Anti-CTLA4 antibody and method for producing and using the same

Abstract

This specification provides for antibodies (or their antigen-binding fragments) that conjugate to human CTLA4 (e.g., antibodies that bind to human CTLA4) and have enhanced effector function, masked antibodies that bind to human CTLA4 (e.g., activatable antibodies), nucleic acid molecules encoding them, pharmaceutical compositions thereof, and methods for their therapeutic use (e.g., for the treatment of cancer).

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of International Application No. PCT / CN2023 / 088000, filed on April 13, 2023, the entire contents of which are incorporated herein by reference. Submission of Sequence Listing in ASCII Text File

[0002] The content of the electronic sequence list (695402001840SEQLIST.xml, size: 223,607 bytes, creation date: April 12, 2023) is incorporated herein by reference in its entirety.

[0003] The present disclosure relates to antibodies that bind to human cytotoxic T - lymphocyte antigen 4 (CTLA4), masked antibodies (e.g., activatable antibodies) that bind to human CTLA4, nucleic acids encoding them, pharmaceutical compositions thereof, and their therapeutic uses.

Background Art

[0004] CTLA4 is a member of the immunoglobulin (Ig) superfamily of proteins that act to downregulate T cell activation and maintain immunogenic homeostasis. In vivo antibody-mediated blockade of CTLA4 has been shown to enhance the anti-cancer immune response in a syngeneic mouse model of prostate cancer (Kwon et al. (1997) Pro c Natl Acad Sci USA, 94(15):8099-103). Furthermore, blockade of CTLA4 function has been shown to enhance the anti-tumor T cell response at various stages of tumor growth in tumor-bearing mice (Yang et al. (1997) Cancer Res 57(18):4036-41, Hurwitz et al. (1998) Pro c Natl Acad Sci USA 95(17):10067-7). However, the conversion from preclinical animal models to human safety is often insufficient, making the development of antibody-based therapeutics suitable for human use still challenging. Therefore, there is a need for cross-reactive anti-CTLA4 antibodies between humans and experimental animals (e.g., mice, monkeys, rats) to enable animal model studies while simultaneously providing suitable human therapeutic candidates. Furthermore, since CTLA4 is expressed on healthy cells such as T effector cells, CTLA4 can adversely affect healthy cells and cause dangerous side effects such as immune checkpoint inhibitor-associated diabetes (Akturk, HK, et al. "Immune checkpoint inhibitor-induced type 1 diabetes: a systematic review and meta-analysis." Diabetic Medicine 36.9 (2019): 1075-1081). Thus, there is a need for the development of safer anti-CTLA4 antibodies that are active only in specific situations, such as the protease-rich tumor microenvironment. [Overview of the project]

[0005] In one embodiment, an isolated antibody conjugated to human CTLA4, provided herein, the antibody comprises a heavy chain comprising a heavy chain variable region (VH) and a light chain comprising a light chain variable region (VL), wherein the heavy chain comprises a heavy chain constant region comprising a human IgG1 Fc region, the heavy chain constant region comprising one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, wherein the numbering of the residues follows EU numbers. In some embodiments, the isolated antibody induces antibody-dependent cell-mediated cytotoxicity (ADCC) against CTLA4-expressing human cells or human Treg cells, and the ADCC activity of the isolated antibody is higher than that of a control antibody containing a human IgG1 Fc region without substitution, and / or the ADCC activity of the isolated antibody is higher than that of ipilimumab.

[0006] In some embodiments of this model, VH includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 1, CDR-H2 containing the amino acid sequence of SEQ ID NO: 2, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 3; VL includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 4, CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 6. In a particular embodiment, VH includes the amino acid sequence of SEQ ID NO: 74, and / or VL includes the amino acid sequence of SEQ ID NO: 75. In one embodiment, the heavy chain includes the amino acid sequence of SEQ ID NO: 91, and the light chain includes the amino acid sequence of SEQ ID NO: 90. In another embodiment, the heavy chain includes the amino acid sequence of SEQ ID NO: 91 without a C-terminal lysine, and the light chain includes the amino acid sequence of SEQ ID NO: 90. In other embodiments of this model, VH includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 12, CDR-H2 containing the amino acid sequence of SEQ ID NO: 13, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 14; VL includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 15, CDR-L2 containing the amino acid sequence of SEQ ID NO: 16, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 17. In a particular embodiment, VH includes the amino acid sequence of SEQ ID NO: 66, and / or VL includes the amino acid sequence of SEQ ID NO: 67. In yet another embodiment of this model, VH includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 44, CDR-H2 containing the amino acid sequence of SEQ ID NO: 45, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 53; VL includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 54, CDR-L2 containing the amino acid sequence of SEQ ID NO: 16, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 55. In a particular embodiment, VH comprises the amino acid sequence of SEQ ID NO: 84, and / or VL comprises the amino acid sequence of SEQ ID NO: 85. In yet another embodiment of this aspect, VH comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 44, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 45, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 46, and VL comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 47, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48.In a particular embodiment, VH comprises the amino acid sequence of SEQ ID NO: 80, and / or VL comprises the amino acid sequence of SEQ ID NO: 81.

[0007] In some embodiments of this model, the antibody comprises two heavy chains and two light chains, each human IgG1 Fc domain of the heavy chains comprising an S298A substitution, an E333A substitution, and a K334A substitution. In some embodiments of this model, the antibody comprises two heavy chains and two light chains, each human IgG1 Fc domain of the heavy chains comprising an S298A substitution, an E333A substitution, a K334A substitution, and a K326A substitution. In some embodiments of this model, the antibody comprises two heavy chains and two light chains, each human IgG1 Fc domain of the heavy chains comprising an F243L substitution, an R292P substitution, a Y300L substitution, and a P396L substitution. In some embodiments of this model, the antibody comprises two heavy chains and two light chains, each human IgG1 Fc domain of the heavy chains comprising an F243L substitution, an R292P substitution, a Y300L substitution, a V305I substitution, and a P396L substitution. In some embodiments of this model, the antibody comprises two heavy chains and two light chains, each human IgG1 Fc domain of the heavy chains comprising an F243L substitution, an R292P substitution, a Y300L substitution, a L235V substitution, and a P396L substitution. In some embodiments of this model, the antibody comprises two heavy chains and two light chains, each human IgG1 Fc domain of the heavy chains comprising an S239D substitution, an I332E substitution, and an A330L substitution. In some embodiments of this model, the antibody comprises two heavy chains and two light chains, each human IgG1 Fc domain of the heavy chains comprising an S239D substitution, and an I332E substitution.

[0008] In another embodiment, a masked antibody, a masking peptide (MP), and an antibody that bind to human CTLA4 are provided herein, wherein the antibody comprises a heavy chain comprising a heavy chain variable region (VH) and a light chain comprising a light chain variable region (VL), the MP being ligated to the N-terminus of the VH or VL, and the MP comprising a masking unit (MU) and a linkage unit (LU) from the N-terminus to the C-terminus, the MU comprising an amino acid sequence selected from the list of SEQ ID NOs: 146-151. In some embodiments of this embodiment, the VH comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and the VL comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In a particular embodiment, the VH comprises the amino acid sequence of SEQ ID NO: 74, and / or the VL comprises the amino acid sequence of SEQ ID NO: 75. In other embodiments of this model, VH includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 12, CDR-H2 containing the amino acid sequence of SEQ ID NO: 13, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 14; VL includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 15, CDR-L2 containing the amino acid sequence of SEQ ID NO: 16, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 17. In a particular embodiment, VH includes the amino acid sequence of SEQ ID NO: 66, and / or VL includes the amino acid sequence of SEQ ID NO: 67. In yet another embodiment of this model, VH includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 44, CDR-H2 containing the amino acid sequence of SEQ ID NO: 45, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 53; VL includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 54, CDR-L2 containing the amino acid sequence of SEQ ID NO: 16, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 55. In a particular embodiment, VH comprises the amino acid sequence of SEQ ID NO: 84, and / or VL comprises the amino acid sequence of SEQ ID NO: 85.In yet another embodiment of this aspect, VH includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 44, CDR-H2 containing the amino acid sequence of SEQ ID NO: 45, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 46; VL includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 47, CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 48. In certain embodiments, VH includes the amino acid sequence of SEQ ID NO: 80, and / or VL includes the amino acid sequence of SEQ ID NO: 81. In some embodiments, the masked antibody is a Fab fragment, scFv, F(ab′)2 fragment, or Fv fragment.

[0009] In some embodiments of this model, the masking peptide (MP) further comprises an N-terminal unit (NU) ligated to the N-terminus of the MU. In some embodiments, the N-terminal unit is approximately 1 to 10 amino acid residues long. In certain embodiments, the N-terminal unit comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 143 to 145.

[0010] In some embodiments of this model, the linkage unit (LU) does not include a cleavage site. In some embodiments, the LU includes a linker. In certain embodiments, the linker includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 155-161. In certain embodiments, the linker includes the amino acid sequence of SEQ ID NO: 159.

[0011] In some embodiments of this model, the masked antibody is an activatable antibody. In some embodiments where the antibody is an activatable antibody, the LU includes at least a first cleavage site (C1). In some embodiments, the first cleavage site (C1) is a protease cleavage site of a protease selected from the group consisting of urokinase-type plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, tobacco etch virus (TEV) protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE. In certain embodiments, the first cleavage site (C1) comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 152-154. In some embodiments where the antibody is an activatable antibody, the LU further comprises a second cleavage site (C2). In some embodiments, the second cleavage site (C2) is a protease cleavage site of a protease selected from the group consisting of urokinase-type plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, tobacco 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 a particular embodiment, the second cleavage site (C2) includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 152-154.In certain embodiments, the second cleavage site (C2) includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 152-154. In some embodiments, the first and second cleavage sites are different. In some embodiments, where the antibody is an activatable antibody, the LU further includes a first linker. In certain embodiments, the first linker includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 155-161. In some embodiments, the LU further includes a second linker (L2). In certain embodiments, the second linker includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 155-161. In some embodiments, the LU includes a first cleavage site (C1), a first linker (L1), a second cleavage site (C2), and a second linker (L2) from the N-terminus to the C-terminus (i.e., a C1-L1-C2-L2 configuration). In other embodiments, LU includes a first linker (L1), a first cleavage site (C1), and a second linker (L2) from the N-terminus to the C-terminus (i.e., an L1-C1-L2 configuration). In some embodiments, LU includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 99 to 103. In a particular embodiment, MU includes the amino acid sequence of SEQ ID NO: 146, and LU includes the amino acid sequence of SEQ ID NO: 99. In a particular embodiment, MU includes the amino acid sequence of SEQ ID NO: 147, and LU includes the amino acid sequence of SEQ ID NO: 99. In a particular embodiment, MU includes the amino acid sequence of SEQ ID NO: 148, and LU includes the amino acid sequence of SEQ ID NO: 99. In a particular embodiment, MU includes the amino acid sequence of SEQ ID NO: 149, and LU includes the amino acid sequence of SEQ ID NO: 99. In a particular embodiment, MU includes the amino acid sequence of SEQ ID NO: 150, and LU includes the amino acid sequence of SEQ ID NO: 99. In a particular embodiment, MU comprises the amino acid sequence of SEQ ID NO: 148, and LU comprises the amino acid sequence of SEQ ID NO: 100. In a particular embodiment, MU comprises the amino acid sequence of SEQ ID NO: 148, and LU comprises the amino acid sequence of SEQ ID NO: 101. In a particular embodiment, MU comprises the amino acid sequence of SEQ ID NO: 148, and LU comprises the amino acid sequence of SEQ ID NO: 102.In certain embodiments, MU includes the amino acid sequence of SEQ ID NO: 148, and LU includes the amino acid sequence of SEQ ID NO: 101. In certain embodiments, MU includes the amino acid sequence of SEQ ID NO: 151, and LU includes the amino acid sequence of SEQ ID NO: 100. In certain embodiments, MU includes the amino acid sequence of SEQ ID NO: 151, and LU includes the amino acid sequence of SEQ ID NO: 101. In certain embodiments, MU includes the amino acid sequence of SEQ ID NO: 150, and LU includes the amino acid sequence of SEQ ID NO: 101. In certain embodiments, MU includes the amino acid sequence of SEQ ID NO: 150, and LU includes the amino acid sequence of SEQ ID NO: 102. In certain embodiments, MU includes the amino acid sequence of SEQ ID NO: 150, and LU includes the amino acid sequence of SEQ ID NO: 102. In certain embodiments, MU includes the amino acid sequence of SEQ ID NO: 150, and LU includes the amino acid sequence of SEQ ID NO: 103. In certain embodiments, MU includes the amino acid sequence of SEQ ID NO: 150, and LU includes the amino acid sequence of SEQ ID NO: 103. In some embodiments, the masking peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 162-177.

[0012] In certain embodiments of this model, the masked antibody comprises two heavy chains and two light chains, wherein each human IgG1 Fc domain of the heavy chains contains one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, where residue numbering is according to EU numbering. In some embodiments of this model, the masked antibody comprises two heavy chains and two light chains, wherein each human IgG1 Fc domain of the heavy chains contains the following substitutions: S298A substitution, E333A substitution, and K334A substitution. In some embodiments of this embodiment, the masked antibody comprises two heavy chains and two light chains, with each human IgG1 Fc domain of the heavy chain comprising the following substitutions: S298A substitution, E333A substitution, K334A substitution, and K326A substitution. In some embodiments of this embodiment, the masked antibody comprises two heavy chains and two light chains, with each human IgG1 Fc domain of the heavy chain comprising the following substitutions: F243L substitution, R292P substitution, Y300L substitution, and P396L substitution. In some embodiments of this embodiment, the masked antibody comprises two heavy chains and two light chains, with each human IgG1 Fc domain of the heavy chain comprising the following substitutions: F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution. In some embodiments of this model, the masked antibody comprises two heavy chains and two light chains, with each human IgG1 Fc domain of the heavy chains comprising the following substitutions: F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution. In some embodiments of this model, the masked antibody comprises two heavy chains and two light chains, with each human IgG1 Fc domain of the heavy chains comprising the following substitutions: S239D substitution, I332E substitution, and A330L substitution. In some embodiments of this model, the masked antibody comprises two heavy chains and two light chains, with each human IgG1 Fc domain of the heavy chains comprising the following substitutions: S239D substitution and I332E substitution.In some embodiments, each heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 104-105, and each light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 106-121. In a particular embodiment, each heavy chain comprises the amino acid sequence of SEQ ID NO: 105, and the light chain comprises the amino acid sequence of SEQ ID NO: 121. In some embodiments, the masked antibody induces antibody-dependent cell-mediated cytotoxicity (ADCC) against CTLA4-expressing human cells or human Treg cells, and the ADCC activity of the isolated antibody is higher than that of a control antibody containing a human IgG1 Fc region without substitutions, and / or the ADCC activity of the isolated antibody is higher than that of ipilimumab.

[0013] In another embodiment, provided herein are isolated polynucleotides encoding one or more polypeptide chains of either an isolated antibody or a masked antibody as described herein. In yet another embodiment, provided herein are vectors comprising the polynucleotides. In some embodiments, the vector is an expression vector and / or a presentation vector. In yet another embodiment, provided herein are host cells comprising the polynucleotides or the vector. In some embodiments, the host cell is a eukaryotic cell. In a particular embodiment, the host cell is a Chinese hamster ovary (CHO) cell. In yet another embodiment, a method is provided for producing either an antibody or a masked antibody as described herein, the method comprising culturing the host cell under conditions suitable for producing the antibody or an activatable antibody. In some embodiments, the method further comprises recovering the antibody or masked antibody produced by the cells.

[0014] In yet another embodiment, the herein provides a pharmaceutical composition comprising an isolated or masked antibody as described herein and a pharmaceutically acceptable carrier.

[0015] In yet another aspect, this Specified provides a method for treating or delaying the progression of cancer in subjects requiring such treatment, the method comprising administering an effective amount of either an isolated antibody or a masked antibody as described herein to the subject, or a pharmaceutical composition comprising either an isolated antibody or a masked antibody as described herein.

[0016] In yet another embodiment, a method for reducing the size of a solid tumor in a subject who needs to reduce the size of a solid tumor, the solid tumor being approximately 400 to 1000 mm3 in size, is provided herein, and the method comprises administering to a subject an effective amount of either an isolated antibody or a masked antibody as described herein, or a pharmaceutical composition comprising either an isolated antibody or a masked antibody as described herein.

[0017] In yet another embodiment, the Specified provides a method for reducing off-target toxicity in a subject having cancer requiring such reduction, the method comprising administering to a subject an effective amount of either an isolated antibody or a masked antibody as described herein, or a pharmaceutical composition comprising either an isolated antibody or a masked antibody as described herein. In some embodiments, the tumor is a colorectal tumor or a lung tumor. In some embodiments, the tumor is a cold tumor. In certain embodiments, the cold tumor is selected from the group consisting of pancreatic cancer, ovarian cancer, and head and neck cancer.

[0018] In some embodiments of any of the methods described herein, the method further comprises administering an effective amount of at least one additional therapeutic agent to a subject. In some embodiments, the at least one additional therapeutic agent is selected from the group consisting of viral gene therapy, immune checkpoint inhibitors, targeted therapies, radiotherapy, vaccine therapy, and chemotherapy. In certain embodiments, the at least one additional therapeutic agent is an immune checkpoint inhibitor. In certain embodiments, the at least one additional therapeutic agent is a PD-1 inhibitor. In some embodiments of any of the methods described herein, the method comprises administering an effective amount of the antibody, the masked antibody, or the pharmaceutical composition to the subject before or after surgery to remove a solid tumor in the subject. In yet another embodiment, a method is provided herein for inducing phagocytosis of cells expressing CTLA4, the method comprising contacting cells expressing CTLA4 with an effective amount of either the isolated antibody or the masked antibody described herein.

[0019] In some embodiments, the phagocytosis is antibody-dependent cell-mediated phagocytosis (ADCP). In certain embodiments, the antibody or masked antibody has a semi-maximal effective concentration (EC50) of less than 3 nM for inducing ADCP in cells expressing CTLA4.

[0020] It should be understood that one, some, or all of the characteristics of the various embodiments described above and herein may be combined to form other embodiments of the Disclosure. These and other embodiments of the Disclosure will be apparent to those skilled in the art. These and other embodiments of the Disclosure are further described by embodiments for carrying out subsequent inventions. [Brief explanation of the drawing]

[0021] [Figure 1] This shows the functional representation of scFv targeting CTLA4 on yeast, as determined by flow cytometry.

[0022] [Figure 2] An example of the selection process of an activatable antibody targeting human CTLA4 is shown. A yeast library displaying fusion proteins was subjected to FACS-based screening several times.

[0023] [Figure 3] The binding affinity of CTLA4-activatable antibody clones in scFv format, including CTLA4-activatable antibody clone BC3864, compared to the scFv fragment of the target antibody without the masking peptide, with the masking peptide intact or cleaved by TEV protease.

[0024] [Figure 4A-4D] It is a figure comparing the masking efficiency of an exemplary CTLA4-activatable antibody against recombinant human CLTA4-hisFc, measured by enzyme-linked immunosorbent assay (ELISA), with the parental antibody TY21580.

[0025] [Figure 5A-5B] The activities of CTLA4-activatable antibodies TY24649 and TY24652 after removal of the masking peptide are shown. Figure 5A shows the reducing CE-SDS results of activatable antibodies TY24649 and TY24652 untreated and treated with protease MMP9. Figure 5B shows the binding of activatable antibodies TY24649 and TY24652 treated with protease MMP9, determined by ELISA, compared to the parental antibody TY21580.

[0026] [Figure 6A-6B] The size exclusion chromatography (SEC) profiles of exemplary activatable antibodies under accelerated stress conditions are shown. Figure 6A shows the SEC profiles of activatable antibodies TY24649 and TY24652 after repeating freeze-thaw 6 cycles compared to control conditions. Figure 6B shows the SEC profiles of exemplary activatable antibodies after storage at 40 °C for up to 28 days compared to control conditions.

[0027] [Figure 7] This shows the hydrophobic interaction chromatography (HIC) profiles of exemplary activatable antibodies compared to the parent antibody TY21580 and the commercially available drug Tecentriq.

[0028] [Figure 8] This shows the effects of ELISA-measured isotype control antibodies, parent antibodies TY21580 and TY24040, or exemplary target-activatable antibodies TY24649, TY24652, and TY22404 on IL-2 secretion from SEA-induced human peripheral blood mononuclear cell (PBMC) activation.

[0029] [Figure 9] This shows the antibody-dependent cell-mediated cytotoxicity (ADCC) activity before and after cleavage of isotype control antibodies, parental antibodies TY21580 and TY24040, or exemplary activatable antibodies TY24649, TY24652, and TY22404 against HEK293F cells that transiently overexpress human CTLA4, as measured by an ADCC reporter gene assay.

[0030] [Figure 10] This figure shows the CLTA4 blocking activity of isotype control antibodies, parent antibodies TY21580 and TY24040, or exemplary activatable antibodies TY24649, TY24652 and TY22404 before and after cleavage, as determined by a CTLA4 blocking bioassay.

[0031] [Figure 11] This study demonstrates the binding activity of parental and activatable anti-CTLA-4 antibodies to cell surface human CTLA4 expressed on HEK293F cells. Concentration-dependent binding curves for isotype control antibodies, parental antibodies TY21580 and TY24040, or exemplary activatable antibodies TY24649 and TY24652 before and after cleavage were determined by flow cytometry assay.

[0032] [Figure 12] This study shows the binding affinity of wild-type and ADCC-enhancing mutant Fc cells to the human, monkey, and mouse-derived Fcγ receptor CD16. Activated TY24649 with wild-type Fc and activated TY24652 with mutant Fc were tested using Biacore.

[0033] [Figure 13A-13C] This shows the in vivo antitumor effects of the parent antibody TY21580, isotype control antibodies, or exemplary CTLA4-activatable antibodies TY24649 and TY24652, and the uncleaved antibody TY24851 in an MC38 syngeneic mouse colorectal tumor model. Figure 13A shows the tumor growth curves for different treatment groups and the individual tumor growth curves for a group of female C57BL / 6 mice with MC38-encompassing tumors. In the upper panel, the data points represent group-mean error bars and represent SEM. In the lower panel, the curves represent tumor growth in individual mice in each treatment group. TY21580, TY24649, TY24652, and TY24851 were administered at 10 mg / kg twice weekly for a total of four doses. Figure 13B shows the tumor growth curves for different treatment groups and the individual tumor growth curves for a group of female C57BL / 6 mice with MC38-encompassing tumors. In the upper panel, the data points represent group-mean error bars and represent SEM. In the panel below, the curves represent tumor growth in individual mice in each treatment group. TY21580, TY24649, TY24652, and TY24851 were administered at a dose of 2 mg / kg twice weekly for a total of four doses. Figure 13C shows the tumor growth curves of different treatment groups for a group of female C57BL / 6 mice with MC38 colonized tumors. The data points represent group-mean error bars and represent SEM. TY21580 and TY24652 were administered at 0.05, 0.2, or 1 mg / kg twice weekly for four doses.

[0034] [Figure 14A-14C]This shows the in vivo antitumor effects of isotype control antibodies, parental antibody TY21580, or exemplary CTLA4-activatable antibodies TY24649 and TY24652, and non-cleaved antibody TY24851 in a CT26 syngeneic mouse colorectal tumor model. The tumor growth curves for different treatment groups of female C57BL / 6 mice with established CT26 tumors are shown. Data points represent group means, and error bars represent standard error (SEM). Figure 14A shows the tumor growth curves after administration of TY21580, TY24649, TY24652, and TY24851 twice weekly at 5 mg / kg for four doses. The upper panel shows the mean for each group, and the lower panel shows the tumor volume per mouse in each group. Figure 14B shows the tumor growth curves after administration of TY21580, TY24649, TY24652, and TY24851 at 1 mg / kg twice weekly for four doses. The upper panel shows the average for each group, and the lower panel shows the tumor volume of individual mice in each group. Figure 14C shows the tumor growth curves when TY21580 was administered at 0.5 mg / kg and TY24652 at 0.1, 0.5, and 2.5 mg / kg twice a week for four doses.

[0035] [Figure 15A-B] This shows the in vivo antitumor effects of isotype control antibodies, parental antibody TY21580, and activatable antibodies TY24649 and TY24652 in a Lewis synzygous mouse liver tumor model. The tumor growth curves for different treatment groups of female C57BL / 6 mice with Lewis-type colonized tumors are shown. Data points represent the group mean, and error bars represent the standard error (SEM). Figure 15A shows the tumor growth curves when TY21580, TY24649, and TY24652 were administered at 10 mg / kg twice weekly for four times. Figure 15B shows the tumor growth curves when TY21580, TY24649, and TY24652 were administered at 2 mg / kg twice weekly for four times.

[0036] [Figure 16] This shows the pharmacokinetic analysis of the parent antibody TY21580 and the activatable antibodies TY24649, TY24652, and TY22404.

[0037] [Figure 17] This shows the plasma concentration-time profiles of activatable anti-CTLA-4 antibodies TY24649 and TY24652 in cynomolgus monkeys after intravenous administration of 30 mg / kg twice weekly.

[0038] [Figure 18] This study demonstrates the effect of anti-CTLA4 antibodies on stimulating peripheral lymphocyte subtypes. The parent antibody TY21580 and activatable antibodies TY22404, TY24649, and TY24652 were administered weekly via IV at a dose of 30 mg / kg in two separate doses to cynomolgus monkeys. Peripheral blood samples were collected pre- and during treatment, and lymphocyte subtypes were examined by flow cytometry using specific markers. The percentages of central memory CD4+ T cells, proliferative central memory CD4+ T cells, and proliferative central memory CD8+ T cells were significantly affected on day 14 after administration with anti-CTLA4 antibodies.

[0039] [Figure 19] This study demonstrates the effects of parental and activatable anti-CTLA4 antibodies combined with anti-PD-1 antibodies on inducing diabetes in NOD / LtJ mice. Blood glucose levels in each mouse were monitored and plotted over time.

[0040] [Figure 20] This shows antibody-dependent cell-mediated phagocytosis (ADCP) of cells overexpressing CTLA-4, induced by an anti-CTLA4 antibody and measured using a luciferase assay.

[0041] [Figure 21A]This study demonstrates the in vivo antitumor effects of activatable anti-CTLA4 antibody TY24652, anti-CD137 antibody AG10131, and combinations thereof in an MC38 syngeneic mouse colorectal tumor model. Tumor growth curves for different treatment groups are shown for a group of female C57BL / 6 mice with established MC38 tumors. Data points represent the group mean, and error bars represent SEM. 0.2 mg / kg TY24652, 10 mg / kg AG10131, and combinations thereof were administered twice weekly for four weeks.

[0042] [Figure 21B] This study demonstrates the in vivo antitumor effects of activatable anti-CTLA4 antibody TY24652, anti-PD-1 antibody, and combinations thereof in a CT26 syngeneic mouse colorectal tumor model. Tumor growth curves are shown for different treatment groups of female C57BL / 6 mice with established CT26 tumors. Data points represent group mean values, and error bars represent SEM values. Mice were administered 0.5 mg / kg TY24652, 5 mg / kg anti-PD-1 antibody, and combinations thereof twice weekly for four weeks.

[0043] [Figures 22A-22E] Figure 22A shows the reduction of Treg cells in tumor-infiltrating lymphocytes (TILs), peripheral lymphocytes (PMBCs), and spleen in a CT26 syngeneic mouse colorectal tumor model induced by the activatable anti-CTLA4 antibody TY24652. Parentheses with one asterisk (*) and three asterisks (***) indicate a significant difference between TY24652 and isotype controls at p<0.001 and p<0.05, respectively. "ns" in parentheses indicates no significant difference between TY24652 and isotype controls. Figure 22A shows the proportion of Tregs among CD4+ T cells. Figure 22B shows the CD8+ T to Treg ratio. Figure 22C shows the CTLA4 expression level of Tregs. Figure 22D shows the proportion of CD4+ T cells to total T cells. Figure 22E shows the proportion of CD8+ T cells to total T cells.

[0044] [Figures 23A-23B] Figure 23A shows the plasma concentration-time profile of the total form of anti-CTLA4 antibody after a single intravenous or subcutaneous injection of 0.5 mg / kg into miniature pigs. Figure 23B shows the plasma concentration-time profile of miniature pigs that received intravenous injection.

[0045] [Figure 24] This study demonstrates the ADCC reporter activity of various anti-CTLA4 antibodies with different Fc mutations compared to the parent antibody TY21580, ipilimumab, and isotype controls.

[0046] [Figure 25] This shows the in vivo antitumor effects of isotype control antibodies, parental antibody TY21580, and antibodies with Fc mutations TY24037, TY24039, and TY24040 in an MC38 colon cancer model. Data points represent the group mean, and error bars represent SEM.

[0047] [Figure 26A-C] This shows the in vivo antitumor effects of isotype control antibodies, ipilimumab, TY21580, and TY24040 in an MC38 colorectal cancer model. Figure 26A shows the mean tumor volume up to day 20 in mice treated with 0.5 mg / kg or 5 mg / kg of antibody; error bars indicate standard error (SEM). Figure 26B shows the tumor volume of mice administered 0.5 mg / kg of ipilimumab, TY21580, and TY24040 compared to the isotype control. Figure 26C shows the tumor volume of individual mice administered 5 mg / kg of ipilimumab, TY21580, and TY24040 compared to the isotype control. In Figures 26B-26C, the inserted numbers indicate the percentage of mice that survived beyond day 40 of the study.

[0048] [Figures 27A-27B]Figure 27A shows Treg depletion in the tumor microenvironment of an MC38 colon cancer model induced by isotype control, ipilimumab, TY21580, and TY24040. Figure 27A shows the percentage of Treg cells among CD4+ T cells in mouse tumors. Asterisked brackets indicate statistically significant differences for both TY21580 and TY24040 compared to the isotype control. Figure 27B shows the number of Tregs per million living cells. Asterisked brackets indicate statistically significant differences for each of ipilimumab, TY21580, and TY24040 compared to the isotype control. ** indicates P<0.01, * indicates P<0.05.

[0049] [Figure 28A-C] This report demonstrates the in vivo antitumor effects of the isotype control antibodies ipilimumab, TY21580, and TY24040 in suppressing the growth of large, established tumors (approximately 1,500 mm3) in an MC38 colon cancer model. Figure 28A shows the mean tumor volume up to day 20 in mice administered 0.5 mg / kg or 5 mg / kg of the antibody; error bars indicate standard error (SEM). Figure 28B shows the tumor volume of mice administered 0.5 mg / kg of ipilimumab, TY21580, and TY24040 compared to the isotype control. Figure 28C shows the tumor volume of individual mice administered 5 mg / kg of ipilimumab, TY21580, and TY24040 compared to the isotype control.

[0050] [Figures 29A-29D]Positron emission tomography-computed tomography (PET / CT) scans of mice injected with the indicated antibodies are shown. Animals were imaged at specified time points after injection. H22-CTLA4 tumors are located on the left side of the animals, and H22 tumors are located on the right side. Signal intensity reflects the percentage of injected volume per gram of tissue (%ID / g), as indicated by the key on the right. Figure 29A shows TY21580, Figure 29B shows TY22404, Figure 29C shows TY24652, and Figure 29D shows TY24851.

[0051] [Figures 30A-30D] This graph shows the average %ID / g in the indicated tissues of mice injected with the indicated antibody. Different bars within each tissue sample reflect the data at the indicated time point. Figure 30A shows TY21580, Figure 30B shows TY22404, Figure 30C shows TY24652, and Figure 30D shows TY24851.

[0052] [Figure 31] This shows the effects of various control antibodies and anti-CTLA-4 antibodies on IL-2 secretion from SEA-activated human peripheral blood mononuclear cells (PBMCs). AFU indicates an afcosylated mutant. An asterisk indicates an antibody containing an Fc-enhancing mutant.

[0053] [Figure 32] This document describes an experimental design for evaluating various control antibodies and anti-CTLA-4 antibodies in an MC38 syngeneic colorectal cancer tumor mouse model. Mice were administered the antibodies twice, every two weeks. AFUs exhibit afucosylated mutations.

[0054] [Figure 33]This study demonstrates the in vivo antitumor effects of isotype control antibodies ipilimumab, TY21580, and TY24040, as well as TY21580-AFU, which suppress the growth of established tumors (approximately 50-100 mm3) in an MC38 colon cancer model. Figure 33A shows the mean tumor volume up to day 22 in mice administered 0.5 mg / kg of antibody; error bars represent standard error (SEM). AFU indicates an afucosylated mutant. Figure 33B shows the tumor volume of individual mice treated with ipilimumab, TY21580, TY21580-AFU, and TY24040 at 0.5 mg / kg.

[0055] [Figure 34] This document describes an experimental design for evaluating various control antibodies and anti-CTLA-4 antibodies in an MC38 syngeneic colorectal cancer mouse model. Mice with large, established tumors (approximately 200-300 mm3) were administered 1 mg / kg of isotype or anti-CTLA-4 antibody once every 3 days, three times a week (q3dx3). AFUs showed afucosylated mutants.

[0056] [Figure 35]Figure 35A shows Treg depletion in the tumor microenvironment of an MC38 colon cancer model by isotype control, ipilimumab, TY21580, TY21580-AFU, and TY24040. * indicates P<0.05. Figure 35A shows a fluorescence-activated cell sorting (FACS) plot of CD4+ cells. Figure 35B shows the percentage of Treg cells among CD4+ T cells in mouse tumors. Asterisked brackets indicate statistically significant differences between TY21580 and TY21580-AFU, and between TY21580, TY21580-AFU, and TY24040 compared to isotype control and ipilimumab. Figure 35C shows the number of Tregs per million cells. Parentheses with asterisks indicate statistically significant differences in ipilimumab, TY21580, TY21580-AFU, and TY24040 compared to isotype controls. Figure 35D shows the percentage of splenic Treg cells compared to mouse CD4+ T cells. Error bars represent SEM. AFU indicates afucosylated mutants. [Modes for carrying out the invention]

[0057] To provide anti-CTLA4 antibodies with potent antitumor activity and reduced off-target effects, we have developed a series of masked anti-CTLA4 antibodies (including activatable anti-CTLA4 antibodies), each containing an N-terminal masking peptide and an IgG1 Fc region. While we do not intend to be bound by any theory or hypothesis, the antitumor activity of the antibodies is at least twofold: 1) blocking CTLA4 sequestration by CD80 and CD86 ligands to stimulate CD28-mediated downstream cell signaling, and 2) killing target cells via enhanced IgG1Fc-mediated effector function such as ADCC and ADCP. The masking peptide is designed to compete with the target binding site of the anti-CTLA4 antibody, thereby inhibiting on-target / off-tumor activity in healthy tissue and enabling antitumor activity in the tumor microenvironment (TME).

[0058] Masked antibodies are designed to mask their antigen-binding site with a masking unit in a masking peptide, preventing them from binding to the antibody's target in healthy tissue. Masked antibodies may also be activatable, in which case the masking unit is designed to unmask the antibody in a TME with specific activation conditions, allowing the antigen-binding site to bind to its target. For example, the masking peptide of an activatable antibody may contain a cleavable site that, when cleaved by a protease, removes the masking unit from the antibody, activating it to bind to CTLA4. In normal tissues or circulation with low CTLA4 expression levels, the masking unit of an activatable anti-CTLA4 antibody functions to block binding to CTLA4, mitigating off-target effects associated with anti-CTLA4 antibodies. However, in TME, when the local CTLA4 concentration is high, the masking antibody can bind to CTLA4 on target cells in the following cases: when masking units are present (i.e., inactive in the case of an activatable antibody), when some masking units of the masking antibody are cleaved (i.e., partially activated in the case of an activatable antibody), and / or when the masking units are completely cleaved (i.e., fully activated in the case of an activatable antibody). Furthermore, in TME, where increased protease activity has been reported, the masking units of the activatable antibody are cleaved, enhancing the binding of the activated anti-CTLA4 antibody to CTLA4 on target cells. Thus, the masked anti-CTLA4 antibody described herein exhibits reduced toxicity to normal cells compared to existing anti-CD47 antibodies, both through inhibiting CTLA4 sequestration by CD80 and CD86 ligands and through enhanced ADCC / ADCP effector function. I. General techniques

[0059] The techniques and procedures described or referenced herein are generally well understood by those skilled in the art and refer to conventional methodologies, such as Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (FMAusubel, et al. eds., (2003)); the series Methods in Enzymeology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GRTaylor eds. (1995)); Harlow and Lane, eds. (1988); Antibodies, A Laboratory Manual, and Animal Cell Culture (RI Freshney, ed. (1987)); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Labora tory Notebook(JECellis,ed.,1998)Academ ic Press, Animal Cell Culture(RIFreshne y),ed.,1987), Introduction to Cell and Ti ssue Culture(JPMather and PERoberts, 1998)Plenum Press, Cell and Tissue Cultur e:Laboratory Procedures(A.Doyle,JBGriffiths,and DG Newell, eds., 1993-8) J. Wiley and Sons, Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.), Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos, eds., 1987), PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994), Current Protocols in Immunology (JEColigan et al., eds., 1991), Short Protocols in Molecular Biology (Wile y and Sons, 1999), Immunobiology (CA Janew ay and P. Travers, 1997), Antibodies (P. Finc h, 1997), Antibodies: A Practical Approach ( D. Catty., ed., IRL Press, 1988-1989), Monoclonal Antibodies: A Practical Approach (PS hepherd and C. Dean, eds., Oxford University Press, 2000), Using The methodology is generally based on widely used methods described in *Antibodies: A Laboratory Manual* (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999), *The Antibodies* (M. Zanetti and JDCapra, eds., Harbor Academic Publishers, 1995), and *Cancer: Principles and Practice of Oncology* (VT DeVita et al., eds., JBLippincott Company, 1993). II. Definition

[0060] Before describing this disclosure in detail, it should be understood that this disclosure is not limited to any particular composition or biological system, and is, needless to say, diverse. It should also be understood that the terms used herein are intended solely to describe specific embodiments and are not intended to be limiting.

[0061] As used herein, unless the context makes it clear, the singular forms “a,” “an,” and “the” include multiple references. For example, a reference to “molecule” may include any combination of two or more such molecules.

[0062] As used herein, the term “approximately” refers to the normal margin of error for each value, as readily understood by those skilled in the art. References to “approximately” values ​​or parameters herein include (and describe) embodiments relating to the value or parameter itself.

[0063] It should be understood that the aspects and embodiments of the disclosure described herein include "including," "consisting of," and "essentially consisting of."

[0064] As used herein, the term "and / or" is intended to include phrases such as "A and / or B" as encompassing both A and B, A or B, A (alone), and B (alone). Similarly, as used herein, the term "and / or" is intended to include phrases such as "A, B, and / or C" as encompassing each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0065] The term "amino acid" refers to natural and synthetic amino acids, as well as amino acid analogs and amino acid mimes that function similarly to natural amino acids. Natural amino acids are those encoded by the genetic code, and those modified later, such as hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. The term "amino acid analog" refers to compounds that have the same basic chemical structure as natural amino acids, but the C-terminal carboxyl group, N-terminal amino group, or side-chain functional group is chemically modified to a different functional group. The term "amino acid mime" refers to chemical compounds that have a different structure from the general chemical structure of amino acids, but function similarly to natural amino acids. As used herein, the 20 conventional amino acids and their abbreviations follow conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, ESGolub and DRGren, Eds., Sinauer Assomoticates, Sunderland, Mass. (1991)).

[0066] The terms “polypeptide,” “protein,” and “peptide” are used interchangeably herein and may refer to polymers of two or more amino acids.

[0067] As used interchangeably herein, “polynucleotide” or “nucleic acid” refers to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or analogs thereof, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase or by synthetic reactions. Polynucleotides may include modified nucleotides, such as methylated nucleotides and their analogs. Modifications to the nucleotide structure, where present, may be conjugated before or after the assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may include post-synthesis modifications, such as conjugation to labels. Other types of modifications include, for example, "caps" that replace one or more naturally occurring nucleotides with analogs, internucleotide modifications such as those by non-charged bonds (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), those involving suspension portions of proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those involving insertors (e.g., acridine, psoralen, etc.), those involving chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.), those involving alkylating agents, those involving modifying bonds (e.g., alpha-anomeric nucleic acids, etc.), and the unmodified form of polynucleotides. Furthermore, any of the hydroxyl groups normally present in the sugar may be substituted with, for example, a phosphonic acid group or a phosphate group, protected with a standard protecting group, or activated to prepare additional binding to additional nucleotides, or conjugated to a solid or semi-solid support. The 5' and 3' terminal OH groups can be phosphorylated or substituted with amines or organic capping groups of 1 to 20 carbon atoms. Other hydroxyls can be derivatized with standard protecting groups.Polynucleotides may also contain analogous forms of ribose or deoxyribose sugars commonly known in the art, such as 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-, or 2'-azidol-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xylose, or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acrylic acid analogs, and basic nucleoside analogs such as methylriboside. One or more phosphodiester bonds may be replaced with alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which the phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H, or a substituted or unsubstituted alkyl (1-20C) (optionally including an ether (-O-) linkage), aryl, alkenyl, cycloalkyl, cycloalkenyl, or aralgyl. Not all links in the polynucleotide need to be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[0068] The term “isolated nucleic acid” refers to a nucleic acid molecule of genomic, cDNA, or synthetic origin, or a combination thereof, that has been isolated from other nucleic acid molecules present in the nucleic acid’s natural source. For example, with respect to genomic DNA, the term “isolated” includes nucleic acid molecules that have been isolated from the chromosome to which the genomic DNA is naturally bound. Preferably, “isolated” nucleic acids do not contain sequences naturally adjacent to the nucleic acid (i.e., sequences located at the 5’ and 3’ ends of the nucleic acid of interest).

[0069] The term "antibody" is used in its broadest sense herein and specifically includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, masked antibodies (e.g., activatable antibodies), multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., single-chain variable fragments or scFv) insofar as they exhibit the desired biological activity.

[0070] The term "antibody" is a recognized term in the art and may refer 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 isotype of the H chains. Each heavy chain has a variable region (abbreviated herein as VH) and a constant region at its N-terminus. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain has a variable region (abbreviated herein as VL) and a constant region at its other end. The light chain constant region consists of one domain, CL. VL is parallel to VH, and CL is parallel to the first constant domain (CH1) of the heavy chain. The pairing of VH and VL together forms a single antigen-binding site. IgM antibodies consist of five basic heterotetrameric units accompanied by a further polypeptide called a J chain, and therefore contain 10 antigen-binding sites, whereas secreted IgA antibodies can form [a different structure].

[0071] As used herein, the terms “hypervariable region” or “HVR” refer to each region of the antibody variable domain whose sequence is hypervariable. HVRs may form structurally defined loops (“hypervariable loops”). Generally, natural quadruple-chain antibodies contain six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs are flanked by more conserved regions called framework regions (FRs). Each VH and VL consists of three HVRs and four FRs, arranged in the order FR1, HVR1, FR2, HVR2, FR3, HVR3, FR4 from the amino terminus to the carboxy terminus. HVRs generally contain amino acid residues derived from the hypervariable loops and / or “complementarity-determining regions” (CDRs), the CDRs being the most sequence-variable and / or involved in antigen recognition. Exemplary hypervariable loops are located at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3). (Chothia and Lesk, J.Mol.Biol.196:901-917 (1987)). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) are located at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)). Except for the CDR1 of VH, CDRs generally contain amino acid residues that form a hypervariable loop. CDRs also contain "specificity-determining residues" or "SDRs," which are residues that come into contact with the antigen. SDRs are contained within a region of the CDR called a shortened CDR or a-CDR.Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) are located at amino acid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)).

[0072] Table I below provides exemplary CDR definitions according to various algorithms known in the art. TIFF2026514030000001.tif95170

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

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

[0075] The term "antibody derivative" or "derivative" of an antibody refers to a molecule that contains the amino acid sequence of an antibody linked to an additional molecular entity, and that can bind to the same antigen (e.g., CTLA4) to which the antibody binds. The amino acid sequence included in the antibody derivative may be the full-length heavy chain, full-length light chain, any part(s) of the full-length heavy chain, any part(s) of the full-length light chain, any other fragment(s) of the antibody, or a complete antibody. The additional molecular entity may be a chemical molecule or a biomolecule. Examples of additional molecular entities include chemical groups, amino acids, peptides, proteins (enzymes, antibodies, etc.), and chemical compounds. The additional molecular entity may have any of the following uses: as a detection agent, label, marker, pharmaceutical, or therapeutic agent. The amino acid sequence of the antibody may be bound or linked to the additional molecular entity by chemical coupling, gene fusion, non-covalent association, etc. The term "antibody derivative" includes chimeric antibodies, humanized antibodies, and molecules derived from modifications of the amino acid sequence of CTLA4 antibodies, such as conservative substitutions, additions, and insertions of amino acids.

[0076] The term "antigen-binding fragment" or "antigen-binding moiety" of an antibody refers to one or more parts of the antibody that possess the ability to bind to an antigen (e.g., CTLA4) to which the antibody binds. Examples of antibody "antigen-binding fragments" include (i) the Fab fragment, which is a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) the F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by disulfide crosslinks at a hinge region; (iii) the Fd fragment, which consists of VH and CH1 domains; (iv) the Fv fragment, which consists of the VL and VH domains of a single arm of the antibody; (v) the dAb fragment, which consists of the VH domain (Ward et al., Nature 341:544-546 (1989)); and (vi) an isolated complementarity-determining region (CDR).

[0077] The term "masked antibody" refers to an antibody or its antigen-binding fragment that contains a peptide that interferes with, disrupts, reduces, blocks, inhibits, or competes with the binding of the antibody or its antigen-binding fragment's target-binding portion (TBM) to its target. Masked antibodies can be produced by linking a masking peptide to the TBM of the antibody or its antigen-binding fragment.

[0078] The term "activatable antibody" refers to a masked antibody or its antigen-binding fragment that exhibits a first binding affinity to a target in an unactivated (e.g., inhibited, masked, and / or uncleaved) state and a second binding affinity to a target in an activated (e.g., uninhibited, unmasked, and / or cleaved) state, with the second binding affinity being higher than the first. Activatable antibodies can be produced by ligating a masking peptide containing an activatable component (e.g., a cleavage site) to the target-binding moiety (TBM) of the antibody or its antigen fragment. Activatable antibodies are described, for example, in U.S. Patent Publication 2019 / 0241886 and U.S. Patent Publication 2021 / 0207126, the contents of which are incorporated herein by reference in their entirety.

[0079] The term "Target-binding moiety (TBM)" refers to the structural portion of the antigen-binding portion of an antibody that binds to the target antigen. The TBM may include any combination of VH and VL, such as any VH or VL described herein.

[0080] A “masking peptide” refers to a structural portion of a masked antibody (e.g., an activatable antibody) that inhibits the binding of TBM to a target antigen, and typically includes a masking unit (MU) and a linkage unit (LU) from the N-terminus to the C-terminus. The C-terminus of the masking peptide is typically linked to the N-terminus of the VH or VL of the masked antibody (e.g., an activatable antibody). In some embodiments, the masking peptide or a portion thereof (e.g., a masking unit (MU)) interferes with or inhibits the binding of TBM to a target so efficiently that the binding of TBM to the target is very low and / or below the detection limit (e.g., the binding cannot be detected by ELISA or flow cytometry assays). The masked antibody (e.g., an activatable antibody) described herein may include one or more linkers located, for example, in the LU, between the MU and LU, between the LU and VH or VL, or between the hinge region of the TBM and Fc. The terms “masking unit” and “masking portion” are used interchangeably herein. The terms "linkage unit" and "linkage unit portion" are used interchangeably in this application.

[0081] The LU of a masking peptide may contain a first cleavage site. The cleavage site generally contains a cleavable amino acid sequence and functions, for example, as a substrate for an enzyme and / or cysteine-cysteine ​​pair capable of forming a reducible disulfide bond. Therefore, when terms such as “cleavage,” “cleavable,” and “cleaved” are used in relation to a cleavage site, the terms encompass, for example, enzymatic cleavage by a protease and cleavage of the disulfide bond between cysteine-cysteine ​​pairs via reduction of the disulfide bond, which may occur upon exposure to a reducing agent. The amino acid sequence of the cleavage site may overlap with or be contained within the MU. An activatable antibody contains a cleavage site configured to mediate the activation of the antibody. For example, if the cleavage site of an activatable antibody is intact (e.g., containing a cysteine-cysteine ​​disulfide bond that has not been cleaved by the corresponding enzyme and / or reduced), the masking peptide or its portion may interfere with or inhibit the binding of the TBM to its target.

[0082] The term "masking efficiency" refers to the efficiency with which a masking peptide inhibits the binding of the TBM to the target antigen. Masking efficiency can be measured as the difference or ratio of properties (e.g., binding affinity to the target antigen) or activity (e.g., inhibition of binding between the target antigen and ligand) of a masked antibody (e.g., an activatable antibody) having a TBM and a masking peptide, compared to a corresponding unmasked antibody ("parent antibody") having the same TBM but lacking the masking peptide. In activatable antibodies, masking efficiency can be measured as the difference or ratio of properties (e.g., binding affinity to the target antigen) or activity (e.g., inhibition of binding between the target antigen and ligand) of an activatable antibody having a TBM and a masking peptide, compared to an unactivated (e.g., inhibited, masked, and / or uncleaved) activatable antibody, compared to an activated (e.g., uninhibited, unmasked, and / or cleaved) activatable antibody, or compared to a parent antibody having the same TBM but lacking the masking peptide. For example, masking efficiency can be measured by dividing the EC50 of an activatable antibody that binds to the target antigen in an unactivated (e.g., inhibited, masked, and / or uncleaved) state by the EC50 or KD of an activatable antibody that binds to the target antigen in an activated (e.g., uninhibited, unmasked, and / or cleaved) state, or by dividing it by the EC50 or KD of a parent antibody that binds to the target antigen. The EC50 value can be measured, for example, by an ELISA assay as described in Example 3, or, for example, by a Jurkat NFAT reporter assay as described in U.S. Patent Application Publication No. 20210207126 A1. The KD value can be measured, for example, by surface plasmon resonance using any of the systems described herein.

[0083] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted immunoglobulins bound to Fc receptors (FcRs) present on specific cytotoxic cells (e.g., NK cells, neutrophils, and macrophages) specifically bind these cytotoxic effector cells to antigen-carrying target cells, which are then killed by cytotoxins. Primary cells that mediate ADCC, such as NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To evaluate the ADCC activity of the target molecule, an in vitro ADCC assay can be performed, for example, as described in U.S. Patent No. 5,500,362 or 5,821,337 or U.S. Patent No. 6,737,056 (Presta). Effector cells useful for such assays include PBMC cells and NK cells. Alternatively, the ADCC activity of the target molecule may be evaluated in vivo in an animal model, for example, as disclosed in Clynes et al. Proc. Natl. Acad. Sci. (USA) 95:652-656 (1998). In this specification, “ADCC-enhancing mutation” refers to a mutation, such as an amino acid substitution in an antibody, that enhances the level of ADCC induced by the antibody.

[0084] As used herein, the term “antibody-dependent cell-mediated phagocytosis” or “ADCP” refers to a mechanism of cellular immune defense in which effector cells of the immune system (e.g., macrophages or T lymphocytes) phagocytose target cells to which specific antibodies are bound on membrane surface antigens. Methods for evaluating ADCP activity are known in the art, including, for example, the assay described in Example 14 of this application. As used herein, “ADCP-enhancing mutation” refers, for example, an amino acid substitution in an antibody that increases the level of ADCP induced by the antibody.

[0085] The term "binding molecule" includes (1) antibodies, (2) antigen-binding fragments of antibodies, and (3) derivatives of antibodies, as defined herein.

[0086] The term "CTLA4" as used in this application includes human CTLA4 (e.g., UniProt accession number P16410), and its variants, isoforms, and species homologs (e.g., mouse CTLA4 (UniProt accession number P09793), rat CTLA4 (UniProt accession number Q9Z1A7), canine CTLA4 (UniProt accession number Q9XSI1), cynomolgus monkey CTLA4 (UniProt accession number G7PL88), etc.). Therefore, as defined and disclosed herein, binding molecules (e.g., antibodies or masked antibodies (e.g., activatable antibodies)) may also bind to CTLA4 from non-human species. In other cases, binding molecules may be perfectly specific to human CTLA4 and may not exhibit cross-reactivity with species or other types.

[0087] The terms “CTLA4 antibody” and “anti-CTLA4 antibody” are used interchangeably herein and refer to antibodies capable of binding to human CTLA4, as defined in [reference].

[0088] The term "chimeric antibody" refers to an antibody that contains amino acid sequences derived from different animal species, such as one that has a variable region derived from a human antibody and a constant region of mouse immunoglobulin.

[0089] The term "competing for binding" refers to the interaction between two antibodies in binding to their respective target epitopes. A primary antibody competes with a secondary antibody for binding if the binding of the primary antibody to its congener epitope is detected to a certain degree in the presence of the secondary antibody compared to the binding of the primary antibody in the absence of the secondary antibody. Alternatively, it may, though not necessarily, be true, if the binding of the secondary antibody to its epitope is also detected to a certain degree in the presence of the primary antibody. That is, a primary antibody can inhibit the binding of the secondary antibody to its respective epitope without the secondary antibody inhibiting the primary antibody's binding to that epitope. However, if each antibody detectably inhibits the binding of the other antibody to its congener epitope, the antibodies are said to "cross-compete" with each other for the binding of their respective epitope(s).

[0090] The term "epitope" refers to the portion of an antigen to which an antibody (or its antigen-binding fragment) binds. Epitopes can be formed from both continuous or discontinuous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from continuous amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes can contain a variety of amino acids in a specific spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, X-ray crystallography, two-dimensional nuclear magnetic resonance, deuterium-hydrogen exchange combined with mass spectrometry, or site-directed mutagenesis, or all methods used in combination with computational modeling of the complex structure of the antigen, its binding antibody, and its variants (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996)). Once the desired epitope of an antigen is determined, an antibody against that epitope can be generated, for example, using the techniques described herein. Antibody generation and characterization can elucidate information about the desired epitope. From this information, antibodies can be competitively screened for binding to the same epitope. An approach to achieve this is to conduct a cross-competition study to find antibodies that bind competitively to each other, i.e., antibodies that compete for binding to the antigen. A high-throughput process for “binning” antibodies based on cross-competition is described in PCT publication number WO03 / 48731.

[0091] The term "germline" refers to the nucleotide sequences of antibody genes and gene segments that are passed from parent to offspring via germ cells. Germline sequences are distinct from nucleotide sequences that encode antibodies of mature B cells, which are altered by recombination and high-frequency mutation events during the maturation process of B cells.

[0092] The term "glycosylation site" refers to an amino acid residue recognized by eukaryotic cells as a site for sugar addition. Typical amino acids to which carbohydrates such as oligosaccharides add are asparagine (N-linked), serine (O-linked), and threonine (O-linked). Specific addition sites are typically indicated by an amino acid sequence referred to herein as the "glycosylation site sequence." The glycosylation site sequences for N-linked glycosylation are as follows: -Asn-X-Ser- or -Asn-X-Thr-, where X is any standard amino acid other than proline. The terms "N-linked" and "O-linked" refer to chemical groups that function as addition sites between a sugar molecule and an amino acid residue. N-linked sugars add via an amino group, while O-linked sugars add via a hydroxyl group. The term "glycan occupancy" refers to the presence of a sugar moiety linked to the glycosylation site (i.e., the glycan moiety is occupied). If there are at least two potential glycosylation sites on a polypeptide, then any of the following sites may be occupied by a sugar moiety: none (occupied by a 0-glycan site), one (occupied by a 1-glycan site), or both (occupied by a 2-glycan site).

[0093] The term “host cell” refers to a cell line that can be manipulated to produce a target protein, protein fragment, or peptide. Host cells include, but are not limited to, cultured cells, such as mammalian cultured cells derived from rodents (rats, mice, guinea pigs, or hamsters) such as CHO, BHK, NSO, SP2 / 0, YB2 / 0; human cells (e.g., HEK293F cells, HEK293T cells); or human tissues or hybridoma cells, yeast cells, insect cells (e.g., S2 cells), bacterial cells (e.g., E. coli cells); and cells contained within transgenic animals or cultured tissues. The term encompasses not only specific target cells but also their offspring. Such offspring may not be identical to the parent cells because certain modifications can occur in subsequent generations due to either mutation or environmental influences, but they still fall within the scope of the term “host cell.”

[0094] A "human antibody" is an antibody produced by a human or human cell, or derived from a non-human source that utilizes the human antibody repertoire, possessing an amino acid sequence that corresponds to another human antibody coding sequence. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0095] The term "humanized antibody" refers to a chimeric antibody that contains amino acid residues derived from a human antibody sequence. A humanized antibody may contain some or all of a CDR or HVR derived from a non-human animal or synthetic antibody, while the antibody framework and constant region contain amino acid residues derived from a human antibody sequence.

[0096] The term “exemplary antibody” refers to the antibodies described in this disclosure and named as listed in Tables 2 and 3, and any one of the six CDRs and / or VH and VLs of the antibodies listed in Tables 2 and 3. These antibodies may be of any class (e.g., IgA, IgD, IgE, IgG, and IgM). Thus, each antibody identified above encompasses antibodies of all five classes having the same amino acid sequence for the VL and VH regions. Furthermore, antibodies of the IgG class may be of any subclass (e.g., IgG1, IgG2, IgG3, and IgG4). Thus, each antibody identified above in the IgG subclass encompasses antibodies of all four subclasses having the same amino acid sequence for the VL and VH regions. The amino acid sequences of the heavy chain constant regions of human antibodies in the five classes and four IgG subclasses are known in the art.

[0097] An “isolated” antibody or conjugated molecule (e.g., a masked antibody) is one that has been isolated from its natural environment. In some embodiments, the antibody is purified to a purity of 95% or greater than 99%, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For an overview of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromato gr. B 848:79-87 (2007).

[0098] The term "Ka" refers to the association rate constant of a specific binding molecule-antigen interaction, while the term "kd" refers to the dissociation rate constant of a specific binding molecule-antigen interaction.

[0099] The term "KD" refers to the equilibrium dissociation constant of a particular antibody-antigen interaction. This is derived from the kd to ka ratio (i.e., kd / ka) and is expressed as molar concentration (M). KD is used as a measure of the affinity of an antibody to its binding partner. A smaller KD indicates that the antibody binds more strongly or that the affinity between the antibody and the antigen is higher. For example, an antibody with a nanomolar (nM) dissociation constant will bind more strongly to a particular antigen than an antibody with a micromolar (μM) dissociation constant. The KD value of an antibody can be determined using methods that are well established in the art. One method for determining the KD of an antibody is by using surface plasmon resonance, typically by using a biosensor system such as the Biacore® system. For example, an assay procedure using the BIACORE® system (BIAcore assay) is described in at least Example 3 of this disclosure.

[0100] The term “prevent” or “prevent” in relation to a particular disease state in mammals refers to preventing or delaying the onset of the disease, or preventing the manifestation of its clinical or asymptomatic symptoms.

[0101] As used herein, “sequence identity” between two polypeptide sequences refers to the percentage of amino acids that are identical between the sequences. The amino acid sequence identity of polypeptides can conventionally be determined using known computer programs such as Bestfit, FASTA, or BLAST (see, for example, Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132:185-219 (2000); Altschul et al., J. Mol. Biol. 215:403-410 (1990); Altschul et al., Nucelic Acids Res. 25:3389-3402 (1997)). When using Bestfit or other sequence alignment programs to determine whether a particular sequence is, for example, 95% identical to a reference amino acid sequence, the parameters are set such that the percentage of identity is calculated over the entire length of the reference amino acid sequence, and a homology gap of up to 5% of the total number of amino acid residues in the reference sequence is permitted. This aforementioned method for determining the percentage of identity between polypeptides is applicable to all proteins, fragments, or variants disclosed herein.

[0102] As used herein, the terms “binding,” “binding,” “specifically binding,” “specifically binding to,” or “specific to” refer to measurable and reproducible interactions, such as binding between a target and an antibody, which determines the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody that binds to a target (which may be an epitope), or specifically binds to it, is an antibody that binds to this target more easily and / or for a longer period of time with higher affinity, binding strength, than it would to other targets. In one embodiment, the degree of antibody binding to an unrelated target is less than about 10% of the antibody binding to the target, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less. In certain embodiments, the antibody specifically binds to an epitope on a protein that is conserved between proteins of different species. In another embodiment, specific binding may include, but does not require, exclusive binding.

[0103] The terms “to treat,” “to cure,” or “to treat” a particular disease state in a mammal refer to producing a desirable or beneficial effect in the mammal having the disease state. Desirable or beneficial effects may include a reduction in the frequency or severity of one or more symptoms of the disease (e.g., tumor growth and / or metastasis, or other effects mediated by the number and / or activity of immune cells), or the cessation or inhibition of further progression of the disease, condition, or disorder. In the context of cancer treatment in mammals, desirable or beneficial effects may include inhibition of further growth or spread of cancer cells, death of cancer cells, inhibition of cancer recurrence, reduction of cancer-related pain, or improvement in the survival rate of the mammal. Effects may be subjective or objective. For example, if the mammal is a human, the human may report improved energy or vitality, or reduced pain, as subjective symptoms of improvement or response to treatment. Alternatively, a clinician may report a reduction in tumor size or tumor volume based on physical examination, laboratory values, tumor markers, or radiographic findings. Some clinical laboratory signs that clinicians may observe for a response to treatment include normalization of tests such as white blood cell count, red blood cell count, platelet count, erythrocyte sedimentation rate, and various enzyme levels. In addition, clinicians may observe a decrease in detectable tumor markers. Alternatively, other tests such as sonograms, nuclear magnetic resonance (MRN) studies, and positron emission tomography (PET) studies can be used to assess objective improvement.

[0104] The term "cold tumor" refers to a tumor in which T cell infiltration is minimal or nonexistent, making it resistant to both spontaneous antitumor immune responses and immunotherapy. The lack of T cell infiltration in cold tumors can be caused by several factors, including, but not limited to, the absence or deficiency of tumor antigen presentation and the absence of T cell activation. Cancers considered cold tumors include, but are not limited to, brain cancer (e.g., glioblastoma), breast cancer, head and neck cancer, ovarian cancer, pancreatic cancer, and prostate cancer.

[0105] The term "vector" refers to a nucleic acid molecule capable of transporting foreign nucleic acid molecules. The foreign nucleic acid molecule is ligated to the vector nucleic acid molecule by recombination techniques such as ligation or recombination. This allows the foreign nucleic acid molecule to be grown, selected, manipulated, or expressed in a host cell or organism. Vectors can be plasmids, phages, transposons, cosmids, chromosomes, viruses, or virions. One type of vector, upon introduction into a host cell, integrates into the host cell's genome and thereby replicates alongside 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 bacterial origins of replication and episomal mammalian vectors). A specific type of vector capable of inducing the expression of expressible foreign nucleic acids to which they are operably ligated is generally called an "expression vector." Expression vectors typically have a regulatory sequence that drives the expression of expressible foreign nucleic acids. Simpler vectors, known as "transcription vectors," are capable of transcription only, not translation: they can replicate in target cells but not express. The term "vector" encompasses all types of vectors, regardless of their function. Vectors that can induce the expression of expressible nucleic acids to which they are operably linked are generally called "expression vectors." Other examples of "vectors" may include presentation vectors (e.g., vectors that direct the expression and presentation of polypeptides encoded on the surface of viruses or cells (such as bacterial cells, yeast cells, insect cells, and / or mammalian cells)).

[0106] As used herein, “subject,” “patient,” or “individual” may refer to a human or a non-human animal. “Non-human animal” may refer to any animal not classified as human, such as a domesticated animal, livestock, or zoo animal, a sporting animal, a pet animal (e.g., a dog, horse, cat, or cow), and an animal used in research. Research animals may refer to, but are not limited to, nematodes, arthropods, vertebrates, mammals, frogs, rodents (e.g., mice or rats), fish (e.g., zebrafish or pufferfish), birds (e.g., chickens), dogs, cats, and non-human primates (e.g., rhesus macaques, crab-eating macaques, chimpanzees, etc.). In some embodiments, the subject, patient, or individual is a human.

[0107] “Effective dose” means the minimum effective amount in terms of dosage and duration required to achieve one or more desired or indicated effects, including therapeutic or preventive outcomes. An effective dose may be provided in one or more doses. For the purposes of this disclosure, an effective dose of an antibody, drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve a preventive or therapeutic treatment. As understood in clinical contexts, an effective dose of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition (e.g., an effective dose when administered as monotherapy or in combination therapy). Thus, “effective dose” may be considered in the context of administration of one or more therapeutic agents, and a single agent may be considered to be given in an effective dose if, when combined with one or more other agents, a desired outcome can or does not occur. III. Binding molecules that bind to human CTLA4 with enhanced effector function

[0108] This 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 molecules that bind to human CTLA4 have enhanced effector function. In particular, this specification provides an isolated antibody or antigen-binding fragment thereof that binds to human CTLA4 and includes a human IgG1 Fc region comprising one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, wherein the numbering of the residues follows EU numbers.

[0109] The S239D and I332E substitutions are described, for example, in Lazar GA, Dang W, Karki S, et al. Engineered antibody Fc variants with enhanced effector function. Proc Natl Acad Sci US A. 2006;103 (11): 4005-4010. doi: 10.1073 / pnas. 0508123103. As described by Lazar et al., antibodies with the S239D and / or I332E substitutions have enhanced binding affinity to the Fcγ receptor (FcγR) and enhanced ability to promote antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP). The position of S239D and the substitution of I332E are provided according to their EU numbers. Both S239 and I332 are located within the CH2 domain of the Fc region.

[0110] Because CTLA4 is expressed in healthy cells such as T effector cells, CTLA4 antibodies known in this field, such as ipilimumab, lack an Fc region with enhanced effector function (such as ADCC and ADCP) to avoid toxicity to healthy cells. To leverage the ADCC and ADCP effector function of the IgG1 Fc region, particularly the region with S239D and I332E substitutions, the inventors have created a masked antibody that selectively binds to CTLA4 only in specific environments, such as the tumor microenvironment (TME). Thus, the present invention provides an antibody that binds to CTLA4 and exhibits enhanced ADCC and ADCP effector function without sacrificing safety.

[0111] As described in detail in the Examples section of this specification, IgG1 Fc regions with ADCC and / or ADCP-enhancing substitutions, including the S239D and I332E substitutions, were introduced into antibodies that bind to human CTLA4 and activatable antibodies that bind to human CTLA4. As shown in Figures 9, 20, and 24, introducing targeted substitutions into the IgG1 Fc regions of the parental and activatable antibodies resulted in improved ability to induce ADCC and ADCP compared to the FDA-approved anti-CTLA4 antibody ipilimumab. As shown in Figure 10, introducing the S239D and I332E substitutions into the activatable antibody also improved its ability to stimulate CD28-mediated downstream cell signaling by blocking CTLA4 sequestration of CD80 and CD86 ligands. In three different animal cancer models, the parental antibody (TY21580), the parental antibody with S239D and I332E substitutions (TY24040), and the activatable antibodies with S239D and I332E substitutions (TY24851 and TY24652) significantly inhibited tumor growth (Figures 13A-15B and 25). Anti-CTLA4 antibodies with F243L, R292P, Y300L, V305I, and P396L Fc region substitutions (TY24037) and S239D, I332E, and A330L Fc region substitutions (TY24039) also showed enhanced tumor growth inhibition compared to the parental antibody (TY21580) lacking Fc region substitutions (Figure 25). In particular, Figure 19 shows that the parental anti-CTLA4 antibody TY21580 increased immune checkpoint inhibitor-associated diabetes when combined with an anti-PD-1 antibody, but the activatable antibodies TY24652 and TY22404 did not. The activatable antibody TY22404 is described in U.S. Publication No. 2021 / 0207126, which is incorporated in its entirety by reference. These results demonstrate the remarkable efficacy of the anti-CTLA4 antibodies of this disclosure in the treatment of cancer by leveraging ADCC and ADCP activity without sacrificing safety.

[0112] Any one of the antibodies that bind to human CTLA4 described herein may contain a human IgG1 Fc region comprising one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, wherein the numbering of the residues follows EU numbers. For example, any one of the anti-CTLA4 antibodies described herein may contain a human IgG1 Fc region, and each IgG1 Fc domain of the human IgG1 Fc region comprises: each human IgG1 in the heavy chain The Fc domain includes: i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viiii) S239D substitution and I332E substitution, where the numbering of the residues follows the EU number. Exemplary antibodies that bind to human CTLA4 are described, for example, in U.S. Patent Application Publication No. 20210207126A1, which is incorporated herein by reference in its entirety.In some embodiments, an antibody conjugating to human CTLA4, as described in U.S. Patent Application Publication No. 20210207126A1, is provided, wherein the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, for human IgG1 (For example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0113] In some embodiments, the anti-CTLA4 antibody described herein is afucosylated. In some embodiments, the anti-CTLA4 antibody of this disclosure comprises a first polypeptide and a second polypeptide, each comprising VH (e.g., two heavy chains); and a third polypeptide and a fourth polypeptide, each comprising VL (e.g., two light chains). In certain embodiments, the anti-CTLA4 antibody comprises a first polypeptide comprising VH and a first Fc domain (i.e., a first heavy chain), a second polypeptide comprising VH and a second Fc domain (i.e., a second heavy chain), and a third polypeptide and a fourth polypeptide, each comprising VL (e.g., two light chains). In certain embodiments, the first Fc domain and the second Fc domain are the same. In other embodiments, the first Fc domain and the second Fc domain are different. In some embodiments, both the first Fc domain and the second Fc domain are IgG1 Fc domains (e.g., human IgG1 Fc domains). The first and second IgG1 Fc domains may be the same IgG1 Fc domain or different IgG1 Fc domains. In some embodiments, the first and second IgG1 Fc domains each include one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; ii i) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), where the numbering of the residues follows EU numbers. In certain embodiments, the first and second IgG1 Fc domains each contain the same substitution.In some embodiments, the first polypeptide and the second polypeptide are heavy chains, and / or the third polypeptide and the fourth polypeptide are light chains.

[0114] In some embodiments, the anti-CTLA4 antibody of the present disclosure comprises two heavy chains and two light chains, wherein each human IgG1 Fc domain of the heavy chain contains one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, and the numbering of the residues follows EU numbers. In some embodiments, each human IgG1 Fc domain of the heavy chain contains S298A substitution, E333A substitution, and K334A substitution, and the numbering of the residues follows EU numbers. In some embodiments, each human IgG1 Fc domain in the heavy chain includes an S298A substitution, an E333A substitution, a K334A substitution, and a K326A substitution, with the numbering of the residues following EU numbers. In some embodiments, each human IgG1 Fc domain in the heavy chain includes an F243L substitution. In some embodiments, each human IgG1 Fc domain in the heavy chain includes an F243L substitution, an R292P substitution, a Y300L substitution, and a P396L substitution, with the numbering of the residues following EU numbers. In some embodiments, each human IgG1 Fc domain in the heavy chain includes an F243L substitution, an R292P substitution, a Y300L substitution, a V305I substitution, and a P396L substitution, with the numbering of the residues following EU numbers. In some embodiments, each human IgG1 Fc domain of the heavy chain includes an F243L substitution, an R292P substitution, a Y300L substitution, an L235V substitution, and a P396L substitution, with the residue numbering following EU numbers. In some embodiments, each human IgG1 Fc domain of the heavy chain includes an S239D substitution, an I332E substitution, and an A330L substitution, with the residue numbering following EU numbers. In some embodiments, each human IgG1 Fc domain of the heavy chain includes an S239D substitution and an I332E substitution, with the residue numbering following EU numbers. In certain embodiments, the Fc domains of the two heavy chains are the same. In other embodiments, the Fc domains of the two heavy chains are different.In some embodiments, the anti-CTLA4 antibody is a masked antibody (e.g., an activatable antibody), and each of the two light chains constitutes a masking unit (MU)-linkage unit (LU)-VL structure from the N-terminus to the C-terminus.

[0115] In some embodiments, the anti-CTLA4 antibody of the present disclosure comprises a first polypeptide and a second polypeptide, one or both of which comprise an antibody heavy chain variable region (VH) and a heavy chain constant region (e.g., an Fc domain) from the N-terminus to the C-terminus, wherein the heavy chain constant region comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution (e.g., i) ii) substitution of S298A, E333A, and K334A; ii) substitution of S298A, E333A, K334A, and K326A; iii) substitution of F243L; iv) substitution of F243L, R292P, Y300L, and P396L; v) substitution of F243L, R292P, Y300L, V305I, and P396L; vi) substitution of F243L, R292P, Y300L, L235V, and P396L; vii) substitution of S239D, I332E, and A330L; or viii) substitution of S239D and I332E), where the numbering of the residues follows EU numbers.In some embodiments, the anti-CTLA4 antibody comprises a first polypeptide and a second polypeptide, each polypeptide comprising an antibody heavy chain variable region (VH) and a heavy chain constant region (e.g., an Fc domain) from the N-terminus to the C-terminus, wherein the heavy chain constant region includes one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution (e.g., i) S298A substitution, E333A substitution ii) substitution of S298A, E333A, K334A, and K326A; iii) substitution of F243L; iv) substitution of F243L, R292P, Y300L, and P396L; v) substitution of F243L, R292P, Y300L, V305I, and P396L; vi) substitution of F243L, R292P, Y300L, L235V, and P396L; vii) substitution of F243L, R292P, Y300L, L235V, and P396L; vii) substitution of S239D, I332E, and A330L; or viiii) substitution of S239D and I332E), wherein the numbering of the residues follows EU numbers. In some embodiments, the heavy chain constant region is the IgG1 Fc domain (e.g., the human IgG1 Fc domain). In certain embodiments, the IgG1 Fc domains of the first and second polypeptides (e.g., human IgG1 Fc domains) dimerize to form an IgG1 Fc region (e.g., human IgG1 Fc region). In certain embodiments, the first and second heavy chain constant regions (e.g., human IgG1 Fc domains) are identical. In other embodiments, the first and second heavy chain constant regions (e.g., human IgG1 Fc domains) are different. In some embodiments, the first and second heavy chain constant regions (e.g., human IgG1 Fc domains) each contain an S239D substitution and / or an I332E substitution. In some embodiments, the anti-CTLA4 antibody further comprises a third polypeptide and a fourth polypeptide, each containing VL.In some embodiments, the anti-CTLA4 antibody is a masked antibody (e.g., an activatable antibody) comprising a third polypeptide and a fourth polypeptide, each polypeptide having a masking unit (MU)-linkage unit (LU)-VL structure from the N-terminus to the C-terminus. In some embodiments, the first and second polypeptides are heavy chains, and / or the third and fourth polypeptides are light chains.

[0116] In some embodiments, the anti-CTLA4 antibody may be: a) CDR-H1 containing an amino acid sequence selected from SEQ ID NOs: 1, 7, 12, 18, 23, 29, 34, 38, 44, 49, and 60, or a variant thereof containing up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; CDR-H2 containing an amino acid sequence selected from SEQ ID NOs: 2, 8, 13, 19, 24, 30, 39, 45, 56, and 61, or a variant thereof containing up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and CDR-H3 containing an amino acid sequence selected from SEQ ID NOs: 3, 9, 14, 20, 25, 31, 35, 40, 46, 50, 53, 57, and 62, or up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions. A variant thereof containing amino acid substitutions; and / or b) CDR-L1 containing an amino acid sequence selected from SEQ ID NOs: 4, 10, 15, 21, 26, 32, 36, 41, 47, 51, 54, 58, and 63, or a variant thereof containing up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; CDR-L2 containing an amino acid sequence selected from SEQ ID NOs: 5, 16, 27, and 63, or a variant thereof containing up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and CDR-L3 containing an amino acid sequence selected from SEQ ID NOs: 6, 11, 17, 22, 28, 33, 37, 43, 48, 52, and 59, or a variant thereof containing up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions. In some embodiments, the anti-CTLA4 antibody comprises one, two, three, four, five, or all six CDRs shown for any of the exemplary anti-CTLA4 antibodies listed in Table 2 below.In some embodiments, the anti-CTLA4 antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, and is used in human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number. TIFF2026514030000002.tif255164TIFF2026514030000003.tif174170

[0117] In some embodiments, the anti-CTLA4 antibody includes a) a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, and 88, and / or b) a light chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, and 89. In some embodiments, the anti-CTLA4 antibody includes 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. 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, and 88, 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. 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, and 89. In some embodiments, the anti-CTLA4 antibody comprises the heavy chain variable region and light chain variable region of any of the exemplary anti-CTLA4 antibodies listed in Table 3 below. In some embodiments, the anti-CTLA4 antibody comprises one, two, or all three CDRs of the heavy chain variable region and / or one, two, or all three CDRs of the light chain variable region shown for any of the exemplary anti-CTLA4 antibodies listed in Table 3 below.In some embodiments, the anti-CTLA4 antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, and is used in human IgG1 The Fc domain includes (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows EU numbers. In certain embodiments, the first and second IgG1 Fc domains are the same (i.e., they contain the same substitutions). In other embodiments, the first and second IgG1 Fc domains are different. TIFF2026514030000004.tif255170TIFF2026514030000005.tif255170TIFF2026514030000006.tif178170

[0118] In some embodiments, the antibody that binds to human CTLA4 comprises one, two, three, four, five, or six CDRs of antibody TY21580 as shown in Table 2. In some embodiments, the antibody comprises the VH and / or VL of antibody TY21580 as shown in Table 3. In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution for human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0119] In some embodiments, an antibody that binds to human CTLA4 contains a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 74. In certain embodiments, the VH sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the amino acid sequence of SEQ ID NO: 74, but retains the same ability to bind to human CTLA4 as the antibody containing SEQ ID NO: 74. In certain embodiments, a total of 1 to 13 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 74. In certain embodiments, the substitutions, insertions, or deletions occur in a region outside the CDR (i.e., within the FR). In certain embodiments, VH comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions.In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution in human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0120] In another embodiment, an antibody is provided that binds to human CTLA4, the antibody comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 75. In certain embodiments, the VL sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the amino acid sequence of SEQ ID NO: 75, but retains the same ability to bind to human CTLA4 as the antibody comprising SEQ ID NO: 75. In certain embodiments, a total of 1 to 11 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 75. In certain embodiments, the substitutions, insertions, or deletions occur in a region outside the CDR (i.e., within the FR). In certain embodiments, VL comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 4, or a variant thereof containing up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, or a variant thereof containing up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 6, or a variant thereof containing up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions.In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution in human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0121] In one embodiment, the antibody that binds to human CTLA4 includes VH, which contains the amino acid sequence of SEQ ID NO: 74, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%, or 100%) sequence identity with SEQ ID NO: 74; and VL, which contains the amino acid sequence of SEQ ID NO: 75, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%, or 100%) sequence identity with SEQ ID NO: 75, wherein the antibody contains one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, and is used with human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0122] In another embodiment, an antibody that binds to human CTLA4 is provided, wherein the antibody includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 1, or VH containing a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; CDR-H2 containing the amino acid sequence of SEQ ID NO: 2, or a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and CDR-H3 containing the amino acid sequence of SEQ ID NO: 3, or a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and CDR-L1 containing the amino acid sequence of SEQ ID NO: 4, or up to 5 (e.g., 1, 2, 3, 4, or 5) VL containing a variant thereof containing an amino acid substitution, CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, or a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 6, or a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, the antibody containing one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution in human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0123] In another embodiment, an antibody that binds to human CTLA4 is provided, comprising VH CDR1, VH CDR2, and VH CDR3, each containing the amino acid sequences of VH CDR1, CDR2, and CDR3 having the sequence described in SEQ ID NO: 74; and VL CDR1, VL CDR2, and VL CDR3, each containing the amino acid sequences of VL CDR1, CDR2, and CDR3 having the sequence described in SEQ ID NO: 75, wherein the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, and is used in human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0124] In some embodiments, the antibody that binds to human CTLA4 includes the light chain and / or heavy chain of antibody TY24040. In some embodiments, the antibody that binds to human CTLA4 includes a light chain containing the amino acid sequence described in SEQ ID NO: 90 below. In some embodiments, the antibody that binds to human CTLA4 includes a light chain containing an amino acid sequence having at least 80%, 81%, 82%, 3%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 90. In some embodiments, the antibody that binds to human CTLA4 includes a heavy chain containing the amino acid sequence described in SEQ ID NO: 91 below. In some embodiments, the antibody that binds to human CTLA4 comprises a heavy chain containing an amino acid sequence having at least 80%, 81%, 82%, 3%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 91. In some embodiments, the antibody that binds to human CTLA4 comprises a heavy chain containing the amino acid sequence described in SEQ ID NO: 91, excluding the C-terminal lysine residue. TY24040 Light Chain (Sequence No. 90) DIQLTQSPSSLSASVGDRVTITCRASQSVRGRFLAWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSSSWPPTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TY24040 heavy chain (SEQ ID NO: 91) EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWSWIRQAPGKGLEWLARIDDDKYYSTLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARSYVYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPEEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0125] In some embodiments, the antibody that binds to human CTLA4 comprises one, two, three, four, five, or six CDRs of antibody TY21585 as shown in Table 2. In some embodiments, the antibody comprises the VH and / or VL of antibody TY21585 as shown in Table 3. In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution for human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0126] In some embodiments, an antibody that binds to human CTLA4 contains a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 64. In certain embodiments, the VH sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the amino acid sequence of SEQ ID NO: 64, but retains the same ability to bind to human CTLA4 as the antibody containing SEQ ID NO: 64. In certain embodiments, a total of 1 to 13 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 64. In certain embodiments, the substitutions, insertions, or deletions occur in a region outside the CDR (i.e., within the FR). In certain embodiments, VH comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 9, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions.In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution in human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0127] In another embodiment, an antibody is provided that binds to human CTLA4, the antibody comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 65. In certain embodiments, the VL sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the amino acid sequence of SEQ ID NO: 65, but retains the same ability to bind to human CTLA4 as the antibody comprising SEQ ID NO: 65. In certain embodiments, a total of 1 to 11 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 65. In certain embodiments, the substitutions, insertions, or deletions occur in a region outside the CDR (i.e., within the FR). In certain embodiments, VL comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 10, or a variant thereof containing up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, or a variant thereof containing up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 11, or a variant thereof containing up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions.In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution in human IgG1 Including the Fc region (e.g., i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or. viii) S239D substitution and I332E substitution), the numbering of the residues follows the EU numbering.

[0128] In one embodiment, the antibody that binds to human CTLA4 includes VH, which contains the amino acid sequence of SEQ ID NO: 64, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%, or 100%) sequence identity with SEQ ID NO: 64; and VL, which contains the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%, or 100%) sequence identity with SEQ ID NO: 65, wherein the antibody contains one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, and is used with human IgG1 The Fc region is included (for example, i) one or more substitutions selected from the group consisting of S298A substitution, E333A substitution, and K334A substitution, ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution, iii) F243L substitution, iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution, v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution, vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution, vii) S239D substitution, I332E substitution, and A330L substitution, or viiii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0129] In another embodiment, an antibody that binds to human CTLA4 is provided, wherein the antibody includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 7, or VH containing a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; CDR-H2 containing the amino acid sequence of SEQ ID NO: 8, or a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and CDR-H3 containing the amino acid sequence of SEQ ID NO: 9, or a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and CDR-L1 containing the amino acid sequence of SEQ ID NO: 10, or up to 5 (e.g., 1, 2, 3, 4, or 5) VL containing a variant thereof containing an amino acid substitution, CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, or a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 11, or a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, the antibody containing one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution in human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0130] In another embodiment, an antibody that binds to human CTLA4 is provided, the antibody comprising VH CDR1, VH CDR2, and VH CDR3, each comprising the amino acid sequences of VH CDR1, CDR2, and CDR3 having the sequence described in SEQ ID NO: 64; and VL CDR1, VL CDR2, and VL CDR3, each comprising the amino acid sequences of VL CDR1, CDR2, and CDR3 having the sequence described in SEQ ID NO: 65, the antibody comprising one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution for human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0131] In some embodiments, the antibody that binds to human CTLA4 comprises one, two, three, four, five, or six CDRs of antibody TY21586 as shown in Table 2. In some embodiments, the antibody comprises the VH and / or VL of antibody TY21586 as shown in Table 3. In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution for human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0132] In some embodiments, an antibody that binds to human CTLA4 contains a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 66. In certain embodiments, the VH sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the amino acid sequence of SEQ ID NO: 66, but retains the same ability to bind to human CTLA4 as the antibody containing SEQ ID NO: 66. In certain embodiments, a total of 1 to 13 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 66. In certain embodiments, the substitutions, insertions, or deletions occur in a region outside the CDR (i.e., within the FR). In certain embodiments, VH comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions.In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution in human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0133] In another embodiment, an antibody is provided that binds to human CTLA4, the antibody comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 67. In certain embodiments, the VL sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the amino acid sequence of SEQ ID NO: 67, but retains the same ability to bind to human CTLA4 as the antibody comprising SEQ ID NO: 67. In certain embodiments, a total of 1 to 11 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 67. In certain embodiments, the substitutions, insertions, or deletions occur in a region outside the CDR (i.e., within the FR). In certain embodiments, VL comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions.In some embodiments, the antibody includes one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), where the numbering of the residues follows EU numbers.

[0134] In one embodiment, the antibody that binds to human CTLA4 includes VH, which contains the amino acid sequence of SEQ ID NO: 66, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%, or 100%) sequence identity with SEQ ID NO: 66; and VL, which contains the amino acid sequence of SEQ ID NO: 67, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%, or 100%) sequence identity with SEQ ID NO: 67, wherein the antibody contains one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, and is used with human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0135] In another embodiment, an antibody that binds to human CTLA4 is provided, wherein the antibody includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 12, or VH containing a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; CDR-H2 containing the amino acid sequence of SEQ ID NO: 13, or a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and CDR-H3 containing the amino acid sequence of SEQ ID NO: 14, or a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and CDR-L1 containing the amino acid sequence of SEQ ID NO: 15, or up to 5 (e.g., 1, 2, 3, 4, or 5) VL containing the amino acid substitution of the same variant, CDR-L2 containing the amino acid sequence of SEQ ID NO: 16, or its variant containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 17, or its variant containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, the antibody containing one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, and human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0136] In another embodiment, an antibody that binds to human CTLA4 is provided, the antibody comprising VH CDR1, VH CDR2, and VH CDR3, each containing the amino acid sequences of VH CDR1, CDR2, and CDR3 having the sequence described in SEQ ID NO: 66; and VL CDR1, VL CDR2, and VL CDR3, each containing the amino acid sequences of VL CDR1, CDR2, and CDR3 having the sequence described in SEQ ID NO: 67. The antibody comprises CDR3, and the antibody includes one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution and P396L substitution (for example, i) S298A substitution, E333A substitution and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution and K326A substitution; ii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), where the numbering of the residues follows EU numbers.

[0137] In some embodiments, the antibody that binds to human CTLA4 comprises one, two, three, four, five, or six CDRs of antibody TY21587 as shown in Table 2. In some embodiments, the antibody comprises the VH and / or VL of antibody TY21587 as shown in Table 3. In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution for human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0138] In some embodiments, an antibody that binds to human CTLA4 contains a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 68. In certain embodiments, the VH sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the amino acid sequence of SEQ ID NO: 68, but retains the same ability to bind to human CTLA4 as the antibody containing SEQ ID NO: 68. In certain embodiments, a total of 1 to 13 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 68. In certain embodiments, the substitutions, insertions, or deletions occur in a region outside the CDR (i.e., within the FR). In certain embodiments, VH comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions.In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution in human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0139] In another embodiment, an antibody is provided that binds to human CTLA4, the antibody comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 69. In certain embodiments, the VL sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the amino acid sequence of SEQ ID NO: 69, but retains the same ability to bind to human CTLA4 as the antibody comprising SEQ ID NO: 69. In certain embodiments, a total of 1 to 11 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 69. In certain embodiments, the substitutions, insertions, or deletions occur in a region outside the CDR (i.e., within the FR). In certain embodiments, VL comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 21, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions.In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution in human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0140] In one embodiment, the antibody that binds to human CTLA4 includes VH, which contains the amino acid sequence of SEQ ID NO: 68, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%, or 100%) sequence identity with SEQ ID NO: 68; and VL, which contains the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%, or 100%) sequence identity with SEQ ID NO: 69, wherein the antibody contains one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, and is used with human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0141] In another embodiment, an antibody that binds to human CTLA4 is provided, wherein the antibody includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 18, or VH containing a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; CDR-H2 containing the amino acid sequence of SEQ ID NO: 19, or a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and CDR-H3 containing the amino acid sequence of SEQ ID NO: 20, or a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and CDR-L1 containing the amino acid sequence of SEQ ID NO: 21, or up to 5 (e.g., 1, 2, 3, 4, or 5) VL containing the amino acid substitution of the same variant, CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, or its variant containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 22, or its variant containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, the antibody containing one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution in human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0142] In another embodiment, an antibody that binds to human CTLA4 is provided, the antibody comprising VH CDR1, VH CDR2, and VH CDR3, each comprising the amino acid sequences of VH CDR1, CDR2, and CDR3 having the sequence described in SEQ ID NO: 68; and VL CDR1, VL CDR2, and VL CDR3, each comprising the amino acid sequences of VL CDR1, CDR2, and CDR3 having the sequence described in SEQ ID NO: 69, the antibody comprising one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution for human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0143] In some embodiments, the antibody that binds to human CTLA4 comprises one, two, three, four, five, or six CDRs of antibody TY21588 as shown in Table 2. In some embodiments, the antibody comprises the VH and / or VL of antibody TY21588 as shown in Table 3. In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution for human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0144] In some embodiments, an antibody that binds to human CTLA4 contains a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 70. In certain embodiments, the VH sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the amino acid sequence of SEQ ID NO: 70, but retains the same ability to bind to human CTLA4 as the antibody containing SEQ ID NO: 70. In certain embodiments, a total of 1 to 13 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 70. In certain embodiments, the substitutions, insertions, or deletions occur in a region outside the CDR (i.e., within the FR). In certain embodiments, VH comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 23, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 24, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions.In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution in human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0145] In another embodiment, an antibody is provided that binds to human CTLA4, the antibody comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 71. In certain embodiments, the VL sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the amino acid sequence of SEQ ID NO: 71, but retains the same ability to bind to human CTLA4 as the antibody comprising SEQ ID NO: 71. In certain embodiments, a total of 1 to 11 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 71. In certain embodiments, the substitutions, insertions, or deletions occur in a region outside the CDR (i.e., within the FR). In certain embodiments, VL comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 26, or a variant thereof containing up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 27, or a variant thereof containing up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 28, or a variant thereof containing up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions.In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution in human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0146] In one embodiment, the antibody that binds to human CTLA4 includes VH, which contains the amino acid sequence of SEQ ID NO: 70, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%, or 100%) sequence identity with SEQ ID NO: 70; and VL, which contains the amino acid sequence of SEQ ID NO: 71, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%, or 100%) sequence identity with SEQ ID NO: 71, wherein the antibody contains one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, and is used with human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0147] In another embodiment, an antibody that binds to human CTLA4 is provided, wherein the antibody includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 23, or VH containing a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; CDR-H2 containing the amino acid sequence of SEQ ID NO: 24, or a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and CDR-H3 containing the amino acid sequence of SEQ ID NO: 25, or a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and CDR-L1 containing the amino acid sequence of SEQ ID NO: 26, or up to 5 (e.g., 1, 2, 3, 4, or 5) VL containing a variant of the same containing the amino acid substitution of, CDR-L2 containing the amino acid sequence of SEQ ID NO: 27, or a variant of the same containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 28, or a variant of the same containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, the antibody containing one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution in human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0148] In another embodiment, an antibody that binds to human CTLA4 is provided, the antibody comprising VH CDR1, VH CDR2, and VH CDR3, each comprising the amino acid sequences of VH CDR1, CDR2, and CDR3 having the sequence described in SEQ ID NO: 70; and VL CDR1, VL CDR2, and VL CDR3, each comprising the amino acid sequences of VL CDR1, CDR2, and CDR3 having the sequence described in SEQ ID NO: 71, the antibody comprising one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution for human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0149] In some embodiments, the antibody that binds to human CTLA4 comprises one, two, three, four, five, or six CDRs of antibody TY21589 as shown in Table 2. In some embodiments, the antibody comprises the VH and / or VL of antibody TY21589 as shown in Table 3. In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution for human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0150] In some embodiments, an antibody that binds to human CTLA4 contains a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 72. In certain embodiments, the VH sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the amino acid sequence of SEQ ID NO: 72, but retains the same ability to bind to human CTLA4 as the antibody containing SEQ ID NO: 72. In certain embodiments, a total of 1 to 13 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 72. In certain embodiments, the substitutions, insertions, or deletions occur in a region outside the CDR (i.e., within the FR). In certain embodiments, VH comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 29, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 30, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 31, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions.In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution in human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0151] In another embodiment, an antibody is provided that binds to human CTLA4, the antibody comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 73. In certain embodiments, the VL sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the amino acid sequence of SEQ ID NO: 73, but retains the same ability to bind to human CTLA4 as the antibody comprising SEQ ID NO: 73. In certain embodiments, a total of 1 to 11 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 73. In certain embodiments, the substitutions, insertions, or deletions occur in a region outside the CDR (i.e., within the FR). In certain embodiments, VL comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 32, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 33, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions.In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution in human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0152] In one embodiment, the antibody that binds to human CTLA4 includes VH, which contains the amino acid sequence of SEQ ID NO: 72, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%, or 100%) sequence identity with SEQ ID NO: 72; and VL, which contains the amino acid sequence of SEQ ID NO: 73, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%, or 100%) sequence identity with SEQ ID NO: 73, wherein the antibody contains one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, and is used with human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution, or viiii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0153] In another embodiment, an antibody that binds to human CTLA4 is provided, wherein the antibody includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 29, or VH containing a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; CDR-H2 containing the amino acid sequence of SEQ ID NO: 30, or a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and CDR-H3 containing the amino acid sequence of SEQ ID NO: 31, or a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and CDR-L1 containing the amino acid sequence of SEQ ID NO: 32, or up to 5 (e.g., 1, 2, 3, 4, or 5) VL containing the amino acid substitution of the same variant, CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, or its variant containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 33, or its variant containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, the antibody containing one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, and human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0154] In another embodiment, an antibody that binds to human CTLA4 is provided, the antibody comprising VH CDR1, VH CDR2, and VH CDR3, each comprising the amino acid sequences of VH CDR1, CDR2, and CDR3 having the sequence described in SEQ ID NO: 72; and VL CDR1, VL CDR2, and VL CDR3, each comprising the amino acid sequences of VL CDR1, CDR2, and CDR3 having the sequence described in SEQ ID NO: 73, the antibody comprising one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution for human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution, or viiii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0155] In some embodiments, the antibody that binds to human CTLA4 comprises one, two, three, four, five, or six CDRs of antibody TY21591 as shown in Table 2. In some embodiments, the antibody comprises the VH and / or VL of antibody TY21591 as shown in Table 3. In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution for human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0156] In some embodiments, the antibody that binds to human CTLA4 includes a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 76. In certain embodiments, the VH sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the amino acid sequence of SEQ ID NO: 76, but retains the same ability to bind to human CTLA4 as the antibody containing SEQ ID NO: 76. In certain embodiments, a total of 1 to 13 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 76. In certain embodiments, the substitutions, insertions, or deletions occur in a region outside the CDR (i.e., within the FR). In certain embodiments, VH comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 35, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions. In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution in human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0157] In another embodiment, an antibody is provided that binds to human CTLA4, the antibody comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 77. In certain embodiments, the VL sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the amino acid sequence of SEQ ID NO: 77, but retains the same ability to bind to human CTLA4 as the antibody comprising SEQ ID NO: 77. In certain embodiments, a total of 1 to 11 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 77. In certain embodiments, the substitutions, insertions, or deletions occur in a region outside the CDR (i.e., within the FR). In certain embodiments, VL comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 36, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions.In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution in human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0158] In one embodiment, the antibody that binds to human CTLA4 includes VH, which contains the amino acid sequence of SEQ ID NO: 76, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%, or 100%) sequence identity with SEQ ID NO: 76; and VL, which contains the amino acid sequence of SEQ ID NO: 77, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%, or 100%) sequence identity with SEQ ID NO: 77, wherein the antibody contains one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, and is used with human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viiii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0159] In another embodiment, an antibody that binds to human CTLA4 is provided, wherein the antibody includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 34, or VH containing a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; CDR-H2 containing the amino acid sequence of SEQ ID NO: 19, or a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and CDR-H3 containing the amino acid sequence of SEQ ID NO: 35, or a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and CDR-L1 containing the amino acid sequence of SEQ ID NO: 36, or up to 5 (e.g., 1, 2, 3, 4, or 5) VL containing the amino acid substitution of the same variant, CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, or its variant containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 37, or its variant containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, the antibody containing one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, and human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0160] In another embodiment, an antibody that binds to human CTLA4 is provided, the antibody comprising VH CDR1, VH CDR2, and VH CDR3, each comprising the amino acid sequences of VH CDR1, CDR2, and CDR3 having the sequence described in SEQ ID NO: 76; and VL CDR1, VL CDR2, and VL CDR3, each comprising the amino acid sequences of VL CDR1, CDR2, and CDR3 having the sequence described in SEQ ID NO: 77, the antibody comprising one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution for human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0161] In some embodiments, the antibody that binds to human CTLA4 comprises one, two, three, four, five, or six CDRs of antibody TY21686 as shown in Table 2. In some embodiments, the antibody comprises the VH and / or VL of antibody TY21686 as shown in Table 3. In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution for human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0162] In some embodiments, an antibody that binds to human CTLA4 contains a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 78. In certain embodiments, the VH sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the amino acid sequence of SEQ ID NO: 78, but retains the same ability to bind to human CTLA4 as the antibody containing SEQ ID NO: 78. In certain embodiments, a total of 1 to 13 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 78. In certain embodiments, the substitutions, insertions, or deletions occur in a region outside the CDR (i.e., within the FR). In certain embodiments, VH comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 39, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 40, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions.In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, and Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution in human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0163] In another embodiment, an antibody is provided that binds to human CTLA4, the antibody comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 79. In certain embodiments, the VL sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the amino acid sequence of SEQ ID NO: 79, but retains the same ability to bind to human CTLA4 as the antibody comprising SEQ ID NO: 79. In certain embodiments, a total of 1 to 11 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 79. In certain embodiments, the substitutions, insertions, or deletions occur in a region outside the CDR (i.e., within the FR). In certain embodiments, VL comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 41, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 42, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 43, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions.In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution in human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0164] In one embodiment, the antibody that binds to human CTLA4 includes VH, which contains the amino acid sequence of SEQ ID NO: 78, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%, or 100%) sequence identity with SEQ ID NO: 78; and VL, which contains the amino acid sequence of SEQ ID NO: 79, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%, or 100%) sequence identity with SEQ ID NO: 79, wherein the antibody contains one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, and is used with human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0165] In another embodiment, an antibody that binds to human CTLA4 is provided, the antibody comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 38, or VH containing a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; CDR-H2 containing the amino acid sequence of SEQ ID NO: 39, or a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; CDR-H3 containing the amino acid sequence of SEQ ID NO: 40, or a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and CDR-L1 containing the amino acid sequence of SEQ ID NO: 41, or up to 5 (e.g., 1, 2, 3, 4, or 5) VL containing the amino acid substitution of the same variant, CDR-L2 containing the amino acid sequence of SEQ ID NO: 42, or its variant containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 43, or its variant containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, the antibody containing one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, and human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0166] In another embodiment, an antibody that binds to human CTLA4 is provided, the antibody comprising VH CDR1, VH CDR2, and VH CDR3, each comprising the amino acid sequences of VH CDR1, CDR2, and CDR3 having the sequence described in SEQ ID NO: 78; and VL CDR1, VL CDR2, and VL CDR3, each comprising the amino acid sequences of VL CDR1, CDR2, and CDR3 having the sequence described in SEQ ID NO: 79, the antibody comprising one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution for human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0167] In some embodiments, the antibody that binds to human CTLA4 comprises one, two, three, four, five, or six CDRs of antibody TY21687 as shown in Table 2. In some embodiments, the antibody comprises the VH and / or VL of antibody TY21687 as shown in Table 3. In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution for human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0168] In some embodiments, an antibody that binds to human CTLA4 contains a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 80. In certain embodiments, the VH sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the amino acid sequence of SEQ ID NO: 80, but retains the same ability to bind to human CTLA4 as the antibody containing SEQ ID NO: 80. In certain embodiments, a total of 1 to 13 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 80. In certain embodiments, the substitutions, insertions, or deletions occur in a region outside the CDR (i.e., within the FR). In certain embodiments, VH comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 44, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 45, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 46, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions.In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution in human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0169] In another embodiment, an antibody is provided that binds to human CTLA4, the antibody comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 81. In certain embodiments, the VL sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the amino acid sequence of SEQ ID NO: 81, but retains the same ability to bind to human CTLA4 as the antibody comprising SEQ ID NO: 81. In certain embodiments, a total of 1 to 11 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 81. In certain embodiments, the substitutions, insertions, or deletions occur in a region outside the CDR (i.e., within the FR). In certain embodiments, VL comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 47, or a variant thereof containing up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, or a variant thereof containing up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 48, or a variant thereof containing up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions.In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution in human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0170] In one embodiment, the antibody that binds to human CTLA4 includes VH, which contains the amino acid sequence of SEQ ID NO: 80, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%, or 100%) sequence identity with SEQ ID NO: 80; and VL, which contains the amino acid sequence of SEQ ID NO: 81, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%, or 100%) sequence identity with SEQ ID NO: 81, wherein the antibody contains one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, and is used with human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0171] In another embodiment, an antibody that binds to human CTLA4 is provided, wherein the antibody includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 44, or VH containing a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; CDR-H2 containing the amino acid sequence of SEQ ID NO: 45, or a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and CDR-H3 containing the amino acid sequence of SEQ ID NO: 46, or a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and CDR-L1 containing the amino acid sequence of SEQ ID NO: 47, or up to 5 (e.g., 1, 2, 3, 4, or 5) VL containing the amino acid substitution of the same variant, CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, or its variant containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 48, or its variant containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, the antibody containing one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution in human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0172] In another embodiment, an antibody that binds to human CTLA4 is provided, the antibody comprising VH CDR1, VH CDR2, and VH CDR3, each comprising the amino acid sequences of VH CDR1, CDR2, and CDR3 having the sequence described in SEQ ID NO: 80; and VL CDR1, VL CDR2, and VL CDR3, each comprising the amino acid sequences of VL CDR1, CDR2, and CDR3 having the sequence described in SEQ ID NO: 81, the antibody comprising one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution for human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0173] In some embodiments, the antibody that binds to human CTLA4 comprises one, two, three, four, five, or six CDRs of antibody TY21689 as shown in Table 2. In some embodiments, the antibody comprises the VH and / or VL of antibody TY21689 as shown in Table 3. In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution for human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0174] In some embodiments, an antibody that binds to human CTLA4 contains a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 82. In certain embodiments, the VH sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the amino acid sequence of SEQ ID NO: 82, but retains the same ability to bind to human CTLA4 as the antibody containing SEQ ID NO: 82. In certain embodiments, a total of 1 to 13 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 82. In certain embodiments, the substitutions, insertions, or deletions occur in a region outside the CDR (i.e., within the FR). In certain embodiments, VH comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 49, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 50, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions.In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution in human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0175] In another embodiment, an antibody is provided that binds to human CTLA4, the antibody comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 83. In certain embodiments, the VL sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the amino acid sequence of SEQ ID NO: 83, but retains the same ability to bind to human CTLA4 as the antibody comprising SEQ ID NO: 83. In certain embodiments, a total of 1 to 11 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 83. In certain embodiments, the substitutions, insertions, or deletions occur in a region outside the CDR (i.e., within the FR). In certain embodiments, VL comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 51, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 52, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions.In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution in human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0176] In one embodiment, the antibody that binds to human CTLA4 includes VH, which contains the amino acid sequence of SEQ ID NO: 82, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%, or 100%) sequence identity with SEQ ID NO: 82; and VL, which contains the amino acid sequence of SEQ ID NO: 83, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%, or 100%) sequence identity with SEQ ID NO: 83, wherein the antibody contains one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, and is used with human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0177] In another embodiment, an antibody that binds to human CTLA4 is provided, wherein the antibody includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 49, or VH containing a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; CDR-H2 containing the amino acid sequence of SEQ ID NO: 19, or a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and CDR-H3 containing the amino acid sequence of SEQ ID NO: 50, or a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and CDR-L1 containing the amino acid sequence of SEQ ID NO: 51, or up to 5 (e.g., 1, 2, 3, 4, or 5) VL containing the amino acid substitution of the same variant, CDR-L2 containing the amino acid sequence of SEQ ID NO: 16, or its variant containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 52, or its variant containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, the antibody containing one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, and human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0178] In another embodiment, an antibody that binds to human CTLA4 is provided, the antibody comprising VH CDR1, VH CDR2, and VH CDR3, each comprising the amino acid sequences of VH CDR1, CDR2, and CDR3 having the sequence described in SEQ ID NO: 82; and VL CDR1, VL CDR2, and VL CDR3, each comprising the amino acid sequences of VL CDR1, CDR2, and CDR3 having the sequence described in SEQ ID NO: 83, the antibody comprising one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution for human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0179] In some embodiments, the antibody that binds to human CTLA4 comprises one, two, three, four, five, or six CDRs of antibody TY21680 as shown in Table 2. In some embodiments, the antibody comprises the VH and / or VL of antibody TY21680 as shown in Table 3. In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution for human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0180] In some embodiments, an antibody that binds to human CTLA4 contains a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 84. In certain embodiments, the VH sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the amino acid sequence of SEQ ID NO: 84, but retains the same ability to bind to human CTLA4 as the antibody containing SEQ ID NO: 84. In certain embodiments, a total of 1 to 13 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 84. In certain embodiments, the substitutions, insertions, or deletions occur in a region outside the CDR (i.e., within the FR). In certain embodiments, VH comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 44, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 45, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 53, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions.In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution in human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0181] In another embodiment, an antibody is provided that binds to human CTLA4, the antibody comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 85. In certain embodiments, the VL sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the amino acid sequence of SEQ ID NO: 85, but retains the same ability to bind to human CTLA4 as the antibody containing SEQ ID NO: 85. In certain embodiments, a total of 1 to 11 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 85. In certain embodiments, the substitutions, insertions, or deletions occur in a region outside the CDR (i.e., within the FR). In certain embodiments, VL comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 54, or a variant thereof containing up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 16, or a variant thereof containing up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 55, or a variant thereof containing up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions.In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution in human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0182] In one embodiment, the antibody that binds to human CTLA4 comprises VH, which contains the amino acid sequence of SEQ ID NO: 84, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%, or 100%) sequence identity with SEQ ID NO: 84; and VL, which contains the amino acid sequence of SEQ ID NO: 85, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%, or 100%) sequence identity with SEQ ID NO: 85, wherein the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, and is used with human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0183] In another embodiment, an antibody that binds to human CTLA4 is provided, wherein the antibody includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 44, or VH containing a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; CDR-H2 containing the amino acid sequence of SEQ ID NO: 45, or a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and CDR-H3 containing the amino acid sequence of SEQ ID NO: 53, or a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and CDR-L1 containing the amino acid sequence of SEQ ID NO: 54, or up to 5 (e.g., 1, 2, 3, 4, or 5) VL containing the amino acid substitution of the same variant, CDR-L2 containing the amino acid sequence of SEQ ID NO: 16, or its variant containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 55, or its variant containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, the antibody containing one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, and human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0184] In another embodiment, an antibody that binds to human CTLA4 is provided, the antibody comprising VH CDR1, VH CDR2, and VH CDR3, each comprising the amino acid sequences of VH CDR1, CDR2, and CDR3 having the sequence described in SEQ ID NO: 84; and VL CDR1, VL CDR2, and VL CDR3, each comprising the amino acid sequences of VL CDR1, CDR2, and CDR3 having the sequence described in SEQ ID NO: 85, the antibody comprising one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution for human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0185] In some embodiments, the antibody that binds to human CTLA4 comprises one, two, three, four, five, or six CDRs of antibody TY21691 as shown in Table 2. In some embodiments, the antibody comprises the VH and / or VL of antibody TY21691 as shown in Table 3. In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution for human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0186] In some embodiments, an antibody that binds to human CTLA4 contains a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 86. In certain embodiments, the VH sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the amino acid sequence of SEQ ID NO: 86, but retains the same ability to bind to human CTLA4 as the antibody containing SEQ ID NO: 86. In certain embodiments, a total of 1 to 13 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 86. In certain embodiments, the substitutions, insertions, or deletions occur in a region outside the CDR (i.e., within the FR). In certain embodiments, VH comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 56, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 57, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions.In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution in human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0187] In another embodiment, an antibody is provided that binds to human CTLA4, the antibody comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 87. In certain embodiments, the VL sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the amino acid sequence of SEQ ID NO: 87, but retains the same ability to bind to human CTLA4 as the antibody comprising SEQ ID NO: 87. In certain embodiments, a total of 1 to 11 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 87. In certain embodiments, the substitutions, insertions, or deletions occur in a region outside the CDR (i.e., within the FR). In certain embodiments, VL comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 58, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 59, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions.In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution in human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0188] In one embodiment, the antibody that binds to human CTLA4 includes VH, which contains the amino acid sequence of SEQ ID NO: 86, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%, or 100%) sequence identity with SEQ ID NO: 86; and VL, which contains the amino acid sequence of SEQ ID NO: 87, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%, or 100%) sequence identity with SEQ ID NO: 87, wherein the antibody contains one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, and is used with human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viiii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0189] In another embodiment, an antibody that binds to human CTLA4 is provided, wherein the antibody includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 7, or VH containing a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; CDR-H2 containing the amino acid sequence of SEQ ID NO: 56, or a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and CDR-H3 containing the amino acid sequence of SEQ ID NO: 57, or a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and CDR-L1 containing the amino acid sequence of SEQ ID NO: 58, or up to 5 (e.g., 1, 2, 3, 4, or 5) VL containing a variant thereof containing an amino acid substitution, CDR-L2 containing the amino acid sequence of SEQ ID NO: 16, or a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 59, or a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, the antibody containing one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution in human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0190] In another embodiment, an antibody that binds to human CTLA4 is provided, the antibody comprising VH CDR1, VH CDR2, and VH CDR3, each comprising the amino acid sequences of VH CDR1, CDR2, and CDR3 having the sequence described in SEQ ID NO: 86; and VL CDR1, VL CDR2, and VL CDR3, each comprising the amino acid sequences of VL CDR1, CDR2, and CDR3 having the sequence described in SEQ ID NO: 87, the antibody comprising one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution for human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0191] In some embodiments, the antibody that binds to human CTLA4 comprises one, two, three, four, five, or six CDRs of antibody TY21692 as shown in Table 2. In some embodiments, the antibody comprises the VH and / or VL of antibody TY21692 as shown in Table 3. In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution for human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0192] In some embodiments, an antibody that binds to human CTLA4 contains a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 88. In certain embodiments, the VH sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the amino acid sequence of SEQ ID NO: 88, but retains the same ability to bind to human CTLA4 as the antibody containing SEQ ID NO: 88. In certain embodiments, a total of 1 to 13 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 88. In certain embodiments, the substitutions, insertions, or deletions occur in a region outside the CDR (i.e., within the FR). In certain embodiments, VH comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 60, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 61, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 62, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions.In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution in human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0193] In another embodiment, an antibody is provided that binds to human CTLA4, the antibody comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 89. In certain embodiments, the VL sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the amino acid sequence of SEQ ID NO: 89, but retains the same ability to bind to human CTLA4 as the antibody comprising SEQ ID NO: 89. In certain embodiments, a total of 1 to 11 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 89. In certain embodiments, the substitutions, insertions, or deletions occur in a region outside the CDR (i.e., within the FR). In certain embodiments, VL comprises one, two, or three CDRs selected from the group consisting of: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 63, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 27, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 43, or a variant thereof comprising up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions.In some embodiments, the antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution in human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0194] In one embodiment, the antibody that binds to human CTLA4 includes VH, which contains the amino acid sequence of SEQ ID NO: 88, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%, or 100%) sequence identity with SEQ ID NO: 88; and VL, which contains the amino acid sequence of SEQ ID NO: 89, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%, or 100%) sequence identity with SEQ ID NO: 89, wherein the antibody contains one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, and is used with human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0195] In another embodiment, an antibody that binds to human CTLA4 is provided, wherein the antibody includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 60, or VH containing a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; CDR-H2 containing the amino acid sequence of SEQ ID NO: 61, or a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and CDR-H3 containing the amino acid sequence of SEQ ID NO: 62, or a variant thereof containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and CDR-L1 containing the amino acid sequence of SEQ ID NO: 63, or up to 5 (e.g., 1, 2, 3, 4, or 5) VL containing the amino acid substitution of the same variant, CDR-L2 containing the amino acid sequence of SEQ ID NO: 27, or its variant containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 43, or its variant containing up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, the antibody containing one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, and human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0196] In another embodiment, an antibody that binds to human CTLA4 is provided, the antibody comprising VH CDR1, VH CDR2, and VH CDR3, each comprising the amino acid sequences of VH CDR1, CDR2, and CDR3 having the sequence described in SEQ ID NO: 88; and VL CDR1, VL CDR2, and VL CDR3, each comprising the amino acid sequences of VL CDR1, CDR2, and CDR3 having the sequence described in SEQ ID NO: 89, the antibody comprising one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution for human IgG1 The Fc region is included (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0197] In some embodiments, the anti-CTLA4 antibody of this disclosure binds to human CTLA4 in the following ways: a) CDR-H1 containing an amino acid sequence selected from SEQ ID NOs: 1, 7, 12, 18, 23, 29, 34, 38, 44, 49, and 60, or a variant thereof containing up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; CDR-H2 containing an amino acid sequence selected from SEQ ID NOs: 2, 8, 13, 19, 24, 30, 39, 45, 56, and 61, or a variant thereof containing up to five (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and CDR-H3 containing an amino acid sequence selected from SEQ ID NOs: 3, 9, 14, 20, 25, 31, 35, 40, 46, 50, 53, 57, and 62, or up to five (e.g., 1, 2, 3) a) variants thereof containing amino acid substitutions 4, 4, or 5); and / or b) CDR-L1 containing an amino acid sequence selected from SEQ ID NOs: 4, 10, 15, 21, 26, 32, 36, 41, 47, 51, 54, 58, and 63, or a variant thereof containing up to five amino acid substitutions (e.g., 1, 2, 3, 4, or 5); CDR-L2 containing an amino acid sequence selected from SEQ ID NOs: 5, 16, 27, and 63, or a variant thereof containing up to five amino acid substitutions (e.g., 1, 2, 3, 4, or 5); and CDR-L3 containing an amino acid sequence selected from SEQ ID NOs: 6, 11, 17, 22, 28, 33, 37, 43, 48, 52, and 59, or a variant thereof containing up to five amino acid substitutions (e.g., 1, 2, 3, 4, or 5). In some embodiments, the anti-CTLA4 antibodies of this disclosure cross-compete for binding to human CTLA4 with antibodies containing one, two, three, four, five, or all six CDRs shown for any of the exemplary antibodies listed in Table 2. In some embodiments, the anti-CTLA4 antibodies of this disclosure cross-compete for binding to human CTLA4 with the following antibodies: a) a heavy chain variable region containing an amino acid sequence selected from SEQ ID NOs. 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, and 88, and / or b) a light chain variable region containing an amino acid sequence selected from SEQ ID NOs. 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, and 89.In some embodiments, the anti-CTLA4 antibody includes 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. 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, and 88, 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. 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, and 89. In some embodiments, the anti-CTLA4 antibodies of this disclosure cross-compete for binding to human CTLA4 with antibodies containing the heavy chain variable region and light chain variable region of any of the exemplary anti-CTLA4 antibodies listed in Table 3. In some embodiments, the anti-CTLA4 antibodies of this disclosure cross-compete for binding to human CTLA4 with antibodies containing one, two, or all three CDRs of the heavy chain variable region and / or one, two, or all three CDRs of the light chain variable region as shown for any of the exemplary anti-CTLA4 antibodies listed in Table 3.

[0198] 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. Also provided herein are one or more anti-CTLA4 antibodies or antigen-binding fragments that cross-compete with one or more of the antibodies or antigen-binding fragments described herein for binding to human CTLA4.

[0199] In some embodiments, the anti-CTLA4 antibody or antigen-binding fragment binds to human, cynomolgus monkey, mouse, rat, and / or canine CTLA4 with a KD 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.). In some embodiments, the anti-CTLA4 antibody or antigen-binding fragment binds to human, cynomolgus monkey, mouse, rat, and / or canine CTLA4 with a KD of approximately 350 nM or less. In some embodiments, the anti-CTLA4 antibody or antigen-binding fragment binds to human CTLA4 with a KD of approximately 100 nM or less. In some embodiments, the anti-CTLA4 antibody or antigen-binding fragment binds to human CTLA4 with a KD of approximately 50 nM or less. In some embodiments, the anti-CTLA4 antibody or antigen-binding fragment binds to human CTLA4 with a KD of approximately 10 nM or less. Methods for measuring the KD of the anti-CTLA4 antibody or antigen-binding fragment can be carried out using any method known in the art, including, for example, surface plasmon resonance, ELISA, isothermal titration calorimetry, filter-bound assay, EMSA, etc. In some embodiments, the KD is measured by surface plasmon resonance or ELISA, as described, for example, in U.S. Patent Application Publication No. 20210207126A1.

[0200] In some embodiments, the anti-CTLA4 antibody or antigen-binding fragment described herein has antagonist activity against human CTLA4. In some embodiments, the anti-CTLA4 antibody or antigen-binding fragment suppresses one or more activities of human CTLA4 when cells expressing human CTLA4 (e.g., human cells) are contacted with the anti-CTLA4 antibody or antigen-binding fragment (e.g., CTLA4 blockade as measured by an increase in reporter gene signal using a CLA4 blockade reporter gene assay).

[0201] In some embodiments, the anti-CTLA4 antibody or antigen-binding fragment is cross-reactive with monkey (e.g., cynomolgus monkey), mouse, rat, and / or canine CTLA4. In some embodiments, the anti-CTLA4 antibody or antigen-binding fragment is cross-reactive with monkey CTLA4. In some embodiments, the anti-CTLA4 antibody or antigen-binding fragment is cross-reactive with mouse CTLA4. In some embodiments, the anti-CTLA4 antibody or antigen-binding fragment is cross-reactive with rat CTLA4. In some embodiments, the anti-CTLA4 antibody or antigen-binding fragment is cross-reactive with canine CTLA4. In some embodiments, the anti-CTLA4 antibody or antigen-binding fragment is cross-reactive with monkey and mouse CTLA4; monkey and rat CTLA4; monkey and canine CTLA4; mouse and rat CTLA4; mouse and canine CTLA4; rat and canine CTLA4; monkey, mouse, and rat CTLA4; monkey, mouse, and canine CTLA4; monkey, rat, and canine CTLA4; mouse, rat, and canine CTLA4; or monkey, mouse, rat, and canine CTLA4. In some embodiments, an anti-CTLA4 antibody or antigen-binding fragment is cross-reactive if it binds to a non-human CTLA4 molecule with a KD of less than approximately 500 nM (e.g., less than approximately 1 nM, less than approximately 10 nM, less than approximately 25 nM, less than approximately 50 nM, less than approximately 75 nM, less than approximately 100 nM, less than approximately 150 nM, less than approximately 200 nM, less than approximately 250 nM, less than approximately 300 nM, less than approximately 350 nM, etc.). 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-bound assays, EMSA, etc. In some embodiments, cross-reactivity is measured by ELISA, for example, as described in U.S. Patent Application Publication No. US20210207126A1.

[0202] In some embodiments, anti-CTLA4 antibodies induce antibody-dependent cytotoxicity (ADCC) in CTLA4-expressing cells (e.g., human cells expressing CTLA4, such as Treg) after the anti-CTLA4 antibody binds to cell-expressed CTLA4. In some embodiments, anti-CTLA4 antibodies induce ADCC in over 10% of cases compared to a control (e.g., isotype control or ipilimumab) (e.g., inducing ADCC in over 10%, over 15%, over 20%, over 25%, over 30%, over 35%, over 40%, etc.). In a particular embodiment, the anti-CTLA4 antibody comprises an IgG1 Fc region (e.g., a human IgG1 Fc region) containing one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, wherein the numbering of the residues follows EU numbers, and the anti-CTLA4 antibody induces the ADCC effect at a rate at least about 10% (e.g., 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%, or at least about 90%) higher than an anti-CTLA4 antibody comprising an Fc region without one or more substitutions. In a particular embodiment, the anti-CTLA4 antibody comprises an IgG1 Fc region (e.g., a human IgG1 Fc region) comprising one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, wherein the numbering of the residues follows EU numbers, and the anti-CTLA4 antibody exhibits an ADCC effect that is at least about 1.5 times (e.g., at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, or at least about 5 times) higher than an anti-CTLA4 antibody comprising an Fc region without substitutions.In some embodiments, the anti-CTLA4 antibody has a semi-maximal effective concentration (EC50) of about 5 nM or less for inducing ADCC in CTLA4-expressing cells (e.g., about 5 nM or less, about 4 nM or less, about 3 nM or less, about 3 nM or less, about 2 nM or less, about 1 nM or less, about 0.5 nM or less, about 0.1 nM or less, or about 0.05 nM or less). Methods for measuring the ADCC effect (e.g., in vitro methods) are known in the art and include, but are not limited to, those described in Example 3 or, for example, in U.S. Patent Application Publication No. 20210207126A1.

[0203] In some embodiments, anti-CTLA4 antibodies induce antibody-dependent cell-mediated phagocytosis (ADCP) effects on CTLA4-expressing cells (e.g., CTLA4-expressing human cells such as Tregs) after the anti-CTLA4 antibody binds to cell-expressed CTLA4. In some embodiments, anti-CTLA4 antibodies induce ADCP effects at over 10% compared to a control (e.g., isotype control or ipilimumab) (e.g., induce ADCP at over 10%, over 15%, over 20%, over 25%, over 30%, over 35%, over 40%, etc.). In certain embodiments in which an anti-CTLA4 antibody includes an IgG1 Fc region (e.g., a human IgG1 Fc region) comprising one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, the numbering of the residues follows EU numbers, and the anti-CTLA4 antibody induces the ADCP effect at a rate at least about 10% (e.g., 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%, or at least about 90%) higher than an anti-CTLA4 antibody comprising an Fc region without substitutions. In a particular embodiment, the anti-CTLA4 antibody includes one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, and includes an IgG1 Fc region (e.g., human IgG1 Fc region), wherein the numbering of the residues follows EU numbers, and the anti-CTLA4 antibody induces the ADCP effect at a rate at least about 1.5 times higher (e.g., at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, or at least about 5 times) higher than an anti-CTLA4 antibody containing an Fc region without substitutions.In some embodiments, the anti-CTLA4 antibody has a semi-maximal effective concentration (EC50) of about 5 nM or less for inducing ADCP in CTLA4-expressing cells (e.g., about 5 nM or less, about 4 nM or less, about 3 nM or less, about 3 nM or less, about 2 nM or less, about 1 nM or less, about 0.5 nM or less, about 0.1 nM or less, or about 0.05 nM or less). Methods for measuring the ADCP of antibodies and antigen-binding fragments are also well known in the art. To evaluate the ADCP activity of the molecule of interest, for example, an in vitro ADCP assay can be performed (see, e.g., Bracher et al., 2007, J. Immunol. Methods 323:160-71). Phagoid cells useful for such assays include peripheral blood mononuclear cells (PBMCs), monocytes purified from PBMCs, or U937 cells differentiated into monocyte lineages. Alternatively or additionally, the ADCP activity of the molecule of interest can be evaluated in vivo, for example, in animal models (see, e.g., Wallace et al., 2001, J.Immunol.Methods 248:167-82).

[0204] In some embodiments, an anti-CTLA4 antibody or antigen-binding fragment can inhibit 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 in contact with the anti-CTLA4 antibody or antigen-binding fragment, compared to the corresponding tumor cells not in contact with the anti-CTLA4 antibody or antigen-binding fragment (or compared to the corresponding tumor cells in contact with an isotype control antibody). In some embodiments, an anti-CTLA4 antibody or antigen-binding fragment can reduce tumor volume in a subject when administered to that subject. In some embodiments, an anti-CTLA4 antibody or antigen-binding fragment can reduce the tumor volume in a subject by at least about 5% (e.g., at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 99%) compared to the initial tumor volume in the subject (e.g., compared to the corresponding tumor in a subject administered with an isotype control antibody before administration of the anti-CTLA4 antibody or antigen-binding fragment). Methods for monitoring tumor cell growth / proliferation, tumor volume, and / or tumor inhibition are known in the art and include, for example, the method described in Example 4, as described in U.S. Patent Application Publication No. 20210207126A1.

[0205] In some embodiments, anti-CTLA4 antibodies or antigen-binding fragments have a therapeutic effect against cancer. In some embodiments, anti-CTLA4 antibodies or antigen-binding fragments alleviate one or more signs or symptoms of cancer. In some embodiments, subjects with cancer achieve partial or complete remission upon administration of anti-CTLA4 antibodies or antigen-binding fragments. Anti-CTLA4 antibody

[0206] In some embodiments, the disclosure provides isolated antibodies that specifically bind to epitopes similar to the ligand-binding sites of human CTLA4. In some embodiments, the antibody specifically binds to an epitope similar to the CD80 binding site of human CTLA4. In some embodiments, the antibody specifically binds to an epitope similar to the CD86 binding site of human CTLA4. In some embodiments, the antibody specifically binds to an epitope containing one or more amino acid residues at the ligand-binding site of human CTLA4 (e.g., 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 ipilimumab epitope. In some embodiments, the epitope does not contain amino acid residues in the CC' loop motif of human CTLA4. In some embodiments, the epitope does not contain the amino acid residues L106 or I108 of human CTLA4. In some embodiments, the antibody specifically binds to an epitope containing the amino acid residues Y105 and L106 of human CTLA4 but not I108, and the amino acid residue numbering follows SEQ ID NO: 134. KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPE(Sequence ID 134)

[0207] In some embodiments, the antibody of this disclosure is a bispecific antibody that binds to a first and a second target, the first target being human CTLA4. In some embodiments, the bispecific antibody binds to a first and a second target, the first target being human CTLA4, and the bispecific antibody comprises all of the CDRs 1, 2, 3, 4, 5, or 6 shown for any of the exemplary antibodies listed in Table 2. In some embodiments, the bispecific antibody binds to a first and a second target, the first target being human CTLA4, and the bispecific antibody comprises the VH and / or VL shown for any of the exemplary antibodies listed in Table 3. 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.

[0208] The anti-CTLA4 antibodies of this disclosure may be produced by any technique known in the art, including conventional monoclonal antibody methodologies, e.g., 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 described in detail herein (see, e.g., Examples 1 and 2). In some embodiments, the anti-CTLA4 antibodies of this disclosure are produced using any of the libraries and / or methods described in U.S. Patent Application Publication US20200362019A1 (which is incorporated herein in its entirety by reference) and / or U.S. Patent Application Publication US20200248336A1 (which is incorporated herein in its entirety by reference).

[0209] Hybridoma production is a very well-established procedure. The common animal line for preparing hybridomas is the mouse line. Immunoprotocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known. One well-known method that may be used to produce the human CTLA4 antibody provided by this disclosure involves the use of the XenoMouse® animal line. The XenoMouse® mouse is an engineered mouse line containing large fragments of human immunoglobulin heavy and light chain loci and deficient in mouse antibody production (see, e.g., Green et al., (1994) Nature Genetics 7:13-21, WO2003 / 040170). The animals are immunized with the CTLA4 antigen. The CTLA4 antigen is isolated and / or purified CTLA4. This may be a fragment of CTLA4, e.g., the extracellular domain of CTLA4. Immunization of animals can be carried out by any method known in the art (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Press, 1990). Methods for immunizing non-human animals such as mice, rats, sheep, goats, pigs, cattle, and horses are well known in the art (see, for example, Harlow and Lane above, and U.S. Patent No. 5,994,619). The CTLA4 antigen 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 (immunostimulatory complex). After immunization of animals with the CTLA4 antigen, immortalized cell lines that produce antibodies are prepared from cells isolated from the immunized animals. After immunization, the animals are sacrificed to immortalize B cells in the lymph nodes and / or spleen.Methods for immortalizing cells include, but are not limited to, introducing oncogenes into cells, flexing cells with oncogenic viruses, culturing cells under conditions that select immortalized cells, subjecting cells to oncogenic or mutant compounds, fusing cells with immortalized cells, such as myeloma cells, and inactivating tumor suppressor genes (see, for example, Harlow and Lane above). When fusion with myeloma cells is used, myeloma cells are preferably non-secreting cell lines that do not secrete immunoglobulin polypeptides. Immortalized cells are screened using CTLA4, a portion thereof, or cells expressing CTLA4. As will be further discussed below, CTLA4 antibody-producing cells, such as hybridomas, are selected and cloned and further screened for desirable properties, including potent proliferation, high antibody production, and desirable antibody characteristics. Hybridomas can be grown in vivo in syngeneic animals, animals lacking an immune system, such as nude mice, or in vitro in cell culture. Methods for selecting, cloning, and growing hybridomas are well known to those skilled in the art.

[0210] The anti-CTLA4 antibodies of this disclosure may also be prepared using phage presentation or yeast presentation methods. Such presentation methods for isolating human antibodies are well-established in the art (see, for example, Knappik, et al. (2000) J.Mol.Biol.296,57-86, Feldhaus et al. (2003) Nat Biotechnol 21: 163-170, and also the methods of Examples 1 and 2 below). antigen binding fragment

[0211] In some other embodiments, the Disclosure provides antigen-binding fragments of any of the CTLA4 antibodies described herein. In some embodiments, the antigen-binding fragment comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution for human IgG1 (For example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0212] The antigen-binding fragment may comprise any sequence of any of the anti-CTLA4 antibodies described herein. In some embodiments, the antigen-binding fragment comprises (1) the light chain of the CTLA4 antibody, (2) the heavy chain of the CTLA4 antibody, (3) the variable region from the light chain of the CTLA4 antibody, (4) the variable region from the heavy chain of the CTLA4 antibody, (5) one or more CDRs of the CTLA4 antibody (e.g., 1, 2, 3, 4, 5, or 6 CDRs), or (6) the amino acid sequences of three CDRs from the light chain and three CDRs from the heavy chain of the CTLA4 antibody.

[0213] In some embodiments, the Disclosure provides antigen-binding fragments of anti-CTLA4 antibodies selected from those listed in Tables 2 and 3, and comprising one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution for human IgG1 Including the Fc region, (for example, i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substitution, R292P substitution, Y300L substitution, and P396L substitution; v) F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution; vi) F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution; vii) S239D substitution, I332E substitution, and A330L substitution, or viiii) S239D substitution and I332E substitution), wherein the numbering of the residues follows the EU number.

[0214] In some embodiments, the antigen-binding fragment of the CTLA4 antibody is a single-chain antibody (scFv) polypeptide comprising (i) a monovalent fragment, the Fab fragment, consisting of VL, VH, CL, and CH1 domains; (ii) a bivalent fragment, the F(ab')2 fragment, containing two Fab fragments linked by disulfide crosslinking at the hinge region; (iii) an Fd fragment, consisting of VH and CH1 domains; (iv) an Fv fragment, consisting of the VL and VH domains of a single arm of the antibody; (v) a dAb fragment, consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); (vi) an isolated CDR; and (vii) a polypeptide, the VL region of the antibody linked to the VH region of the antibody (e.g., Bird et al. (1988) Science 242:423-426, Huston et al. (1988) Proc. Natl. Acad. Sci. USA (See 85:5879-5883) Human IgG1 Fc region

[0215] In some embodiments, the anti-CTLA4 antibody includes a human IgG1 Fc region. In some embodiments, the human IgG1 Fc region includes one or more amino acid substitutions that confer enhanced effector function. In some embodiments, the human IgG1 Fc region includes one or more amino acid substitutions that enhance ADCC. In some embodiments, the human IgG1 Fc region includes one or more amino acid substitutions that reduce fucosylation. In some embodiments, the human IgG1 Fc region includes one or more amino acid substitutions that cause the human IgG1 Fc region to afucosylate. In some embodiments, the human IgG1 Fc region is afucosylated. In some embodiments, the human IgG1 Fc region includes one or more substitutions (e.g., 1, 2, 3, 4, 5, 6, or more) selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, wherein the numbering of the residues follows EU numbers.

[0216] In some embodiments, the human IgG1 Fc region includes S298A substitution, E333A substitution, and K334A substitution, wherein the numbering of the residues follows EU numbers. In certain embodiments, the human IgG1 Fc region includes S298A substitution, E333A substitution, and K334A substitution, and does not include one or more of the L235V substitution, S239D substitution, F243L substitution, R292P substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, and P396L substitution (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or any of these), wherein the numbering of the residues follows EU numbers.

[0217] In some embodiments, the human IgG1 Fc region includes S298A substitution, E333A substitution, K334A substitution, and K326A substitution, wherein the numbering of the residues follows EU numbers. In some embodiments, the human IgG1 Fc region includes S298A substitution, E333A substitution, K334A substitution, and K326A substitution, and does not include one or more of the L235V substitution, S239D substitution, F243L substitution, R292P substitution, Y300L substitution, V305I substitution, A330L substitution, I332E substitution, and P396L substitution (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or any of these), wherein the numbering of the residues follows EU numbers.

[0218] In some embodiments, the human IgG1 Fc region includes the F243L substitution, and the numbering of the residues follows EU numbers. In some embodiments, the human IgG1 Fc region includes the F243L substitution and does not include one or more of the following substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or any of them): L235V substitution, S239D substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, and the numbering of the residues follows EU numbers.

[0219] In some embodiments, the human IgG1 Fc region includes F243L substitution, R292P substitution, Y300L substitution, and P396L substitution, wherein the numbering of the residues follows EU numbers. In some embodiments, the human IgG1 Fc region includes F243L substitution, R292P substitution, Y300L substitution, and P396L substitution, but does not include one or more of the L235V substitution, S239D substitution, S298A substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, and K334A substitution (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or any of these), wherein the numbering of the residues follows EU numbers.

[0220] In some embodiments, the human IgG1 Fc region includes F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution, wherein the numbering of the residues follows EU numbers. In some embodiments, the human IgG1 Fc region includes F243L substitution, R292P substitution, Y300L substitution, V305I substitution, and P396L substitution, and does not include one or more of the L235V substitution, S239D substitution, S298A substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, and K334A substitution (e.g., 1, 2, 3, 4, 5, 6, 7, or any of them), wherein the numbering of the residues follows EU numbers.

[0221] In some embodiments, the human IgG1 Fc region includes F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution, wherein the numbering of the residues follows EU numbers. In some embodiments, the human IgG1 Fc region includes F243L substitution, R292P substitution, Y300L substitution, L235V substitution, and P396L substitution, but does not include one or more of the S239D substitution, S298A substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, and K334A substitution (e.g., 1, 2, 3, 4, 5, 6, 7, or any of them), wherein the numbering of the residues follows EU numbers.

[0222] In some embodiments, the human IgG1 Fc region includes S239D substitution, I332E substitution, and A330L substitution, with the numbering of the residues following EU numbers. In some embodiments, the human IgG1 Fc region includes S239D substitution, I332E substitution, and A330L substitution, but does not include one or more of the L235V substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, E333A substitution, K334A substitution, and P396L substitution (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or any of them), with the numbering of the residues following EU numbers.

[0223] In some embodiments, the human IgG1 Fc region includes S239D substitution and I332E substitution, wherein the numbering of the residues follows EU numbers. In some embodiments, the human IgG1 Fc region includes S239D substitution and I332E substitution, but does not include one or more of the following substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or any of them): L235V substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, E333A substitution, K334A substitution, and P396L substitution, wherein the numbering of the residues follows EU numbers.

[0224] In some embodiments, the anti-CTLA4 antibody comprises one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, and the numbering of the residues follows EU numbers, and the anti-CTLA4 antibody induces antibody-dependent cytotoxicity (ADCC) against CTLA4-expressing human cells or human Treg cells. In some embodiments, the ADCC activity of the anti-CTLA4 antibody is higher than that of a control antibody containing a human IgG1 Fc region without substitutions, and / or the ADCC activity of the anti-CTLA4 antibody is higher than that of ipilimumab. In some embodiments, the ADCC activity of an anti-CTLA4 antibody is higher than that of a control antibody containing a human IgG1 Fc region without substitutions, and / or the ADCC activity of an anti-CTLA4 antibody is higher than that of an afucosylated variant of ipilimumab. In certain embodiments, the anti-CTLA4 antibody contains one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution in the IgG1 Fc region (e.g., human IgG1). The anti-CTLA4 antibody includes an Fc region, the numbering of the residues follows EU numbering, and the ADCC activity of the anti-CTLA4 antibody is 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%, or at least about 90%) greater than that of an anti-CTLA4 antibody containing an Fc region without substitutions (e.g., isotype control or ipilimumab) compared to the ADCC activity of a control antibody.In a particular embodiment, the anti-CTLA4 antibody comprises an IgG1 Fc region (e.g., a human IgG1 Fc region) containing one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, wherein the residue numbering follows EU numbering, and the anti-CTLA4 antibody induces an ADCP effect of about 10% or more (e.g., induces ADCP by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or about 40%) compared to a control (e.g., an isotype control or ipilimumab). In a particular embodiment, the anti-CTLA4 antibody comprises an IgG1 Fc region (e.g., a human IgG1 Fc region) containing one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, wherein the numbering of the residues follows EU numbers, and the anti-CTLA4 antibody induces the ADCC effect at a rate at least about 1.5 times (e.g., at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, or at least about 5 times) higher than an anti-CTLA4 antibody containing an Fc region without substitutions. In some embodiments, the anti-CTLA4 antibody has a semi-maximal effective concentration (EC50) of about 5 nM or less for inducing ADCC in CTLA4-expressing cells (e.g., about 5 nM or less, about 4 nM or less, about 3 nM or less, about 3 nM or less, about 2 nM or less, about 1 nM or less, about 0.5 nM or less, about 0.1 nM or less, or about 0.05 nM or less). Methods for measuring the ADCP of antibodies and antigen-binding fragments are also well known in the art. For example, to evaluate the ADCP activity of a molecule of interest, an in vitro ADCP assay can be performed (see, e.g., Bracher et al., 2007, J. Immunol. Methods 323: 160-71).Phagocytic cells useful for such assays include peripheral blood mononuclear cells (PBMCs), monocytes purified from PBMCs, or U937 cells differentiated into a mononuclear form. Alternatively or additionally, the ADCP activity of the molecule of interest can be evaluated in vivo, for example, in animal models (see, e.g., Wallace et al., 2001, J. Immunol. Methods 248: 167-82).

[0225] In some embodiments, the anti-CTLA4 antibody binds to cellularly expressed CTLA4, and then to CTLA4-expressing cells. The antibody induces antibody-dependent cell-mediated phagocytosis (ADCP) in CTLA4-expressing human cells (e.g., Treg cells). In some embodiments, the anti-CTLA4 antibody induces approximately 10% or more of the ADCP effect compared to a control (e.g., an isotype control or ipilimumab) (e.g., induces ADCP by approximately 10%, 15%, 20%, 25%, 30%, 35%, or 40%). In certain embodiments, the anti-CTLA4 antibody comprises an IgG1 Fc region (e.g., a human IgG1 Fc region) containing one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, wherein the numbering of the residues follows EU numbers, and the anti-CTLA4 antibody induces the ADCP effect at a rate at least about 10% (e.g., 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%, or at least about 90%) higher than an anti-CTLA4 antibody comprising an Fc region without substitutions. In certain embodiments in which an anti-CTLA4 antibody includes an IgG1 Fc region (e.g., a human IgG1 Fc region) containing one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, the numbering of the residues follows EU numbers, and the anti-CTLA4 antibody induces the ADCP effect at a rate at least about 1.5 times (e.g., at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, or at least about 5 times) greater than an anti-CTLA4 antibody containing an Fc region without substitutions.In some embodiments, the anti-CTLA4 antibody has a semi-maximal effective concentration (EC50) of about 5 nM or less for inducing ADCP in CTLA4-expressing cells (e.g., about 5 nM or less, about 4 nM or less, about 3 nM or less, about 3 nM or less, about 2 nM or less, about 1 nM or less, about 0.5 nM or less, about 0.1 nM or less, or about 0.05 nM or less). Methods for measuring the ADCP of antibodies and antigen-binding fragments are also well known in the art. For example, to evaluate the ADCP activity of a molecule of interest, an in vitro ADCP assay can be performed (see, e.g., Bracher et al., 2007, J. Immunol. Methods 323: 160-71). Phagocytic cells useful for such assays include peripheral blood mononuclear cells (PBMCs), monocytes purified from PBMCs, or U937 cells differentiated into a mononuclear type. Alternatively or additionally, the ADCP activity of the molecule of interest can be evaluated in vivo, for example, in animal models (see, e.g., Wallace et al., 2001, J. Immunol. Methods 248: 167-82). Anti-CTLA4 antibody containing the human IgG1 Fc region

[0226] In some embodiments, an isolated antibody conjugated to human CTLA4, provided herein, comprises a heavy chain containing a heavy chain variable region (VH) and a light chain containing a light chain variable region (VL), the heavy chain comprising a heavy chain constant region containing a human IgG1 Fc region, the heavy chain constant region comprising one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, wherein the numbering of the residues follows EU numbers. In some embodiments, the antibody comprises two heavy chains and two light chains, where each heavy chain contains human IgG1 The Fc domain includes: i) S298A substitution, E333A substitution, and K334A substitution; ii) S298A substitution, E333A substitution, K334A substitution, and K326A substitution; iii) F243L substitution; iv) F243L substi...

Claims

1. An isolated antibody that binds to human CTLA4, wherein the antibody comprises a heavy chain containing a heavy chain variable region (VH) and a light chain containing a light chain variable region (VL), the heavy chain comprising a heavy chain constant region containing a human IgG1 Fc region, the heavy chain constant region comprising one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, wherein the numbering of the residues follows EU numbers.

2. The above, 1) VH includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 1, CDR-H2 containing the amino acid sequence of SEQ ID NO: 2, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 3; VL includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 4, CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 6; 2) VH includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 12, CDR-H2 containing the amino acid sequence of SEQ ID NO: 13, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 14; VL includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 15, CDR-L2 containing the amino acid sequence of SEQ ID NO: 16, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 17; 3) VH includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 44, CDR-H2 containing the amino acid sequence of SEQ ID NO: 45, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 53; VL includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 54, CDR-L2 containing the amino acid sequence of SEQ ID NO: 16, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 55; or 4) The antibody according to claim 1, wherein VH comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 44, CDR-H2 containing the amino acid sequence of SEQ ID NO: 45, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 46, and VL comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 47, CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

48.

3. The isolated antibody according to claim 1 or 2, wherein 1) VH comprises the amino acid sequence of SEQ ID NO: 74 and / or VL comprises the amino acid sequence of SEQ ID NO: 75; 2) VH comprises the amino acid sequence of SEQ ID NO: 66 and / or VL comprises the amino acid sequence of SEQ ID NO: 67; 3) VH comprises the amino acid sequence of SEQ ID NO: 84 and / or VL comprises the amino acid sequence of SEQ ID NO: 85; or 4) VH comprises the amino acid sequence of SEQ ID NO: 80 and / or VL comprises the amino acid sequence of SEQ ID NO:

81.

4. The isolated antibody according to any one of claims 1 to 3, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 91, and the light chain comprises the amino acid sequence of SEQ ID NO:

90.

5. The isolated antibody according to any one of claims 1 to 3, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 91 without a C-terminal lysine, and the light chain comprises the amino acid sequence of SEQ ID NO:

90.

6. The antibody comprises two heavy chains and two light chains, and each human IgG1 Fc domain of the heavy chain contains the following: i) Replacement of S298A, E333A, and K334A; ii) Replacement of S298A, E333A, K334A, and K326A; iii) F243L substitution; iv) Replacement of F243L, R292P, Y300L, and P396L; v) Replacement of F243L, R292P, Y300L, V305I, and P396L; vi) Replace F243L, R292P, Y300L, L235V, and P396L; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) An isolated antibody according to any one of claims 1 to 5, comprising S239D substitution and I332E substitution.

7. The isolated antibody according to claim 6, wherein each of the human IgG1 Fc domains comprises an S239D substitution and an I332E substitution.

8. The isolated antibody according to claim 6, wherein each of the human IgG1 Fc domains comprises an S239D substitution, an I332E substitution, and an A330L substitution.

9. The isolated antibody according to claim 6, wherein each of the human IgG1 Fc domains comprises an F243L substitution, an R292P substitution, a Y300L substitution, a V305I substitution, and a P396L substitution.

10. The isolated antibody according to any one of claims 1 to 9, wherein the isolated antibody induces antibody-dependent cell-mediated cytotoxicity (ADCC) against CTLA4-expressing human cells or human Treg cells, the ADCC activity of the isolated antibody is higher than that of a control antibody containing a human IgG1 Fc region without substitution, and / or the ADCC activity of the isolated antibody is higher than that of ipilimumab.

11. A masked antibody that binds to human CTLA4, a masking peptide (MP), and an antibody, wherein the antibody comprises a heavy chain comprising a heavy chain variable region (VH) and a light chain comprising a light chain variable region (VL), the MP being linked to the N-terminus of the VH or VL, the MP comprising a masking unit (MU) and a linkage unit (LU) from the N-terminus to the C-terminus, the MU comprising an amino acid sequence selected from the list of SEQ ID NOs: 146-151, and the, 1) VH includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 1, CDR-H2 containing the amino acid sequence of SEQ ID NO: 2, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 3; VL includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 4, CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 6; 2) VH includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 12, CDR-H2 containing the amino acid sequence of SEQ ID NO: 13, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 14; VL includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 15, CDR-L2 containing the amino acid sequence of SEQ ID NO: 16, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 17; 3) VH includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 44, CDR-H2 containing the amino acid sequence of SEQ ID NO: 45, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 53; VL includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 54, CDR-L2 containing the amino acid sequence of SEQ ID NO: 16, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 55; or 4) VH includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 44, CDR-H2 containing the amino acid sequence of SEQ ID NO: 45, and CDR-H3 containing the amino acid sequence of SEQ ID NO:

46. VL includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 47, CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

48.

12. The masked antibody according to claim 11, wherein the MP further comprises an N-terminal unit (NU) ligated to the N-terminus of the MU.

13. The masked antibody according to claim 12, wherein the N-terminal unit has a length of about 1 to 10 amino acid residues.

14. The masked antibody according to claim 12, wherein the N-terminal unit comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 143 to 145.

15. The masked antibody according to any one of claims 11 to 14, wherein the LU does not contain a cleavage site.

16. The masked antibody according to claim 15, wherein the LU comprises a linker.

17. The masked antibody according to claim 16, wherein the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 155 to 161.

18. The masked antibody according to claim 16, wherein the linker comprises the amino acid sequence of SEQ ID NO:

159.

19. The masked antibody according to any one of claims 11 to 14, wherein the masked antibody is an activatable antibody.

20. The masked antibody according to claim 19, wherein the LU comprises at least a first cleavage site (C1).

21. The first cleavage site (C1) contains urokinase-type plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, tobacco etch virus (TEV) protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, The masked antibody according to claim 20, wherein the masked antibody is a protease cleavage site of a protease selected from the group consisting of ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE.

22. The masked antibody according to claim 20 or 21, wherein the first cleavage site (C1) comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 152 to 154.

23. The masked antibody according to any one of claims 20 to 22, wherein the LU further comprises a second cleavage site (C2).

24. The masked antibody according to claim 23, wherein the second cleavage site (C2) is a protease cleavage site of a protease selected from the group consisting of urokinase-type plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, tobacco etch virus (TEV) protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE.

25. The masked antibody according to claim 23 or 24, wherein the second cleavage site (C2) comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 152 to 154.

26. A masked antibody according to any one of claims 23 to 25, wherein the first and second cleavage sites are different.

27. The masked antibody according to any one of claims 19 to 26, wherein the LU further comprises a first linker.

28. The masked antibody according to claim 27, wherein the first linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 155 to 161.

29. The masked antibody according to claim 27 or 28, wherein the LU further comprises a second linker (L2).

30. The masked antibody according to claim 29, wherein the second linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 155 to 161.

31. The aforementioned LU extends from the N-terminus to the C-terminus: 1) First cut portion (C1), first linker (L1), second cut portion (C2), and second linker (L2); or 2) A masked antibody according to any one of claims 27 to 30, comprising a first linker (L1), a first cleavage site (C1), and a second linker (L2).

32. The masked antibody according to any one of claims 11 to 31, wherein the LU comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 99 to 103.

33. The above, MU contains the amino acid sequence of SEQ ID NO: 146, and LU contains the amino acid sequence of SEQ ID NO: 99; MU contains the amino acid sequence of SEQ ID NO: 147, and LU contains the amino acid sequence of SEQ ID NO: 99; MU contains the amino acid sequence of SEQ ID NO: 148, and LU contains the amino acid sequence of SEQ ID NO: 99; MU contains the amino acid sequence of SEQ ID NO: 149, and LU contains the amino acid sequence of SEQ ID NO: 99; MU contains the amino acid sequence of SEQ ID NO: 150, and LU contains the amino acid sequence of SEQ ID NO: 99; MU contains the amino acid sequence of SEQ ID NO: 148, and LU contains the amino acid sequence of SEQ ID NO: 100; MU contains the amino acid sequence of SEQ ID NO: 148, and LU contains the amino acid sequence of SEQ ID NO: 101; MU contains the amino acid sequence of SEQ ID NO: 148, and LU contains the amino acid sequence of SEQ ID NO: 102; MU contains the amino acid sequence of SEQ ID NO: 148, and LU contains the amino acid sequence of SEQ ID NO: 101; MU contains the amino acid sequence of SEQ ID NO: 151, and LU contains the amino acid sequence of SEQ ID NO: 100; MU contains the amino acid sequence of SEQ ID NO: 151, and LU contains the amino acid sequence of SEQ ID NO: 101; MU contains the amino acid sequence of SEQ ID NO: 150, and LU contains the amino acid sequence of SEQ ID NO: 101; MU contains the amino acid sequence of SEQ ID NO: 150, and LU contains the amino acid sequence of SEQ ID NO: 102; MU contains the amino acid sequence of SEQ ID NO: 150, and LU contains the amino acid sequence of SEQ ID NO: 102; MU contains the amino acid sequence of SEQ ID NO: 150, and LU contains the amino acid sequence of SEQ ID NO: 103; or A masked antibody according to any one of claims 11 to 31, wherein MU comprises the amino acid sequence of SEQ ID NO: 150, and LU comprises the amino acid sequence of SEQ ID NO:

103.

34. The masked antibody according to any one of claims 11 to 33, wherein the masking peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 162 to 177.

35. The masked antibody according to any one of claims 11 to 34, wherein 1) VH comprises the amino acid sequence of SEQ ID NO: 74 and / or VL comprises the amino acid sequence of SEQ ID NO: 75; 2) VH comprises the amino acid sequence of SEQ ID NO: 66 and / or VL comprises the amino acid sequence of SEQ ID NO: 67; 3) VH comprises the amino acid sequence of SEQ ID NO: 84 and / or VL comprises the amino acid sequence of SEQ ID NO: 85; or 4) VH comprises the amino acid sequence of SEQ ID NO: 80 and / or VL comprises the amino acid sequence of SEQ ID NO:

81.

36. The masked antibody according to any one of claims 11 to 35, wherein the masked antibody is a Fab fragment, scFv, F(ab')2 fragment, or Fv fragment.

37. The masked antibody according to any one of claims 11 to 35, wherein the antibody comprises two heavy chains and two light chains, and each human IgG1 Fc domain of the heavy chain contains one or more substitutions selected from the group consisting of L235V substitution, S239D substitution, F243L substitution, R292P substitution, S298A substitution, Y300L substitution, V305I substitution, K326A substitution, A330L substitution, I332E substitution, E333A substitution, K334A substitution, and P396L substitution, wherein the residue numbering is according to EU numbering.

38. Each of the aforementioned human IgG1 Fc domains includes the following: i) Replacement of S298A, E333A, and K334A; ii) Replacement of S298A, E333A, K334A, and K326A; iii) F243L substitution; iv) Replacement of F243L, R292P, Y300L, and P396L; v) Replacement of F243L, R292P, Y300L, V305I, and P396L; vi) Replace F243L, R292P, Y300L, L235V, and P396L; vii) S239D substitution, I332E substitution, and A330L substitution; or viii) S239D substitution and I332E substitution, the masked antibody according to claim 37.

39. The masked antibody according to claim 38, wherein each of the human IgG1 Fc domains comprises an S239D substitution and an I332E substitution.

40. The masked antibody according to claim 38, wherein each of the human IgG1 Fc domains comprises an S239D substitution, an I332E substitution, and an A330L substitution.

41. The masked antibody according to claim 38, wherein each of the human IgG1 Fc domains comprises an F243L substitution, an R292P substitution, a Y300L substitution, a V305I substitution, and a P396L substitution.

42. The masked antibody according to any one of claims 37 to 41, wherein each heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 104 to 105, and each light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 106 to 121.

43. The masked antibody according to claim 42, wherein each heavy chain comprises the amino acid sequence of SEQ ID NO: 105, and each light chain comprises the amino acid sequence of SEQ ID NO:

121.

44. The masked antibody according to any one of claims 37 to 43, wherein the masked antibody induces antibody-dependent cell-mediated cytotoxicity (ADCC) against CTLA4-expressing human cells or human Treg cells, the ADCC activity of the isolated antibody is higher than that of a control antibody containing a human IgG1 Fc region without substitution, and / or the ADCC activity of the isolated antibody is higher than that of ipilimumab.

45. Isolated polynucleotides encoding one or more polypeptide chains of an isolated antibody according to any one of claims 1 to 10 or a masked antibody according to any one of claims 11 to 44.

46. A vector comprising the polynucleotide described in claim 45.

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

48. A host cell comprising a polynucleotide or vector according to any one of claims 45 to 47.

49. The host cell according to claim 48, wherein the host cell is a eukaryotic cell.

50. The host cell according to claim 48 or claim 49, wherein the host cell is a Chinese hamster ovary (CHO) cell.

51. A method for producing an antibody or a masked antibody, comprising culturing a host cell according to any one of claims 48 to 50 under conditions suitable for the production of the antibody or an activatable antibody.

52. The method according to claim 51, further comprising recovering the antibody or masked antibody produced by the cells.

53. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 10 or a masked antibody according to any one of claims 11 to 44, and a pharmaceutically acceptable carrier.

54. A method for treating cancer or delaying its progression in a subject requiring such treatment, comprising administering to the subject an effective amount of an antibody according to any one of claims 1 to 10, a masked antibody according to any one of claims 11 to 44, or a pharmaceutical composition according to claim 53.

55. A method for reducing the size of a solid tumor in a subject requiring reduction in size, the method comprising administering to the subject an effective amount of an antibody according to any one of claims 1 to 10, a masked antibody according to any one of claims 11 to 44, or a pharmaceutical composition according to claim 53, wherein the solid tumor has a size of about 400 to 1000 mm³.

56. A method for reducing on-target off-tumor toxicity in a target of cancer requiring reduction, wherein the method comprises an effective amount of the masked antibody according to any one of claims 11 to 44 in the target.

57. The method according to any one of claims 54 to 56, wherein the tumor is a colorectal tumor or a lung tumor.

58. The method according to any one of claims 54 to 57, wherein the tumor is a cold tumor.

59. The method according to claim 58, wherein the cold tumor is selected from the group consisting of pancreatic cancer, ovarian cancer, and head and neck cancer.

60. The method according to any one of claims 54 to 59, further comprising administering an effective amount of at least one additional therapeutic agent to the subject.

61. The method according to claim 60, wherein the at least one additional therapeutic agent is selected from the group consisting of viral gene therapy, immune checkpoint inhibitors, targeted therapy, radiotherapy, vaccine therapy, and chemotherapy.

62. The method according to claim 61, wherein the at least one additional therapeutic agent is an immune checkpoint inhibitor.

63. The method according to claim 62, wherein the at least one additional therapeutic agent is a PD-1 inhibitor.

64. The method according to any one of claims 54 to 63, wherein the method comprises administering an effective amount of the antibody, the masked antibody, or the pharmaceutical composition to the subject before or after surgery to remove a solid tumor in the subject.

65. A method for inducing phagocytosis in cells expressing CTLA4, comprising contacting the cells expressing CTLA4 with an effective amount of the antibody described in any one of claims 1 to 10, or the masked antibody described in any one of claims 11 to 44.

66. The method according to claim 65, wherein the phagocytosis is antibody-dependent cell-mediated phagocytosis (ADCP).

67. The method according to any one of claims 65 to 66, wherein the antibody or masked antibody has a half-maximal effective concentration (EC50) of less than 3 nM for inducing ADCP in cells expressing CTLA4.

68. A method for depleting regulatory T cells, comprising administering an effective amount of an antibody according to any one of claims 1 to 10 or a pharmaceutical composition according to claim 53 to a subject.

69. The masked antibody according to any one of claims 11 to 35, wherein the antibody comprises a human IgG1 Fc region, and the human IgG1 Fc region is afucosylated.

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