Anti-CTLA4 antibodies and methods of making and using same

Abstract

The present invention provides 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, pharmaceutical compositions thereof, and therapeutic methods of use thereof (e.g., for the treatment of cancer).

Description

[0001] This application is a divisional application of the invention application with an application date of February 2, 2019, a Chinese application number of 201980022531.1, and an invention title of "Anti-CTLA4 Antibodies and Methods for Their Preparation and Use".

[0002] Cross-reference to related applications

[0003] This application claims the priority benefit of International Application No. PCT / CN2018 / 075064, filed on February 2, 2018, which is incorporated herein by reference in its entirety.

[0004] Submission of the Sequence Listing as an ASCII text file

[0005] The content of the following submission in ASCII text file format is incorporated herein by reference in its entirety: Sequence Listing in computer-readable form (CRF) (file name: 695402000541SEQLIST.TXT, date of record: February 1, 2019, size: 102 KB). Technical field

[0006] The present 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, their pharmaceutical compositions, and their therapeutic uses. Background art

[0007] CTLA4 is a member of the immunoglobulin (Ig) protein superfamily that functions to downregulate T cell activation and maintain immunogenic homeostasis. Antibody-mediated CTLA4 blockade in vivo has been shown to enhance the anti-cancer immune response in an allogeneic murine prostate cancer model (Kwon et al. (1997) Proc Natl Acad Sci USA, 94(15):8099–103). In addition, blocking CTLA4 function has been shown to enhance the anti-tumor T cell response in tumor-bearing 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 therapeutic agents suitable for human use remains difficult because the translation from preclinical animal models to human safety is often poor. Thus, there is a need for anti-CTLA4 antibodies that are cross-reactive between different species such as humans and experimental animals (e.g., mice, monkeys, rats, etc.) to enable parallel animal model studies and provide suitable human therapeutic candidates. In addition, there is a need to develop safer anti-CTLA4 antibodies that are active only in certain situations such as in protease-rich tumor microenvironments.

[0008] All references cited herein, including patent applications, patent publications, non-patent literature, and UniProtKB / Swiss-Prot / GenBank accession numbers, are incorporated herein by reference in their entirety to the extent as if each individual reference had been specifically and individually indicated to be incorporated by reference. SUMMARY OF THE INVENTION

[0009] To meet the above and other needs, antibodies (e.g., cross-reactive antibodies) that bind to human CTLA4 and antigen-binding fragments thereof are disclosed herein. The anti-CTLA4 antibodies or antigen-binding fragments thereof of the present disclosure have at least one (e.g., one, some, or all) of the following functional properties: (a) a K of 500 nM or less DBind to human, cynomolgus monkey, mouse, rat, and / or dog CTLA4; (b) have antagonistic activity against human CTLA4; (c) do 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) have cross-reactivity with cynomolgus monkey, mouse, rat, and / or dog CTLA4; (e) induce ADCC effects (e.g., on Tregs); (f) activate human PBMCs (e.g., stimulate the secretion of IL-2 and / or IFNγ); (g) be capable of inhibiting tumor cell growth and establishing immune memory against tumor cells; (h) have a therapeutic effect on cancer; and (i) block the binding of human CTLA4 to human CD80 and / or human CD86 (see Examples 1-5 below).

[0010] Disclosed herein are precision / case-dependent activatable antibodies that bind to human CTLA4 when in the active form rather than the inactive form, i.e., the activatable antibodies bind to CTLA4 (active) only after cleaving the cleavable moiety (CM) to remove the masking moiety (MM). In some embodiments, the identified masking moiety (MM) described herein is capable of efficiently masking antibody activity and / or reducing or completely inhibiting antigen binding, while in some embodiments, there are no chemically labile residues methionine and / or tryptophan. Additionally, the activatable antibodies identified and described herein are as efficient as their parental antibodies in treating various cancer types, while having significantly reduced cytotoxicity in susceptible animals (NOD mice).

[0011] Accordingly, in one aspect, provided herein is an anti-CTLA4 antibody (e.g., a human antibody) that binds to human CTLA4 and has cross-reactivity with CTLA4 polypeptides from at least one non-human animal selected from the group consisting of cynomolgus monkey, mouse, rat, and dog. In some embodiments, the antibody binds to cynomolgus monkey CTLA4 and mouse CTLA4. In some embodiments combinable with any previous embodiment, the antibody has a dissociation constant (K D ) 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) for binding to human CTLA4, cynomolgus monkey CTLA4, mouse CTLA4, rat CTLA4, and / or dog CTLA4. In some embodiments, K DMeasured by surface plasmon resonance (SPR). In some embodiments, binding of the antibody to CTLA4 induces antibody-dependent cell cytotoxicity (ADCC) against CTLA4-expressing cells. In some embodiments, binding of the antibody to CTLA4 induces ADCC against Treg cells. In some embodiments, binding of the anti-CTLA4 antibody described herein induces antibody-dependent cell cytotoxicity (ADCC) against CTLA4-expressing human cells or human Treg cells, wherein in vitro, the ADCC activity of the anti-CTL4 antibody is higher than the ADCC activity of ipilimumab, and wherein both antibodies comprise a wild-type human IgG1 Fc region. In some embodiments, binding of the anti-CTLA4 antibody described herein induces antibody-dependent cell cytotoxicity (ADCC) against CTLA4-expressing human cells or human Treg cells, wherein in vitro, the ADCC activity of the anti-CTLA4 antibody is two-fold or higher than the ADCC activity of ipilimumab, and wherein both antibodies comprise a wild-type human IgG1 Fc region. In some embodiments, in vitro, the EC50 of the ADCC activity of the anti-CTL4 antibody is 50% or less of the EC50 of the ADCC activity of ipilimumab. Assays for measuring ADCC activity are described in Examples 3 and 15. In some embodiments, in a mouse cancer model, the anti-CTLA4 antibody selectively depletes Treg cells in the tumor microenvironment (e.g., reduces the percentage of Treg cells in tumor-infiltrating lymphocytes) compared to PBMCs or spleen. See, e.g., Example 18.

[0012] In some embodiments, which can be combined with any of the previous embodiments, the antibody specifically binds to an epitope comprising amino acid residues at the ligand-binding site of human CTLA4, such as the CD80 and / or CD86 binding site of human CTLA4. In some embodiments, the antibody specifically binds to an epitope similar to the ligand-binding site of human CTLA4, such as the CD80 and / or CD86 binding site of human CTLA4. In some embodiments, the antibody specifically binds to an epitope comprising amino acid residues Y105 and L106 of human CTLA4, wherein the numbering of the amino acid residues is according to SEQ ID NO:207. In some embodiments, the antibody does not bind to residue I108 of human CTLA4, wherein the numbering of the amino acid residues is 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 an IC50 higher than that of ipilimumab for blocking the binding of CD80 and / or CD86 to human CTLA4. In some embodiments, in an assay in which CD86 or CD80 is plate-bound while CTLA4 is in solution or CTLA4 is displayed on the cell surface, the anti-CTLA4 antibody has an IC50 that is 3.5-fold or higher (including 3.9-fold or higher) that of ipilimumab for blocking the binding of CD80 and / or CD86 to human CTLA4. See Example 13, Tables 23, Figures 57A - 57D and Figure 58 . Assays for testing the blocking activity (ligand competition) and IC50 of the antibody are described in Examples 3 and 13.

[0013] In some embodiments, which can be combined with any of the previous embodiments, 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), where X1 is F or Y, X2 is D or G, and X3 is A, G, or W; Formula (II): YSIX1SGX2X3WX4WI (SEQ ID NO:2), where 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), where 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: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 wherein the HVR-H3 comprises an amino acid sequence according to a formula selected from the group consisting of: Formula (VII): ARX1X2X3X4FDX5 (SEQ ID 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; Formula (VIII): ARX1GX2GYFDX3 (SEQ ID NO:8), where X1 is D or L, X2 is F or Y, and X3 is V or Y; Formula (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; Formula (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 said HVR-L1 comprises an amino acid sequence according to a formula selected from the 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: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 said 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 said HVR-L3 comprises an amino acid sequence according to a formula selected from the group consisting of: 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:16), wherein X1 is S or Y, X2 is D or Y, and X3 is Q or Y;Sum 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 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-29, HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 30-39, HVR-H3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 40-52, HVR-L1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-65, HVR-L2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 66-69, and HVR-L3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 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-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 having the amino acid sequence of SEQ ID NO:22, HVR-H2 having the amino acid sequence of SEQ ID NO:34, HVR-H3 having the amino acid sequence of SEQ ID NO:44, HVR-L1 having the amino acid sequence of SEQ ID NO:57, HVR-L2 having the amino acid sequence of SEQ ID NO:66, and HVR-L3 having the amino acid sequence of SEQ ID NO:74; f) HVR-H1 having the amino acid sequence of SEQ ID NO:23, HVR-H2 having the amino acid sequence of SEQ ID NO:35, HVR-H3 having the amino acid sequence of SEQ ID NO:45, HVR-L1 having the amino acid sequence of SEQ ID NO:58, HVR-L2 having the amino acid sequence of SEQ ID NO:66, and HVR-L3 having the amino acid sequence of SEQ ID NO:75; g) HVR-H1 having the amino acid sequence of SEQ ID NO:24, HVR-H2 having the amino acid sequence of SEQ ID NO:32, HVR-H3 having the amino acid sequence of SEQ ID NO:46, HVR-L1 having the amino acid sequence of SEQ ID NO:59, HVR-L2 having the amino acid sequence of SEQ ID NO:66, and HVR-L3 having the amino acid sequence of SEQ ID NO:76; h) HVR-H1 having the amino acid sequence of SEQ ID NO:25, HVR-H2 having the amino acid sequence of SEQ ID NO:36, HVR-H3 having the amino acid sequence of SEQ ID NO:47, HVR-L1 having the amino acid sequence of SEQ ID NO:60, HVR-L2 having the amino acid sequence of SEQ ID NO:69, and HVR-L3 having the amino acid sequence of SEQ ID NO:77; i) HVR-H1 having the amino acid sequence of SEQ ID NO:26, HVR-H2 having the amino acid sequence of SEQ ID NO:37, HVR-H3 having the amino acid sequence of SEQ ID NO:48, HVR-L1 having the amino acid sequence of SEQ ID NO:61, HVR-L2 having the amino acid sequence of SEQ ID NO:66, and HVR-L3 having the amino acid sequence of 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, 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; or m) 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. In some embodiments combinable with any previous 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 combinable with any previous 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 to 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 to the amino acid sequence of SEQ ID NO:95;b) a heavy chain variable region comprising 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 to the amino acid sequence of SEQ ID NO:83, and a light chain variable region comprising 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 to the amino acid sequence of SEQ ID NO:96; c) a heavy chain variable region comprising 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 to the amino acid sequence of SEQ ID NO:84, and a light chain variable region comprising 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 to the amino acid sequence of SEQ ID NO:97; d) a heavy chain variable region comprising 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 to the amino acid sequence of SEQ ID NO:85, and a light chain variable region comprising 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 to the amino acid sequence of SEQ ID NO:98; e) a heavy chain variable region comprising 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 to the amino acid sequence of SEQ ID NO:86, and a light chain variable region comprising 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 to the amino acid sequence of SEQ ID NO:99; f) a 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 to the amino acid sequence of SEQ ID NO:87, and a 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 to the amino acid sequence of SEQ ID NO:100;g) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:88 or a variant thereof having at least about 90% (such as at least about 92%, 95%, 98%, 99% or greater) sequence identity to the amino acid sequence of SEQ ID NO:88, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:101 or a variant thereof having at least about 90% (such as at least about 92%, 95%, 98%, 99% or greater) sequence identity to the amino acid sequence of SEQ ID NO:101; h) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:89 or a variant thereof having at least about 90% (such as at least about 92%, 95%, 98%, 99% or greater) sequence identity to the amino acid sequence of SEQ ID NO:89, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:102 or a variant thereof having at least about 90% (such as at least about 92%, 95%, 98%, 99% or greater) sequence identity to the amino acid sequence of SEQ ID NO:102; i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:90 or a variant thereof having at least about 90% (such as at least about 92%, 95%, 98%, 99% or greater) sequence identity to the amino acid sequence of SEQ ID NO:90, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:103 or a variant thereof having at least about 90% (such as at least about 92%, 95%, 98%, 99% or greater) sequence identity to the amino acid sequence of SEQ ID NO:103; j) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:91 or a variant thereof having at least about 90% (such as at least about 92%, 95%, 98%, 99% or greater) sequence identity to the amino acid sequence of SEQ ID NO:91, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:104 or a variant thereof having at least about 90% (such as at least about 92%, 95%, 98%, 99% or greater) sequence identity to the amino acid sequence of SEQ ID NO:104; k) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:92 or a variant thereof having at least about 90% (such as at least about 92%, 95%, 98%, 99% or greater) sequence identity to the amino acid sequence of SEQ ID NO:92, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:105 or a variant thereof having at least about 90% (such as at least about 92%, 95%, 98%, 99% or greater) sequence identity to the amino acid sequence of SEQ ID NO:105;l) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 93 or a variant thereof having at least about 90% (such as at least about 92%, 95%, 98%, 99% or greater) sequence identity to the amino acid sequence of SEQ ID NO: 93, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 106 or a variant thereof having at least about 90% (such as at least about 92%, 95%, 98%, 99% or greater) sequence identity to the amino acid sequence of SEQ ID NO: 106; or m) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 94 or a variant thereof having at least about 90% (such as at least about 92%, 95%, 98%, 99% or greater) sequence identity to the amino acid sequence of SEQ ID NO: 94, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 107 or a variant thereof having at least about 90% (such as at least about 92%, 95%, 98%, 99% or greater) sequence identity to the amino acid sequence of SEQ ID NO: 107.;

[0014] In some embodiments, the anti-CTLA4 antibodies described herein comprise 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 said HVR-H1 comprises the amino acid sequence of SEQ ID NO:23, or said HVR-H2 comprises the amino acid sequence of SEQ ID NO:35, or said 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 said HVR-L1 comprises the amino acid sequence of SEQ ID NO:58, or said HVR-L2 comprises the amino acid sequence of SEQ ID NO:66, or said 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 comprise one, two, or three conservative 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 to 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 to the amino acid sequence of SEQ ID NO:100.

[0015] In some embodiments, which can be combined with any of the previous embodiments, 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, which can be combined with any of the previous embodiments, 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 a human IgG1 or a variant with enhanced ADCC activity. In some embodiments, the antibody comprises a human IgG1 with reduced (or non-) fucosylation. In some embodiments, the antibody is a human antibody.

[0016] Other aspects of the disclosure relate to an antibody that competes or cross-competes with any of the antibodies described herein for binding to human CTLA4. Also provided herein are antibodies that bind to the same epitope and / or substantially the same epitope as any of the antibodies described herein.

[0017] Other aspects of the disclosure relate to an activatable antibody comprising: a) a first polypeptide that comprises, from the N-terminus to the C-terminus, a masking moiety (MM), a cleavable moiety (CM), and a target binding moiety (TBM), wherein the MM comprises, according to formula (XVIII): X m CX n CZ o (SEQ ID NO:134) amino acid sequence, where m is 2-10, n is 3-10, and o is 1-10, where 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 where 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; wherein when the CM is not cleaved, the MM inhibits the binding of the activatable antibody to human CTLA4; wherein the CM comprises at least a first cleavage site; and wherein the TBM comprises an antibody heavy chain variable region (VH); and b) a second polypeptide that comprises an antibody light chain variable region (VL); and wherein when the CM is cleaved, the activatable antibody binds to human CTLA4 through the VH and the VL. In some embodiments, m is 3-10.

[0018] Other aspects of the disclosure relate to an activatable antibody comprising: a) a polypeptide that comprises, from the N-terminus to the C-terminus, a masking moiety (MM), a cleavable moiety (CM), and a target binding moiety (TBM), wherein the MM comprises, according to formula (XVIII): X m CX n CZ oThe amino acid sequence of (SEQ ID NO:134), where m is 2 - 10, n is 3 - 10, and o is 1 - 10, where 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 where 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; wherein when the CM is not cleaved, the MM inhibits the binding of the activatable antibody to human CTLA4; wherein the CM comprises at least a first cleavage site; and wherein the TBM comprises an antibody light chain variable region (VL); and b) a second polypeptide, the second polypeptide comprising 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.

[0019] Other aspects of the present disclosure relate to an activatable antibody comprising: a polypeptide that, from the N - terminus to the C - terminus, comprises a masking moiety (MM), a cleavable moiety (CM), and a target - binding moiety (TBM), wherein the MM comprises, according to formula (XVIII): X m CX n CZ o The amino acid sequence of (SEQ ID NO:134), where m is 2 - 10, n is 3 - 10, and o is 1 - 10, where 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 where 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; wherein when the CM is not cleaved, the MM inhibits the binding of the activatable antibody to human CTLA4; wherein the CM comprises at least a first cleavage site; wherein the TBM, from the N - terminus to the C - terminus, comprises an antibody light chain variable region (VL) and 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.

[0020] Other aspects of the present disclosure relate to an activatable antibody comprising: a polypeptide that, from the N - terminus to the C - terminus, comprises a masking moiety (MM), a cleavable moiety (CM), and a target - binding moiety (TBM), wherein the MM comprises, according to formula (XVIII): X m CX n CZ oThe amino acid sequence of (SEQ ID NO:134), where m is 2 - 10, n is 3 - 10, and o is 1 - 10, where 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 where 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; 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 comprises, from the N-terminus to the C-terminus, a variable region of the antibody heavy chain (VH) and a variable region of the antibody light chain (VL); and wherein when the CM is cleaved, the activatable antibody binds to human CTLA4 through the VH and the VL.

[0021] 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 combinable with any previous embodiment, each X is not M, W, or C. In some embodiments combinable with any previous embodiment, X in formula (XVIII) m each X in 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 combinable with any previous embodiment, X in formula (XVIII) n each X in 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 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), where m is 2 - 10, and where 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, each X is not M, W, or C. In some embodiments, each X 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 combinable with any previous embodiment, the masking moiety (MM) comprises an amino acid sequence selected from SEQ ID NO: 141 - 147. In some embodiments combinable with any previous embodiment, MM further comprises an additional amino acid sequence at its N-terminus. In some embodiments, the additional amino acid sequence comprises the amino acid sequence of SEQ ID NO: 148.

[0022] In some embodiments, which can be combined with any previous 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, Tobacco Etch Virus (TEV) protease, plasmin, thrombin, factor X, PSA, PSMA, Cathepsin D, Cathepsin K, Cathepsin S, ADAM10, ADAM12, ADAMTS, Caspase-1, Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10, Caspase-11, Caspase-12, Caspase-13, Caspase-14, and TACE. In some embodiments, which can be combined with any previous embodiment, the CM further comprises 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 NOs: 156-163. In some embodiments, which can be combined with any previous embodiment, the CM further comprises a second cleavage site. In some embodiments, the second cleavage site is at the C-terminus of L1. In some embodiments, the second 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, Tobacco Etch Virus (TEV) protease, plasmin, thrombin, factor X, PSA, PSMA, Cathepsin D, Cathepsin K, Cathepsin S, ADAM10, ADAM12, ADAMTS, Caspase-1, Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10, Caspase-11, Caspase-12, Caspase-13, Caspase-14, and TACE. In some embodiments, the first cleavage site and the second cleavage site are different. In some embodiments, which can be combined with any previous embodiment, the CM further comprises 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 NOs: 156-163.In some embodiments, which can be combined with any of the previous embodiments, the CM also includes a third linker (L3) at the N-terminus of the first cleavage site. In some embodiments, which can be combined with any of the previous embodiments, the CM includes at least a first protease cleavage site and is cleaved with 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 etch virus (TEV) protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE.

[0023] In some embodiments, which can be combined with any of the previous embodiments, the activatable antibody includes a masking moiety (MM) and a cleavable moiety (CM) containing an amino acid sequence according to formula (XXIX): EVGSYX1X2X3X4X5X6CX7X8X9X10X11X12CX13X14SGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 164), where 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, which can be combined with any of the previous embodiments, the activatable antibody includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 165-179.

[0024] In some embodiments, which can be combined with any previous embodiment, VL comprises HVR-L1 having the amino acid sequence of SEQ ID NO:58, HVR-L2 having the amino acid sequence of SEQ ID NO:66, and HVR-L3 having the amino acid sequence of SEQ ID NO:75. In some embodiments, which can be combined with any previous 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 to the amino acid sequence of SEQ ID NO:100. In some embodiments, which can be combined with any previous embodiment, VH comprises HVR-H1 having the amino acid sequence of SEQ ID NO:23, HVR-H2 having the amino acid sequence of SEQ ID NO:35, and HVR-H3 having the amino acid sequence of SEQ ID NO:45. In some embodiments, which can be combined with any previous 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 to the amino acid sequence of SEQ ID NO:87.

[0025] Other aspects of the disclosure relate to a pharmaceutical composition comprising any antibody and / or activatable antibody described herein and a pharmaceutically acceptable carrier.

[0026] Other aspects of the disclosure relate to polynucleotides encoding any antibody and / or activatable antibody described herein. In some embodiments, the polynucleotide comprises a sequence selected from SEQ ID NOs: 108-133.

[0027] Other aspects of the disclosure relate to a vector comprising any polynucleotide described herein. In some embodiments, the vector is an expression vector and / or a display vector.

[0028] Other aspects of the disclosure relate to a host cell comprising any polynucleotide and / or vector 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.

[0029] Other aspects of the disclosure relate to a method of producing an antibody or activatable antibody, the method comprising culturing any host cell described herein under conditions suitable for producing the antibody or activatable antibody. In some embodiments, the method further comprises recovering the antibody or activatable antibody produced by the cell.

[0030] Other aspects of the present disclosure relate to a method of treating cancer or delaying the progression of cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of any of the antibodies, activatable antibodies, and / or pharmaceutical compositions 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 the present disclosure relate to a method of reducing the size of a solid tumor in a subject in need thereof, wherein the solid tumor has a size of about 400-1000 mm 3 in size, the method comprising administering to the subject an effective amount of any of the antibodies, activatable antibodies, and / or pharmaceutical compositions described herein. In some embodiments, the solid tumor has a size of about 400-800 mm 3Dimensions. In some embodiments, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent. In some embodiments, the at least one additional therapeutic agent is selected from the group consisting of viral gene therapy, immune checkpoint inhibitors, targeted therapies, radiation therapy, 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, the DNA alkylating platinum derivative cisplatin, 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, pembrolizumab, radiation therapy, single-dose radiation, fractionated radiation, focal radiation, whole-organ radiation, IL-12, IFNα, GM-CSF, chimeric antigen receptor, adoptively transferred T cells, anti-cancer vaccines, and oncolytic viruses. In some embodiments, the method comprises administering to the subject an effective amount of the anti-CTLA4 antibody, activating antibody, or pharmaceutical composition described herein before or after surgery to remove a tumor in the subject. In some embodiments, the anti-CD137 antibody comprises 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 to 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 to the sequence of SEQ ID NO:230.

[0031]

[0032] 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 the present disclosure. These and other aspects of the present disclosure will become apparent to those skilled in the art. These and other embodiments of the present disclosure are further described by the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figures 1A - 1B Shows binding of the antibody to CTLA4 as measured by ELISA. Figure 1A Shows binding of the indicated antibody to human CTLA4. Figure 1B Shows binding of the indicated antibody to canine CTLA4.

[0034] Figure 2 Shows species cross-reactivity of the indicated antibody, isotype control, or vehicle (PBSA) with HEK293F cells transiently overexpressing empty vector (pIRES), or murine or human CTLA4 as measured by flow cytometry.

[0035] Figure 3 Shows binding of antibody TY21580 or isotype control to activated human, monkey, and murine T cells as measured by flow cytometry.

[0036] Figures 4A - 4C Shows specificity of the antibody for CTLA4 as measured by flow cytometry. Figure 4A Shows binding of the indicated antibody or isotype control to HEK293F cells transiently overexpressing human PD-1, CTLA4, LAG3, TIM3, B7-H3, or empty vector (293F). Figure 4BShow the binding of the indicated antibody, isotype control, or vehicle (PBSA) to HEK293F cells transiently overexpressing human CD95, CD120a, OX40, CD40, CTLA4, or empty vector (pIRES). Figure 4C Show the binding of the indicated antibody, isotype control, or vehicle (PBSA) to HEK293F cells transiently overexpressing human TIM3, CTLA4, PD-L1, LAG3, BTLA, VISTA, PD-L2, ICOS, B7-H4, PD-1, B7-H3, or empty vector (pIRES).

[0037] Figures 5A - 5D Show the blocking ability of the antibody, as determined by ELISA. Figure 5A Show the ability of antibodies TY21687, TY21689, TY21680, and TY21691 to block the binding of human CD80 to human CTLA4. Figure 5B Show the ability of antibodies TAC2114, TY21585, TY21587, TY21588, TY21589, TY21580, and TY21591 to block the binding of human CD80 to human CTLA4. Figure 5C Show the ability of antibodies TY21687, TY21689, TY21680, and TY21691 to block the binding of human CD86 to human CTLA4. Figure 5D Show the ability of antibodies TAC2114, TY21585, TY21587, TY21588, TY21589, TY21580, and TY21591 to block the binding of human CD86 to human CTLA4.

[0038] Figures 6A - 6B Show the blocking ability of the antibody, as determined by FACS. Figure 6A Show the ability of the indicated antibody, isotype control, or vehicle (PBSA) to block the binding of human CD80 to HEK293F cells transiently overexpressing human CTLA4. Figure 6B Show the ability of the indicated antibody, isotype control, or vehicle (PBSA) to block the binding of human CD86 to HEK293F cells transiently overexpressing human CTLA4.

[0039] Figure 7 Show the ability of the indicated antibody to bind to FcRn, as determined by surface plasmon resonance (SPR).

[0040] Figures 8A - 8B Show the activation of human peripheral blood mononuclear cells (PBMC) achieved by antibody TY21580 or isotype control, as measured by ELISA. Figure 8AShows the effect on IL-2 secretion from CD3-stimulated human PBMCs treated with antibody TY21580 or isotype control. Figure 8B Shows the effect on IFNγ secretion from CD3-stimulated human PBMCs treated with antibody TY21580 or isotype control.

[0041] Figure 9 Shows the effect on IL-2 secretion from human PBMCs treated with antibody TY21580 in the presence or absence of anti-CD3 antibody, as measured by ELISA.

[0042] Figure 10 Shows the effect on IFNγ secretion, as measured by ELISA, from human dendritic cells (DCs) co-cultured with allogeneic CD4 + T cells in the presence of antibody TY21580, isotype control, or anti-PD-1 antibody.

[0043] Figures 11A - 11B Shows the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of exemplary antibodies against HEK293F cells transiently overexpressing human CTLA4, as determined by lactate dehydrogenase (LDH) release assay. Figure 11A Shows the ADCC activity of antibody TY21580 or isotype control against HEK293F cells transiently overexpressing human CTLA4 and incubated with human natural killer (NK) cells. Figure 11B Shows the ADCC activity of antibody TY21580, TAC2114, or isotype control against HEK293F cells transiently overexpressing human CTLA4 and incubated with human NK cells.

[0044] Figures 12A - 12B Shows the ADCC activity of exemplary antibodies against human Tregs isolated from two donors, as determined by calcein-AM release assay. Figure 12A Shows the ADCC activity of antibody TY21580, TAC2114, or isotype control against human Treg cells (from donor #96) incubated with human NK cells. Figure 12B Shows the ADCC activity of antibody TY21580, TAC2114, or isotype control against human Treg cells (from donor #12) incubated with human NK cells.

[0045] Figure 13 Shows the complement-dependent cytotoxicity (CDC) activity of antibody TY21580 or isotype control against HEK293F cells transiently overexpressing human CTLA4, as determined by calcein-AM release assay.

[0046] Figure 14 Show the CDC activity of antibody TY21580 or isotype control against activated human CD4 + T cells, as measured by calcein-AM release assay.

[0047] Figures 15A - 15C Show the in vivo anti-tumor efficacy of antibody TY21580 or isotype control in the MC38 syngeneic mouse colorectal tumor model. Figure 15A Show the tumor growth curves of different treatment groups of female C57BL / 6 mice bearing MC38-established tumors. Data points represent group means; error bars represent SEM. Figure 15B Show the individual tumor growth curves for each test group. Figure 15C Show the rechallenge study indicating durable memory of immunity against MC38 tumor cells.

[0048] Figure 16 Show the in vivo anti-tumor efficacy of antibody TY21580 or isotype control in the CT26 syngeneic mouse colorectal tumor model. Show the tumor growth curves of different treatment groups of female C57BL / 6 mice bearing CT26-established tumors. Data points represent group means; error bars represent SEM.

[0049] Figure 17 Show the in vivo anti-tumor efficacy of antibody TY21586, TY21580 or isotype control in the H22 syngeneic mouse liver tumor model. Show the tumor growth curves of different treatment groups of female C57BL / 6 mice bearing H22-established tumors. Data points represent group means; error bars represent SEM.

[0050] Figure 18 Show the in vivo anti-tumor efficacy of antibodies TY21580, TY21687, TY21687, TY21691 and TY21580 or isotype control in the Lewis syngeneic mouse lung tumor model. Show the tumor growth curves of different treatment groups of female C57BL / 6 mice bearing Lewis cell-established tumors. Data points represent group means; error bars represent SEM.

[0051] Figure 19 Show the in vivo anti-tumor efficacy of antibody TY21580 or isotype control in the PAN02 syngeneic mouse pancreatic tumor model. Show the tumor growth curves of different treatment groups of female C57BL / 6 mice bearing PAN02-established tumors. Data points represent group means; error bars represent SEM.

[0052] Figures 20A - 20BShow the in vivo anti-tumor efficacy of monotherapies of antibody TY21580, anti-CD137 antibody or isotype control, and the TY21580 + anti-CD137 antibody combination therapy in the 3LL syngeneic mouse lung tumor model. Figure 20A Show the tumor growth curves of different treatment groups of female C57BL / 6 mice bearing tumors established with 3LL. Data points represent group means; error bars represent SEM. Figure 20B Show the individual tumor growth curves for each test group.

[0053] Figure 21 Show a rechallenge study indicating durable memory of immunity against H22 mouse liver tumor cells. Mice with complete responses in the TY21580 treatment group were subcutaneously rechallenged with H22 tumor cells on the opposite flanks on day 59. Naïve mice were also inoculated with H22 tumor cells simultaneously.

[0054] Figure 22 Show the time course of the blood concentration of the indicated antibodies administered intravenously to female BALB / c mice at a concentration of 10 mg / kg, as determined by ELISA.

[0055] Figure 23 Show the time course of the blood concentration of the indicated antibodies administered intravenously to cynomolgus monkeys at a concentration of 10 mg / kg, as determined by ELISA.

[0056] Figure 24 Show a comparison of the time course of the blood concentration of the indicated antibodies administered intravenously to cynomolgus monkeys at a concentration of 10 mg / kg with the appearance of anti-drug antibodies (ADA) in these monkeys, as determined by ELISA.

[0057] Figures 25A - 25B Show the mean spleen weights of male and female BALB / c mice after repeated intraperitoneal administration of antibody TY21580 or vehicle control. Figure 25A Show the mean spleen weights of male BALB / c mice after repeated intraperitoneal administration of antibody TY21580 or vehicle control on days 1, 4, 7, and 11. Figure 25B Show the mean spleen weights of female BALB / c mice after repeated intraperitoneal administration of antibody TY21580 or vehicle control on days 1, 4, 7, and 11.

[0058] Figure 26 Show the histopathology of BALB / c mice after repeated intraperitoneal administration of antibody TY21580 or vehicle control on days 1, 4, 7, and 11.

[0059] Figures 27A - 27B Show the stability of exemplary antibodies after storage at high concentration.Figure 27A Shows the size exclusion chromatography (SEC) profile of antibody TY21586 after storage at >100 mg / mL. Figure 27B Shows the SEC profile of antibody TY21580 after storage at >100 mg / mL.

[0060] Figure 28 Shows the SEC profile of exemplary antibodies under accelerated stress conditions.

[0061] Figure 29 Shows a schematic diagram of the self-blocking peptide selection process using Fab fragments displayed on the yeast surface with an anti-CTLA4 antibody.

[0062] Figure 30 Shows a schematic diagram of the self-blocking peptide selection process using scFv fragments displayed on the yeast surface with an anti-CTLA4 antibody.

[0063] Figures 31A - 31B Shows the functional display of CTLA4-targeting Fab and scFv on yeast, as determined by flow cytometry. Figure 31A Shows the functional display of CTLA4-targeting Fab on the surface of yeast. Figure 31B Shows the functional display of CTLA4-targeting scFv on the surface of yeast.

[0064] Figure 32 Shows an exemplary selection process for an activatable antibody targeting human CTLA4. A yeast library displaying a fusion protein is subjected to several rounds of FACS-based screening.

[0065] Figures 33A - 33B Shows the CTLA4 binding affinity of exemplary CTLA4-activatable antibody clones, as determined by flow cytometry. Figure 33A Shows the binding affinity of CTLA4-activatable antibody clones in scFv form compared to an scFv fragment of the target antibody without a masking peptide, including cases where the masking peptide of CTLA4-activatable antibody clone B13287 is intact or the masking peptide is cleaved by TEV protease. Figure 33B Shows the CTLA4 binding affinity of CTLA4-activatable antibody clones in Fab form compared to a Fab fragment of the target antibody without a masking peptide, including cases where the masking peptide of CTLA4-activatable antibody clone B13189 is intact or the masking peptide is cleaved by TEV protease.

[0066] Figures 34A - 34B Shows the masking efficiency of exemplary CTLA4-activatable antibodies TY22401, TY22403, TY22402, and TY22404 compared to the parental antibody TY21580.Figure 34A Shows the association and dissociation curves of the indicated activatable antibodies compared to the parental antibody TY21580, as measured by the ForteBio system. Figure 34B Graph showing the relative ratio of bound activatable antibodies compared to the parental antibody TY21580.

[0067] Figures 35A - 35B Shows the masking efficiency of exemplary CTLA4 activatable antibodies against recombinant human CTLA4-Fc, as measured by ELISA. Figure 35A Shows the first batch of ELISA data indicating the binding of CTLA4 activatable antibodies TY22401, TY22402, TY22403, TY22404 to recombinant human CTLA4-Fc compared to the parental antibody TY21580. Figure 35B Shows the binding of CTLA4 activatable antibodies TY22563, TY22564, TY22565, TY22566 to recombinant human CTLA4-Fc compared to the parental antibody TY21580.

[0068] Figures 36A - 36B Shows the activity of CTLA4 activatable antibody TY22404 after removal of the masking peptide. Figure 36A Shows the SDS-PAGE results of activatable antibody TY22404 without treatment, treated with protease uPA, or treated with 5 or 10 units of protease MMP-9. Figure 36B Shows the binding of activatable antibody TY22404 without treatment, treated with protease uPA, or treated with protease MMP-9 compared to the parental antibody TY21580, as measured by ELISA.

[0069] Figures 37A - 37C Shows the size exclusion chromatography (SEC) profiles of exemplary activatable antibodies under accelerated stress conditions. Figure 37A Shows the SEC profile of activatable antibody TY22402 after six freeze-thaw cycles compared to control conditions. Figure 37B Shows the SEC profile of activatable antibody TY22402 after seven days at 50 °C compared to control conditions. Figure 37C Shows the percentage of the SEC main peak area of exemplary activatable antibodies after seven days at 50 °C, after storage at 40 °C for up to 28 days, or after six freeze-thaw cycles compared to control conditions.

[0070] Figure 38 Shows the percentage of the SEC main peak area of activatable antibodies TY22401 and TY22402 after storage at approximately 8 mg / mL or >150 mg / mL.

[0071] Figure 39 Shows the masking efficiency of untreated activatable antibodies TY21580, TY22401, TY22402, and TY22566 incubated at pH 3.7 for 30 minutes or incubated at pH 3.7 for 1 hour, as measured by the ForteBio system.

[0072] Figures 40A - 40B Shows the activation of human peripheral blood mononuclear cells (PBMCs) achieved by isotype control antibodies, parental antibody TY21580, or exemplary CTLA4-activatable antibodies TY22401, TY22402, or TY22404, as measured by ELISA. Figure 40A Shows the effect on IL-2 secretion of CD3-sensitized human PBMCs stimulated with isotype control antibodies, parental antibody TY21580, and exemplary CTLA4-activatable antibodies TY22401, TY22402, or TY22404. Figure 40B Shows the effect on IFNγ secretion of CD3-sensitized human PBMCs stimulated with isotype control antibodies, parental antibody TY21580, and exemplary CTLA4-activatable antibodies TY22401, TY22402, or TY22404.

[0073] Figure 41 Shows the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of isotype control antibodies, parental antibody TY21580, or exemplary activatable antibodies TY22401, TY21580, or TY22404 against HEK293F cells transiently overexpressing human CTLA4, as measured by the ADCC reporter gene assay.

[0074] Figures 42A - 42B Shows the in vivo antitumor efficacy of parental antibody TY21580, isotype control antibody, or exemplary CTLA4-activatable antibodies TY22401, TY22402, or TY22566 in the MC38 syngeneic mouse colorectal tumor model. Figure 42A Shows the tumor growth curves of different treatment groups of female C57BL / 6 mice bearing MC38-established tumors. Data points represent group means; error bars represent SEM. Figure 42B Shows the individual tumor growth curves of the groups treated with TY21580, TY22401, TY22402, and TY22566.

[0075] Figure 43Show the in vivo anti-tumor efficacy of an isotype control antibody, the parental antibody TY21580, or one of three activatable antibodies in a CT26 syngeneic mouse colorectal tumor model. Tumor growth curves of different treatment groups of female C57BL / 6 mice bearing CT26-established tumors are shown. Data points represent group means; error bars represent SEM.

[0076] Figure 44 Show the in vivo anti-tumor efficacy of an isotype control antibody, the parental antibody TY21580, or one of three activatable antibodies in an H22 syngeneic mouse liver tumor model. Tumor growth curves of different treatment groups of female C57BL / 6 mice bearing H22-established tumors are shown. Data points represent group means; error bars represent SEM.

[0077] Figures 45A - 45B Show the in vivo anti-tumor efficacy of the parental antibody TY21580, an isotype control antibody, and exemplary activatable antibodies TY22401, TY22402, or TY22566 in a 3LL syngeneic mouse lung tumor model. Figure 45A Show the tumor growth curves of different treatment groups of female C57BL / 6 mice bearing 3LL-established tumors. Data points represent group means; error bars represent SEM. Figure 45B Show the individual tumor growth curves of the groups treated with TY21580, TY22401, TY22402, and TY22566.

[0078] Figures 46A - 46C Show the time course of the blood concentration of the test article (TA) administered intravenously at a concentration of 10 mg / kg to female BALB / c mice, as determined by ELISA. Figure 46A Show the time course of the blood concentration of the activatable antibody TY22401 administered intravenously at a concentration of 10 mg / kg to female BALB / c mice compared to the parental antibody TY21580. Figure 46B Show the time course of the blood concentration of the activatable antibody TY22402 administered intravenously at a concentration of 10 mg / kg to female BALB / c mice compared to the parental antibody TY21580. Figure 46C Show the time course of the blood concentration of the activatable antibody TY22404 administered intravenously at a concentration of 10 mg / kg to female BALB / c mice compared to the parental antibody TY21580.

[0079] Figure 47 Show the repeated-dose toxicity of an isotype control antibody, the parental antibody TY21580, and exemplary activatable antibodies TY22566, TY22401, and TY22402 using a NOD mouse model. The percentage of survival over 20 days for each treatment group is shown.

[0080] Figures 48A - 48C Shows the mean spleen weight of BALB / c mice after repeated intraperitoneal administration of the indicated activatable antibody. Figure 48A Shows the mean spleen weight of BALB / c mice after repeated intraperitoneal administration of the activatable antibody TY22402, the parental antibody TY21580, or an isotype control on days 1, 4, 7, and 11. Figure 48B Shows the mean spleen weight of BALB / c mice after repeated intraperitoneal administration of the activatable antibody TY22566, the parental antibody TY21580, or an isotype control on days 1, 4, 7, and 11. Figure 48C Shows the mean spleen weight of BALB / c mice after repeated intraperitoneal administration of the activatable antibody TY22401, the parental antibody TY21580, or an isotype control on days 1, 4, 7, and 11.

[0081] Figure 49 Shows the size exclusion chromatography (SEC) profile of the indicated activatable antibody after seven days at 50 °C compared to a control condition.

[0082] Figure 50 Shows 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 a control condition.

[0083] Figure 51 Shows the size exclusion chromatography (SEC) profile of the indicated activatable antibody after six freeze-thaw cycles compared to a control condition.

[0084] Figure 52 Shows the percentage of the SEC main peak ratio of the indicated activatable antibody after storage at >115 mg / mL.

[0085] Figure 53 Shows an overview of the stability data.

[0086] Figure 54 Depicts a multiple sequence alignment of a portion of human and mouse CTLA4, in which the contact residues between human CTLA4 and either CD80, CD86, or ipilimumab (based on two crystal structures) are located. The contacting amino acids are shaded in gray, the key contacting amino acids are boxed, the dimer interface amino acids are indicated by dots, and the amino acids that differ between mouse CTLA4 and human CTLA4 are underlined and shown in bold. From top to bottom, the sequences shown are represented by SEQ ID NO:203 - 208.

[0087] Figure 55A Depicts the interaction between human CTLA4 and its ligand CD80. Figure 55BDepicts the interaction between human CTLA4 and its ligand CD86. Figure 55C Depicts the structural alignment between human CTLA4 and murine CTLA4. Human CTLA4 is colored in black and murine CTLA4 is colored in white.

[0088] Figures 56A - 56E Depicts the results from epitope mapping experiments, showing TY21580 ( Figure 56A ), ipilimumab ( Figure 56B ), human CD80 ( Figure 56C ), human CD86 ( Figure 56D ) and murine CD86 ( Figure 56E ) binding capabilities to human CTLA4, murine CTLA4 and CTLA4 mutants according to flow cytometry.

[0089] Figures 57A - 57D Depicts the effect of TY21580 and ipilimumab on receptor-ligand binding blockade between human CTLA4 and CD80 or CD86. Figure 57A and Figure 57B Show the binding curves of human CD80 ( Figure 57A ) or CD86 ( Figure 57B ) to plate-bound human recombinant CTLA4 protein in the presence of serial dilutions of TY21580, ipilimumab or isotype control antibody, as measured by ELISA. Figures 57C - 57D Show the binding curves of human recombinant CTLA4 protein to plate-bound human CD80 ( Figure 57C ) or CD86 ( Figure 57D ) in the presence of serial dilutions of TY21580, ipilimumab or isotype control antibody, as measured by ELISA.

[0090] Figure 58 Depicts the reporter signal activation mediated by CTLA4 blockade of the CD28 pathway by anti-CTLA4 antibodies. Jurkat / CTLA4 and aAPC / Raji cells were co-cultured in the presence of serial dilutions of anti-CTLA4 antibody, with human IgG1 anti-HEL antibody as isotype control. After overnight incubation, the luminescence signal was measured with Bio-luciferase substrate and the relative luciferase units (RLU) were normalized to the blank control. Results are presented as mean RLU fold ± SEM. Experiments were performed in triplicate. Note: When fitting the curve, data points at the highest concentration of TY21580 (500 μg / mL) were excluded from the analysis because a significant hook effect was observed at this time.

[0091] Figure 59Depict the activation of ADCC reporter signal transduction achieved by anti-CTLA4 antibody. 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 as an isotype control. After incubation for 6 hours, the luminescence signal was measured using ONE-Glo luciferase substrate. The relative luciferase units (RLU) were normalized to the blank control, and the results are presented as mean RLU ± SEM. Experiments were performed in triplicate.

[0092] Figure 60A and Figure 60B Depict the tumor growth curve of MC38 tumor-bearing mice treated with anti-CTLA4 antibody. Figure 60A Depict the group-average tumor growth over time in MC38 tumor-bearing mice treated with isotype control antibody (1 mg / kg, BIW), TY21580 (1 mg / kg or 0.2 mg / kg, BIW), or ipilimumab (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 Depict the tumor growth over time in individual MC38 tumor-bearing mice treated with isotype control antibody (group-1), TY21580 (group-2 and group-3), or ipilimumab (group-4 and group-5).

[0093] Figure 61A and Figure 61B Depict the effects of TY21580 and ipilimumab on the intratumoral regulatory T (Treg) cell levels in subcutaneous MC38 tumors from mice treated with TY21580 or ipilimumab. Figure 61A Show the percentage of regulatory T (Treg) cells (CD4+CD25+) among CD4+ T cells isolated from tumors. Figure 61B Depict the ratio of cytotoxic T lymphocytes (CD8+ T cells) to Treg cells (i.e., CD8+ / Treg ratio) in subpopulations 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 62A and Figure 62B Depict the effects of TY21580 and ipilimumab on the intratumoral regulatory T (Treg) cell levels in subcutaneous CT26 tumors from mice treated with TY21580 or ipilimumab. Figure 62AShow the percentage of regulatory T (Treg) cells (CD4+CD25+) among CD4+ T cells isolated from tumors. Figure 62B Depict the ratio of cytotoxic T lymphocytes (CD8+ T cells) to Treg cells (i.e., CD8+ / Treg ratio) in subpopulations 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.

[0095] Figure 63 Depict FOXP3 in mice bearing CT26 tumors treated with isotype control antibody or TY21580 + CD4 + The expression level of CTLA4 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.

[0096] Figures 64A - 64D Depict the tumor growth curves of mice bearing murine H22 hepatocellular carcinoma treated with TY21580 or isotype control antibody. Figure 64A Depict when the tumor reaches 500 mm 3 or 800 mm 3 start TY21580 treatment, or when the tumor reaches 500 mm 3 start isotype control antibody treatment of the group mean tumor growth. Data points represent the mean of tumors from 8 mice / group, and error bars represent the standard error of the mean (SEM). Figures 64B - 64D Depict the individual tumor growth in each mouse. Figure 64B Depict the tumor growth in mice treated with isotype control antibody, where treatment starts when the tumor reaches 500 mm 3 when treatment starts; Figure 64C Depict the tumor growth in mice treated with TY21580, where treatment starts when the tumor reaches 500 mm 3 when treatment starts; Figure 64D Depict the tumor growth in mice treated with TY21580, where treatment starts when the tumor reaches 800 mm 3 when treatment starts.

[0097] Figure 65A and Figure 65BDepict the masking efficiency of exemplary activatable antibodies containing masking peptides of variable lengths compared to the parental antibody TY21580. The masking efficiency was determined using an ELISA-based method. Figure 65A and Figure 65B Represent two experimental settings using the same experimental method to test various activatable anti-CTLA4 antibodies.

[0098] Figure 66 Depict the masking efficiency of exemplary activatable antibodies containing cleavage peptides of different lengths compared to the parental antibody TY21580. The masking efficiency was determined using an ELISA-based method. Detailed Description

[0099] I. General Techniques

[0100] The techniques and procedures described or referenced herein are generally well understood by those skilled in the art and are generally employed by those skilled in the art using conventional methods such as those widely utilized as described in the following: Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd Edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (F.M. Ausubel et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.); PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. 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 (J.E. Cellis ed, 1998) Academic Press; Animal Cell Culture (R.I. Freshney ed, 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths and D.G. Newell eds, 1993-8) J.Wiley and Sons; Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell eds); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P.Edited by Calos, 1987); PCR: The Polymerase Chain Reaction, (Edited by Mullis et al., 1994); Current Protocols in Immunology (Edited by J.E. Coligan et al., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. 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: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (Edited by M. Zanetti and J.D. Capra, Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (Edited by V.T. DeVita et al., J.B. Lippincott Company, 1993).

[0101] II. Definitions

[0102] Before describing the present disclosure in detail, it should be understood that the present disclosure is not limited to specific compositions or biological systems that can undoubtedly vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting.

[0103] As used herein, unless the context clearly dictates otherwise, the singular forms "a / an" and "the" include plural referents. Thus, for example, reference to "a molecule" optionally includes a combination of two or more of said molecules, and so on.

[0104] As used herein, the term "about" refers to the usual error range that is readily known to a person skilled in the art for the corresponding value. References in this disclosure to "about" a value or parameter include (and describe) embodiments that are directed to that value or parameter per se.

[0105] It should be understood that aspects and embodiments of the disclosure described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0106] As used herein, the term "and / or" in phrases such as "A and / or B" is intended to include both A and B; A or B; A alone; and B alone. Similarly, the term "and / or" as used herein in phrases such as "A, B, and / or C" is intended to encompass 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.

[0107] The term "amino acid" refers to naturally occurring amino acids, synthetic amino acids, and amino acid analogs and mimetics that act in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later 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 in which the C-terminal carboxyl group, the N-terminal amino group, or a side chain functional group has been chemically modified to another functional group. The term "amino acid mimetic" refers to a compound that has a general chemical structure different from that of an amino acid, but that acts in a manner similar to a naturally occurring amino acid.

[0108] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See, e.g., Immunology—A Synthesis (2nd ed., E.S. Golub and D.R. Gren eds., Sinauer Associates, Sunderland, Mass. (1991)).

[0109] The terms "polypeptide," "protein," and "peptide" are used interchangeably herein and can refer to a polymer of two or more amino acids.

[0110] "Polynucleotide" or "nucleic acid", which may be used interchangeably herein, refers to a polymer of nucleotides of any length and includes DNA and RNA. Nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs 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 analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interspersed with non-nucleotide components. Polynucleotides may contain one or more modifications made after synthesis, such as conjugation to a label. Other types of modifications include, for example, "caps"; substitution of one or more of the naturally occurring nucleotides with analogs; internucleotide modifications such as those having uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and those having charged linkages (e.g., phosphorothioates, dithiophosphates, etc.), those containing pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those having intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylating agents, those having modified linkages (e.g., α-anomeric nucleic acids, etc.); and unmodified forms of one or more polynucleotides. In addition, any hydroxyl group normally present in the sugar may be replaced, for example, by a phosphonate group, a phosphate group, protected by a standard protecting group, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to a solid or semi-solid support. The 5' and 3' terminal OHs may be phosphorylated or replaced with an amine or an organic capping group moiety having 1 to 20 carbon atoms. Other hydroxyl groups may also be derivatized to obtain standard protecting groups. Polynucleotides may also contain analogous forms of ribose or deoxyribose that are 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-anomeric sugars; epimeric sugars such as arabinose, xylose or lyxose; pyranoses; furanoses; sedoheptuloses; acyclic analogs and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages 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 ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO or CH2 ("acetal"), where each R or R' is independently H or an optionally substituted or unsubstituted alkyl (1-20 C) containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or aralkyl. Not all linkages in a polynucleotide need be the same.The foregoing applies to all polynucleotides mentioned herein, including RNA and DNA.

[0111] The term "isolated nucleic acid" refers to a nucleic acid molecule or combination thereof from a genomic, cDNA, or synthetic source, which is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid. For example, with respect to genomic DNA, the term "isolated" includes a nucleic acid molecule that is separated from the chromosome to which the genomic DNA is naturally associated. Preferably, an "isolated" nucleic acid is free of sequences that naturally flank the nucleic acid, i.e., sequences located at the 5' and 3' termini of the target nucleic acid.

[0112] The term "antibody" is used herein in the broadest sense and specifically encompasses 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), provided that they exhibit the desired biological activity.

[0113] In some embodiments, the term "antibody" refers to an antigen-binding protein (i.e., immunoglobulin) having a basic four-polypeptide chain structure consisting of two identical heavy (H) chains and two identical light (L) chains. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each heavy chain has a variable region (abbreviated herein as V H ) at the N-terminus, followed by a constant region. The heavy chain constant region contains three domains, namely C H1 , C H2 , and C H3 . Each light chain has a variable region (abbreviated herein as V I ) at the N-terminus, followed by a constant region at its other end. The light chain constant region contains one domain, namely C L . V L aligns with V H , and C L aligns with the first constant domain (CH1) of the heavy chain. V H and V L 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, and thus contain 10 antigen-binding sites, while secreted IgA antibodies can polymerize to form a multivalent aggregate containing 2 - 5 basic 4-chain units and the J chain.

[0114] V H region and V LThe region can be further subdivided into hypervariable regions, known as hypervariable regions (HVRs), based on structural and sequence analysis. The HVRs are interspersed with more conserved regions known as framework regions (FWs) (see, for example, Chen et al. (1999) J. Mol. Biol. (1999) 293, 865-881). Each V H and V L is composed of three HVRs and four FWs arranged in the following order from the amino terminus to the carboxy 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.

[0115] The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody can mediate immunoglobulin binding to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq). Within the light and heavy chains, the variable and constant regions are joined by a "J" region having about 12 or more amino acids, and the heavy chain also includes a "D" region having about 10 or more amino acids (see, for example, Fundamental Immunology Chapter 7 (Paul, W. ed., 2nd ed. Raven Press, N.Y). (1989)).

[0116] L chains from any vertebrate species can be assigned to one of two distinct types called κ and λ based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the constant domain (CH) of the heavy chain of the antibody, the antibody can be assigned to different classes or isotypes. There are five antibody classes: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated as α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu), respectively. The IgG antibody class can be further classified into four subclasses according to γ heavy chains Y1-Y4, namely IgG1, IgG2, IgG3, and IgG4..

[0117] The term "antibody derivative" or "derivative of an antibody" refers to a molecule that is capable of binding the same antigen (e.g., CTLA4) to which the antibody binds and that comprises an amino acid sequence of the antibody linked to an additional molecular entity. The amino acid sequence of the antibody contained in the antibody derivative can be the full-length heavy chain of the antibody, the full-length light chain of the antibody, 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 a 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, marker, drug, or therapeutic agent. The amino acid sequence of the antibody can be attached or linked to the additional molecular entity by chemical conjugation, genetic fusion, non-covalent association, or otherwise. The term "antibody derivative" also encompasses chimeric antibodies, humanized antibodies, and molecules obtained by modifications of the amino acid sequence of the CTLA4 antibody such as conservative amino acid substitutions, additions, and insertions.

[0118] The term "antigen-binding fragment" or "antigen-binding portion" of an antibody refers to one or more portions of an antibody that retain the ability to bind the antigen (e.g., CTLA4) to which the antibody binds. Examples of "antigen-binding fragments" of an antibody include (i) Fab fragments, which are monovalent fragments consisting of V L 、V H 、C L and C H1 domains; (ii) F(ab′)2 fragments, which are bivalent fragments comprising two Fab fragments linked at the hinge region by a disulfide bridge; (iii) Fd fragments consisting of V H and C H1 domains; (iv) Fv fragments consisting of the V L and V H domains of a single arm of the antibody, (v) dAb fragments (Ward et al., Nature 341:544-546 (1989)), which consist of V H domains; and (vi) isolated complementarity-determining regions (CDRs).

[0119] The term "binding molecule" encompasses (1) antibodies, (2) antigen-binding fragments of antibodies, and (3) antibody derivatives, each as defined herein.

[0120] 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.). Thus, a binding molecule (e.g., an antibody or an activatable antibody) as defined and disclosed herein can also bind CTLA4 from species other than human. In other cases, the binding molecule can be fully specific for human CTLA4 and may not exhibit species cross-reactivity or other types of cross-reactivity.

[0121] The term "CTLA4 antibody" refers to an antibody as defined herein that is capable of binding human CTLA4.

[0122] The term "chimeric antibody" refers to an antibody that contains amino acid sequences derived from different animal species, such as those having variable regions derived from human antibodies and murine immunoglobulin constant regions.

[0123] The term "competitive binding" refers to the interaction of two antibodies when they bind to a binding target. If the binding of a first antibody to its cognate epitope is detectably reduced in the presence of a second antibody compared to its binding in the absence of the second antibody, then the first antibody competes for binding with the second antibody. An alternative scenario where the binding of the second antibody to its epitope is also detectably reduced in the presence of the first antibody may or may not be the case. That is, the first antibody can 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 the other antibody to its cognate epitope, whether to the same extent, greater, or lesser, the antibodies are said to "cross-compete" for binding to one or more of their corresponding epitopes.

[0124] The term "epitope" refers to the part of an antigen that is bound by an antibody (or its antigen-binding fragment). Epitopes can be formed by either contiguous amino acids or non-contiguous amino acids that are juxtaposed by the tertiary folding of a protein. Epitopes formed by contiguous amino acids are generally retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are generally lost upon treatment with denaturing solvents. Epitopes can include various numbers of amino acids in a unique spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, x-ray crystallography, two-dimensional nuclear magnetic resonance, a combination of deuterium and hydrogen exchange with mass spectrometry, or site-directed mutagenesis, or all methods in combination with computational modeling of the antigen and its complex structure with its binding antibody and its variants (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Volume 66, edited by G.E. Morris (1996)). Once the desired epitope of an antigen is determined, antibodies specific for that epitope can be generated, for example, using the techniques described herein. The generation and characterization of antibodies can also elucidate information about the desired epitope. Based on this information, it is then possible to competitively screen for antibodies that bind to the same epitope. One method for achieving this is to perform cross-competition studies to identify antibodies that compete with each other for binding to the antigen, i.e., the antibodies compete for binding to the antigen. A high-throughput method for "sorting" antibodies based on their cross-competition is described in PCT Publication No. WO 03 / 48731.

[0125] The term "germline" refers to the nucleotide sequences of antibody genes and gene segments as they are passed from parent to offspring through the germ cells. Germline sequences differ from the nucleotide sequences encoding antibodies in mature B cells, which have been altered during the process of B cell maturation by recombination and hypermutation events.

[0126] The term "glycosylation site" refers to the amino acid residue that is recognized by a eukaryotic cell as the site of attachment for a sugar residue. The amino acids at which carbohydrates such as oligosaccharides are attached are generally asparagine (N-linked), serine (O-linked), and threonine (O-linked) residues. Specific attachment sites are generally indicated by an amino acid sequence referred to herein as the "glycosylation site sequence". The glycosylation site sequence for N-linked glycosylation is: -Asn-X-Ser- or -Asn-X-Thr-, where X can be any conventional amino acid other than proline. The terms "N-linked" and "O-linked" refer to the chemical groups that serve as the attachment sites between the sugar molecule and the amino acid residue. N-linked sugars are attached through an amino group; O-linked sugars are attached through a hydroxyl group. The term "glycan occupancy" refers to the presence of a carbohydrate moiety attached to a glycosylation site (i.e., the glycan site is occupied). When there are at least two potential glycosylation sites on a polypeptide, none (0 glycan site occupancy), one (1 glycan site occupancy), or two (2 glycan site occupancy) sites can be occupied by a carbohydrate moiety.

[0127] The term "host cell" refers to a cell 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, HEK293T cells; or human tissues or hybridoma cells, yeast cells, insect cells (e.g., S2 cells), bacterial cells (e.g., Escherichia coli (E. coli) cells), and cells contained within transgenic animals or cultured tissues. The term encompasses not only the particular subject cells but also the progeny of such cells. Since certain modifications may occur in subsequent generations due to mutation or environmental influences, the progeny may not be identical to the parental cells but are still included within the scope of the term "host cell".

[0128] A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell or derived from a non-human source that utilizes a human antibody repertoire or other human antibody-encoding sequences. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0129] The term "humanized antibody" refers to a chimeric antibody that contains amino acid residues derived from human antibody sequences. A humanized antibody may contain some or all of the CDRs or HVRs from a non-human animal or synthetic antibody, while the framework regions and constant regions of the antibody contain amino acid residues derived from human antibody sequences.

[0130] The term "exemplary antibody" refers to any of the antibodies described in the present disclosure and designated as those listed in Tables A and B, and any antibody that contains the 6 HVRs and / or VH and VL of the antibodies listed in Tables A and B. These antibodies can be in any class (e.g., IgA, IgD, IgE, IgG, and IgM). Thus, each of the antibodies identified above encompasses antibodies having the same V L region and V H region amino acid sequence in all five classes. In addition, antibodies in the IgG class can be in any subclass (e.g., IgG1, IgG2, IgG3, and IgG4). Thus, each of the antibodies identified above in the IgG subclass encompasses antibodies having the same V L region and V H region amino acid sequence in all four subclasses. The amino acid sequences of the heavy chain constant regions of human antibodies in the five classes and in the four IgG subclasses are known in the art.

[0131] An "isolated" antibody or binding molecule (e.g., an activating antibody) is an antibody or binding molecule that has been separated from the components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0132] The term "K a " refers to the association rate constant of a particular binding molecule-antigen interaction, where the term "k d " refers to the dissociation rate constant of a particular binding molecule-antigen interaction.

[0133] The term "K D " refers to the equilibrium dissociation constant of a particular antibody-antigen interaction. It is obtained from the ratio of k d to k a (i.e., k d / k a ) and is expressed as molar concentration (M). K D is used as a measure of the affinity of an antibody for binding to its binding partner. The smaller the K D , the more tightly the antibody binds, or the higher the affinity between the antibody and the antigen. For example, an antibody with a nanomolar (nM) dissociation constant binds more tightly to a particular antigen than an antibody with a micromolar (μM) dissociation constant. The K D value of an antibody can be determined using well-established methods in the art. One method for determining the K D of an antibody is by using surface plasmon resonance (SPR), typically utilizing a biosensor system, such as system. For example, the assay procedure using a BIACORE TM system (BIAcore assay) is described in at least Example 3 of the present disclosure.

[0134] The term "mammal" refers to any animal species of the class Mammalia. Examples of mammals include: humans; laboratory animals such as rats, mice, hamsters, rabbits, non-human primates, and guinea pigs; domestic animals such as cats, dogs, cows, sheep, goats, horses, and pigs; and captive wild animals such as lions, tigers, elephants, etc.

[0135] The term "prevent (prevent / preventing)" with respect to a disease condition in a mammal refers to preventing or delaying the onset of the disease, or preventing the manifestation of clinical or subclinical symptoms of the disease.

[0136] As used herein, "sequence identity" between two polypeptide sequences indicates the percentage of amino acids that are the same between the sequences. Amino acid sequence identity of polypeptides can be routinely determined using known computer programs such as Bestfit, FASTA, or BLAST (see, e.g., 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., Nucelic 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, the parameters are set such that the percentage identity is calculated over the full length of the reference amino acid sequence and gaps in homology are allowed to account for up to 5% of the total number of amino acid residues in the reference sequence. This method, mentioned above in determining the percentage identity between polypeptides, is applicable to all proteins, fragments, or variants thereof disclosed herein.

[0137] As used herein, the terms "bind", "specifically bind", or "is specific for" refer to a measurable and reproducible interaction such as binding between a target and an antibody, which determines the presence of the target in the presence of a heterogeneous population of molecules including biomolecules. For example, an antibody that binds or specifically binds a target (which can be an epitope) is an antibody that binds this target with greater affinity, avidity, more readily, and / or for a longer duration compared to its binding to other targets. In one embodiment, the degree of binding of the antibody to an irrelevant target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds a target has a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM. In certain embodiments, the antibody specifically binds an epitope that is conserved between proteins from different species. In another embodiment, specific binding can include, but does not require, exclusive binding.

[0138] The term "treat / treating / treatment" with respect to a disease condition in a mammal means to effect a desired or beneficial result in the mammal having the disease condition. A desired or beneficial result can include a decrease 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 arrest or inhibition of the further development of the disease, disorder or condition. In the case of treating cancer in a mammal, a desired or beneficial result can include inhibiting further growth or spread of cancer cells, causing cancer cells to die, inhibiting cancer recurrence, alleviating pain associated with cancer, or improving the survival period of the mammal. The result can be subjective or objective. For example, if the mammal is a human, the human 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 detect a decrease in tumor size or tumor burden based on physical examination, laboratory parameters, tumor markers or radiological study results. Some laboratory signs of response to treatment that a clinician may observe include normalization of tests such as white blood cell count, red blood cell count, platelet count, erythrocyte sedimentation rate and various enzyme level tests. Additionally, a clinician may observe a decrease in detectable tumor markers. Alternatively, other tests can be used to evaluate objective improvement, such as ultrasound scans, magnetic resonance tests and positron emission tests.

[0139] The term "vector" refers to a nucleic acid molecule capable of transporting a foreign nucleic acid molecule. The foreign nucleic acid molecule is ligated to the vector nucleic acid molecule by recombinant techniques such as ligation or recombination. This allows for the propagation, selection, further manipulation, or expression of the foreign nucleic acid molecule in a host cell or organism. Vectors can be plasmids, phages, transposons, cosmids, chromosomes, viruses, or virus particles. One type of vector can integrate into the genome of the host cell after introduction into the host cell and thus replicate along with the host genome (e.g., non - episomal mammalian vectors). Another type of vector is capable of autonomous replication in the host cell into which it is introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Another specific type of vector that can direct the expression of an expressible foreign nucleic acid to which it is operably linked is commonly referred to as an "expression vector". Expression vectors typically have control sequences that drive the expression of the expressible foreign nucleic acid. A simpler vector called a "transcription vector" can only be transcribed but not translated: they can replicate in target cells but not express. The term "vector" encompasses all types of vectors regardless of their function. Vectors that can direct the expression of an expressible nucleic acid to which they are operably linked are commonly referred to as "expression vectors". Other examples of "vectors" can include display vectors (e.g., vectors that direct the expression and display of an encoded polypeptide on the surface of a virus or a cell such as a bacterial cell, yeast cell, insect cell, and / or mammalian cell).

[0140] As used herein, "subject", "patient", or "individual" can refer to a human or non - human animal. "Non - human animal" can refer to any animal not classified as human, such as domestic animals, farm animals or zoo animals, sport animals, companion 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 embodiments, the subject, patient, or individual is human.

[0141] "Effective amount" means at least the following amount: at the necessary dosage and for the necessary period of time, said amount being effective to achieve one or more desired or indicated effects including therapeutic or prophylactic outcomes. The effective amount may be provided in one or more administrations. For the purposes of the present disclosure, an effective amount of an antibody, drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve a prophylactic or therapeutic treatment. As is understood in a clinical setting, the 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., as an effective amount administered in the form of monotherapy or combination therapy). Thus, an "effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be administered in an effective amount if, in combination with one or more other agents, a desired outcome is achieved or has been achieved.

[0142] III. Binding Molecules That Bind to Human CTLA4

[0143] The present disclosure relates in part to isolated binding molecules that bind to 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 the HVRs, 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, the present disclosure relates to a binding molecule that binds to human CTLA4 and 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) a K of 500 nM or less DBind to human, cynomolgus monkey, mouse, rat, and / or dog CTLA4; (b) have antagonistic activity against human CTLA4; (c) do 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) have cross-reactivity with monkey, mouse, rat, and / or dog CTLA4; (e) induce ADCC effects (e.g., on Tregs); (f) activate human PBMCs (e.g., stimulate the secretion of IL-2 and / or IFNγ); (g) be capable of inhibiting tumor cell growth; (h) have a therapeutic effect on cancer; and (i) block 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 antibodies described herein have lower activity in blocking the binding of CD80 and / or CD86 to human CTLA4 compared to ipilimumab. See Figure 57C and Figure 57D and Figure 58 . In some embodiments, the anti-CTLA4 antibodies described herein selectively deplete Treg cells in the tumor microenvironment compared to Treg depletion in PBMCs or the spleen. In some embodiments, the anti-CTLA4 antibodies described herein have higher Treg depletion activity in the tumor microenvironment compared to ipilimumab. See Figures 61A - 61B 、 Figures 62A - 62B and Figure 63 . The present disclosure also provides one or more anti-CTLA4 antibodies or antigen-binding fragments that cross-compete for binding to human CTLA4 with one or more of the antibodies or antigen-binding fragments described herein.

[0144] In some embodiments, the antibody or antigen-binding fragment has a K D bind to human, cynomolgus monkey, mouse, rat, and / or dog CTLA4. In some embodiments, the antibody or antigen-binding fragment has a K DBinds to human, cynomolgus monkey, mouse, rat, and / or dog CTLA4. In some embodiments, the antibody or antigen-binding fragment has a K of about 100 nM or less D Binds to human CTLA4. In some embodiments, the antibody or antigen-binding fragment has a K of about 50 nM or less D Binds to human CTLA4. In some embodiments, the antibody or antigen-binding fragment has a K of about 10 nM or less D Binds to human CTLA4. The method for measuring the K of the antibody or antigen-binding fragment D can be carried out using any method known in the art, including, for example, surface plasmon resonance, ELISA, isothermal titration calorimetry, filter binding assay, EMSA, etc. In some embodiments, the K D is measured by surface plasmon resonance or ELISA (see, for example, Example 3 below).

[0145] In some embodiments, the antibody or antigen-binding fragment described herein has antagonist 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 represses one or more activities of human CTLA4 (e.g., CTLA4 blockade, as measured by an increase in reporter gene signal using a CLA4 blockade reporter gene assay).

[0146] In some embodiments, the antibody or antigen-binding fragment is cross-reactive with monkey (e.g., cynomolgus monkey), mouse, rat, and / or dog CTLA4. 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 monkey and mouse CTLA4; monkey and rat CTLA4; monkey and dog CTLA4; mouse and rat CTLA4; mouse and dog CTLA4; rat and dog CTLA4; monkey, mouse, and rat CTLA4; monkey, mouse, and dog CTLA4; monkey, rat, and dog CTLA4; mouse, rat, and dog CTLA4; or monkey, mouse, rat, and dog CTLA4. In some embodiments, if the antibody or antigen-binding fragment has a K 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 combined with a non-human CTLA4 molecule, the antibody or antigen-binding fragment has cross-reactivity. Methods for measuring antibody cross-reactivity are known in the art and include, but are not limited to, surface plasmon resonance, ELISA, isothermal titration calorimetry, filter binding assays, EMSA, etc. In some embodiments, cross-reactivity is measured by ELISA (see, for example, Example 3 below).

[0147] 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 and include, but are not limited to, the methods described in Example 3 below. In some embodiments, relative to a control (e.g., an isotype control or ipilimumab), the antibody induces an ADCC effect of more than about 10% (e.g., induces an 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.).

[0148] In some embodiments, the antibody or antigen-binding fragment is capable of inhibiting tumor cell growth and / or proliferation. In some embodiments, relative to the corresponding tumor cells not contacted with the antibody or antigen-binding fragment (or relative to the corresponding tumor cells contacted with an isotype control antibody), 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%). 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 the tumor volume in the subject. In some embodiments, 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 an isotype control antibody), the antibody or antigen-binding fragment is capable of reducing the tumor volume in the 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%). Methods for monitoring tumor cell growth / proliferation, tumor volume, and / or tumor inhibition are known in the art and include, for example, the methods described in Example 4 below.

[0149] In some embodiments, the antibody or antigen-binding fragment has a therapeutic effect on cancer. In some embodiments, the antibody or antigen-binding fragment alleviates one or more signs or symptoms of cancer. In some embodiments, a subject suffering from cancer experiences partial or complete remission when the antibody or antigen-binding fragment is administered.

[0150] In another aspect, the present disclosure provides an isolated antibody that competes or cross-competes with any illustrative antibody of the present disclosure, such as TY21585, TY21586, TY21587, TY21588, TY21589, TY21580, TY21591, TY21686, TY21687, TY21689, TY21680, TY21691, and / or TY21692, for binding to human CTLA4. In a particular embodiment, the present disclosure provides an isolated antibody that competes or cross-competes with any illustrative antibody of the present disclosure for binding to the same epitope on human CTLA4. The ability of an antibody to compete or cross-compete with another antibody for binding 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 the present disclosure can be allowed to bind to human CTLA4 under saturating conditions, and then the ability of a test antibody to bind CTLA4 is measured. If the test antibody is able to bind CTLA4 simultaneously with the illustrative antibody, then the test antibody binds a different epitope from the illustrative antibody. However, if the test antibody is not able to bind CTLA4 simultaneously, then the test antibody binds the same epitope, an overlapping epitope, or an epitope that is closely adjacent to the epitope bound by the illustrative antibody. This experiment can be performed using various methods such as ELISA, RIA, FACS, or surface plasmon resonance.

[0151] 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 and human CD80, human CTLA4 and human CD86). In some embodiments, the antibody or antigen-binding fragment blocks the binding between CTLA4 and its ligand in vitro. In some embodiments, the antibody or antigen-binding fragment has a half-maximal inhibitory concentration (IC 50 ) 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.) for blocking the binding of CTLA4 to CD80 and / or CD86. In some embodiments, the antibody or antigen-binding fragment has a half-maximal inhibitory concentration (IC 50)。In some embodiments, when provided at a concentration of about 100 nM or greater (such as 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 / completely blocks" means that the antibody or antigen-binding fragment is capable of reducing the binding between a first protein and a second protein by at least about 80% (such as 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 a first protein (such as human CTLA4) and a second protein (such as human CD80 or human CD86) are known in the art and include, but are not limited to, BIAcore analysis, ELISA assays, and flow cytometry (see, for example, Example 3 below). In some embodiments, compared to ipilimumab, the anti-CTLA4 antibodies described herein have lower activity in blocking ligand binding.

[0152] CTLA4 antibody

[0153] In some aspects, the present disclosure provides an isolated antibody that binds to human CTLA4. In some embodiments, the antibody has a Kd of 1000 nM or less (such as 50 nM or less, 10 nM or less). D Binds to human CTLA4, as measured by surface plasmon resonance. In some embodiments, the antibody has cross-reactivity with at least one non-human species selected from cynomolgus monkeys, mice, rats, and dogs.

[0154] In some aspects, the present 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 comprising one or more amino acid residues in the ligand-binding site of human CTLA4 (such as the CD80 and / or CD86-binding site). In some embodiments, the antibody specifically binds to an epitope on human CTLA4 that is different from the epitope of ipilimumab. In some embodiments, the epitope does not comprise the amino acid residues in the CC' loop motif of human CTLA4. In some embodiments, the epitope does not comprise the amino acid residues L106 or I108 of human CTLA4. In some embodiments, the antibody specifically binds to an epitope comprising the amino acid residues Y105 and L106 but not I108 of human CTLA4, wherein the numbering of the amino acid residues is according to SEQ ID NO:207.

[0155] KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQ ADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLR AMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPE(SEQ ID NO:207)

[0156] In one aspect, the present disclosure provides an isolated 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 HVR-H1 comprises an amino acid sequence according to a formula selected from the following: Formula (I): X1TFSX2YX3IHWV (SEQ ID NO:1), where X1 is F or Y, X2 is D or G, and X3 is A, G, or W; Formula (II): YSIX1SGX2X3WX4WI (SEQ ID NO:2), where 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), where X1 is G or S; HVR-H2 comprises an amino acid sequence according to a formula selected from the following: Formula (IV): IGX1IX2HSGSTYYSX3SLKSRV (SEQ ID 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 HVR-H3 comprises an amino acid sequence according to a formula selected from the following: Formula (VII): ARX1X2X3X4FDX5 (SEQ ID 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; Formula (VIII): ARX1GX2GYFDX3 (SEQ ID NO:8), where X1 is D or L, X2 is F or Y, and X3 is V or Y; Formula (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 Formula (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 / or b) wherein the light chain variable region comprises HVR-L1, HVR-L2, and HVR-L3, wherein HVR-L1 comprises an amino acid sequence according to a formula selected from the following: Formula (XI): RASQX1X2X3SX4LX5 (SEQ ID NO:11), where 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; said HVR-L2 comprises an amino acid sequence according to Formula (XIV): X1ASX2X3X4X5GX6 (SEQ ID NO:14), where 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 said HVR-L3 comprises an amino acid sequence according to a formula selected from the following: Formula (XV): YCX1X2X3X4X5X6PX7T (SEQ ID 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; 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.;

[0157] In some embodiments, the antibody comprises: a) an HVR-H1 comprising an amino acid sequence selected from SEQ ID NOs: 18-29; an HVR-H2 comprising an amino acid sequence selected from SEQ ID NOs: 30-39; and an HVR-H3 comprising an amino acid sequence selected from SEQ ID NOs: 40-52; and / or b) an HVR-L1 comprising an amino acid sequence selected from SEQ ID NOs: 53-65; an HVR-L2 comprising an amino acid sequence selected from SEQ ID NOs: 66-69; and an HVR-L3 comprising an amino acid sequence selected from SEQ ID NOs: 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 in Table A below.

[0158] Table A: Anti-CTLA4 HVR Sequences

[0159]

[0160]

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

[0162] In some embodiments, the antibody comprises: a) a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 82-94; and / or b) a light chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 95-107. In some embodiments, the antibody comprises a heavy chain variable region comprising 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 to a sequence selected from SEQ ID NOs: 82-94, and / or a light chain variable region comprising 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 to a sequence selected from SEQ ID NOs: 95-107. In some embodiments, the antibody comprises the heavy chain variable region and the light chain variable region of any of the exemplary antibodies described in Table B below. In some embodiments, 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 sequences of anti-CTLA4 variable regions

[0164]

[0165]

[0166]

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

[0168] In some embodiments, the antibodies of the present disclosure cross-compete for binding to human CTLA4 with antibodies comprising: a) an HVR-H1 containing an amino acid sequence selected from SEQ ID NOs: 18-29; an HVR-H2 containing an amino acid sequence selected from SEQ ID NOs: 30-39; and an HVR-H3 containing an amino acid sequence selected from SEQ ID NOs: 40-52; and / or b) an HVR-L1 containing an amino acid sequence selected from SEQ ID NOs: 53-65; an HVR-L2 containing an amino acid sequence selected from SEQ ID NOs: 66-69; and an HVR-L3 containing an amino acid sequence selected from SEQ ID NOs: 70-81. In some embodiments, the antibodies of the present disclosure cross-compete for binding to human CTLA4 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 the present disclosure cross-compete for binding to human CTLA4 with antibodies comprising: a) a heavy chain variable region containing an amino acid sequence selected from SEQ ID NOs: 82-94; and / or b) a light chain variable region containing an amino acid sequence selected from SEQ ID NOs: 95-107. In some embodiments, the antibodies of the present disclosure cross-compete for binding to human CTLA4 with antibodies comprising a VH and / or a VL shown for any of the exemplary antibodies described in Table B.

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

[0170] In some embodiments, the antibodies of the present disclosure are bispecific antibodies that bind a first target and a second target, wherein the first target is human CTLA4. In some embodiments, the bispecific antibody binds a first target and a second target, wherein the first target is human CTLA4, and wherein the bispecific antibody comprises a) an HVR-H1 comprising an amino acid sequence selected from SEQ ID NOs: 18-29; an HVR-H2 comprising an amino acid sequence selected from SEQ ID NOs: 30-39; and an HVR-H3 comprising an amino acid sequence selected from SEQ ID NOs: 40-52; and / or b) an HVR-L1 comprising an amino acid sequence selected from SEQ ID NOs: 53-65; an HVR-L2 comprising an amino acid sequence selected from SEQ ID NOs: 66-69; and an HVR-L3 comprising an amino acid sequence selected from SEQ ID NOs: 70-81. In some embodiments, the bispecific antibody binds 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 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 comprising an amino acid sequence selected from SEQ ID NOs: 82-94; and / or b) a light chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 95-107. In some embodiments, the bispecific antibody binds a first target and a second target, wherein the first target is human CTLA4, and wherein the bispecific antibody comprises a VH and / or a VL shown for any of the exemplary antibodies described in Table B. In some embodiments, 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 of the present disclosure can be produced by any technique known in the art, including conventional monoclonal antibody methods such as standard somatic cell hybridization techniques (see, e.g., Kohler and Milstein, Nature 256:495 (1975)), viral or oncogenic transformation of B lymphocytes, or recombinant antibody techniques as detailed herein (see, e.g., Examples 1 and 2). In some embodiments, the antibodies of the present disclosure are produced using any library and / or method described in PCT application number PCT / CN2017 / 098333 (incorporated herein by reference in its entirety) and / or PCT application number PCT / CN2017 / 098299 (incorporated herein by reference in its entirety).

[0172] Hybridoma generation is an extremely well-established procedure. A common animal system used for preparing hybridomas is the murine system. Immunization protocols and techniques for isolating immunized spleen cells for fusion are known in the art. The fusion partners (e.g., murine myeloma cells) and the fusion procedure are also known. A well-known method that can be used to prepare the human CTLA4 antibodies provided by the present disclosure involves using XenoMouse TM animal system. XenoMouse TMA mouse is a large fragment containing the human immunoglobulin heavy and light chain loci, and an engineered mouse strain that is defective in mouse antibody production (see, e.g., Green et al., (1994) Nature Genetics 7:13-21; WO2003 / 040170). An animal is immunized with a CTLA4 antigen. The CTLA4 antigen is an isolated and / or purified CTLA4. It can be a fragment of CTLA4, such as the extracellular domain of CTLA4. Immunization of the animal can be carried out by any method known in the art (see, e.g., 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, e.g., Harlow and Lane (supra) and U.S. Patent No. 5,994,619). The CTLA4 antigen can be administered with an adjuvant to stimulate an immune response. Exemplary adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptide), or ISCOM (immunostimulating complex). After immunizing the animal with the CTLA4 antigen, an antibody-producing immortalized cell line is prepared from cells isolated from the immunized animal. After 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 oncogenes into them, infecting them with oncogenic viruses, culturing them under conditions that select for immortalized cells, subjecting them to carcinogenic or mutagenic compounds, fusing them with immortalized cells such as myeloma cells, and inactivating tumor suppressor genes (see, e.g., Harlow and Lane (supra)). If fusion with myeloma cells is used, the myeloma cells preferably do not secrete immunoglobulin polypeptides (non-secreting cell lines). Immortalized cells are screened using CTLA4, a portion thereof, or cells expressing CTLA4. Antibody-producing cells such as hybridomas that produce CTLA4 antibodies are selected, cloned, and further screened for desirable characteristics, including robust growth, high antibody production, and desirable antibody characteristics, as further discussed below. Hybridomas can be amplified 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 amplifying hybridomas are well known to those of ordinary skill in the art.

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

[0174] Antigen-binding fragment

[0175] In some other aspects, the present disclosure provides antigen-binding fragments of any of the CTLA4 antibodies described herein.

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

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

[0178] In some embodiments, the antigen-binding fragment of a CTLA4 antibody comprises: (i) a Fab fragment, which is a monovalent fragment composed of V L 、V H 、C L and C H 1 domains; (ii) an F(ab′)2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment composed of V H and C H 1 domains; (iv) an Fv fragment composed of the V L and V H domains of a single arm of the antibody; (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which is composed of a V H domain; (vi) an isolated CDR, and (vii) a single-chain antibody (scFv), which is a polypeptide comprising the V L region of the antibody linked to the V HPolypeptides of the region (see, e.g., 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 aspects, the present disclosure provides derivatives of any of the CTLA4 antibodies described herein.

[0181] In some embodiments, the antibody derivatives are obtained by modifying the amino acid sequence of an illustrative antibody of the present disclosure (e.g., the "parent antibody") while maintaining the overall molecular structure of the amino acid sequence of the parent antibody. The amino acid sequence of any region of the parent antibody chain can be modified, such as the framework region, the HVR region, or the constant region. The types of modifications include substitutions, insertions, deletions, or combinations thereof of one or more amino acids of the parent antibody.

[0182] In some embodiments, the antibody derivative comprises a V that is 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% identical to the amino acid sequence set forth in any of SEQ ID NOs: 82-107 L or V HRegion. In some embodiments, the antibody derivative comprises an HVR-H1 amino acid sequence region that is 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% identical to the amino acid sequence set forth in any one 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%, 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% identical to the amino acid sequence set forth in any one 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%, 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% identical to the amino acid sequence set forth in any one 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%, 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% identical to the amino acid sequence set forth in any one 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%, 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% identical to the amino acid sequence set forth in any one 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%, 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% identical to the amino acid sequence set forth in any one 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 conservative or non-conservative substitutions of the amino acid sequence set forth in any of SEQ ID NOs: 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 substitutions encompass both conservative substitutions and non-conservative substitutions. The term "conservative amino acid substitution" means that one amino acid is replaced by another amino acid, where the two amino acids have similarities in certain physicochemical properties, such as the polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphiphilic nature of the residues involved. For example, substitutions can generally be made within each of the following groups: (a) non-polar (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 the HVRs, framework regions or constant regions. In one embodiment, the present disclosure provides a V containing an illustrative antibody of the present disclosure H and V LHVR sequences, but antibody derivatives having framework sequences different from those of the illustrative antibodies. The framework sequences can be obtained from public DNA databases including germline antibody gene sequences or public references. For example, the germline DNA sequences of human heavy and light chain variable region genes can be found in the Genbank database or the "VBase" human germline sequence database (Kaba et al., Sequences of Proteins of Immunological Interest, 5th edition, U.S. Department 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)). Framework sequences useful for constructing antibody derivatives include those that are structurally similar to the framework sequences used by the illustrative antibodies of the present disclosure. For example, the HVR-H1, HVR-H2, and HVR-H3 sequences and the HVR-L1, HVR-L2, and HVR-L3 sequences of the illustrative antibodies can be grafted onto a framework region having the same sequence as the framework region found in the germline immunoglobulin gene from which the framework sequence is derived, or the HVR sequences can be grafted onto a framework 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 an illustrative antibody of the present disclosure. In one example, one or more HVRs from one or more illustrative antibodies are combined with the HVRs of an antibody from a non-human animal such as a mouse or a rat. In another example, all of the HVRs of the chimeric antibody are derived from one or more illustrative antibodies. In some particular embodiments, the chimeric antibody comprises one, two, or three HVRs from the heavy chain variable region of an illustrative antibody and / or one, two, or three HVRs from the light chain variable region of an illustrative antibody. Chimeric antibodies 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 in the HVR regions of the chains. 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 evaluated in in vitro or in vivo assays known in the art. Typically, conservative substitutions are introduced. The mutations can be amino acid additions and / or deletions. Additionally, typically up to one, two, three, four, or five residues within the HVR regions are altered. In some embodiments, the antibody derivative contains 1, 2, 3, or 4 amino acid substitutions in the heavy chain HVR and / or in the light chain HVR. In another embodiment, the amino acid substitution is such that one or more cysteines in the antibody are changed to another residue, such as, but not limited to, alanine or serine. The cysteine can be a canonical or non-canonical cysteine. In one embodiment, the antibody derivative has 1, 2, 3, or 4 conservative amino acid substitutions in the heavy chain HVR region relative to the amino acid sequence of the illustrative antibody.

[0188] The framework residues within the V H and / or V L regions can also be modified. Generally, the framework variants are prepared to reduce the immunogenicity of the antibody. One method is to "back-mutate" one or more framework residues to the corresponding germline sequence. An antibody that has undergone somatic cell mutations can contain framework residues that are different from the germline sequence from which the antibody is derived. The residues can be identified by comparing the antibody framework sequence with the germline sequence from which the antibody is derived. To return the framework region sequences to their germline configuration, the somatic mutations can be "back-mutated" to the germline sequence by, for example, site-directed mutagenesis or PCR-mediated mutagenesis.

[0189] In addition, modifications can also be made in 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 antibody-dependent cell-mediated cytotoxicity. In one example, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is altered, for example, increased or decreased. This method is further described in U.S. Patent No. 5,677,425. Altering the number of cysteine residues in the hinge region of CH1, for example, to facilitate the assembly of the light and heavy chains or to 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] In addition, the antibodies of the present disclosure can be modified according to conventional experiments known in the art to alter its potential glycosylation sites or patterns. In another aspect, the present disclosure provides derivatives of CTLA4 antibodies that contain at least one mutation in the variable region of the light or heavy chain that alters the glycosylation pattern in the variable region. Such antibody derivatives can have increased affinity and / or altered specificity with respect to antigen binding. The mutations can 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, the present disclosure provides derivatives of CTLA4 antibodies that have a potential N-linked glycosylation site at asparagine in the heavy chain variable region, wherein the potential N-linked glycosylation site in one heavy chain variable region is removed. In another embodiment, the present disclosure provides derivatives of CTLA4 antibodies that have a potential N-linked glycosylation site at asparagine in the heavy chain variable region, wherein the potential N-linked glycosylation sites in both 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, the present disclosure provides an antibody derivative comprising a CTLA4 antibody or an antigen-binding fragment thereof as described herein linked to an additional molecular entity. Examples of additional molecular entities include pharmaceutical agents, peptides or proteins, detection agents or labels, and antibodies.

[0192] In some embodiments, the antibody derivative comprises an antibody of the present disclosure conjugated to a pharmaceutical agent. Examples of pharmaceutical agents include cytotoxic agents or other cancer therapeutic agents and radioisotopes. Specific examples of cytotoxic agents include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as analogs or homologs thereof.Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine), alkylating agents (e.g., mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (previously known as daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (previously known as actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine). Examples of radioisotopes that can be conjugated to an antibody for diagnostic or therapeutic use include, but are not limited to, iodine. 131 , indium 111 , yttrium 90 and lutetium 177Methods for conjugating antibodies to pharmaceutical agents are known in the art, such as using various linker technologies. Examples of linker types include hydrazones, thioethers, esters, disulfides, and peptide-containing linkers. For further discussion of linkers and methods for conjugating therapeutic agents to antibodies, see, e.g., 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 that is a multimeric form of a CTLA4 antibody, such as an antibody dimer, trimer, or higher-order oligomer of monomeric antibodies. The individual monomers within the antibody multimer can be the same or different. In addition, the individual antibodies within the multimer can have the same or different binding specificities. Multimerization of the antibody can be achieved by natural aggregation of the antibody. For example, a certain percentage of a purified antibody preparation (e.g., purified IgG4 molecules) spontaneously forms protein aggregates containing antibody homodimers as well as other higher-order antibody multimers. Alternatively, antibody homodimers can be formed by chemical ligation techniques known in the art, such as by using crosslinking agents. Suitable crosslinking agents include those having heterobifunctionality, i.e., those having two distinct reactive groups separated by an appropriate spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester, 4-(maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester, and S-acetylthioacetic acid N-succinimide ester), or those having homobifunctionality (such as dibutyl succinate). The linker can be obtained commercially, for example, from Pierce Chemical Company, Rockford, IL. Antibody multimerization can also be achieved by recombinant DNA techniques known in the art.

[0194] Examples of other antibody derivatives provided by the present disclosure include single-chain antibodies, mini-bifunctional antibodies, domain antibodies, nanobodies, and monobodies. A "single-chain antibody" (scFv) consists of a single polypeptide chain containing a V L domain linked to a V H domain, where the V L domain and the VH The domains pair to form monovalent molecules. Single-chain antibodies can be prepared according to methods known in the art (see, e.g., Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). “Mini-bifunctional antibodies” consist of two chains, each chain containing a heavy-chain variable region linked to a light-chain variable region on the same polypeptide chain, joined by a short peptide linker, where the two regions on the same chain do not pair with each other, but pair with complementary domains on the other chain to form a bispecific molecule. Methods for preparing mini-bifunctional antibodies are known in the art (see, e.g., Holliger P. et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448 and Poljak R.J. et al., (1994) Structure 2:1121-1123). Domain antibodies (dAbs) are small functional binding units of antibodies, corresponding to the variable regions of the heavy or light chains of antibodies. Domain antibodies are well-expressed in bacterial, yeast, and mammalian cell systems. Other details of domain antibodies and methods for their production are known in the art (see, e.g., U.S. Patent Nos. 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 generally contain a single variable domain and two constant domains (CH2 and CH3), and retain the antigen-binding ability of the original antibody. Nanobodies can be prepared by methods known in the art (see, e.g., U.S. Patent Nos. 6,765,087, U.S. Patent No. 6,838,254, WO 06 / 079372). Single antibodies consist of one light chain and one heavy chain of an IgG4 antibody. Single antibodies can be prepared by removing the hinge region of the IgG4 antibody. Other details of single antibodies and methods for preparing them can be found in WO2007 / 059782.

[0195] IV. Activable Binding Polypeptides Targeting CTLA4

[0196] The present disclosure also pertains in part to precision / situation-dependent activatable binding polypeptides (i.e., activatable antibodies) that bind to human CTLA4, including activatable antibodies comprising any anti-CTLA4 antibody described herein (e.g., anti-CTLA4 antibodies, anti-CTLA4 antibody binding fragments, and / or anti-CTLA4 antibody derivatives); 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 a better safety margin, as evaluated by changes in spleen weight. The change in spleen size with increasing dose of the administered drug is used as a benchmark for evaluating the safety margin of the drug candidate being used. As Figures 48A - 48B shown, the activatable anti-CTLA4 antibodies described herein have a better safety margin relative to the parental antibody (an antibody without a masking moiety).

[0197] In some embodiments, the activatable antibody of the present disclosure comprises: (a) a masking moiety (MM); (b) a cleavable moiety (CM); and (c) a target-binding moiety (TBM). In some embodiments, the MM is any masking moiety described herein. In some embodiments, the CM is any cleavable moiety described herein. In some embodiments, the TBM is any target-binding moiety described herein (e.g., a target-binding moiety (TBM) comprising an antibody light chain variable region and / or an antibody heavy chain variable region such as 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 capable of binding to its target (e.g., human CTLA4 or human CD137).

[0198] In some embodiments, the activatable antibody comprises: (a) a polypeptide that comprises, from the N-terminus to the C-terminus, a masking moiety (MM), a cleavable moiety (CM), and a target-binding moiety (TBM), wherein the MM is any masking moiety described herein, the CM is any cleavable moiety 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 that comprises, from the N-terminus to the C-terminus, a masking moiety (MM), a cleavable moiety (CM), and a target-binding moiety (TBM), wherein the MM is any masking moiety described herein, the CM is any cleavable moiety 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, an activatable antibody comprises: a polypeptide that, from the N-terminus to the C-terminus, comprises a masking moiety (MM), a cleavable moiety (CM), and a target binding moiety (TBM), wherein the MM is any masking moiety described herein, the CM is any cleavable moiety 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 polypeptide,” “ABP,” or “activatable antibody” include a polypeptide 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 a target. In some embodiments, the TBM comprises an antigen binding domain (ABD) of an antibody or an antibody fragment thereof (such as any antibody or antigen binding fragment described herein). In some embodiments, the antigen binding domain comprises a heavy chain variable region that contains one, two, or three of the heavy chain variable region hypervariable regions (HVRs) described herein, and a light chain variable region that contains one, two, or three of the light chain variable region HVRs described herein (such as 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 that contains any heavy chain variable region sequence described herein, and a light chain variable region that contains any light chain variable region sequence described herein (such as the heavy chain variable region sequence and / or the light chain variable region sequence shown in Table B). In some embodiments, the TBM (such as comprising 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 that binds the target in the absence of the MM. In some embodiments, the VH and the VL are covalently linked, such as in an scFv. In some embodiments, the VH and the VL are not covalently linked. In some embodiments, the VH and the 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, an activatable antibody comprises a polypeptide having the following structure from the N-terminus to the C-terminus: 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, an activatable antibody comprises a polypeptide having the following structure from the N-terminus to the C-terminus: masking moiety (MM) - cleavable moiety (CM) - VL - VH (e.g., a scFv). In some embodiments, an activatable antibody comprises a polypeptide having the following structure from the N-terminus to the C-terminus: 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, an activatable antibody comprises a polypeptide having the following structure from the N-terminus to the C-terminus: masking moiety (MM) - cleavable moiety (CM) - VH - VL (e.g., a scFv).

[0203] CM generally includes an amino acid sequence that is cleavable, e.g., serves as a substrate for an enzyme, and / or cysteine-cysteine pairings capable of forming reducible disulfide bonds. Thus, when the terms "cleavage", "cleavable", "cleaved", etc. are used in connection with CM, the terms encompass, e.g., enzymatic cleavage effected by a protease, and disruption of the disulfide bond between cysteine-cysteine pairings by reduction of the disulfide bond that can be effected by exposure to a reducing agent.

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

[0205] In some embodiments, the masking moiety (MM) interferes with, hinders, reduces the ability of, prevents, inhibits, or competes with the target-binding moiety for binding to its target (e.g., an "inactive" activatable antibody). In some embodiments, the masking moiety (MM) interferes with, hinders, reduces, prevents, inhibits, or competes with the target-binding moiety for binding to its target only when the polypeptide has not 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 polypeptide within the cleavage moiety. In some embodiments, activation induces a conformational change in the polypeptide (e.g., displacement of the masking moiety (MM)), such that the masking moiety no longer prevents the activatable antibody from binding to its target. In some embodiments, the masking moiety (MM) interferes with, hinders, reduces the ability of, prevents, inhibits, or competes with the target-binding moiety for binding to its target only when the cleavable moiety (CM) has not been cleaved by one or more proteases that effect cleavage within the cleavable moiety (CM). In some embodiments, prior to activation, the masking moiety (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, the masking efficiency is measured as the difference in the affinity (prior to activation) of the activatable antibody comprising the masking moiety (MM) for binding to its target relative to the affinity of the polypeptide lacking the masking moiety for binding to its target (e.g., the difference in the affinity for a target antigen such as CTLA4 of an activatable antibody comprising the masking moiety (MM) (prior to activation) relative to the parental antibody lacking the masking moiety (MM), or the difference in the affinity for a target antigen such as CTLA4 of an activatable antibody comprising the masking moiety (MM) (prior to activation) relative to the affinity of the activatable antibody for the target antigen after activation). In some embodiments, the masking efficiency is measured by dividing the EC 50 (prior to activation) of the activatable antibody comprising the masking moiety (MM) by the EC 50 of the parental antibody (e.g., by measuring the EC 50; see, for example, the method of Example 8). In some embodiments, the masking efficiency is measured as the difference in the affinity of an activatable antibody comprising a masking moiety (MM) for its target before activation relative to the affinity of the activatable antibody comprising a masking moiety (MM) for its target after activation (e.g., the difference in the affinity of the activatable antibody for a target antigen such as CTLA4 before activation relative to the activatable antibody after activation). In some embodiments, the masking moiety (MM) binds to the target binding moiety (TBM) and prevents the activatable antibody from binding its target (e.g., an “inactive” activatable antibody). In some embodiments, the masking moiety (MM) has a dissociation constant for binding to the target binding moiety (TBM) that is greater than the dissociation constant of the target binding moiety (TBM) for its target.

[0206] In some embodiments, after the activatable antibody has been activated (e.g., activated by treatment with one or more proteases that cleave within the cleavable moiety (CM), 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 an enzyme or protein ligand, etc.), the masking moiety (MM) does not interfere with, impede, reduce the ability of, prevent, inhibit, or compete 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, impede, reduce the ability of, prevent, inhibit, or compete with the target binding moiety (TBM) for binding to its target. In some embodiments, after activation, the masking moiety (MM) has a masking efficiency of at most about 1.75 (e.g., at most about 1.75, at most about 1.5, at most about 1.4, at most about 1.3, at most about 1.2, at most about 1.1, at most about 1.0, at most about 0.9, at most about 0.8, at most about 0.7, at most about 0.6, or at most about 0.5, etc.) (e.g., the relative affinity of the activatable antibody after activation compared to the affinity of the parental antibody).

[0207] In some embodiments, the activatable antibodies of the present disclosure contain a pair of cysteine residues at fixed positions to ensure that the activatable antibodies have a constrained conformation, and / or a masking moiety (MM) with few or no chemically labile residues such as methionine or tryptophan. Advantageously, including a pair of cysteine residues at fixed positions ensures that the activatable antibodies have a constrained conformation, thus tending to exhibit increased binding affinity and / or specificity. In addition, the activatable antibodies of the present disclosure include 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 activatable antibodies of the present disclosure are situation-dependent (e.g., are activated (able to bind their targets only) in certain situations such as in a protease-rich tumor microenvironment). In some embodiments, the activatable antibodies of the present disclosure provide improved safety over more conventional non-activatable antibodies (e.g., exhibit reduced toxicity, do not induce significant changes in the weights of many organs, do not alter liver histopathology, hematology, and / or blood biochemistry, etc.). In some embodiments, compared to more conventional non-activatable antibodies, the activatable antibodies of the present disclosure have improved pharmacokinetic properties (e.g., have a longer in vivo half-life).

[0209] Anti-CTLA4 activatable antibody activity

[0210] In some embodiments, the present disclosure relates to activatable antibodies that bind human CTLA4 when in the active form (e.g., the activatable antibodies are inactive before cleavage in the cleavable moiety (e.g., by one or more proteases) and active after cleavage in the cleavable moiety (e.g., by one or more proteases)). In some embodiments, when in the active form, the activatable antibodies have 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) a K of 500 nM or less, such as about 10 nM or less DBind to human, cynomolgus monkey, mouse, rat, and / or dog CTLA4; (b) have antagonistic activity against human CTLA4; (c) do 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) have cross-reactivity with monkey, mouse, rat, and / or dog CTLA4; (e) induce ADCC effects (e.g., on Tregs); (f) activate human PBMCs (e.g., stimulate the secretion of IL-2 and / or IFNγ); (g) be capable of inhibiting tumor cell growth; (h) have a therapeutic effect on cancer; and (i) inhibit the binding of human CTLA4 to human CD80 and / or human CD86. Also provided herein are one or more activating antibodies that compete or cross-compete with one or more of the activating antibodies targeting CTLA4 and / or anti-CTLA4 antibodies described herein for binding to human CTLA4.

[0211] In some embodiments, when in an inactive form, the activating antibody has a K of about 500 nM or greater D Bind to human, cynomolgus monkey, mouse, rat, and / or dog CTLA4. In some embodiments, when in an active form, the activating antibody has a K 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 Bind to human, cynomolgus monkey, mouse, rat, and / or dog CTLA4. In some embodiments, when in an active form, the activating antibody has a K of about 350 nM or less D Bind to human, cynomolgus monkey, mouse, rat, and / or dog CTLA4. In some embodiments, when in an active form, the activating antibody has a K of about 100 nM or less D Bind to human CTLA4. In some embodiments, when in an active form, the activating antibody has a K of about 50 nM or less D Bind to human CTLA4. In some embodiments, when in an active form, the activating antibody has a K of about 10 nM or less D Bind to human CTLA4. Measure the K of the activating antibody DThe method can be carried out using any method known in the art, including, for example, by surface plasmon resonance, ELISA, isothermal titration calorimetry, filter 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 activatable antibody does not have antagonist activity against human CTLA4. In some embodiments, when in an active form, the activatable antibody has antagonist activity against human CTLA4 (e.g., inducing an ADCC effect (such as against Tregs), activating PBMCs (such as by activating, inducing, and / or stimulating the secretion of IL-2 and / or IFNγ), blocking the binding of human CTLA4 to human CD80 and / or human CD86, etc.). In some embodiments, when in an active form, the activatable antibody represses one or more activities of human CTLA4 (e.g., when cells expressing human CTLA4 (such as human cells) are contacted with the activatable antibody, repressing one or more activities of human CTLA4).

[0213] In some embodiments, when in an inactive form, the activatable antibody does not cross-react with cynomolgus monkey (e.g., cynomolgus macaque), mouse, rat, and / or dog CTLA4. In some embodiments, when in an active form, the activatable antibody cross-reacts with cynomolgus monkey (e.g., cynomolgus macaque), mouse, rat, and / or dog CTLA4. In some embodiments, when in an active form, the activatable antibody cross-reacts with cynomolgus monkey CTLA4. In some embodiments, when in an active form, the activatable antibody cross-reacts with mouse CTLA4. In some embodiments, when in an active form, the activatable antibody cross-reacts with rat CTLA4. In some embodiments, when in an active form, the activatable antibody cross-reacts with dog CTLA4. In some embodiments, when in an active form, the activatable antibody cross-reacts with cynomolgus monkey and mouse CTLA4; cynomolgus monkey and rat CTLA4; cynomolgus monkey and dog CTLA4; mouse and rat CTLA4; mouse and dog CTLA4; rat and dog CTLA4; cynomolgus monkey, mouse, and rat CTLA4; cynomolgus monkey, mouse, and dog CTLA4; cynomolgus monkey, rat, and dog CTLA4; mouse, rat, and dog CTLA4; or cynomolgus monkey, mouse, rat, and dog CTLA4. In some embodiments, when in an active form, the activatable binding polypeptide has 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 and include, but are not limited to, surface plasmon resonance, ELISA, isothermal titration calorimetry, filter binding assays, EMSA, etc.

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

[0215] In some embodiments, the activatable antibody is capable of inhibiting tumor cell growth and / or proliferation. In some embodiments, 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%) when the tumor cells are contacted with the activatable antibody relative to the corresponding tumor cells not contacted with the activatable antibody (or relative to the corresponding tumor cells contacted with an isotype control antibody). In some embodiments, when the activatable antibody is administered to a subject, the activatable antibody is capable of reducing the tumor volume in the subject. In some embodiments, the activatable antibody is capable of reducing the tumor volume in the 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 activatable antibody; compared to the corresponding tumor in a subject administered an isotype control antibody). Methods for monitoring tumor cell growth / proliferation, tumor volume, and / or tumor inhibition are known in the art and include, for example, the methods described in the following examples.

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

[0217] In some embodiments, the present disclosure provides an isolated activatable antibody that, when in its active form, competes or cross-competes for binding to human CTLA4 with an antibody comprising: a) HVR-H1 having the amino acid sequence of SEQ ID NO:23; HVR-H2 having the amino acid sequence of SEQ ID NO:35; and HVR-H3 having the amino acid sequence of SEQ ID NO:45; and / or b) HVR-L1 having the amino acid sequence of SEQ ID NO:58; HVR-L2 having the amino acid sequence of SEQ ID NO:66; and HVR-L3 having the amino acid sequence of SEQ ID NO:75. In some embodiments, the present disclosure provides an isolated activatable antibody that, when in its active form, competes or cross-competes for binding to human CTLA4 with an antibody comprising: a) a heavy chain variable region having the amino acid sequence of SEQ ID NO:87; and / or b) a light chain variable region having the amino acid sequence of SEQ ID NO:100. The ability of the activatable antibody to compete or cross-compete for binding with the 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 allowed to bind to human CTLA4 under saturating conditions, and then the ability of the test activatable antibody (when in its active form) to bind CTLA4 is measured. If the test activatable antibody is able to bind CTLA4 simultaneously with the antibody, then the test activatable antibody binds to a different epitope than the antibody. However, if the test activatable antibody is not able to bind CTLA4 simultaneously, then the test activatable antibody binds to the same epitope, an overlapping epitope, or an epitope that is closely adjacent to the epitope bound by the antibody. This experiment can be performed using various methods such as ELISA, RIA, FACS, or surface plasmon resonance.

[0218] In some embodiments, an activatable antibody (when in an inactive form) does not inhibit the binding between CTLA4 and one or more of its binding partners (e.g., human CTLA4 and human CD80, human CTLA4 and human CD86). In some embodiments, an activatable antibody (when in an active form) inhibits the binding between CTLA4 and one or more of its binding partners (e.g., human CTLA4 and human CD80, human CTLA4 and human CD86). In some embodiments, an activatable antibody inhibits the binding between CTLA4 and its ligand in vitro. In some embodiments, for inhibiting the binding of CTLA4 to CD80 and / or CD86, an activatable antibody has a half-maximal inhibitory concentration (IC 50 ) 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.). In some embodiments, for inhibiting the binding of CTLA4 to CD80 and / or CD86, an activatable antibody has a half-maximal inhibitory concentration (IC 50 ) of about 100 nM or less. In some embodiments, when provided at a concentration 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.), an activatable antibody completely inhibits the binding of human CTLA4 to CD80 and / or CD86. As used herein, the term "complete inhibiting / completely inhibits" means that an activatable antibody is capable of reducing the binding between a first protein and a 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 polypeptide to inhibit the binding of a first protein (e.g., human CTLA4) and a second protein (e.g., human CD80 or human CD86) are known in the art and include, but are not limited to, BIAcore analysis, ELISA assays, and flow cytometry.

[0219] Masking moiety (MM)

[0220] In some embodiments, the present disclosure relates to an activatable antibody comprising a masking moiety (MM). In some embodiments, the masking moiety (MM) comprises, according to formula (XVIII): X m CX n CZ oThe amino acid sequence of (SEQ ID NO:134), where m is 2 - 10, n is 3 - 10, and o is 1 - 10, where 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 where 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, X is not W, M, and / or C. In some embodiments, X in formula (XVIII) m each X 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 in formula (XVIII) n each X 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 a polypeptide encoded by a polynucleotide sequence according to formula (XX): (NNK) m TGY(NNK) n TGY(NHC) o (SEQ ID NO:136), where each N is independently A, G, T, or C, where each K is independently T or G, where each Y is independently T or C, and where each H is independently A, T, or C.

[0221] In some embodiments, the masking moiety (MM) comprises, according to formula (XIX): Z m CZ n CZ o (SEQ ID NO:135) of the amino acid sequence, where 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 embodiments, 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 embodiments, m is 6 - 8. In some embodiments, m is 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, m is 6.

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

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

[0225] In some embodiments, the masking moiety (MM) comprises an amino acid sequence according to formula (XXI): Z6CX6CZ2 (SEQ ID NO:137), where 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 where 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 moiety (MM) comprises an amino acid sequence according to formula (XXII): Z6CX8CZ2 (SEQ ID NO:138), where 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 where 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), where 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.

[0228] In some embodiments, the masking moiety (MM) comprises an amino acid sequence according to formula (XXIV): (Z6)C(Z8)C(Z2)(SEQ ID NO:140), where 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 a masking moiety (MM) containing a sequence selected from the group consisting of X m CPDHPYPC XX(SEQ ID NO:181), X m CDAFYPYCXX(SEQ ID NO:182), X m CDSHYPYCXX(SEQ ID NO:183), and X m CVPYYYACXX(SEQ ID NO:184), where m is 2 - 10, and where 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 moiety (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 embodiments, MM comprises the sequence 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 moiety (MM) comprises an amino acid sequence selected from NFVADSCPDHPYPCSA (SEQ ID NO: 141), IVHHSDCDAFYPYCDS (SEQ ID NO: 142), YSAYPACDSHYPYCNS (SEQ ID NO: 143), PNPSSDCVPYYYACAY (SEQ ID NO: 144), YSAYPACDSHYPYCQS (SEQ ID NO: 145), PQPSSDCVPYYYACAY (SEQ ID NO: 146), and PNPA SDCVPYYYACAY (SEQ ID NO: 147).

[0230] In some embodiments, any masking moiety (MM) described herein may further comprise 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 tag, flag tag, maltose binding protein, and glutathione-S-transferase tag), detection tags (such as tags that can be detected photometrically (e.g., red or green fluorescent protein, etc.)), tags having detectable enzyme activity (such as alkaline phosphatase, etc.), tags containing a secretion sequence, a leader sequence, and / or a stabilization sequence, protease cleavage sites (such as furin cleavage site, TEV cleavage site, thrombin cleavage site), etc. In some embodiments, the one or more additional amino acid sequences are at the N-terminus of the masking moiety (MM). In some embodiments, the additional amino acid sequence comprises the sequence EVGSY (SEQ ID NO: 148) 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 moiety (CM), prior to undergoing a (local) pH change (increase or decrease), prior to a temperature shift (increase or decrease), prior to contact with a second molecule such as a small molecule or protein ligand, etc.), the masking moiety binds to the target binding moiety (TBM) and inhibits the activatable antibody from binding its target, but after activation (e.g., after treatment with one or more proteases that cleave within the cleavable moiety (CM), after undergoing a (local) pH change (increase or decrease), after a temperature shift (increase or decrease), after contact with a second molecule such as a small molecule or protein ligand, etc.), it does not bind to the TBM and / or inhibits the activatable antibody from binding its target. In some embodiments, when the CM is not cleaved, the masking moiety (MM) inhibits the binding of the activatable antibody to its target, but when the CM is cleaved, it does not inhibit the binding of the activatable antibody to its target. In some embodiments, the masking moiety (MM) has a dissociation constant for binding to the TBM that is greater than the dissociation constant of the activatable antibody for its target (when in the active form) (e.g., at least about 1.5-fold greater, at least about 2-fold greater, at least about 2.5-fold greater, at least about 3-fold greater, at least about 3.5-fold greater, at least about 4-fold greater, at least about 4.5-fold greater, at least about 5-fold greater, at least about 10-fold greater, at least about 100-fold greater, at least about 500-fold greater, etc.).

[0232] Cleavable moiety (CM)

[0233] In some embodiments, the present disclosure relates to an activatable antibody comprising a cleavable moiety (CM). In some embodiments, the cleavable moiety (CM) is cleaved and / or disrupted by treatment with one or more proteases that cleave within the cleavable moiety (CM), by a pH change (increase or decrease), by a temperature shift (increase or decrease), and / or by contact with a second molecule such as a small molecule or protein ligand, etc.

[0234] In some embodiments, the cleavable moiety (CM) comprises 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 can be recognized and / or cleaved by any protease known in the art (e.g., a protease known to co-localize with the target of the activatable antibody comprising the CM) can be used, including, for example, protease cleavage sites recognized and / or cleaved by: urokinase-type plasminogen activator (uPA); matrix metalloproteinases (e.g., MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-19, MMP-20, MMP-23, MMP-24, MMP-26, and / or MMP-27); tobacco etch virus (TEV) protease; plasmin; thrombin; PSA; PSMA; ADAMS / ADAMTS (e.g., ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMDEC1, ADAMTS1, ADAMTS4, and / or ADAMTS5); caspases (e.g., caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, and / or caspase-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); metalloproteases (e.g., meprin, neprilysin, PSMA, and / or BMP-1); serine proteases (e.g., activated protein C, cathepsin A, cathepsin G, chymase, and / or coagulation factor proteases such as FVIIa, FIXa, FXa, FXIa, FXIIa); elastase; granzyme B; arginase; HtrA1; human neutrophil elastase;Lactoferrin; marapsin; NS3 / 4A; PACE4; tPA; tryptase; type II transmembrane serine protease (TTSP) (such as DESC1, DPP-4, FAP, Hepsin, Matriptase-2, MT-SP1 / Matriptase, TMPRSS2, TMPRSS3, and / or TMPRSS4); and the like. In some embodiments, the first protease cleavage site is a cleavage site of a protease selected from the following: uPA, MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, TEV protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE. In some embodiments, the first protease cleavage site is a cleavage site of a protease selected from the following: 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 masking moiety (MM) and a cleavable moiety (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 masking moiety (MM) and a cleavable moiety (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 an amino acid selected from 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.

[0237] In some embodiments, the activatable antibody comprises a masking moiety (MM) and a cleavable moiety (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 the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P.

[0238] In some embodiments, the activatable antibody comprises a masking moiety (MM) and a cleavable moiety (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 moiety (CM) further comprises a first linker (L1). In some embodiments, the first linker (L1) is at the C-terminus of a first cleavage site (CS1) (e.g., a first protease cleavage site). In some embodiments, the cleavable moiety (CM) comprises the following structure from the N-terminus to the C-terminus: (CS1)-L1.

[0240] Any suitable linker known in the art can be used (e.g., a flexible linker), including, for example: glycine polymers (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 polymers (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), GSGSG (SEQ ID NO:160), GSGGG (SEQ ID NO:161), GGGSG (SEQ ID NO:162) and / or GSSSG (SEQ ID NO:163); glycine-alanine polymers; alanine-serine polymers; etc. The linker sequence can have any length, such as from about 1 amino acid (e.g., glycine or serine) to about 20 amino acids (e.g., a 20-amino acid glycine polymer or glycine-serine polymer), from about 1 amino acid to about 15 amino acids, from about 3 amino acids to about 12 amino acids, from about 4 amino acids to about 10 amino acids, from about 5 amino acids to about 9 amino acids, from about 6 amino acids to about 8 amino acids, etc. In some embodiments, the length of the linker is any one 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 NOs: 159-163. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:156 or 157.

[0241] In some embodiments, the cleavable moiety (CM) further comprises at least a second cleavage site (e.g., at least second, at least third, at least fourth, at least fifth, etc.). In some embodiments, the cleavable moiety (CM) further comprises a second cleavage site (CS2). In some embodiments, the second cleavage site is a second protease cleavage site. The second protease cleavage site can 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 at the C-terminus of the first linker (L1). In some embodiments, the cleavable moiety (CM) comprises the following structure from the N-terminus to the C-terminus: (CS1)-L1-(CS2).

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

[0243] Exemplary MM-CM sequences

[0244] In some embodiments, the activatable antibody of the present 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), where 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 the present disclosure comprise the following amino acid sequences: EVGSYDALHYACPPDYYACYY SGRSAGGGGTENLYFQGSGGS (SEQ ID NO:165); EVGSYNSY HAYCPHPLYPCTASGRSAGGGGTENLYFQGSGGS (SEQ ID NO:166); EVGSYASSAVLCVTAYFSCNSSGRSAGGGGTENLYFQGSG GS (SEQ ID NO:167); EVGSYNFVADSCPDHPYPCSASGRSAGG GGSPLGLAGSGGS (SEQ ID NO:168); EVGSYNFVADSCPDHPYPCSASGRSAGGGGTENLYFQGSGGS (SEQ ID NO:169); EVGSY IVHHSDCDAFYPYCDSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO:170); EVGSYIVHHSDCDAFYPYCDSSGRSAGGGGTENLYFQG SGGS (SEQ ID NO:171); EVGSYYSAYPACDSHYPYCNSSGRSA GGGGSPLGLAGSGGS (SEQ ID NO:172); EVGSYYSAYPACDSHYPYCNSSGRSAGGGGTENLYFQGSGGS (SEQ ID NO:173); EVGSYPNPSSDCVPYYYACAYSGRSAGGGGSPLGLAGSGGS (SEQ ID NO:174); EVGSYPNPSSDCVPYYYACAYSGRSAGGGGTEN LYFQGSGGS (SEQ IDNO:175); EVGSYYSAYPACDSHYPYCQS SGRSAGGGGSPLGLAGSGGS (SEQ ID NO:176); EVGSYYSAYPACDSHYPYCNSAGRSAGGGGSPLGLAGSGGS (SEQ ID NO:177); EVGSYPQPSSDCVPYYYACAYSGRSAGGGGSPLGLAGSGGS (SEQ ID NO:178); and / or EVGSYPNPASDCVPYYYACAYSGRSA GGGGSPLGLAGSGGS (SEQ ID NO:179). In some embodiments, the polypeptides of the present 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), where 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, where n is 1 - 11, and where 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.

[0246] Target - binding moiety (TBM)

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

[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 can be a κ or λ light chain. The antibody heavy chain can 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 an IgG1, IgG2, IgG3, or IgG4 subclass. Using methods known in the art, the antibody heavy chains described herein can be converted from one class or subclass to another class or subclass.

[0249] Any one or more of the target - binding moieties (TBMs) described herein can be combined with: any of the HVR sequences 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 of the heavy - chain variable region sequences and / or light - chain variable region sequences described herein (e.g., the heavy - chain variable region sequences and / or light - chain variable region sequences shown in Table B above); and / or any of the antibodies described herein.

[0250] In some embodiments, the target binding moiety (TBM) comprises the sequence of one or more of the anti-CTLA4 antibodies described herein, the anti-CTLA4 antibodies including antibodies described with respect to specific amino acid sequences of HVRs, variable regions (VL, VH), and / or light and heavy chains (e.g., IgG1, IgG2, IgG4). In some embodiments, the target binding moiety (TBM) comprises an antibody light chain variable region that comprises HVR-L1 having the amino acid sequence RASQSVRGRFLA (SEQ ID NO:58), HVR-L2 having the amino acid sequence DASNRATGI (SEQ ID NO:66), and / or HVR-L3 having the amino acid sequence YCQQSSSWPPT (SEQ ID NO:75). In some embodiments, the target binding moiety (TBM) comprises an antibody light chain variable region that comprises 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 to the sequence of SEQ ID NO:100. In some embodiments, the target binding moiety (TBM) comprises an antibody heavy chain variable region that comprises HVR-H1 having the amino acid sequence YYSISSGYHWSWI (SEQ ID NO:23), HVR-H2 having the amino acid sequence LARIDWDDDKYYSTSLKSRL (SEQ ID NO:35), and / or HVR-H3 having the amino acid sequence ARSYVYFDY (SEQ ID NO:45). In some embodiments, the target binding moiety (TBM) comprises an antibody heavy chain variable region that comprises 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 to the sequence of SEQ ID NO:87. In some embodiments, the target binding moiety (TBM) comprises: a) an antibody light chain variable region that comprises HVR-L1 having the amino acid sequence RASQSVRGRFLA (SEQ ID NO:58), HVR-L2 having the amino acid sequence DASNRATGI (SEQ ID NO:66), and / or HVR-L3 having the amino acid sequence YCQQSSSWPPT (SEQ ID NO:75); and b) an antibody heavy chain variable region that comprises HVR-H1 having the amino acid sequence YYSISSGYHWSWI (SEQ ID NO:23), HVR-H2 having the amino acid sequence LARIDWDDDKYYSTSLKSRL (SEQ ID NO:35), and / or HVR-H3 having the amino acid sequence ARSYVYFDY (SEQ ID NO:45).In some embodiments, the target binding moiety (TBM) comprises an antibody light chain variable region having the amino acid sequence of SEQ ID NO: 100 and an antibody heavy chain variable region having the amino acid sequence of SEQ ID NO: 87.

[0251] Activable binding polypeptide properties

[0252] In some embodiments, the activable binding polypeptides (i.e., activable antibodies) of the present disclosure comprise: (a) a masking moiety (MM), (b) a cleavable moiety, and (c) a target binding moiety. In some embodiments, the masking moiety (MM) binds to the target binding moiety (TBM) of the activable antibody and reduces or inhibits the binding of the activable binding moiety to CTLA4 (e.g., human CTLA4) compared to the corresponding binding polypeptide lacking the masking moiety and / or compared to the parental antibody binding to CTLA4 (e.g., human CTLA4).

[0253] In some embodiments, an "activable" binding polypeptide is a binding polypeptide that exhibits a first level of binding to CTLA4 when in an inhibited, masked, and / or uncleaved state and a second level of binding 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, after cleavage within the cleavable moiety (e.g., by one or more proteases), the activable binding polypeptide has an increased opportunity to access CTLA4.

[0254] In some embodiments, the activable antibodies of the present disclosure are generally considered to be "activable" binding polypeptides when the binding affinity of the polypeptide for CTLA4 (e.g., human CTLA4) increases by at least about 2-fold (e.g., at least about 2-fold, at least about 2.5-fold, at least about 3, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 250-fold, at least about 500-fold, at least about 750-fold, or at least about 1000-fold or more) after activation of the activable antibody compared to before activation of the activable antibody (e.g., after activation by treatment with one or more proteases that cleave within the cleavable moiety (CM), after activation by a change in pH (increase or decrease), after activation by a change in temperature (increase or decrease), after activation by contact with a second molecule (such as a small molecule), etc.). In some embodiments, if after "activation", the EC of the activable antibody 50Reduced 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 Examples below), then the activatable antibodies of the present disclosure are generally considered "activatable". In some embodiments, if after treatment with a protease that cleaves within the cleavable moiety (CM), the EC of the polypeptide 50 Reduced to at least about 1 / 2 (e.g., as measured by ELISA or FACS assay; see Examples below), then the activatable antibodies of the present disclosure are generally considered "activatable".

[0255] In some embodiments, when the masking moiety (MM) binds to the target binding moiety (TBM) of the activatable antibody, the K of the activatable antibody with respect to CTLA4 D is when the masking moiety (MM) is not bound to the target binding moiety (TBM) (e.g., after "activation" of the activatable antibody (such as after protease treatment to effect cleavage within the cleavable moiety (CM))) and / or is the K of the parental antibody with respect to CTLA4 D about 2 (e.g., about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 25, about 50, about 75, about 100, about 250, about 500, about 750 or about 1000 or more) times greater. Methods for measuring affinity are known in the art and include, for example, the methods described in the Examples below.

[0256] In some embodiments, relative to when the masking moiety is not bound to the target binding moiety (e.g., after "activation" of the activatable antibody (such as after protease treatment to effect cleavage within the cleavable moiety (CM))), and / or relative to the K of the parental antibody with respect to CTLA4 D when the masking moiety binds to the target binding moiety of the activatable antibody, the K of the activatable antibody with respect to CTLA4 DReduced by at least about 25% (such as 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 and include, for example, the methods described in the following examples.

[0257] In some embodiments, the masking moiety sterically hinders the binding of the activatable antibody to CTLA4 and / or allosterically hinders the binding of the activatable antibody to CTLA4. In some embodiments, the masking moiety does not comprise the amino acid sequence of the natural binding partner of the activatable antibody and / or the parental antibody.

[0258] In some embodiments, the dissociation constant of the masking moiety for the target binding moiety is greater than the dissociation constant of the activatable antibody for CTLA4 (when activated). In some embodiments, the dissociation constant of the masking moiety for the target binding moiety is about 2 (such as about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 25, about 50, about 75, about 100, about 250, about 500, about 750, or about 1000 or more) times greater than the dissociation constant of the activatable antibody for CTLA4 (when activated). In some embodiments, the dissociation constant of the masking moiety for the target binding moiety is approximately equal to the dissociation constant of the activatable antibody for 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 antibodies of the present disclosure are conjugated to a pharmaceutical agent. Examples of pharmaceutical agents include cytotoxic or other cancer therapeutic agents and radioisotopes. Specific examples of cytotoxic agents include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, didehydroxymethylepoxyquinomicin, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as analogs or homologs thereof. Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (previously called daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (previously called actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine). Examples of radioisotopes that can be conjugated to an antibody for diagnostic or therapeutic use include, but are not limited to, iodine 131 , indium 111 , yttrium 90 , and lutetium 177 . Methods for conjugating polypeptides to pharmaceutical agents are known in the art, such as using various linker technologies. Examples of linker types include hydrazones, thioethers, esters, disulfides, and peptide-containing linkers. For further discussion of linkers and methods for conjugating 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 Acids, Vectors, Host Cells, and Recombinant Methods for Generating CTLA4 Antibodies and / or Precision / Situation-Dependent Activatable Antibodies

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

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

[0264] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding an amino acid sequence set forth in any one of SEQ ID NOs: 18-107 or consists of said nucleotide sequence.

[0265] In some embodiments, the nucleic acid molecule comprises the nucleotide sequence described in Table C below or consists of said nucleotide sequence.

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

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273] The nucleic acids of the present disclosure can be obtained using any suitable molecular biology techniques. For antibodies expressed by hybridomas, the cDNAs encoding the light and heavy chains of the antibodies prepared by the hybridomas can be obtained by PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display techniques), the nucleic acids encoding the antibodies can be recovered from the libraries.

[0274] Encoding VH The isolated DNA of the V region can be converted into a full-length heavy chain gene by operably linking the V-encoding DNA to another DNA molecule encoding the heavy chain constant regions (CH1, CH2, and CH3). The sequences of the 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 covering these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region. For the Fab fragment heavy chain gene, the V- H encoding DNA can be operably linked to another DNA molecule encoding only the CH1 constant region of the heavy chain. H The isolated DNA of the V region can be converted into a full-length light chain gene (as well as the Fab light chain gene) by operably linking the V-encoding DNA to another DNA molecule encoding the light chain constant region CL. The sequences of the 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 a κ or λ constant region.

[0275] To create the scFv gene, the V- L encoding DNA fragment and the V- L encoding DNA fragment are operably linked to another fragment encoding a flexible linker, such as a fragment encoding the amino acid sequence (Gly4-Ser)3, so that the V-

[0276] and V- H sequences can be expressed as a continuous single-chain protein, in which the V- L region and the V- H and V- L regions are joined by the flexible linker (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)). L region and the V- H region are joined by the flexible linker (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] The present disclosure also provides a vector comprising a nucleic acid molecule described herein. In some embodiments, the vector is an expression vector or a display vector (such as 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 a portion of a light or heavy chain (such as a CDR or HVR; a light chain variable region or a heavy chain variable region), a full-length light or heavy chain, a polypeptide comprising a portion or full-length of a heavy or light chain, or the amino acid sequence of an antibody derivative or 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, provided herein is a vector, wherein 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 a heavy chain variable region as described herein and a light chain variable region as described herein.

[0278] To express the binding molecules of the present disclosure, DNA encoding a partial or full-length light and heavy chain is inserted into an expression vector such that the DNA molecule is operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" means that the antibody gene is linked to the vector such that the transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the DNA molecule. The expression vector and expression control sequences are selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors, or the two genes can be inserted into the same expression vector. The antibody genes are inserted into the expression vector by any suitable method (such as ligation of complementary restriction sites on the antibody gene fragment and the vector, or DNA ligation based on homologous recombination). The light chain variable region and the heavy chain variable region of the antibodies described herein can be used to create full-length antibody genes of any antibody isotype and subclass by inserting them into an expression vector that already encodes the heavy chain constant region and the light chain constant region of the desired isotype and subclass such that the V H segment is operably linked to one or more C H segments within the vector, and the V L segment is operably linked to the C L segment within the vector. Additionally or 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 such that the signal peptide is in-frame linked to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin).

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

[0280] In addition to the antibody chain gene and regulatory sequences, the expression vector may also carry additional sequences, such as sequences that regulate the replication of the vector in a host cell (e.g., an origin of replication) and selectable marker genes. Selectable marker genes facilitate the selection of host cells into which the vector has been introduced (see, for example, U.S. Patent Nos. 4,399,216, 4,634,665, and 5,179,017, all by Axel et al.). For example, typically, selectable marker genes confer resistance to drugs such as G418, hygromycin, or methotrexate on 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 (for G418 selection).

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

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

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

[0284] Eukaryotic hosts suitable for transformation include yeast, insects (such as S2 cells), and mammalian cells. Mammalian host cells for expressing the binding molecules of the present 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)), NS0 myeloma cells, COS cells, HEK293F cells, HEK293T cells, and Sp2 cells. In particular, for use with NS0 myeloma or CHO cells, another expression system is the GS (glutamine synthetase) gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP 338,841. In some embodiments, the antibodies of the present disclosure are produced in CHO cells. In some embodiments, the antibodies of the present disclosure are modified and do not include a C-terminal lysine residue (e.g., the C-terminal lysine residue of the heavy chain of the antibody described herein is removed (such as 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 expression of the antibody in the host cell or secretion of the antibody into the medium in which the host cell is growing. The antibody can be recovered from the medium using any suitable protein purification method known in the art (e.g., protein A chromatography and / or ion exchange chromatography).

[0285] VI. Compositions

[0286] In other aspects, the present disclosure provides a composition comprising a binding molecule (e.g., an antibody or an activatable antibody) provided by the present 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, the present disclosure provides a composition comprising a binding molecule (e.g., an antibody or an activatable antibody) provided by the present disclosure and a pharmaceutically acceptable carrier, wherein the binding molecule comprises a variable domain containing the HVR amino acid sequences disclosed herein, and wherein the composition comprises at most about 11%, 10%, 8%, 5%, 3%, or 2% of the binding molecule (e.g., an antibody or an activatable antibody) glycosylated at asparagine of the amino acid sequence, compared to the total amount of the binding molecule (e.g., an antibody or an activatable antibody) present in the composition. In another embodiment, the composition comprises at least about 2% of the binding molecule (e.g., an antibody or an activatable antibody) glycosylated at asparagine of the amino acid sequence, compared to the total amount of the binding molecule (e.g., an antibody or an activatable antibody) present in the composition.

[0288] The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in a formulation for delivering a binding molecule. The carrier can be a glidant, binder, coating agent, disintegrant, filler or diluent, preservative (such as an antioxidant, antibacterial agent or antifungal agent), sweetening agent, absorption delaying agent, wetting agent, emulsifying agent, buffering agent, 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, buffering agents, buffered saline, and isotonic agents such as sugars, polyols, sorbitol, and sodium chloride.

[0289] The composition can 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 infusible solutions), microemulsions, liposomes, dispersions or suspensions. Examples of solid dosage forms include tablets, pills, capsules, microcapsules, and powders. A particular form of the 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. A sterile solution can be prepared by incorporating the antibody in the required amount into a suitable carrier, followed by sterile microfiltration. Generally, a dispersion is prepared by incorporating the binding molecule (e.g., an antibody or an activatable antibody) into a sterile vehicle containing a basic dispersion medium and other carriers. In the case of sterile powders for the preparation of sterile liquids, the preparation methods include vacuum drying and freeze drying (lyophilization) to produce a powder of the active ingredient plus any additional desired ingredients from its previously sterile filtered solution. The various dosage forms of the composition can be prepared by conventional techniques known in the art.

[0290] The relative amount of the binding molecule (e.g., antibody or activatable antibody) included in the composition will vary depending on many factors, such as the specific binding molecule and carrier used, the dosage form, and the desired release and pharmacokinetic characteristics. The amount of the binding molecule (e.g., antibody or activatable antibody) in a single dosage form will generally be the amount that produces a therapeutic effect, but can also be a smaller amount. Generally, this amount will be in the range of about 0.01% to about 99%, about 0.1% to about 70%, or about 1% to about 30% relative to the total weight of the dosage form.

[0291] In addition to the binding molecule (e.g., antibody or activatable antibody), one or more additional therapeutic agents may also be included in the composition. Examples of additional therapeutic agents are described hereinbelow. The suitable amount of the 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 agent and carrier used, the dosage form, and the desired release and pharmacokinetic characteristics. The amount of the additional therapeutic agent included in a single dosage form will generally be the amount of the agent that produces a therapeutic effect, but can also be a smaller amount.

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

[0293] VII. Uses of Binding Molecules and Pharmaceutical Compositions

[0294] The binding molecules (e.g., antibody or activatable antibody) and pharmaceutical compositions provided by the present disclosure can be used for treatment, diagnosis, or other purposes, such as modulating the immune response, treating cancer, enhancing the efficacy of other cancer therapies, enhancing the efficacy of vaccines, or treating autoimmune diseases. Accordingly, in other aspects, the present disclosure provides methods of using the binding molecules (e.g., antibody or activatable antibody) or pharmaceutical compositions. In one aspect, the present disclosure provides a method of treating a disorder in a mammal, the method comprising administering to the mammal in need of treatment an effective amount of the binding molecule (e.g., antibody or activatable antibody) or composition provided by the present disclosure. The binding molecule (e.g., antibody or activatable antibody) can be a CTLA4 antibody (e.g., a human anti-human CTLA4 antibody) or a CTLA4 activatable antibody. In some embodiments, the mammal is a human.

[0295] In some embodiments, the disorder is cancer. A variety of cancers can be treated or prevented with the methods, uses, compositions or agents provided by the present disclosure.Examples of said cancers include lung cancers such as bronchial carcinoma (e.g., squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma), alveolar cell carcinoma, bronchial adenoma, chondromatous hamartoma (non-cancerous), and sarcoma (cancerous); heart cancers such as myxoma, fibroma, and rhabdomyoma; bone cancers such as osteochondroma, chondroma, chondroblastoma, chondromyxoid fibroma, osteoid osteoma, giant cell tumor, chondrosarcoma, multiple myeloma, osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, Ewing's tumor (Ewing's sarcoma), and reticulum cell sarcoma; brain cancers such as glioma (e.g., glioblastoma multiforme), anaplastic astrocytoma, astrocytoma, oligodendroglioma, medulloblastoma, chordoma, schwannoma, ependymoma, meningioma, pituitary adenoma, pinealoma, osteoma, hemangioblastoma, craniopharyngioma, chordoma, germinoma, teratoma, dermoid cyst, and hemangioma; cancers in the digestive system such as leiomyoma, epidermoid carcinoma, adenocarcinoma, leiomyosarcoma, gastric adenocarcinoma, intestinal lipoma, intestinal neurofibroma, intestinal fibroma, colorectal polyps, and colorectal cancer; liver cancers such as hepatocellular adenoma, hemangioma, hepatocellular carcinoma, fibrolamellar carcinoma, cholangiocarcinoma, hepatoblastoma, and angiosarcoma; kidney cancers such as renal adenocarcinoma, renal cell carcinoma, adrenocortical adenoma, and transitional cell carcinoma of the renal pelvis; bladder cancer; blood cancers such as acute lymphocytic (lymphoblastic) leukemia, acute myeloid (myelocytic, myelogenous, myeloblastic, myelomonocytic) leukemia, chronic lymphocytic leukemia (e.g., Sezary syndrome and hairy cell leukemia), chronic myeloid (myelogenous, myeloblastic, granulocytic) leukemia, 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; 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 carcinoma), ovarian cancer (ovarian carcinoma), vulvar cancer, vaginal cancer, fallopian tube cancer, and hydatidiform mole; thyroid cancer (including papillary, follicular, anaplastic, or medullary carcinoma); pheochromocytoma (adrenal); 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, the present disclosure provides a method of 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 a composition provided by the present 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 grammatical variations thereof means stimulating, provoking, increasing, improving, or strengthening any response of the immune system of a mammal. The immune response can be a cellular response (i.e., mediated by cells, such as by cytotoxic T lymphocytes) or a humoral response (i.e., mediated by antibodies), and can 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 evaluated using many 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-bearing animals, antibody production, immune cell proliferation, expression of cell surface markers, and cytotoxicity. Generally, the methods of the present disclosure enhance the immune response achieved by a mammal as compared to the immune response achieved by an untreated mammal or a mammal not treated using the recited method.

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

[0298] A wide variety of cancer therapeutics can be used in combination with the binding molecules (e.g., antibodies or activatable antibodies) provided by the present disclosure. Those of ordinary skill 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 the present disclosure, such as antibodies or activatable antibodies, and will not be limited to the forms of therapies set forth herein. Examples of the types 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 viral gene therapy, immune checkpoint inhibitor, targeted therapy, radiotherapy, vaccination therapy, and / or chemotherapeutic agent.

[0299] The term "chemotherapeutic agent" refers to a chemical or biological substance that can cause the death of cancer cells or interfere with the growth, division, repair, and / or function of cancer cells. Examples of chemotherapeutic agents include those disclosed in WO 2006 / 129163 and US20060153808, 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), nemorubicin, idarubicin (IDAMYCIN PFS, ZAVEDOS), mitoxantrone (DHAD, NOVANTRONE), dactinomycin (actinomycin D, COSMEGEN), plicamycin (MITHRACIN), mitomycin (MUTAMYCIN), and bleomycin (BLENOXANE), vinorelbinetartrate (NAVELBINE), vinblastine (VELBAN), vincristine (ONCOVIN), and vindesine (ELDISINE); (3) Antimetabolites such as capecitabine (XELODA), 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);Capecitabine (Xeloda), raltitrexed (TOMUDEX), tegafur-uracil (UFTORAL), and gemcitabine (Gemzar); (5) Taxanes, such as docetaxel (TAXOTERE), paclitaxel (TAXOL); (6) Platinum drugs, such as cisplatin (PLATINOL), carboplatin (PARAPLATIN), and oxaliplatin (ELOXATIN); (7) Topoisomerase inhibitors, such as irinotecan (CAMPTOSAR), topotecan (HYCAMTIN), etoposide (ETOPOPHOS, VEPESSID, TOPOSAR), and teniposide (VUMON); (8) Epipodophyllotoxin (podophyllotoxin derivatives), such as etoposide (ETOPOPHOS, VEPESSID, TOPOSAR); (9) Folic acid derivatives, such as leucovorin (WELLCOVORIN); (10) Nitrosourea, such as carmustine (BiCNU), lomustine (CCNU);(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), imatinib mesylate (GLEEVEC), genefitinib, lapatinib, sorafenib, thalidomide, sunitinib (SUTENT), axitinib, rituximab (RITUXAN, MABTHERA), trastuzumab (HERCEPTIN), cetuximab (ERBITUX), bevacizumab (AVASTIN), and ranibizumab (LUCENTIS), lym-1 (ONCOLYM), the antibody against insulin-like growth factor-1 receptor (IGF-1R) disclosed in WO2002 / 053596; (12) 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 (13) Proteasome inhibitors, such as bortezomib (VELCADE).;

[0300] The term "immunotherapeutic agent" refers to a chemical or biological substance that enhances the immune response of a mammal. Examples of immunotherapeutic agents include: Bacillus Calmette-Guérin (BCG); cytokines, such as interferons; 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), ibritumomab tiuxetan (ZEVALIN), panitumumab (VECTIBIX), rituximab (Rituxan, MabThera), trastuzumab (Herceptin), tositumomab (BEXXAR), ipilimumab (YERVOY), tremelimumab, CAT-3888, agonist antibodies against the OX40 receptor (such as those disclosed in WO2009 / 079335), agonist antibodies against 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 "hormonal therapeutic agent" refers to a chemical or biological substance that inhibits or eliminates the production of a hormone, or inhibits or counteracts the effect of a hormone on the growth and / or survival of cancerous cells. Examples of such agents suitable for the methods herein include those disclosed in US20070117809. Examples of specific hormonal therapeutic 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 of the present disclosure can also be used in combination with non-pharmacological hormonal therapies such as (1) surgical methods that remove all or a portion of an organ or gland involved in the production of a hormone such as the ovaries, testes, adrenal glands, and pituitary gland, and (2) radiation therapy, wherein an organ or gland of a patient is subjected to radiation in an amount sufficient to inhibit or eliminate the production of the targeted hormone.

[0302] In some embodiments, the additional therapeutic agent is one or more of the following: pomalidomide, lenalidomide, thalidomide, pomalidomide, thalidomide, the DNA alkylating platinum derivative cisplatin, 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, pembrolizumab, radiation therapy, single-dose radiation, fractionated radiation, focal radiation, whole-organ radiation, IL-12, IFNα, GM-CSF, chimeric antigen receptor, adoptively transferred T cells, anti-cancer vaccines, and oncolytic viruses.

[0303] Combination therapies for treating cancer also encompass the combination of a binding molecule (e.g., an antibody or an activatable antibody) with surgery for removing a tumor. The binding molecule (e.g., an antibody or an activatable antibody) can be administered to a mammal before, during, or after surgery.

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

[0305] The binding molecules (e.g., an antibody or an activatable antibody) and compositions provided by the present disclosure can be administered by any suitable enteral administration route or parenteral administration route. The term "enteral administration route" refers to administration through any part of the gastrointestinal tract. Examples of enteral routes include oral, transmucosal, buccal, and rectal routes, or intragastric routes. "Parenteral administration route" refers to an administration route other than the enteral route. Examples of parenteral administration routes include intravenous, intramuscular, intradermal, intraperitoneal, intratumoral, intravesical, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intratracheal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal, subcutaneous, or topical administration. The binding molecules (e.g., an antibody or an activatable antibody) and compositions of the present disclosure can be administered using any suitable method, such as by oral ingestion, nasogastric tube, gastrostomy tube, injection, infusion, implantable infusion pump, and osmotic pump. The suitable administration route and method can vary depending on many factors, such as the specific binding molecule (e.g., an antibody or an activatable antibody) used, the desired absorption rate, the specific formulation or dosage form used, the type or severity of the condition being treated, the specific site of action, and the condition of the patient, and can be readily selected by those skilled in the art.

[0306] An "effective amount" of a binding molecule (such as an antibody or an activatable antibody) can refer to an amount effective to achieve a predetermined therapeutic purpose. For example, in the case of enhancing an immune response, an "effective amount" can be any amount effective to stimulate, elicit, increase, improve, or enhance any response of the immune system of a mammal. In the case of treating a disease, an "effective amount" can be any amount sufficient to cause any desirable or beneficial effect in the mammal being treated. In particular, in the case of treating cancer, examples of desirable or beneficial effects include inhibiting the further growth or spread of cancer cells, causing cancer cells to die, inhibiting the recurrence of cancer, alleviating pain associated with cancer, or improving the survival period of a mammal. The therapeutically effective amount of a binding molecule (such as an antibody or an activatable antibody) is generally in the range of about 0.001 to about 500 mg per kg body weight of a mammal, and more typically in the range of about 0.01 to about 100 mg / kg. For example, the amount can be about 0.3 mg, 1 mg, 3 mg, 5 mg, 10 mg, 50 mg, or 100 mg per kg body weight of a mammal. In some embodiments, the therapeutically effective amount of a binding molecule (such as an antibody or an activatable antibody) is in the range of about 0.01 - 30 mg per kg body weight of a mammal. In some other embodiments, the therapeutically effective amount of a binding molecule (such as an antibody or an activatable antibody) is in the range of about 0.05 - 15 mg per kg body weight of a mammal. The precise dosage 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 disorder to be treated, the particular binding molecule (such as an antibody or an activatable antibody) employed, the route of administration, the time of administration, the duration of treatment, the particular additional therapies employed, the age, sex, weight, condition, general health, and prior medical history of the patient being treated, and similar factors well known in the medical arts.

[0307] The binding molecule (such as an antibody or an activatable antibody) or composition is typically administered at multiple times. The interval between single doses can be, for example, daily, weekly, monthly, every three months, or annually. An exemplary treatment regimen requires administration once a week, 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 dosage regimens for a binding molecule (such as an antibody or an activatable antibody) include intravenous administration of 1 mg per kg body weight or 3 mg per kg body weight, using one of the following dosing schedules: (i) every four weeks for six doses, followed by every three months; (ii) every three weeks; (iii) 3 mg per kg body weight, once, followed by every three weeks, 1 mg per kg body weight.

[0308] VIII. Kit

[0309] In another aspect, the present disclosure provides a kit comprising a binding molecule (e.g., an antibody or an activatable antibody) and / or a composition of the present disclosure. In some embodiments, the kit further comprises a package insert comprising instructions for using the binding molecule (e.g., an antibody or an activatable antibody) and / or the composition. In some embodiments, the kit further comprises one or more buffers, e.g., for storing, transferring, administering, or otherwise using the binding molecule (e.g., an antibody or an activatable antibody) and / or the composition. In some embodiments, the kit further comprises one or more containers for storing the binding molecule (e.g., an antibody or an activatable antibody) and / or the composition.

[0310] The foregoing written description is considered to be sufficient to enable one of ordinary skill in the art to practice the present disclosure. The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way. Indeed, various modifications of the present disclosure will become apparent to those of ordinary skill in the art in light of the foregoing description and fall within the scope of the appended claims.

[0311] Examples

[0312] Example 1: Generation of Primary Fab Specifically Binding to Human CTLA4

[0313] A proprietary phagemid library (see PCT application number PCT / CN2017 / 098333, which is incorporated herein by reference in its entirety, and also see PCT application number PCT / CN2017 / 098299, which is 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 last 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 hits whose ELISA signal was at least twice that of 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 by the ForteBio Octet RED96 system. Briefly, an AHC sensor (anti - human IgG Fc capture - dip - and - read biosensor) was used to capture recombinant human CTLA4 - Fc (Sino Biological, 11159 - H03H) and immersed into wells containing purified Fab diluted to 10 μg / mL in kinetic buffer (PBS buffer containing 0.02% Tween 20, 0.1% BSA). The acquired ForteBio data was processed with Data Acquisition software 7.1, and the kinetic data was fitted to a 1:1 Langmuir binding model. The candidate list was curated to 234 Fab hits with both ELISA - positive hits and unique sequences. Following the criteria of K D response signal R > 0.1, R 2 > 0.9, the list was further curated to 43 target hits. The affinity and kinetic parameters (background - subtracted) of these hits are shown in Table 1 below.

[0314] Table 1: Affinity of selected Fabs for 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 hits (2 μg / mL) was added and incubated for 1 hour. After washing the wells 3 - 4 times, serial dilutions of human, cynomolgus monkey, or mouse CTLA4 antigen fused to human FC fragment were added and incubated for 1 hour. After washing, HRP-labeled goat anti-human FC antibody was diluted 1:2000 in PBS and added to each well for 1 hour incubation. The plates were washed three times and incubated with TMB substrate for 3 - 5 minutes at room temperature. Absorbance was measured at 450 nm after terminating the reaction. The species cross-reactivity of each of the tested Fabs is summarized in Table 2 below.Of note, this analysis identified Fabs with different cross-reactivities: the results indicate that hits B13873, B15700, B15704, B15706, B15709, B15711, B15712, B15715, B15720, B15725, B15723, B15731, B15732, B15735, B15736, B15744, B15760, B16083 and B15188 bind to human, monkey and mouse CTLA4; hits B15188, B15190, B15701, B15729, B15733, B15742, B15747, B15743, B15751, B15752, B15753 and B18157 bind to human and monkey CTLA4 and weakly bind to mouse CTLA4; hits B13878, B14242, B15189, B15491, B15673, B15694, B15696, B15699, B15702, B15705, B15710, B15716, B15717, B15719, B15721, B15722, B15724, B15728, B15734, B15737, B15738, B15739, B15740, B15745, B15746, B15749, B15750, B15754, B15756, B15757, B15759 and B15762 bind to human CTLA4 but not to mouse CTLA4; hit B15688 binds to human and mouse CTLA4 but not to monkey CTLA4; and hits 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 Fabs 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 to 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 hits cloned as IgG1 antibodies

[0325]

[0326]

[0327] Following the manufacturer's protocol, each pair of plasmids encoding the antibody heavy and light chains was transiently transfected into 293F cells. The supernatants of the cells transfected with the plasmids encoding antibodies TY21585, TY21586, TY21587, TY21588, TY21589, TY21580, or TY21591 were collected, clarified by centrifugation and filtration, and the resulting IgG was purified by standard protein A affinity chromatography (MabSelect SuRe, GE Healthcare). The protein was eluted and neutralized, and buffer exchange was performed into 20 mM PB buffer (20 mM NaH2PO4, 150 mM NaCl, pH 7.0). Protein concentration was determined by ultraviolet spectrophotometry, and IgG purity was analyzed by SDS-PAGE or SEC-HPLC under denaturing, reducing, and non-reducing conditions.

[0328] The supernatants of the cells transfected with the plasmids encoding antibodies TY21687, TY21689, TY21680, TY21691, or TY21692 were collected, clarified by centrifugation and filtration, and the resulting IgG was purified by standard protein A affinity chromatography (MabSelect SuRe, GE Healthcare). The protein was eluted and neutralized, and buffer exchange was performed into 20 mM histidine buffer (20 mM histidine, 3.5 mL 6M HCl, pH 5.5). Protein concentration was determined by ultraviolet spectrophotometry, and IgG purity was analyzed by SDS-PAGE or SEC-HPLC under denaturing, reducing, and non-reducing conditions.

[0329] Example 3: In vitro functional characterization of IgG-converted antibodies

[0330] Using Biacore according to the manufacturer's guidelines TM The Biacore T200 instrument (Biacore AB, Uppsala, Sweden) was used to examine the binding affinity and kinetics of antibodies against human, cynomolgus monkey, and mouse CTLA4 by surface plasmon resonance (SPR) analysis (Table 4). According to the instructions of the amine coupling kit (GE Biacore #BR-1000-50), the anti-human IgG (Fc) antibody from the human antibody capture kit (GE BR-1008-39) was immobilized on a CM5 chip by coupling its amine group to the carboxylated surface of the sensor chip. The immobilized anti-human IgG (Fc) antibody was 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 ipilimumab. Binding was measured at six different concentrations (3.13, 6.25, 12.5, 25, 50, and 100 nM diluted in running buffer) and a flow rate of 30 μl / min was used. The running buffer used was HBS-EP (100 mM HEPES, 1.5 M sodium chloride, 0.05% surfactant P20, pH 7.6). Using the Biacore T200 evaluation software (Biacore AB) according to the manufacturer's guidelines, the association and dissociation curves were fitted to a 1:1 Langmuir binding model. As shown in Table 4 below, all tested antibodies were able to bind human and cynomolgus monkey CTLA4, and all antibodies except TY21591, TY21689, and TAC2114 were also able to bind mouse CTLA4.

[0331] Table 4: Binding affinity of IgG1 antibodies for human, cynomolgus monkey, and mouse CTLA4

[0332]

[0333] NC = No cross-reactivity

[0334] Next, certain IgG antibodies were tested for binding to soluble human ( Figure 1A , Table 5A) or canine ( Figure 1B, the ability of CTLA4. Prepare 1 μg / mL of human CTLA4 fused to the human FC fragment, or canine CTLA4 fused to a His tag, and use it to coat an ELISA plate overnight at 2 - 8 °C. After blocking, add 100 μL of serially diluted IgG antibody and incubate at 37 °C for 1 hour. Wash the plate four times, then incubate with an HRP - anti - human IgG (Fab - specific) antibody (1:6000 dilution) at 37 °C for 1 hour. Wash the plate four times again and incubate with TMB substrate at room temperature for 15 minutes. Measure the absorbance at 450 nm after terminating the reaction. Analyze the data using nonlinear fitting with Graphpad Prism 6. As Figures 1A - 1B shown in Table 5A - Table 5B, all tested antibodies bind to human CTLA4 and, except for antibodies TY21586 and TAC2114, also bind to canine CTLA4. Of concern, TY21580 binds both human CTLA4 and dog CTLA4 with the highest affinity, with K D values of 0.27 and 0.49, respectively.

[0335] Table 5A: ELISA for human CTLA4

[0336]

[0337] Table 5B: ELISA for canine CTLA4

[0338]

[0339] ND = Not detected

[0340] The affinity of the antibodies for human and mouse CTLA4 transiently expressed on the surface of HEK293F cells was also evaluated ( Figure 2 ). Briefly, HEK293F cells were transfected with plasmids 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, transfer the mammalian cell suspension (2x10 5 / well) to an Eppendorf tube, centrifuge, discard the supernatant, and resuspend the cells in 1 mL of PBSA (to achieve a density of 4×10 6cells / mL), and added to a 96-well plate. Serial 3-fold dilutions of the test antibodies (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) were pipetted into the 96-well plate, incubated for 1 hour on ice (protected from light), the cells were washed with pre-cooled 1xPBSA buffer, and then incubated on ice for 30 minutes with Alexa 647-conjugated mouse anti-human FC antibody. The cells were then washed once and subsequently analyzed by flow cytometry ( CytoFlex). As Figure 2 shown, all test antibodies were able to bind human CTLA4 expressed on the surface of HEK293F cells, and all antibodies except TY21589 bound to mouse CTLA4 exposed on the mouse cell surface. TY21580 bound to 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 affinities 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 then the sensor was contacted with IgG transition hits at a concentration of 15 μg / mL (diluted in 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. Using ForteBio Data Analysis 7.1 (Pall, USA) according to the manufacturer's guidelines, the association and dissociation curves were fitted relative to a 1:1 Langmuir binding model. As shown in Table 6 below, all test antibodies were able to bind rat CTLA4.

[0342] Table 6: Binding affinities of test antibodies for 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 activated human, monkey, and mouse T cells was tested. Human PBMCs were freshly isolated from the blood of a healthy donor (#106) by density gradient centrifugation using 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 antibody and anti-CD28 antibody. Briefly, anti-CD3 antibody (clone: OKT3, BioLegend) at 0.2 μg per well in 200 μL was coated overnight in 96-well plates at 4 °C. After washing, T cells suspended in RPMI-1640 containing 10% FBS and 1% penicillin / streptomycin were added to the plates. 5x10E5 T cells in 200 μL were added to each well of the 96-well plates. Next, 1 μL of anti-human CD28 antibody (clone: 28.2, BD) was added to achieve a final concentration of 5 μg / mL. The T cells were incubated for 96 hours, and then the binding of TY21580 to T cells was determined by flow cytometry analysis ( Figure 3 ). The T cells were stained with APC-labeled TY21580 or human IgG1 (isotype control) at 37 °C for 2 hours. After washing, the cells were analyzed on a CytoFLEX flow cytometer (Beckman Coulter), and the data were analyzed using FlowJo software. As shown in Figure 3 , TY21580 bound to activated CD4+ and CD8+ human T cells, while the control IgG showed no binding. In addition, APC-TY21580 showed no binding to resting T cells (data not shown).

[0346] Monkey PBMCs were freshly isolated from the blood of naïve cynomolgus monkeys by density gradient centrifugation using Histopaque-1077 (Sigma). Monkey T cells were isolated from PBMCs using a non-human primate total T cell isolation kit (Miltenyi Biotec) and subsequently stimulated with anti-CD3 antibody and anti-CD28 antibody. Briefly, anti-CD3 antibody (clone: SP34, BD) at 0.2 μg per well in 200 μL was coated overnight in 96-well plates at 4 °C. After washing, T cells suspended in RPMI-1640 containing 10% FBS and 1% penicillin / streptomycin were added to the plates. 2x10E5 T cells in 200 μL were added to each well of the 96-well plates. Next, 1 μL of anti-human CD28 antibody (clone: 28.2, BD) was added to achieve a final concentration of 5 μg / mL. The T cells were incubated for 72 hours, and the binding of TY21580 to T cells was determined by flow cytometry analysis ( Figure 3)。The T cells were stained with APC-labeled TY21580 or human IgG1 (isotype control) at 37 °C for 2 hours. After washing, the cells were analyzed on a CytoFLEX flow cytometer (Beckman Coulter), and the data were analyzed using FlowJo software. As Figure 3 shown, TY21580 binds to activated CD4+ and CD8+ monkey T cells, while the control IgG shows no binding. In addition, APC-TY21580 shows no binding to resting T cells (data not shown).

[0347] Mouse T cells isolated from the spleens of adult BALB / c mice were used to induce CTLA-4 expression. Splenocytes from fresh mouse spleens were used to isolate T cells using the EasySep TM Mouse T Cell Isolation Kit (StemCell Technologies), and then stimulated with anti-mouse CD3 antibody and anti-CD28 antibody. Briefly, the anti-mouse CD3ε antibody (Biolegend) was coated at 0.2 μg per well in 200 μL in a 96-well plate overnight at 4 °C. After washing, mouse T cells suspended in RPMI-1640 containing 10% FBS and 1% penicillin / streptomycin were added to each well of the plate at 5x10E5 T cells in 200 μL. Then, 1 μL of anti-mouse CD28 antibody (eBioscience) was added to reach a final concentration of 5 μg / mL. The mouse T cells were incubated for 72 hours, and then the binding of TY21580 to the T cells was determined by flow cytometry analysis ( Figure 3 )。The T cells were stained with APC-labeled TY21580 or human IgG1 (isotype control) at 37 °C for 2 hours. After washing, the cells were analyzed on a CytoFLEX flow cytometer (Beckman Coulter), and the data were analyzed using FlowJo software. As Figure 3 shown, TY21580 binds to activated mouse CD4+ and CD8+ T cells, while the control IgG shows no binding.

[0348] Binding selectivity of the antibody 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. The transfected cells were washed in pre-chilled 1xPBSA buffer (1.76 mM KH2PO4, 10.14 mM Na2HPO4·12H2O, 2.68 mM KCl, 136.89 mM NaCl, and 1% BSA), and then incubated with 100 nM test antibody on ice for 1 hour. The cells were washed once with staining buffer, and Alexa 647-conjugated mouse anti-human FC antibody was added and incubated in the dark on ice for 30 minutes. The samples were washed once with staining buffer and then analyzed by flow cytometry. TY21585, TY21586, TY21580, TY21687, TY21689, TY21680, and TY21691 ( Figure 4A ) were tested in the case of human CTLA4, PD1, LAG3, Tim3, and B7-H3; TY21585, TY21586, TY21580 ( Figure 4B ) were further tested in the case of human CD95, TNFR1, OX40, and CD40; in addition, TY21586, TY21580 ( Figure 4C ) were tested in the case of human PD-L1, BLTA, VISTA, PDL2, ICOS, and B7-H4. As Figures 4A - 4C shown, all test antibodies specifically bound to human CTLA4 but not to any other test antigen (or parental cells transfected with empty vector).

[0350] Ligand competition binding according to ELISA

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

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

[0353] Ligand competition binding according to flow cytometry

[0354] The ability of the antibodies to block the binding of CTLA4 to its cognate ligands CD80 and CD86 was also tested by flow cytometry. Plasmids encoding full-length human CTLA4 were transiently expressed in HEK293F cells. The cells were washed with staining buffer (PBSA buffer including 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 minutes, 100 nM of biotinylated human CD80-Fc-Bio or CD86-Fc-Bio was added to each well and incubated on ice for an additional 1 hour. The cells were washed once with staining buffer and 100 μL of staining buffer containing Alexa fluor 633-conjugated streptavidin was added and incubated on ice in the dark for 30 minutes. The cells were washed once and analyzed by CytoFlex flow cytometry. As Figure 6A shown, all of the test 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; and TY21585, TY21587, TY21589, TY21591 achieving less effective blocking. TY21589 showed little to no blocking. As Figure 6B shown, all of the test 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 to FcγR

[0356] Next, the binding affinities 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) were tested. Using Biacore according to the manufacturer's guidelines TMThe T200 instrument (Biacore AB, Uppsala, Sweden) was used to examine protein binding by surface plasmon resonance (SPR) analysis. According to the instructions of the amine coupling kit (GE Biacore #BR-1000-50), Protein L (Sino Biological Inc. 11044-H07E) was immobilized on a CM5 chip by coupling it through its amine groups to the carboxylated surface of the sensor chip. The immobilized anti-human IgG (Fc) antibody was used to capture TY21586, TY21580, and TAC2114. Serial concentrations (12.5, 25, 50, 25, 100, and 200 nM) of the FcγR protein diluted in the running buffer were injected at a flow rate of 30 μl / min. The running buffer used was HBS-EP (100 mM HEPES, 1.5 M sodium chloride, 0.05% surfactant P20, pH 7.6). According to the manufacturer's guidelines, the association and dissociation curves were fitted 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 showed similar affinities for binding to FcγR compared to the reference antibody (TAC2114).

[0357] Table 7: Binding of antibodies to FcγR

[0358]

[0359] Binding to FcRn

[0360] According to the manufacturer's guidelines, Biacore TM T200 instrument (Biacore AB, Uppsala, Sweden) was used to examine the binding affinity of the test antibodies for recombinant human FcRn by surface plasmon resonance (SPR) analysis. According to the instructions of the amine coupling kit (GE Biacore #BR-1000-50), human FcRn protein (Sino Biological Inc. 11044-H07E) was immobilized on a CM5 chip by coupling it through its amine groups to the carboxylated surface of the sensor chip. Each 100 nM antibody was diluted in the running buffer (50 mM NaPO4, 150 mM NaCl, and 0.05% (v / v) surfactant 20, pH 6.0) and injected as a sample for 120 seconds at a flow rate of 30 μl / min. As Figure 7As shown, compared to TAC2114, antibodies TY21585, TY21580, TY21591, TY21687, and TY21691 exhibit a higher binding % to FcRn, indicating that the IgG-FcRn complex on the Biacore chip can undergo conformational changes that stabilize the complex compared to the reference antibody (TAC2114). Antibodies TY21586, TY21587, TY21589, TY21689, and TY21680 show low binding %.

[0361] Human PBMC activation

[0362] Preliminary studies showed that TY21580 did not stimulate human T cell activation or proliferation. Since CTLA4 activity on T cells is related to the first signal (TCR / CD3) and the second signal involving B7-CD28 / CTLA-4, human PBMCs were selected and the activity of TY21580 was determined in the presence of a low concentration of anti-CD3 antibody. The anti-CD3 antibody (OKT-3) was coated overnight on a 96-well plate at 4°C. After washing, 1x10 5 freshly isolated human PBMCs were added to each well, followed by the addition of the test article at different concentrations. At 48 hours after stimulation, the induction of IL-2 was measured using the Human IL-2 ELISA Ready-SET-Go (Invitrogen) kit. IFNγ in the supernatant was measured using the Human IFNγ ELISA Ready-SET-Go (Invitrogen) kit. As Figure 8A and Figure 9 shown, in the presence of the anti-CD3 antibody, antibody TY21580 significantly increased the activation of human PBMCs, while TY21580 alone was not active.

[0363] Dendritic cell MLR assay

[0364] In the case of the following three pairs of donors, DC-MLR assays were performed using monocyte-derived DCs and CD4+ T lymphocytes: D42 / D109, D32 / D104, and D104 / D42 ( Figure 10)). To obtain DC cells, 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 in vitro culture for 6 days in RPMI-1640 supplemented with 10% heat-inactivated FBS, 1% penicillin / streptomycin, 20 ng / mL rhGM-CSF, and 20 ng / mL rhIL-4. On day 3, the medium was replaced with fresh medium. On day 6, DC maturation was induced for 24 hours in RPMI-1640 medium 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 selection. The test article was titrated to the corresponding concentration (as Figure 10 shown). The collected DCs (1x10 4 ) were co-cultured with allogeneic CD4+ T cells (1x10 5 ) in the presence or absence of the titrated test article. 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. As Figure 10 shown, antibody TY21580 showed weak activity in the DC-MLR assay using human CD4+ T cells and DCs.

[0365] ADCC activity of antibody TY21580

[0366] HEK293F cells overexpressing human CTLA-4 were used as target cells to evaluate the ADCC activity mediated by TY21580. Human NK cells were freshly isolated from human PBMCs using the human NK isolation kit (StemCell). 1x10 5 NK cells and 1x10 4 HEK293F / hCTLA-4 cells (E:T ratio 10:1) were mixed with different concentrations of the antibody. After incubation for 4 hours, LDH was measured to determine the ADCC activity. Then the lysis % was calculated using the following formula: Lysis % = [(experimental release value) – mean (target + NK)] / [mean (target maximum release value) – mean (only target)] × 100%. As Figures 11A - 11B shown, TY21580 showed stronger ADCC activity compared to the reference antibody (TAC2114). The isotype control showed no ADCC activity in any case.

[0367] ADCC activity was also evaluated using human Treg cells (A, donor #96; B, donor #12) and NK cells (A, donor #99; B, donor #05). To obtain human Treg cells, human PBMCs were freshly isolated from healthy donors and Treg cells were negatively selected using the EASYSEP TM Human Regulatory T Cell Enrichment Kit (StemCell Technologies). The 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, human PBMCs were freshly isolated from another healthy donor and NK cells were isolated using the Human NK Isolation Kit (StemCell Technologies). The human Treg cells were labeled with 10 μM calcein-AM (Invitrogen) at 37 °C for 30 minutes. After washing three times, the labeled Treg cells were mixed with different concentrations of the test article, followed by the addition of NK cells. 1x10 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 incubation for 4 hours, the concentration of calcein-AM in the supernatant was measured to determine ADCC activity using the following formula: % lysis = [(experimental release value) – mean (target + NK)] / [mean (target maximum release value) – mean (target only)] × 100%. As Figures 12A - 12B shown, antibody TY21580 showed stronger ADCC activity compared to the reference antibody (TAC2114). The isotype control showed no ADCC activity.

[0368] CDC activity of TY21580

[0369] HEK293F cells overexpressing human CTLA-4 were labeled with 10 μM calcein-AM (Invitrogen) at 37 °C for 30 minutes. In the wells of a 96-well plate, different concentrations of the antibody were mixed with 1x10 4 labeled cells and 5% normal human serum complement (NHSC, Quidel). After incubation for 5 hours, the calcein-AM in the supernatant was measured to determine CDC activity ( Figure 13 ).

[0370] Freshly isolated human PBMCs were obtained 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) + ionomycin (1 μM) for 20 hours 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 minutes. In the wells of a 96-well plate, different concentrations of the antibody were mixed with 1 x 10 4 labeled human CD4+ T cells and 5% normal human serum complement (NHSC, Quidel). After a 5-hour 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.

[0371] In summary, these results indicate that the antibodies described herein are capable of binding human CTLA4 with high affinity and specificity, and that the antibodies potently block the interaction of CTLA4 with its cognate ligands CD86 and CD80. Cross-reactivity of the antibodies with CTLA4 from multiple species was also shown. In addition, binding to CTLA4 can modulate T cell activation and induce ADCC activity against CTLA4-expressing cells such as Tregs.

[0372] Example 4: In Vivo Characterization of IgG Switch Antibodies

[0373] As described in the above examples, the species cross-reactivity (human and mouse) of the antibodies allowed for the determination of the anti-tumor efficacy of the antibodies 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.

[0374] Anti-Tumor Efficacy in the MC38 Colorectal Tumor Model

[0375] C57BL / 6 mice (n = 8 / group, female, 6 - 8 weeks old) were subcutaneously inoculated with MC38 (NTCC-MC38) murine colon cancer cells. When tumors were established (80 mm 3 ), treatment was initiated by intraperitoneal injection twice a week for three weeks with an isotype control antibody and three different doses of the antibody TY21580. Tumor growth was monitored twice a week and reported as the mean tumor volume ± s.e.m. over time ( Figures 15A - 15C ). As Figure 15A shown, TY21580 demonstrated potent in vivo anti-tumor activity compared to the isotype control antibody, with complete tumor regression at all three doses. AsFigure 15B As shown, up to 60 days after treatment, 8 out of 8 mice in the 10 mg / kg TY21580 group, 7 / 8 in the 2.5 mg / kg TY21580 group, and 6 / 8 in the 0.5 mg / kg TY21580 group remained tumor-free. When the mice in the 10 mg / kg TY21580 group were rechallenged, durable memory of immunity against MC38 tumor cells was demonstrated, as Figure 15C shown.

[0376] Antitumor Efficacy in the CT26 Colorectal Tumor Model

[0377] BALB / c mice (n = 8 / group, female, 7 - 8 weeks old) were subcutaneously inoculated with CT26 (Shanghai Institutes for Biological Sciences) murine colon cancer cells. When tumors were established (70 mm 3 ), treatment was initiated by intraperitoneal injection twice a week with isotype control antibody and two different doses of antibody TY21580. Tumor growth was monitored twice a week and reported as mean tumor volume ± s.e.m. over time. As Figure 16 shown, TY21580 exhibited potent in vivo antitumor activity compared to the isotype control antibody, with nearly 100% inhibition achieved at doses as low as 0.1 - 1 mg / kg.

[0378] Antitumor Efficacy in the H22 Liver Tumor Model

[0379] BALB / c mice (n = 5 / group, female, 7 - 8 weeks old) were subcutaneously inoculated with H22 (China Center for Type Culture Collection) murine hepatoma cells. When tumors were established (60 mm3), treatment was initiated by intraperitoneal injection twice a week with 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 a week and reported as mean tumor volume ± s.e.m. over time. As Figure 17 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. In addition, administration of TY21580 at 1 mg / kg resulted in tumor regression.

[0380] Antitumor Efficacy in the Lewis Lung Tumor Model

[0381] Female C57BL / 6 mice (n = 6 / group, 8 weeks old) were subcutaneously inoculated with Lewis (JenNio Bio, Guandong, China) murine lung cancer cells. When tumors were established (70 mm 3 ), treatment was initiated by intraperitoneal injection twice a week with an isotype control antibody or antibodies TY21580, TY21687, TY21680, or TY21691, all at a dose of 5 mg / kg. Tumor growth was monitored twice a week and reported as mean tumor volume ± s.e.m. over time. As Figure 18 shown, antibodies TY21580, TY21687, and TY21680 showed significant inhibition of tumor growth compared to the isotype control antibody, while antibody TY21691 did not show potent anti-tumor activity.

[0382] Anti-tumor efficacy in the PAN02 pancreatic tumor model

[0383] Female C57BL / 6 mice (n = 8 / group, 6 weeks old) were subcutaneously inoculated with PAN-02 (CAMS Cell Culture Center) murine pancreatic cancer cells. When tumors were established (85 mm 3 ), treatment was initiated by intraperitoneal injection twice a week with an isotype control antibody or antibody TY21580 at three different doses (0.5 mg / kg, 2 mg / kg, 0.5 mg / kg). Tumor growth was monitored twice a week and reported as mean tumor volume ± s.e.m. over time. As Figure 19 shown, TY21580 showed potent anti-tumor activity in a dose-dependent manner compared to the isotype control antibody.

[0384] Anti-tumor efficacy of antibody TY21580 monotherapy or in combination with an anti-CD137 antibody in the 3LL lung tumor model

[0385] Female C57BL / 6 mice (n = 10 / group, 6 - 8 weeks old) were subcutaneously inoculated with 3LL (JCRB) murine lung cancer cells. When tumors were established (75 mm 3) When the tumor size reached [a certain size], treatment was initiated by intraperitoneal injection twice a week with an isotype control antibody, TY21580 (10 mg / kg), an anti-CD137 antibody (10 mg / kg), or a combination of TY21580 and the anti-CD137 antibody. The anti-CD137 antibody is a proprietary monoclonal antibody developed with the ability to bind both human and murine CD137 (see PCT application number PCT / CN2017 / 098332, which is incorporated herein by reference in its entirety). Tumor growth was monitored twice a week and reported as the mean tumor volume ± s.e.m. over time. As Figures 20A - 20B shown, both TY21580 and the anti-CD137 antibody showed potent antitumor activity compared to the isotype control antibody, and the combination inhibited tumor growth to a greater extent than either single monotherapy alone.

[0386] Re-challenge of mice completely responsive to TY21580

[0387] BALB / c mice (n = 8 / group, female, 7 - 8 weeks old) were subcutaneously inoculated with H22 (China Center for Type Culture Collection) murine hepatocarcinoma cells. When the tumors were established (60 mm 3 ) in size, treatment was initiated by intraperitoneal injection twice a week for three weeks with an isotype control antibody or antibody TY21580 at two different doses (1 mg / kg, 10 mg / kg). Tumor growth was monitored twice a week and reported as the mean tumor volume ± s.e.m. over time. Compared to the isotype control antibody, TY21580 at both doses led to complete tumor regression several days after the last dose, and at 60 days post-treatment, the mice remained tumor-free. Then, on day 60, the mice in the 10 mg / kg TY21580 treatment group were subcutaneously re-challenged with H22 tumor cells in the opposite flank, and tumor growth was monitored. As Figure 21 shown, after re-challenge with the same tumor cells, these mice remained tumor-free, indicating the generation of specific antitumor memory in these mice. Meanwhile, a re-challenge control group was established with naïve mice inoculated with the same number of H22 tumor cells, and their tumors grew rapidly.

[0388] Antibody pharmacokinetics

[0389] Pharmacokinetic studies were conducted on antibodies TY21585, TY21586, TY21580, and TY21591 in BALB / c female mice (at approximately 8 weeks of age). By tail vein injection, three mice per group were intravenously injected with the test antibodies at 10 mg / kg. Blood samples (approximately 20 μL each) were collected at 1 hour, 8 hours, 48 hours, 168 hours, 336 hours, and 500 hours after dosing. Blank control blood was collected from three naive female mice without antibody administration. Serum concentrations of each test antibody were determined by ELISA, in which CTLA4-His-Fc was used for capture and HRP-labeled anti-human IgG (Fab specific) antibody (Sigma) was used for detection. As Figure 22 shown, in mice, TY21586 exhibited similar pharmacokinetics to TAC2114, while TY21585, TY21580, and TY21591 were cleared much more rapidly.

[0390] Pharmacokinetic studies were also conducted on TY21586 and TY21580 in naive cynomolgus monkeys. Each antibody was administered to one female and one male monkey at 10 mg / kg by intravenous bolus injection. Serum samples were collected before dosing (0 hour) and at 0.25 hour, 1 hour, 8 hours, 24 hours, 72 hours, 120 hours, 168 hours, 240 hours, 336 hours, 504 hours, and 672 hours after dosing. Serum concentrations of TY21586 and TY21580 were determined by ELISA, in which CTLA4-His-Fc was used for capture and HRP-labeled anti-human IgG (Fab specific) antibody (Sigma) was used for detection. As Figure 23 and Figure 24 shown, compared to TY21586, TY21580 was cleared more rapidly in monkeys, probably due to the rapid increase in anti-drug antibodies observed in these animals.

[0391] Repeated-dose toxicity studies

[0392] A repeated-dose toxicity study of TY21580 was conducted in normal BALB / c mice. On days 1, 4, 7, and 11, vehicle control or antibody TY21580 (at 25 mg / kg or 50 mg / kg) was administered intraperitoneally (10 mL / kg). Five female and five male mice (5 weeks old) were included in each group. The mice were monitored daily for abnormal behavior and symptoms, and food intake and body weight were measured daily. On day 14, the animals were euthanized for postmortem examination and other analyses. Blood was collected from each animal, with up to six blood samples (three males, three females) collected per group for hematology (RBC, platelet, WBC, WBC differential) and / or blood biochemistry (ALT, AST, GLB, ALP, and LDH, etc.) analyses. The following organs were collected from each mouse and weighed: heart, lung, thymus, liver, spleen, kidney, testis, and ovary. Liver samples from six animals (three males, three females) per group were fixed in FFPE. FFPE blocks of liver tissue were prepared, sectioned, and stained with H&E for histopathological analysis.

[0393] During the entire in-life period of the study, no abnormal behavior was observed, or there was no unscheduled animal death. Compared to vehicle treatment, TY21580 did not affect the food intake and body weight of the animals. In addition, postmortem examination did not show any obvious lesions in the mice of the treatment groups at either dose level, with the exception of an increased spleen weight in the TY21580-treated group ( Figures 25A - 25B ). Hematological analysis did not show any significant changes, as indicated by the blood biochemical parameters tested in the mice treated with TY21580. No obvious abnormalities were found in the histopathological sections of the liver from the mice ( Figure 26 ). In summary, in this study, TY21580 was well tolerated, with no significant toxicity observed in the mice.

[0394] In summary, these results indicate that the CTLA4 antibody described herein is extremely safe for mice, has potent anti-tumor activity, and can induce durable immune memory against tumor cells.

[0395] Example 5: Antibody developability profile

[0396] For developability assessment, purified TY21586 and TY21580 were exchanged into a storage buffer (20 mM histidine, pH 5.5). All experiments were conducted in the storage buffer, including solubility, stability under accelerated stress conditions, and differential scanning fluorimetry (DSF) testing. For all SEC-HPLC analyses, a TSKgel column (Tosoh Bioscience G3000SWxl) was used.

[0397] Antibody solubility

[0398] Samples containing antibody TY21586 or TY21580 were formulated at a concentration greater than 100 mg / mL in storage buffer, and the amount of high molecular weight (HMW) protein aggregates was tested (Table 8). The antibody was then adjusted to approximately 12 mg / mL in storage buffer. Samples (12 μg each) were then assayed by SEC-HPLC to detect high molecular weight protein aggregates. As shown in Figure 27, in the case of the antibody formulated at a high concentration (above 100 mg / mL), no significant increase in HMW aggregates was observed over 30 minutes.

[0399] Table 8: Antibody solubility

[0400] Antibody Name Concentration (mg / mL) Aggregation (HMW%) TY21586 197.8 0 TY21580 126.0 +0.10

[0401] Antibody stability under accelerated stress conditions

[0402] Antibody stability was also examined under accelerated stress conditions. The results of these experiments are s...

Claims

1. An anti-CTLA4 antibody, wherein the antibody binds to human CTLA4 and is cross-reactive with cynomolgus monkey, mouse, rat, and dog CTLA4.

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

3. The antibody according to claim 2, wherein said K D is measured by surface plasmon resonance (SPR).

4. The antibody according to any one of claims 1-3, wherein binding of the anti-CTLA4 antibody induces antibody-dependent cellular cytotoxicity (ADCC) against CTLA4-expressing human cells or human Treg cells, and wherein the ADCC activity of the anti-CTL4 antibody is higher than the ADCC activity of ipilimumab.

5. The antibody according to any one of claims 1-4, wherein (a) the antibody specifically binds to an epitope comprising amino acid residues Y105 and L106 of human CTLA4 but not comprising residue I108, wherein the numbering of the amino acid residues is according to SEQ ID NO:207; and / or (b) in an assay where CD80 and / or CD86 are plate-bound or when human CTLA4 is present on the cell surface, the anti-CTLA4 antibody has an IC50 higher than the IC50 of ipilimumab for blocking the binding of CD80 and / or CD86 to human CTLA4.

6. The antibody according to any one of claims 1-5, wherein 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: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. 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 according to a formula selected from the group consisting of: Formula (VII): ARX1X2X3X4FDX5 (SEQ ID 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; Formula (VIII): ARX1GX2GYFDX3 (SEQ ID NO:8), where X1 is D or L, X2 is F or Y, and X3 is V or Y; Formula (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; Formula (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 said HVR-L1 comprises an amino acid sequence according to a formula selected from the group consisting of: Formula (XI): RASQX1X2X3SX4LX5 (SEQ ID NO:11), where 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; wherein said HVR-L2 comprises an amino acid sequence according to Formula (XIV): X1ASX2X3X4X5GX6 (SEQ ID NO:14), where 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 said HVR-L3 comprises an amino acid sequence according to a formula selected from the group consisting of: Formula (XV): YCX1X2X3X4X5X6PX7T (SEQ ID 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; 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.

7. The antibody according to any one of claims 1-3, wherein the HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-29, the HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 30-39, the HVR-H3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 40-52, the HVR-L1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-65, the HVR-L2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 66-69, and the HVR-L3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 70-81.

8. The antibody according to any one of claims 1-3, wherein 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-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, 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, 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; 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, 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; or m) 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.

9. The antibody according to claim 7 or claim 8, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:82-94 or an amino acid sequence having at least 90% sequence identity with a 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 or an amino acid sequence having at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NO:95-107.

10. The antibody according to any one of claims 7-9, wherein the antibody comprises: a) 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; b) 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; c) 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; d) 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; e) 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; f) 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; g) 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; h) 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; i) 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; j) 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; k) 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; l) 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; or m) 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.

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