Those skilled in the art will recognize the existence and development of other cancer therapies that can be used in combination with the methods and binding molecules of this disclosure, such as antibodies or activating antibodies, and will not be limited to those therapeutic forms set forth herein. Examples of additional therapeutic agents that can be used in combination therapies for treating cancer include (1) chemotherapeutic agents, (2) immunotherapeutic agents, and (3) hormonal therapeutic agents. In some embodiments, the additional therapeutic agent is a viral gene therapy, an immune checkpoint inhibitor, a targeted therapy, a radiotherapy, a vaccination therapy, and / or a chemotherapeutic agent.

[0299] The term "chemotherapy agent" refers to a chemical or biological substance that can cause cancer cell death or interfere with the growth, division, repair, and / or function of cancer cells. Examples of chemotherapy agents include those disclosed in WO 2006 / 129163 and US 20060153808, the disclosures of which are incorporated herein by reference. Examples of specific chemotherapeutic agents include: (1) alkylating agents, such as chlorambucil (LEUKERAN), cyclophosphamide (CYTOXAN), ifosfamide (IFEX), mechlorethamine hydrochloride (MUSTARGEN), thiotepa (THIOPLEX), streptozotocin (ZANOSAR), carmustine (BICNU, GLIADEL WAFER), lomustine (CEENU), and dacarbazine (DTIC-DOME);(2) Alkaloids or vinca alkaloids, including cytotoxic antibiotics such as doxorubicin (ADRIAMYCIN), epirubicin (ELLENCE, PHARMORUBICIN), daunorubicin (CERUBIDINE, DAUNOXOME), nemombicin, idarubicin (IDAMYCIN PFS, ZAVEDOS), mitoxantrone (DHAD, NOVANTRONE), and dactinomycin (actinomycin D). D), Cosmeggen, plicamycin (MITHRACIN), mitomycin (MUTAMYCIN), bleomycin (BLENOXANE), vinorelbine tartrate (NAVELBINE), vinblastine (VELBAN), vincristine (ONCOVIN), and vindesine (ELDISINE); (3) antimetabolites, such as capecitabine (XELODA). (3) Cytarabine (CYTOSAR-U), fludarabine (FLUDARA), gemcitabine (GEMZAR), hydroxyurea (HYDRA), methotrexate (FOLEX, MEXATE, TREXALL), nelarabine (ARRANON), trimetrexate (NEUTREXIN), and pemetrexed (ALIMTA); (4) pyrimidine antagonists, such as 5-fluorouracil (5-FU);(5) Taxanes, such as docetaxel (TAXOTERE) and paclitaxel (TAXOL); (6) Platinum drugs, such as cisplatin (PLATINOL) and carboplatin (PARAPLATIN) and oxaliplatin (ELOXATIN); (7) Topoisomerase inhibitors, such as irinotecan (Capecitabine). (7) Topotecan (HYCAMTIN), etoposide (ETOPOPHOS, VEPESSID, TOPOSAR), and teniposide (VUMON); (8) Epipodophyllotoxin (podophyllotoxin derivatives), such as etoposide (VEPESSID, VEPESSID, TOPOSAR); (9) Folic acid derivatives, such as leucovorin (WELLCOVORIN); (10) Nitrosourea, such as carmustine (Biknikov) and lomustine (Systos).(11) Inhibitors of receptor tyrosine kinases, including epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), insulin receptor, insulin-like growth factor receptor (IGFR), hepatocyte growth factor receptor (HGFR), and platelet-derived growth factor receptor (PDGFR), such as gefitinib (Iressa), erlotinib (Tarceva), bortezomib (Velcade), and imatinib mesylate. mesylate (GLEEVEC), gefitinib, lapatinib, sorafenib, thalidomide, sunitinib (SUTENT), axitinib, rituximab (RITUXAN, MABTHERA), trastuzumab (HERCEPTIN), cetuximab (ERBITUX), bevacizumab (AVASTIN) (12) Antibodies against insulin-like growth factor-1 receptor (IGF-1R) disclosed in WO2002 / 053596, such as ranibizumab (LUCENTIS), lym-1 (ONCOLYM), and ranibizumab (LUCENTIS), lym-1 (ONCOLYM), and ranibizumab (LUCENTIS), lym-1 (ONCOLYM), and ranibizumab (LUCENTIS); (13) Angiogenesis inhibitors, such as bevacizumab (Avastin), suramin (GERMANIN), angiostatin, SU5416, thalidomide, and matrix metalloproteinase inhibitors (such as batimastat and marimastat) and those disclosed in WO2002055106; and (14) Proteasome inhibitors, such as bortezomib (Vilcord).

[0300] The term "immunotherapy agent" refers to a chemical or biological substance that enhances the immune response in mammals. Examples of immunotherapy agents include: BCG; cytokines such as interferon; vaccines such as MyVax personalized immunotherapy, Onyvax-P, Oncophage, GRNVAC1, Favld, Provenge, GVAX, Lovaxin C, BiovaxID, GMXX, and NeuVax; and antibodies such as alemtuzumab (CAMPATH), bevacizumab (Avastin), cetuximab (Erbitux), gemtuzunab ozogamicin (MYLOTARG), and ibritumomab. tiuxetan (ZEVALIN), panitumumab (VECTIBIX), rituximab (Rituximab), trastuzumab (Herceptin), tositumomab (BEXXAR), iprimab (YERVOY), tremelimumab, CAT-3888, agonists of the OX40 receptor (such as those disclosed in WO2009 / 079335), agonists of the CD40 receptor (such as those disclosed in WO2003 / 040170), and TLR-9 agonists (such as those disclosed in WO2003 / 015711, WO2004 / 016805, and WO2009 / 022215).

[0301] The term "hormone therapeutic agent" refers to a chemical or biological substance that inhibits or eliminates the production of hormones, or inhibits or resists the effects of hormones on the growth and / or survival of cancer cells. Examples of agents suitable for the methods described herein include those disclosed in US20070117809. Examples of specific hormone therapy agents include tamoxifen (NOLVADEX), toremifene (Fareston), fulvestrant (FASLODEX), anastrozole (ARIMIDEX), exemestane (AROMASIN), letrozole (FEMARA), megestrol acetate (MEGACE), goserelin (ZOLADEX), and leuprolide (LUPRON). The binding molecules disclosed herein can also be used in combination with non-pharmacological hormone therapies such as (1) surgical methods to remove all or part of organs or glands involved in hormone production, such as the ovaries, testes, adrenal glands, and pituitary glands, and (2) radiation therapy, wherein the patient’s organs or glands are subjected to a sufficient amount of radiation to inhibit or eliminate the production of the targeted hormone.

[0302] In some implementations, the additional therapeutic agent is one or more of the following: pomalidomide, lelimid, lenalidomide, pomalidomide, thalidomide, cisplatin (a platinum-containing derivative of DNA alkylation), 5-fluorouracil, cyclophosphamide, anti-CD137 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-CD20 antibody, anti-CD40 antibody, anti-DR5 antibody, anti-CD1d antibody, anti-TIM3 antibody, anti-SLAMF7 antibody, anti-KIR receptor antibody, anti-OX40 antibody, anti-HER2 antibody, anti-ErbB-2 antibody, anti-EGFR antibody, cetuximab, rituximab, trastuzumab, pamumab, radiotherapy, single-dose radiation, fractionated radiation, focal radiation, whole-organ radiation, IL-12, IFNα, GM-CSF, chimeric antigen receptor, inherited metastatic T cells, anticancer vaccines, and oncolytic viruses.

[0303] Combination therapies for cancer treatment also encompass the combination of binding molecules (such as antibodies or activating antibodies) with surgical procedures used to remove tumors. The binding molecules (such as antibodies or activating antibodies) can be administered to mammals before, during, or after the surgery.

[0304] Combination therapies for cancer treatment also encompass combinations of binding molecules (such as antibodies or activatable antibodies) with radiation therapies such as ionizing (electromagnetic) radiation therapy (such as X-rays or gamma rays) and particle beam radiation therapy (such as high-linear-energy radiation). The radiation source can be external or internal to the mammal. The binding molecules (such as antibodies or activatable antibodies) can be administered to the mammal before, during, or after radiation therapy.

[0305] The conjugating molecules (e.g., antibodies or activatable antibodies) and compositions provided by this disclosure can be administered via any suitable enteral or parenteral route. The term "enteral route" refers to administration through any part of the gastrointestinal tract. Examples of enteral routes include oral, transmucosal, buccal, and transrectal routes, or intragastric routes. "Parenteral route" refers to administration other than the enteral route. Examples of parenteral routes include intravenous, intramuscular, intradermal, intraperitoneal, intratumoral, intrabladder, intraarticular, intrasheath, intracystic, intraorbital, intracardiac, tracheal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal, subcutaneous, or topical administration. The conjugating molecules (e.g., antibodies or activatable antibodies) and compositions of this disclosure can be administered using any suitable method, such as oral ingestion, nasogastric tube, gastrostomy tube, injection, infusion, implantable infusion pump, and osmotic pump. Suitable routes and methods of administration can vary depending on many factors, such as the specific binding molecule used (e.g., antibody or activatable antibody), the required absorption rate, the specific formulation or dosage form used, the type or severity of the disease being treated, the specific site of action, and the patient's condition, and can be readily selected by those skilled in the art.

[0306] The term "effective amount" for a binding molecule (e.g., an antibody or an activating antibody) can refer to an amount that effectively achieves a predetermined therapeutic purpose. For example, in the case of enhancing an immune response, an "effective amount" can be any amount that effectively stimulates, arouses, increases, improves, or strengthens any response of the mammalian immune system. In the case of treating a disease, an "effective amount" can be any amount sufficient to produce any desired or beneficial effect in the treated mammal. In particular, in the case of treating cancer, examples of desired or beneficial effects include inhibiting the further growth or spread of cancer cells, causing cancer cell death, inhibiting cancer recurrence, reducing cancer-related pain, or improving the survival of the mammal. Therapeutic effective amounts of binding molecules (e.g., antibodies or activating antibodies) are generally from about 0.001 to about 500 mg per kg of body weight in mammals, and more generally in the range of about 0.01 to about 100 mg / kg. For example, amounts can be about 0.3 mg, 1 mg, 3 mg, 5 mg, 10 mg, 50 mg, or 100 mg per kg of body weight in mammals. In some embodiments, the therapeutically effective dose of the binding molecule (e.g., an antibody or an activating antibody) is in the range of about 0.01-30 mg per kg body weight in mammals. In some other embodiments, the therapeutically effective dose of the binding molecule (e.g., an antibody or an activating antibody) is in the range of about 0.05-15 mg per kg body weight in mammals. The precise dose level to be administered can be readily determined by those skilled in the art and will depend on many factors, such as the type and severity of the condition to be treated, the specific binding molecule used (e.g., an antibody or an activating antibody), the route of administration, the time of administration, the duration of treatment, any specific additional therapies used, the age, sex, weight, condition, general health status, and prior medical history of the patient being treated, and similar factors well known in the medical field.

[0307] The conjugating molecule (e.g., antibody or activatable antibody) or combination is typically administered at multiple intervals. The intervals between single doses may be, for example, daily, weekly, monthly, every three months, or annually. An exemplary treatment regimen requires administration once weekly, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months. Typical dosing regimens for the conjugating molecule (e.g., antibody or activatable antibody) include intravenous administration of 1 mg or 3 mg per kg of body weight, using one of the following dosing schedules: (i) six doses every four weeks, followed by every three months; (ii) every three weeks; (iii) 3 mg per kg of body weight once, followed by 1 mg per kg of body weight every three weeks.

[0308] VIII. Medicine Box

[0309] On the other hand, this document provides a kit containing a binding molecule (e.g., an antibody or an activatable antibody) and / or composition of the present disclosure. In some embodiments, the kit further includes a packaging insert containing instructions on using the binding molecule (e.g., an antibody or an activatable antibody) and / or composition. In some embodiments, the kit further includes one or more buffers, for example for storing, transferring, administering, or otherwise using the binding molecule (e.g., an antibody or an activatable antibody) and / or composition. In some embodiments, the kit further includes one or more containers for storing the binding molecule (e.g., an antibody or an activatable antibody) and / or composition.

[0310] 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.

[0311] Example

[0312] Example 1: Generating primary Fab that specifically binds to human CTLA4

[0313] A proprietary phage plasmid library (see PCT / CN2017 / 098333, incorporated herein by reference in its entirety, and also PCT / CN2017 / 098299, incorporated herein by reference in its entirety) was used for panning against the human CTLA4 antigen. A total of 3–5 rounds of panning were performed. After the final round of panning, single-colony supernatant ELISA was performed to identify primary hits that specifically recognize human CTLA4 (see, for example, UniProt accession number P16410). Primary hits were defined as those whose ELISA signal was at least twice the background ELISA signal. The hits were then sequenced, and unique clones were expressed and purified in *E. coli*. Their affinity for human CTLA4 was measured using a ForteBioOctet RED96 system. In brief, the AHC sensor (anti-human IgG Fc capture impregnation and readout biosensor) was used to capture recombinant human CTLA4-Fc (Sino Biological, 11159-H03H) and impregnate it in wells containing purified Fab diluted to 10 μg / mL in kinetic buffer (PBS buffer containing 0.02% Tween 20 and 0.1% BSA). The acquired ForteBio data were processed using Data Acquisition software 7.1, and the kinetic data were fitted relative to a 1:1 Langmuir binding model. The candidate list was narrowed down to 234 Fab hits with both ELISA positive hits and unique sequences. Following K... D Response signal R > 0.1, R 2 Based on the criterion of >0.9, the list was further refined to 43 target objects. The affinity and kinetic parameters of these objects (excluding background) are shown in Table 1 below.

[0314] Table 1: Affinity of the selected Fab to human CTLA4

[0315]

[0316]

[0317] Next, the species cross-reactivity of various Fab hits was determined by ELISA. Briefly, 100 μL of 1.25 μg / mL anti-human IgG (Fab-specific) antibody (Sigma, I5260) was coated overnight at 4 °C on Maxisorp microplates (Thermo Scientific 446469). After blocking, 100 μL of Fab hit (2 μg / mL) was added and incubated for 1 hour. After washing the wells 3–4 times, serially diluted human, cynomolgus monkey, or mouse CTLA4 antigen fused with the human FC fragment was added and incubated for 1 hour. After washing, HRP-labeled goat anti-human FC antibody was diluted 1:2000 with PBS and added to each well for 1 hour incubation. The plate was washed three times and incubated with TMB substrate at room temperature for 3–5 minutes. The absorbance was measured at 450 nm after the reaction was terminated. The species cross-reactivity of each of the tested Fabs is summarized in Table 2 below.Of particular interest was that this analysis identified Fabs with varying cross-reactivity: the results indicated that the hit compounds B13873, B15700, B15704, B15706, B15709, B15711, B15712, B15715, B15720, B15725, B15723, B15731, B15732, B15735, B15736, B15744, B15760, B16083, and B15188 bind to human... CTLA4 in humans, monkeys, and mice; the targets B15188, B15190, B15701, B15729, B15733, B15742, B15747, B15743, B15751, B15752, B15753, and B18157 bind to CTLA4 in humans and monkeys, and bind weakly to CTLA4 in mice; the targets B13878, B14242, B15189, B15491, B15673, B15694, and B1 5696, B15699, B15702, B15705, B15710, B15716, B15717, B15719, B15721, B15722, B15724, B15728, B15 734, B15737, B15738, B15739, B15740, B15745, B15746, B15749, B15750, B15754, B15756, B15757, B1575 9 and B15762 bind to human CTLA4 but not to mouse CTLA4; hitter B15688 binds to human and mouse CTLA4 but not to monkey CTLA4; and hitters B13874, B13880, B13898, B15187, B15489, B15672, B15695, B15730, B15741, B18153 and B18174 bind to human CTLA4 but not to monkey or mouse CTLA4.

[0318] Table 2: Cross-reactivity of Fab with human, monkey, and mouse CTLA4

[0319]

[0320]

[0321]

[0322] Example 2: IgG Conversion and Expression

[0323] Next, 13 of the selected hits from Example 1 above were converted into human IgG1 antibodies for detailed biophysical and functional characterization (Table 3). The heavy and light chains of Fab hits B15709, B15716, B15722, B15732, B15740, B15744, B15756, B15700, B15711, B15717, B15735, B15736, and B16083 were cloned into the mammalian expression vector pTT5-SPB, respectively. The heavy and light chains of the reference antibody were also cloned into pTT5-SPB.

[0324] Table 3: Fab-hit compounds cloned in the form of IgG1 antibodies

[0325] Antibody (AB) name: Fab Hit Identifier: Same type: TY21585 B15709 hIgG1 TY21586 B15716 hIgG1 TY21587 B15722 hIgG1 TY21588 B15732 hIgG1 TY21589 B15740 hIgG1 TY21580 B15744 hIgG1 TY21591 B15756 hIgG1 TY21687 B15700 hIgG1 TY21688 B15711 hIgG1 TY21689 B15717 hIgG1 TY21680 B15735 hIgG1 TY21691 B15736 hIgG1 TY21692 B16083 hIgG1 TAC2114 refer to hIgG1

[0326] Following the manufacturer's protocol, each pair of plasmids encoding the heavy and light chains of the antibodies was transiently transfected into 293F cells. Supernatants from cells transfected with plasmids encoding antibodies TY21585, TY21586, TY21587, TY21588, TY21589, TY21580, or TY21591 were collected, clarified by centrifugation and filtration, and the resulting IgG was purified using standard protein A affinity chromatography (MabSelect SuRe, GE Healthcare). Proteins were eluted and neutralized, and buffer exchanged to 20 mM PB buffer (20 mM NaH2PO4, 150 mM NaCl, pH 7.0). Protein concentration was determined by UV spectrophotometry, and IgG purity was analyzed by SDS-PAGE or SEC-HPLC under denaturing, reducing, and non-reducing conditions.

[0327] The supernatant of cells transfected with plasmids encoding antibodies TY21687, TY21689, TY21680, TY21691, or TY21692 was collected, clarified by centrifugation and filtration, and the resulting IgG was purified using standard protein A affinity chromatography (MabSelect SuRe, GE Healthcare). Proteins were eluted and neutralized, and buffer exchanged to enter 20 mM histidine buffer (20 mM histidine, 3.5 mL 6 M HCl, pH 5.5). Protein concentration was determined by UV spectrophotometry, and IgG purity was analyzed by SDS-PAGE or SEC-HPLC under denaturing, reducing, and non-reducing conditions.

[0328] Example 3: In vitro functional characterization of antibodies against IgG conversion

[0329] According to the manufacturer's guidelines, use BiacoreTM The T200 instrument (Biacore AB, Uppsala, Sweden) was used to examine the binding affinity and kinetics of antibodies against human, monkey, and mouse CTLA4 using surface plasmon resonance (SPR) analysis (Table 4). Anti-human IgG (Fc) antibodies from the Human Antibody Capture Kit (GE BR-1008-39) were immobilized on the CM5 chip by coupling their amine groups to the carboxylated surface of the sensor chip, according to the instructions of the amine conjugation kit (GE Biacore#BR-1000-50). The immobilized anti-human IgG (Fc) antibodies were used to capture antibodies TY21585, TY21586, TY21580, TY21591, TY21687, TY21689, TY21680, TY21691, TY21692, and TAC2114. TAC2114 has the same amino acid sequence as the commercial antibody ipelamaribalbin. Binding was measured at six different concentrations (3.13, 6.25, 12.5, 25, 50, and 100 nM diluted in run buffer) at a flow rate of 30 μl / min. The run buffer used was HBS-EP (100 mM HEPES, 1.5 M sodium chloride, 0.05% surfactant P20, pH 7.6). Association and dissociation curves were fitted against a 1:1 Langmuir binding model using Biacore T200 evaluation software (Biacore AB) according to the manufacturer's guidelines. As shown in Table 4 below, all tested antibodies were able to bind to human and monkey CTLA4, and all antibodies except TY21591, TY21689, and TAC2114 were also able to bind to mouse CTLA4.

[0330] Table 4: Binding affinity of IgG1 antibody to CTLA4 in humans, monkeys, and mice

[0331]

[0332] NC = No cross-reactivity

[0333] Next, tests were conducted on the binding of certain IgG antibodies to soluble human (Figure IA, Table 5A) or canine ( Figure 1BTable 5B) Capacity of CTLA4. Human CTLA4 fused with the human FC fragment or canine CTLA4 fused with the His tag was prepared at 1 μg / mL and used to coat ELISA plates overnight at 2–8 °C. After blocking, 100 μL of serially diluted IgG antibody was added and incubated at 37 °C for 1 hour. The plate was washed four times and then incubated with HRP-anti-human IgG (Fab-specific) antibody (1:6000 dilution) at 37 °C for 1 hour. The plate was washed four times again and incubated with TMB substrate at room temperature for 15 minutes. The absorbance was measured at 450 nm after the reaction was terminated. Data were analyzed using Graphpad Prism 6 with nonlinear fitting. Figure 1A-Figure 1B As shown in Tables 5A-5B, all tested antibodies bound to human CTLA4, and in addition to antibodies TY21586 and TAC2114, they also bound to canine CTLA4. Of interest, TY21580 bound to both human and canine CTLA4 with the highest affinity, where K... D They are 0.27 and 0.49 respectively.

[0334] Table 5A: ELISA for human CTLA4

[0335]

[0336]

[0337] Table 5B: ELISA for CTLA4 in dogs

[0338]

[0339] ND=not detected

[0340] The antibody affinity for human and mouse CTLA4 transiently expressed on the surface of HEK293F cells was also evaluated. Figure 2 In brief, HEK293F cells were transfected with a plasmid expressing full-length human, monkey, or mouse CTLA4 from a bicistronic IRES vector that also encodes EGFP, and EGFP expression was used to identify transfected cells. After 48 hours, mammalian cell suspensions (2 x 10⁻⁶ cells / mL) were... 5 Transfer the cells (at / well) to Eppendorf tubes, centrifuge, discard the supernatant, and resuspend the cells in 1 mL of PBSA (to achieve a density of 4 × 10⁹ cells / well). 6Cells were added to 96-well plates at 3-fold serial dilutions (15 μg / mL, 5 μg / mL, 1.67 μg / mL, 0.55 μg / mL, 0.185 μg / mL, 0.062 μg / mL, and 0.0309 μg / mL, plus a 0 μg / mL blank control) and pipette into the 96-well plates. Cells were incubated on ice for 1 hour (protected from light), washed with pre-cooled 1xPBSA buffer, and then... The 647-conjugated mouse anti-human FC antibody was incubated together on ice for 30 minutes. The cells were then washed once and subsequently analyzed by flow cytometry. Use CytoFlex for analysis. Figure 2 As shown, all tested antibodies were able to bind to human CTLA4 expressed on the surface of HEK293F cells, and all antibodies except TY21589 bound to mouse CTLA4 exposed on the surface of mouse cells. TY21580 bound to both human and mouse CTLA4 expressed on the cell surface with low nM affinity, while antibodies TY21585 and TY21586 bound to human CTLA4 expressed on the cell surface with high nM affinity.

[0341] The binding affinity and kinetics of antibodies TY21580, TY21687, TY21680, and TY21691 against rat CTLA4 protein were also tested using a ForteBio red 96 instrument (Pall, USA). An SA sensor (Pall, 185019) was used to immobilize biotinylated rat CTLA4 protein fused to human FC, and the sensor was then contacted with IgG conversion targets at a concentration of 15 μg / mL (diluted with KB buffer, i.e., PBS buffer supplemented with 0.02% Tween 20 and 0.1% BSA) for 300 seconds, followed by dissociation in KB buffer for 300 seconds. Association and dissociation curves were fitted against a 1:1 Langmuir binding model using ForteBio Data Analysis 7.1 (Pall, USA) according to the manufacturer's guidelines. As shown in Table 6 below, all tested antibodies were able to bind to rat CTLA4.

[0342] Table 6: Testing the binding affinity of antibodies to rat CTLA4

[0343] Antibody name: <![CDATA[K D (nM):]]> TY21580 0.38 TY21687 0.78 TY21680 0.21 TY21691 0.58

[0344] Binding of IgG to activated T cells

[0345] Next, the ability of IgG to bind to activated human, monkey, and mouse T cells was tested. Human PBMCs were freshly isolated from healthy donor (#106) blood by density gradient centrifugation using a Histopaque-1077 (Sigma). Human T cells were isolated from PBMCs using a Human T Cell Enrichment Kit (StemCell Technologies) and subsequently stimulated with anti-CD3 and anti-CD28 antibodies. Briefly, anti-CD3 antibody (clone: ​​OKT3, BioLegend) was inoculated overnight in 96-well plates at 0.2 μg per well in 200 μL at 4°C. After washing, T cells suspended in RPMI-1640 containing 10% FBS and 1% penicillin / streptomycin were added to the plates. Five 5x10E T cells in 200 μL were added to each well of the 96-well plate. Then, 1 μL of anti-human CD28 antibody (clone: ​​28.2, BD) was added to achieve a final concentration of 5 μg / mL. T cells were incubated for 96 hours, and then the binding of TY21580 to T cells was determined by flow cytometry. Figure 3 T cells were stained with APC-labeled TY21580 or human IgG1 (isotype control) at 37°C for 2 hours. After washing, cells were analyzed on a CytoFLEX flow cytometer (Beckman Coulter), and data were analyzed using FlowJo software. Figure 3 As shown, TY21580 binds to activated CD4+ and CD8+ human T cells, while control IgG shows no binding. Furthermore, APC-TY21580 shows no binding to resting T cells (data not shown).

[0346] Monkey PBMCs were freshly isolated from the blood of primate cynomolgus monkeys using a Histopaque-1077 (Sigma) via density gradient centrifugation. Monkey T cells were isolated from the PBMCs using a non-human primate whole T cell isolation kit (Miltenyi Biotec) and subsequently stimulated with anti-CD3 and anti-CD28 antibodies. Briefly, anti-CD3 antibody (clone: ​​SP34, BD) was inoculated overnight in 96-well plates at 0.2 μg per well in 200 μL at 4°C. After washing, T cells suspended in RPMI-1640 containing 10% FBS and 1% penicillin / streptomycin were added to the plates. 2 x 10⁵ T cells per well in 200 μL were added to each well of the 96-well plate. Then, 1 μL of anti-human CD28 antibody (clone: ​​28.2, BD) was added to achieve a final concentration of 5 μg / mL. T cells were incubated for 72 hours, and the binding of TY21580 to T cells was determined by flow cytometry. Figure 3T cells were stained with APC-labeled TY21580 or human IgG1 (isotype control) at 37°C for 2 hours. After washing, cells were analyzed on a CytoFLEX flow cytometer (Beckman Coulter), and data were analyzed using FlowJo software. Figure 3 As shown, TY21580 binds to activated CD4+ and CD8+ monkey T cells, while control IgG shows no binding. Furthermore, APC-TY21580 shows no binding to resting T cells (data not shown).

[0347] Mouse T cells isolated from the spleen of adult BALB / c mice were used to induce CTLA-4 expression. Spleen cells from fresh mouse spleens were used with EasySep. TM T cells were isolated using a mouse T cell isolation kit (StemCell Technologies) and subsequently stimulated with anti-mouse CD3 and anti-CD28 antibodies. Briefly, anti-mouse CD3ε antibody (Biolegend) was inoculated overnight at 4°C in 200 μL at 0.2 μg per well in 96-well plates. After washing, mouse T cells suspended in RPMI-1640 containing 10% FBS and 1% penicillin / streptomycin were added to each well at a concentration of 5 x 10⁵ T cells per well in 200 μL. Then, 1 μL of anti-mouse CD28 antibody (eBioscience) was added to achieve a final concentration of 5 μg / mL. The mouse T cells were incubated for 72 hours, followed by flow cytometry analysis to determine the binding of TY21580 to the T cells. Figure 3 T cells were stained with APC-labeled TY21580 or human IgG1 (isotype control) at 37°C for 2 hours. After washing, cells were analyzed on a CytoFLEX flow cytometer (Beckman Coulter), and data were analyzed using FlowJo software. Figure 3 As shown, TY21580 binds to activated mouse CD4+ and CD8+ T cells, while control IgG shows no binding.

[0348] Antibody binding selectivity to human CTLA4

[0349] Next, antibody selectivity was examined. Human CTLA4, PD1, LAG3, Tim3, B7H3, CD95, TNFR1, OX40, CD40, PD-L1, BLTA, VISTA, PDL2, ICOS, and B7H4 were transiently overexpressed on the surface of HEK293F cells. Transfected cells were washed in pre-cooled 1xPBSA buffer (1.76 mM KH2PO4, 10.14 mM Na2HPO4·12H2O, 2.68 mM KCl, 136.89 mM NaCl, and 1% BSA), followed by incubation on ice for 1 hour with 100 nM test antibody. Cells were washed once with staining buffer and Alexa was added. 647-conjugated mouse anti-human FC antibody was incubated on ice in the dark for 30 minutes. Samples were washed once with staining buffer and then analyzed by flow cytometry. TY21585, TY21586, TY21580, TY21687, TY21689, TY21680, and TY21691 were tested in human CTLA4, PD1, LAG3, Tim3, and B7-H3 conditions. Figure 4A Further testing of TY21585, TY21586, and TY21580 was conducted in individuals with CD95, TNFR1, OX40, and CD40. Figure 4B In addition, TY21586 and TY21580 were tested in the presence of human PD-L1, BLTA, VISTA, PDL2, ICOS, and B7-H4. Figure 4C ).like Figures 4A-4C As shown, all test antibodies bind specifically to human CTLA4 and not to any other test antigen (or parental cells transfected with an empty vector).

[0350] Based on ligand competitive binding of ELISA

[0351] Next, the ability of the antibody to block the binding of CTLA4 to its homologous ligands CD80 and CD86 was tested by ELISA. Recombinant human CTLA4 (fused with human Fc and His tags) was diluted to 1 μg / mL in carbonate buffer (pH 9.4) and coated onto Maxisorp plates overnight at 4°C. The plates were blocked with PBS supplemented with 2% (w / v) skim milk at 37°C for 1 hour. After washing, 50 μL of biotinylated CD80 (4 μg / mL) and 50 μL of various concentrations of test antibody (2-fold serial dilutions ranging from 200 μg / mL to 1.56 μg / mL) were sequentially added to each well, and incubated at 37°C for 1 hour. The plates were washed four times, and 100 μL of HRP-neutralavirin (1:1000) was added to each well and incubated at 37°C for 1 hour. The plate was washed as previously described, and 50 μL of TMB substrate solution was added and incubated at room temperature for 5 minutes, followed by termination of the reaction with 50 μL of sulfuric acid (2 M). Figures 5A-5B As shown, except for TY21589, all tested antibodies blocked the binding of CTLA4 to CD80.

[0352] Recombinant human CD86 fused with human Fc was diluted to 1 μg / mL in carbonate buffer (pH 9.4) and coated onto Maxisorp plates overnight at 4°C. The plates were blocked with PBS supplemented with 2% (w / v) skim milk at 37°C for 1 hour. After washing, 50 μL of biotinylated human CTLA4 fused with human FC and His tag (2.8 μg / mL) and 50 μL of various concentrations of test antibody (2-fold serial dilutions ranging from 100 μg / mL to 0.78 μg / mL) were sequentially added to each well and incubated at 37°C for 1 hour. The plates were washed four times, and 100 μL of HRP-neutralavirin (1:1000) was added to each well and incubated at 37°C for 1 hour. The plates were washed as previously described, and 50 μL of TMB substrate solution was added and incubated at room temperature for 5 minutes, followed by termination of the reaction with 50 μL of sulfuric acid (2M). like Figures 5C-5D As shown, all tested antibodies blocked the binding of CTLA4 to CD86.

[0353] Based on ligand competitive binding in flow cytometry

[0354] The ability of the antibody to block the binding of CTLA4 to its homologous ligands CD80 and CD86 was also tested by flow cytometry. A plasmid encoding full-length human CTLA4 was transiently expressed in HEK293F cells. Cells were washed with staining buffer (PBSA buffer containing 1.76 mM KH2PO4, 10.14 mM Na2HPO4·12H2O, 2.68 mM KCl, 136.89 mM NaCl, and 1% BSA) and resuspended in staining buffer containing 100 nM of the test antibody. After incubation on ice for 60 min, 100 nM biotinylated human CD80-Fc-Bio or CD86-Fc-Bio was added to each well, and the cells were incubated on ice for another 1 h. Cells were washed once with staining buffer, and 100 μL of staining buffer containing Alexa fluor 633-conjugated streptavidin was added and the cells were incubated on ice in the dark for 30 min. Cells were washed once and analyzed using CytoFlex flow cytometry. Figure 6A As shown, all tested antibodies blocked the binding of CTLA4 to CD80 in a concentration-dependent manner. TY21588 showed the strongest blocking ability, followed by TY21580 and TAC2114 achieving significant blocking; while TY21585, TY21587, TY21589, and TY21591 achieved less effective blocking. TY21589 showed little or no blocking. Figure 6B As shown, all tested antibodies blocked the binding of CTLA4 to CD86 in a concentration-dependent manner. TY21588, TY21589, TY21580, TY21591, and TAC2114 showed the strongest blocking ability; TY21585 and TY21587 achieved less effective blocking.

[0355] Binding with FcγR

[0356] Next, the binding affinity of TY21586, TY21580, and TAC2114 for CD16a(176Phe) (Sino Biological Inc., 10389-H08H), CD16a(176Val, 10389-H08H1), CD32a (Sino Biological Inc., 10374-H08H), CD32b (Sino Biological Inc., 10259-H08H), and CD64 (Sino Biological Inc., 10256-H08H) was tested. Biacore was used according to the manufacturer's guidelines. TMThe T200 instrument (Biacore AB, Uppsala, Sweden) was used to examine protein binding via surface plasmon resonance (SPR) analysis. Protein L (Sino Biological Inc. 11044-H07E) was immobilized on the CM5 chip via its amine group coupled to the carboxylated surface of the sensor chip, according to the amine conjugation kit (GE Biacore #BR-1000-50). Immobilized anti-human IgG (Fc) antibody was used to capture TY21586, TY21580, and TAC2114. Successive concentrations (12.5, 25, 50, 25, 100, and 200 nM) of FcγR protein diluted in run buffer were injected at a flow rate of 30 μl / min. The run buffer used was HBS-EP (100 mM HEPES, 1.5 M sodium chloride, 0.05% surfactant P20, pH 7.6). Following the manufacturer's guidelines, association and dissociation curves were fitted relative to a 1:1 Langmuir binding model using Biacore T200 evaluation software (Biacore AB, Uppsala, Sweden). As shown in Table 7 below, TY21586 and TY21580 exhibited similar affinity for FcγR compared to the reference antibody (TAC2114).

[0357] Table 7: Binding of antibodies to FcγR

[0358]

[0359]

[0360] Combination with FcRn

[0361] According to the manufacturer's guidelines, use Biacore TM The T200 instrument (Biacore AB, Uppsala, Sweden) was used to examine the binding affinity of the test antibody to recombinant human FcRn using surface plasmon resonance (SPR) analysis. Following the instructions of the amine conjugation kit (GE Biacore#BR-1000-50), the human FcRn protein (Sino Biological Inc. 11044-H07E) was immobilized on the CM5 chip via its amine groups coupled to the carboxylated surface of the sensor chip. 100 nM of each antibody was diluted in running buffer (50 mM NaPO4, 150 mM NaCl, and 0.05% (v / v) surfactant 20, pH 6.0) and the sample was injected at a flow rate of 30 μl / min for 120 seconds. Figure 7As shown, antibodies TY21585, TY21580, TY21591, TY21687, and TY21691 exhibited higher binding percentages to FcRn compared to TAC2114. This indicates that the IgG-FcRn complex on the Biacore chip can withstand conformational changes that stabilize the complex compared to the reference antibody (TAC2114). Antibodies TY21586, TY21587, TY21589, TY21689, and TY21680 showed low binding percentages.

[0362] Human PBMC activation

[0363] Preliminary studies showed that TY21580 did not stimulate human T cell activation or proliferation. Since CTLA4 activity on T cells is associated with the first signaling pathway (TCR / CD3) and the second signaling pathway involving B7-CD28 / CTLA-4, human PBMCs were selected, and the activity of TY21580 in the presence of low concentrations of anti-CD3 antibody was measured. Anti-CD3 antibody (OKT-3) was coated overnight at 4°C on 96-well plates. After washing, 1x10⁻⁶ cells were added to each plate. 5 Freshly isolated human PBMCs were added to each well, followed by the addition of test items at different concentrations. Induction of IL-2 was measured 48 hours post-stimulation using the Human IL-2 ELISA Ready-SET-Go (Invitrogen) kit. IFNγ levels in the supernatant were measured using the Human IFNγ ELISA Ready-SET-Go (Invitrogen) kit. Figure 8A and Figure 9 As shown, in the presence of anti-CD3 antibody, antibody TY21580 significantly increased the activation of human PBMCs, while TY21580 alone had no activity.

[0364] Dendritic cell MLR assay

[0365] DC-MLR was performed using mononuclear cell-derived DCs and CD4+ T lymphocytes in the following three donor pairs: D42 / D109, D32 / D104, and D104 / D42. Figure 10To obtain dendritic cells (DCs), PBMCs were isolated from healthy donors by density gradient centrifugation, and CD14+ monocytes were purified from PBMCs using a positive selection commercial kit (StemCell). CD14+ monocytes were skewed to become DCs by culturing in RPMI-1640 supplemented with 10% heat-inactivated FBS, 1% penicillin / streptomycin, 20 ng / mL rhGM-CSF, and 20 ng / mL rhIL-4 for 6 days. On day 3, the medium was replaced with fresh medium. On day 6, DC maturation was induced by continuous 24-hour exposure to RPMI-1640 supplemented with 10% heat-inactivated FBS, 1% penicillin / streptomycin, and 50 ng / mL rhTNF-α. CD4+ T cells were purified from another healthy donor by negative separation. Test items were titrated to the appropriate concentrations (e.g., ...). Figure 10 (As shown in the diagram). This allows for the collection of DC (1x10). 4 ) and allogeneic CD4+ T cells (1x10 5 Co-culture was performed with or without titrated test items. Anti-PD1 antibody was used as a positive control for the DC-MLR assay. On day 5 after co-culture, IFNγ in the supernatant was measured by ELISA using the Human IFNγ Ready-SET-Go ELISA kit. Figure 10 As shown, antibody TY21580 showed weak activity in a DC-MLR assay using human CD4+ T cells and DCs.

[0366] ADCC activity of antibody TY21580

[0367] HEK293F cells overexpressing human CTLA-4 were used as target cells to evaluate TY21580-mediated ADCC activity. Human NK cells were isolated from fresh human PBMCs using a human NK cell isolation kit (StemCell). 1 x 10 5 NK cells and 1x10 4 HEK293F / hCTLA-4 cells (E:T ratio 10:1) were mixed with different concentrations of antibody. After 4 hours of incubation, LDH was measured to determine ADCC activity. The lysis % was then calculated using the following formula: lysis % = [(experimental release value) - average (target + NK)] / [average (maximum target release value) - average (target only)] × 100%. Figures 11A-11B As shown, TY21580 exhibited stronger ADCC activity compared to the reference antibody (TAC2114). The isotype control showed no ADCC activity regardless.

[0368] ADCC activity was also assessed using human Treg cells (A, donor #96; B, donor #12) and NK cells (A, donor #99; B, donor #05). To obtain human Treg cells, fresh human PBMCs were isolated from healthy donors and processed using EASYSEP. TM Human Regulatory T Cell Enrichment Kit (StemCell Technologies) negatively selected Treg cells. Enriched human Treg cells were further expanded by CD3 / CD28 stimulation in the presence of IL-2 and confirmed by CD25 and FOXP3 staining and FACS analysis. To obtain human NK cells, fresh human PBMCs were isolated from another healthy donor, and NK cells were isolated using a Human NK Cell Isolation Kit (StemCell Technologies). Human Treg cells were labeled with 10 μM calcein-AM (Invitrogen) at 37°C for 30 min. After three washes, the labeled Treg cells were mixed with different concentrations of test material, followed by the addition of NK cells. 1 x 10 5 NK cells and 1x10 4 Labeled human Treg cells were added to the wells of a 96-well plate and mixed to produce an E:T ratio of 10:1. After 4 hours of incubation, the concentration of calcein-AM in the supernatant was measured to determine ADCC activity using the following formula: Dissolution % = [(Experimental release value) - Average (target + NK)] / [Average (maximum target release value) - Average (target only)] × 100%. Figures 12A-12B As shown, antibody TY21580 exhibited stronger ADCC activity compared to the reference antibody (TAC2114). The isotype control showed no ADCC activity.

[0369] CDC activity of TY21580

[0370] HEK293F cells overexpressing human CTLA-4 were labeled with 10 μM calcein-AM (Invitrogen) at 37°C for 30 minutes. Different concentrations of antibody were mixed with 1x10 μM calcein-AM in the wells of a 96-well plate. 4 A mixture of labeled cells and 5% normal human serum complement (NHSC, Quidel) was prepared. After 5 hours of incubation, calcein-AM in the supernatant was measured to determine CDC activity. Figure 13 ).

[0371] Human PBMCs were freshly isolated from a healthy donor (donor #57). CD4+ T cells were isolated using the EasySep Human CD4+ T Cell Enrichment Kit (StemCell) and stimulated with PMA (50 ng / mL) + iomycin (1 μM) for 20 h to induce CTLA-4 expression on the cell surface. Activated human CD4+ T cells were then labeled with 10 μM calcein-AM (Invitrogen) at 37°C for 30 min. Different concentrations of antibody were mixed with 1 x 10⁻⁶ ions of 10 ... 4 A mixture of labeled human CD4+ T cells and 5% normal human serum complement (NHSC, Quidel) was prepared. After 5 hours of incubation, calcein-AM in the supernatant was measured to determine CDC activity. Figure 14 TY21580 showed no CDC activity against HEK293F / hCTLA-4 cells or activated human T cells.

[0372] In summary, these results indicate that the antibody described herein can bind to human CTLA4 with high affinity and specificity, and that the antibody efficiently blocks the interaction of CTLA4 with its homologous ligands CD86 and CD80. Cross-reactivity with CTLA4 from multiple species was also demonstrated. Furthermore, binding to CTLA4 modulates T cell activation and induces ADCC activity against CTLA4-expressing cells such as Tregs.

[0373] Example 4: In vivo characterization of antibodies against IgG conversion

[0374] As described in the above embodiments, the species cross-reactivity of the antibody (human and mouse) allows for the determination of the antitumor efficacy of the antibody in multiple syngeneic tumor models, including the MC38 and CT26 colorectal tumor models, the H22 liver tumor model, the PAN02 pancreatic tumor model, and the 3LL lung tumor model.

[0375] Antitumor efficacy in the MC38 colorectal tumor model

[0376] C57BL / 6 mice (n=8 / group, female, 6-8 weeks old) were subcutaneously inoculated with MC38 (NTCC-MC38) mouse colon cancer cells. When tumors (80 mm) were established... 3 Treatment began with intraperitoneal injections twice a week for three weeks, using an isotype control antibody and three different doses of antibody TY21580. Tumor growth was monitored twice a week and reported as mean tumor volume ± sem over time. Figures 15A-15C ).like Figure 15AAs shown, compared to the isotype control antibody, TY21580 exhibited potent in vivo antitumor activity, with complete tumor regression at all three doses. Figure 15B As shown, up to sixty days after treatment, 8 out of 8 mice in the 10 mg / kg TY21580 group, 7 out of 8 mice in the 2.5 mg / kg TY21580 group, and 6 out of 8 mice in the 0.5 mg / kg TY21580 group remained tumor-free. When mice in the 10 mg / kg TY21580 group were rechallenged, durable memory of immunity against MC38 tumor cells was demonstrated, as shown in the figure. Figure 15C As shown in the image.

[0377] Antitumor efficacy in the CT26 colorectal tumor model

[0378] BALB / c mice (n=8 / group, female, 7-8 weeks old) were subcutaneously inoculated with CT26 (Shanghai Institutes for Biological Sciences) mouse colon cancer cells. When tumors (70 mm) were established... 3 Treatment began with intraperitoneal injections twice weekly of an allotype control antibody and two different doses of the antibody TY21580. Tumor growth was monitored twice weekly and reported as mean tumor volume ± sem over time. Figure 16 As shown, TY21580 exhibits potent in vivo antitumor activity compared to the isotype control antibody, achieving almost 100% inhibition at doses as low as 0.1-1 mg / kg.

[0379] Antitumor efficacy in H22 liver tumor model

[0380] BALB / c mice (n=5 / group, female, 7-8 weeks old) were subcutaneously inoculated with H22 (China Center for Type Culture Collection) mouse hepatocellular carcinoma cells. Once tumors (60 mm³) were established, treatment began with intraperitoneal injections twice weekly of either an isotype control antibody, antibody TY21586 at three different doses (0.1 mg / kg, 1 mg / kg, 5 mg / kg), or antibody TY21580 at two different doses (0.1 mg / kg, 1 mg / kg). Tumor growth was monitored twice weekly and reported as mean tumor volume ± sem over time. Figure 17As shown, both TY21580 and TY21586 exhibited potent in vivo antitumor activity in a dose-dependent manner compared to the isotype control antibody. When compared at the same dose, TY21580 was more potent than TY21586 in this tumor model. Furthermore, administration of TY21580 at 1 mg / kg resulted in tumor regression.

[0381] Antitumor efficacy in a Lewis lung tumor model

[0382] Lewis (JenNio Bio, Guandong, China) mouse lung cancer cells were subcutaneously inoculated into C57BL / 6 mice (n=6 / group, female, 8 weeks old). When tumors (70 mm) were established... 3 Treatment was initiated twice weekly via intraperitoneal injection with either an isotype control antibody or antibodies TY21580, TY21687, TY21680, or TY21691, all at a dose of 5 mg / kg. Tumor growth was monitored twice weekly and reported as mean tumor volume ± sem over time. Figure 18 As shown, compared with the isotype control antibody, antibodies TY21580, TY21687 and TY21680 showed significant inhibition of tumor growth, while antibody TY21691 did not show strong antitumor activity.

[0383] Antitumor efficacy in the PAN02 pancreatic tumor model

[0384] PAN-02 (CAMS Cell Culture Center) mouse pancreatic cancer cells were subcutaneously inoculated into C57BL / 6 mice (n=8 / group, female, 6 weeks old). When tumors (85 mm) were established... 3 Treatment began with intraperitoneal injections twice weekly of either an isotype control antibody or antibody TY21580 at three different doses (0.5 mg / kg, 2 mg / kg, and 0.5 mg / kg). Tumor growth was monitored twice weekly and reported as mean tumor volume ± sem over time. Figure 19 As shown, TY21580 exhibits potent antitumor activity in a dose-dependent manner compared to the isotype control antibody.

[0385] Antitumor efficacy of antibody TY21580 monotherapy or in combination with anti-CD137 antibody in a 3LL lung tumor model

[0386] C57BL / 6 mice (n=10 / group, female, 6-8 weeks old) were subcutaneously inoculated with 3LL (JCRB) mouse lung cancer cells. When tumors (75 mm) were established... 3Treatment was initiated twice weekly via intraperitoneal injection with an allotype control antibody, TY21580 (10 mg / kg), anti-CD137 antibody (10 mg / kg), or a combination of TY21580 and anti-CD137 antibody. The anti-CD137 antibody is a proprietary monoclonal antibody developed with the ability to bind both human and mouse CD137 (see PCT application number PCT / CN2017 / 098332, which is incorporated herein by reference in its entirety). Tumor growth was monitored twice weekly and reported as mean tumor volume ± sem over time. Figures 20A-20B As shown, both TY21580 and anti-CD137 antibody exhibited potent antitumor activity compared to the isotype control antibody, and the combination inhibited tumor growth to a greater extent than either single monotherapy.

[0387] Re-energization of mice that fully respond to TY21580

[0388] BALB / c mice (n=8 / group, female, 7-8 weeks old) were subcutaneously inoculated with H22 (China Center for Type Culture Collection) mouse hepatocellular carcinoma cells. When tumors (60 mm) were established... 3 Mice were treated with either an allotype control antibody or TY21580 at two different doses (1 mg / kg and 10 mg / kg) via intraperitoneal injection twice a week for three weeks. Tumor growth was monitored twice a week and reported as mean tumor volume ± sem over time. Compared to the allotype control antibody, TY21580 at both doses resulted in complete tumor regression several days after the last dose, and mice remained tumor-free for 60 days post-treatment. Mice in the 10 mg / kg TY21580 treatment group were then subcutaneously rechallenged with H22 tumor cells in the opposite flank on day 60, and tumor growth was monitored. Figure 21 As shown, these mice remained tumor-free after re-excitation with the same tumor cells, indicating the development of specific anti-tumor memory in these mice. Meanwhile, a re-excitation control group was established using naïve mice inoculated with the same number of H22 tumor cells, and their tumors grew rapidly.

[0389] Antibody drug metabolism kinetics

[0390] Pharmacokinetic studies of antibodies TY21585, TY21586, TY21580, and TY21591 were performed in female BALB / c mice (approximately 8 weeks of age). Three mice in each group were injected intravenously via tail vein with the test antibodies at 10 mg / kg. Blood samples (approximately 20 μL each) were collected at 1, 8, 48, 168, 336, and 500 hours post-administration. Blood samples were collected from three naïve female mice that had not received antibody administration as blank controls. Serum concentrations of each test antibody were determined by ELISA, with CTLA4-His-Fc used for capture and HRP-labeled anti-human IgG (Fab-specific) antibody (Sigma) used for detection. Figure 22 As shown, in mice, TY21586 exhibits similar pharmacokinetics to TAC2114, while TY21585, TY21580, and TY21591 are cleared much faster.

[0391] Pharmacokinetic studies of TY21586 and TY21580 were also conducted in primitive cynomolgus monkeys. Each antibody was administered intravenously at 10 mg / kg to one female and one male monkey. Serum samples were collected before administration (0 h) and at 0.25 h, 1 h, 8 h, 24 h, 72 h, 120 h, 168 h, 240 h, 336 h, 504 h, and 672 h after administration. Serum concentrations of TY21586 and TY21580 were determined by ELISA, with CTLA4-His-Fc used for capture and HRP-labeled anti-human IgG (Fab-specific) antibody (Sigma) used for detection. Figure 23 and Figure 24 As shown, TY21580 was cleared more rapidly in monkeys than TY21586, possibly due to the rapid increase in anti-drug antibodies observed in these animals.

[0392] Repeated-dose toxicity studies

[0393] Repeat-dose toxicity studies of TY21580 were conducted in normal BALB / c mice. The mediator control or antibody TY21580 (at 25 mg / kg or 50 mg / kg) was administered intraperitoneally on days 1, 4, 7, and 11. Each group included five female and five male mice (5 weeks old). Mice were monitored daily for abnormal behavior and symptoms, and food intake and body weight were measured daily. On day 14, animals were euthanized for postmortem examination and other analyses. Blood was collected from each animal, with up to six blood samples collected per group (three males and three females) for hemat...

Claims

1. An anti-CTLA4 antibody, wherein the antibody comprises HVR-H1 consisting of the amino acid sequence of SEQ ID NO:23, HVR-H2 consisting of the amino acid sequence of SEQ ID NO:35, HVR-H3 consisting of the amino acid sequence of SEQ ID NO:45, HVR-L1 consisting of the amino acid sequence of SEQ ID NO:58, HVR-L2 consisting of the amino acid sequence of SEQ ID NO:66, and HVR-L3 consisting of the amino acid sequence of SEQ ID NO:75, and wherein the antibody binds to human CTLA4.

2. The antibody of claim 1, wherein the antibody binds to cynomolgus monkey CTLA4 and mouse CTLA4.

3. The antibody of claim 1 or 2, wherein the antibody has a dissociation constant (K) of about 350 nM or less. D ) binds to human CTLA4, cynomolgus monkey CTLA4, mouse CTLA4, rat CTLA4 and / or dog CTLA4.

4. The antibody of claim 3, wherein the K D Measured by surface plasmon resonance (SPR).

5. The antibody of any one of claims 1-4, wherein binding of the anti-CTLA4 antibody induces antibody-dependent cellular cytotoxicity (ADCC) against CTLA4-expressing human cells or human Treg cells, wherein the ADCC activity of the anti-CTLA4 antibody is higher than the ADCC activity of ipilimumab.

6. The antibody of any one of claims 1 to 5, wherein the antibody comprises: The heavy chain variable region comprises the amino acid sequence of SEQ ID NO:87 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

100.

7. The antibody of any one of claims 1-6, wherein the antibody is an antibody fragment.

8. The antibody of claim 7, wherein the antibody fragment is a Fab, Fab', Fab'-SH, F(ab')2, Fv or scFv fragment.

9. The antibody of any one of claims 1-6, wherein the antibody comprises a human IgG1, IgG2, IgG3 or IgG4 Fc region.

10. A polynucleotide encoding the antibody according to any one of claims 1 to 9.

11. The polynucleotide of claim 10, comprising the sequence shown in SEQ ID NO:

113.

12. A vector comprising the polynucleotide according to claim 10 or claim 11.

13. The vector according to claim 12, wherein the vector is an expression vector and / or a display vector.

14. A host cell comprising the polynucleotide according to claim 10 or 11 or the vector according to claim 12 or 13.

15. The host cell of claim 14, wherein the host cell is a eukaryotic cell.

16. A host cell as claimed in claim 14 or claim 15, wherein the host cell is a Chinese Hamster Ovary (CHO) cell.

17. A method for preparing an antibody, the method comprising culturing the host cell according to any one of claims 14 to 16 under conditions suitable for producing the antibody.

18. The method of claim 17, further comprising recovering the antibody produced by the cell.

19. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier.

20. Use of the antibody of any one of claims 1 to 9 in the preparation of a medicament for treating or delaying the progression of colorectal cancer, liver cancer, pancreatic cancer or lung cancer.

21. The use according to claim 20, wherein the drug is used to make a 3 The size of solid tumors is reduced.

22. The use according to claim 21, wherein the medicament is administered in combination with at least one additional therapeutic agent.

23. The use of claim 22, wherein the at least one additional therapeutic agent is selected from the group consisting of viral gene therapy, immune checkpoint inhibitors, targeted therapy, vaccination therapy, and chemotherapy.

24. The use of claim 22 or claim 23, wherein the at least one additional therapeutic agent is selected from the group consisting of lenalidomide, pomalidomide, thalidomide, DNA alkylated platinum derivatives, 5-fluorouracil, cyclophosphamide, anti-CD137 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CD20 antibodies, anti-CD40 antibodies, anti-DR5 antibodies, anti-CD1d antibodies, anti-TIM3 antibodies, anti-SLAMF7 antibodies, anti-KIR receptor antibodies, anti-OX40 antibodies, anti-HER2 antibodies, anti-EGFR antibodies, IL-12, IFNα, GM-CSF, chimeric antigen receptors, adoptively transferred T cells, anti-cancer vaccines, and oncolytic viruses.

25. The use of claim 24, wherein the at least one additional therapeutic agent is selected from cisplatin, cetuximab, rituximab, trastuzumab and pembrolizumab.

26. The use of claim 21, wherein the medicament is administered in combination with radiation therapy.

27. The use according to claim 26, wherein the radiation therapy is single dose radiation, fractionated radiation, focal radiation or whole organ radiation.

28. The use of any one of claims 20-24, wherein the medicament is administered before or after surgery to remove a solid tumor in the subject.

Images