Anti-CTLA4 antibodies and methods of use thereof

Abstract

To provide anti-CTLA4 protein therapeutics that effectively target tumors without side effects.SOLUTION: Anti-CTLA4 binding proteins (e.g., antibodies, bispecific antibodies, and chimeric receptors), their use in treating and preventing cancer, and compositions and kits comprising the anti-CTLA4 binding proteins are provided.SELECTED DRAWING: Figure 8D

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from U.S. Provisional Application No. 62 / 785,111, filed December 26, 2018, the contents of which are incorporated by reference in their entirety.

[0002] Submission of sequence listing as an ASCII text file The contents of the following submission, in an ASCII text file, are incorporated herein by reference in their entirety: Sequence Listing Computer Readable Form (CRF) (Filename: 737762002540.txt, Date Recorded: December 23, 2019, Size: 39.5 KB).

[0003] FIELD OF THE INVENTION The present invention relates to anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4) binding proteins (e.g., anti-CTLA4 antibodies) and methods related to their use. [Background technology]

[0004] Background of the Invention Cancer is the second leading cause of death in the United States, accounting for more deaths than the next five leading causes (chronic respiratory disease, stroke, accidents, Alzheimer's disease, and diabetes). While significant progress has been made, particularly in targeted therapies, much research remains in this field. Immunotherapy and its branch, immuno-oncology, have yielded viable and exciting treatment options for treating malignancies. In particular, it is now recognized that one hallmark of cancer is immune evasion, and significant efforts have been made to identify targets and develop therapies directed at these targets to reactivate the immune system to recognize and treat cancer. In fact, ipilimumab, an anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4) antibody, has resulted in long-term survival in patients with stage III / IV melanoma. Ipilimumab is an immune checkpoint antagonist that interrupts T cell inhibition by blocking CTLA4, which can lead to depletion of T regulatory cells (Tregs) (Korman, A., et al., 2005. Tumor immunotherapy: preclinical and clinical activity of anti-CTLA4 antibodies. Current Opinion in Investigational Drugs 6:582-591 (Non-Patent Document 1); Quezada et al., J. Exp. Med., 206(8):1717-1725, 2009 (Non-Patent Document 2); Selby et al., Cancer Immunol Res., 1(1);32-42, 2013 (Non-Patent Document 3)). Unfortunately, ipilimumab induces systemic (not tumor-specific) activation of T cell-dependent immune responses that result in potentially life-threatening immune-related adverse effects, often limiting dose and duration of treatment (Weber, JS, et al., 2008. Phase I / II study of ipilimumab for patients with metastatic melanoma. Journal of Clinical Oncology 26:5950-5956). These include enteritis, dermatitis, hypophysitis, uveitis, hepatitis, nephritis, and death.Enterocolitis is the most common major toxicity (affecting approximately 20% of patients). Due to the serious safety risks associated with immune-mediated adverse reactions, the FDA approved ipilimumab with a risk evaluation and mitigation strategy (REMS). Recently, it has been shown that the combination of ipilimumab with a second immune checkpoint modulator targeting PD1 (e.g., nivolumab) significantly increases the efficacy of melanoma immunotherapy compared with ipilimumab alone. However, this increase was associated with an increased frequency of grade 3 / 4 adverse events, affecting more than 50% of patients receiving the combination therapy (Wolchok, JD, et al. 2013. Nivolumab plus ipilimumab in Advanced Melanoma. N Engl J Med (Non-Patent Document 5)).

[0005] These findings illustrate the need for the development of anti-CTLA4 protein therapeutics that effectively target tumors without the side effects associated with certain anti-CTLA4 antibodies, such as ipilimumab. Provided herein are anti-CTLA binding proteins, compositions thereof, and methods of use thereof to address this need.

[0006] All references cited herein, including patent applications, patent publications, and scientific literature, are incorporated by reference in their entirety as if each individual reference was specifically and individually indicated to be incorporated by reference. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Korman, A., et al., 2005. Tumor immunotherapy: preclinical and clinical activity of anti-CTLA4 antibodies. Current Opinion in Investigational Drugs 6:582-591 [Non-patent document 2] Quezada et al.,J.Exp.Med.,206(8):1717-1725,2009 [Non-patent document 3] Selby et al.Cancer Immunol Res.,1(1);32-42,2013 [Non-patent document 4] Weber,JS,et al.,2008.Phase I / II study of ipilimumab for patients with metastatic melanoma.Journal of Clinical Oncology 26:5950-5956 [Non-patent document 5] Wolchok,JD,et al.2013.Nivolumab plus Ipilimumab in Advanced Melanoma.N Engl J Med Summary of the Invention

[0008] Provided herein are anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4) binding proteins, compositions comprising same, and methods of using same.

[0009] Provided herein is an anti-CTLA4 antibody or antigen-binding fragment thereof comprising a light chain variable (VL) domain and a heavy chain variable (VH) domain, wherein the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3; and / or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6; or the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15; and / or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 16, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 18. and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 18, or the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, and / or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 10, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 11, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12, or the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 19, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 20, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 21, and / or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24.

[0010] Provided herein is an anti-CTLA4 antibody or antigen-binding fragment thereof comprising a light chain variable (VL) domain and a heavy chain variable (VH) domain, wherein the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3, and / or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6.

[0011] Provided herein is an anti-CTLA4 antibody or antigen-binding fragment thereof comprising a light chain variable (VL) domain and a heavy chain variable (VH) domain, wherein the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15, and / or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 16, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 18.

[0012] Provided herein is an anti-CTLA4 antibody or antigen-binding fragment thereof comprising a light chain variable (VL) domain and a heavy chain variable (VH) domain, wherein the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, and / or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 10, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 11, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12.

[0013] Provided herein is an anti-CTLA4 antibody or antigen-binding fragment thereof comprising a light chain variable (VL) domain and a heavy chain variable (VH) domain, wherein the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 19, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 20, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 21, and / or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24.

[0014] In some of any of the provided embodiments, the VL domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 25, and the VH domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 26, or the VL domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 30, and the VH domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 31.

[0015] In some of any of the provided embodiments, the VL domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 25, and the VH domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 26. In some of any of the provided embodiments, the VL domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 30, and the VH domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 31.

[0016] In some of any of the provided embodiments, the VL domain comprises the amino acid sequence of SEQ ID NO: 25 and the VH domain comprises the amino acid sequence of SEQ ID NO: 26, or the VL domain comprises the amino acid sequence of SEQ ID NO: 30 and the VH domain comprises the amino acid sequence of SEQ ID NO: 31.

[0017] Provided herein is an anti-CTLA4 antibody or antigen-binding fragment thereof comprising a light chain variable (VL) domain and a heavy chain variable (VH) domain, wherein the VL domain comprises the amino acid sequence of SEQ ID NO: 25 and / or the VH domain comprises the amino acid sequence of SEQ ID NO: 26, or the VL domain comprises the amino acid sequence of SEQ ID NO: 30 and / or the VH domain comprises the amino acid sequence of SEQ ID NO: 31.

[0018] In some of any of the provided embodiments, the VL domain comprises the amino acid sequence of SEQ ID NO: 25, and the VH domain comprises the amino acid sequence of SEQ ID NO: 26. In some of any of the provided embodiments, the VL domain comprises the amino acid sequence of SEQ ID NO: 30, and the VH domain comprises the amino acid sequence of SEQ ID NO: 31.

[0019] In some of any of the provided embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant domain (CH). In some of any of the provided embodiments, the antibody or antigen-binding fragment thereof comprises a CH sequence selected from the group consisting of SEQ ID NOs: 35-38. In some of any of the provided embodiments, the CH comprises the amino acid substitution S239D or I332E or both, wherein the amino acid residues are numbered according to the EU index of Kabat. In some of the provided embodiments, the antibody or antigen-binding fragment thereof comprises the CH sequence of SEQ ID NO: 38.

[0020] In some of any of the embodiments, the antibody or antigen-binding fragment thereof comprises a light chain constant domain (CL). In some of any of the embodiments, the antibody or antigen-binding fragment thereof comprises the CL sequence of SEQ ID NO:39.

[0021] In some of the optional embodiments, the light chain comprises the amino acid sequence of SEQ ID NO: 27 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 29, or the light chain comprises the amino acid sequence of SEQ ID NO: 32 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 34.

[0022] Provided herein is an anti-CTLA4 antibody or antigen-binding fragment thereof, comprising a light chain comprising the amino acid sequence of SEQ ID NO:27, and a heavy chain comprising the amino acid sequence of SEQ ID NO:29.

[0023] Provided herein is an anti-CTLA4 antibody or antigen-binding fragment thereof, comprising a light chain comprising the amino acid sequence of SEQ ID NO:32, and a heavy chain comprising the amino acid sequence of SEQ ID NO:34.

[0024] In some of any of the embodiments, the antibody or antigen-binding fragment thereof is afucosylated or fucose-deficient.

[0025] In some of the embodiments, the anti-CTLA4 antibody or antigen-binding fragment thereof is conjugated to an agent. In some of the embodiments, the agent is an inhibitor of tubulin polymerization, a DNA damaging agent, or a DNA synthesis inhibitor. In some of the embodiments, the agent is a maytansinoid, an auristatin, a pyrrolobenzodiazepine (PBD) dimer, a calicheamicin, a duocarmycin, an indolinobenzodiazepine dimer, or an exatecan derivative Dxd.

[0026] Provided herein is a bispecific antibody or antigen-binding fragment thereof, comprising a first pair of light and heavy chains that specifically bind to CTLA4 and a second pair of light and heavy chains that specifically bind to an antigen, wherein the first pair of light chains comprises a VL domain and the first pair of heavy chains comprises a VH domain, wherein the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3, and / or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6, or the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15, and / or the VH domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16. or the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9; and / or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 10, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 11, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12; or the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 19, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 20, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 21; and / or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24.

[0027] Provided herein is a bispecific antibody or antigen-binding fragment thereof, comprising a first pair of light chains and heavy chains that specifically bind to CTLA4 and a second pair of light chains and heavy chains that specifically bind to an antigen, wherein the first pair of light chains comprises a VL domain and the first pair of heavy chains comprises a VH domain, wherein the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3, and / or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6.

[0028] Provided herein is a bispecific antibody or antigen-binding fragment thereof, comprising a first pair of light chains and heavy chains that specifically bind to CTLA4 and a second pair of light chains and heavy chains that specifically bind to an antigen, wherein the first pair of light chains comprises a VL domain and the first pair of heavy chains comprises a VH domain, wherein the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15, and / or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 16, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 18.

[0029] Provided herein is a bispecific antibody or antigen-binding fragment thereof, comprising a first pair of light chains and heavy chains that specifically bind to CTLA4 and a second pair of light chains and heavy chains that specifically bind to an antigen, wherein the first pair of light chains comprises a VL domain and the first pair of heavy chains comprises a VH domain, wherein the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:9, and / or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO:10, CDR-H2 comprising the amino acid sequence of SEQ ID NO:11, and CDR-H3 comprising the amino acid sequence of SEQ ID NO:12.

[0030] Provided herein is a bispecific antibody or antigen-binding fragment thereof, comprising a first pair of light chains and heavy chains that specifically bind to CTLA4 and a second pair of light chains and heavy chains that specifically bind to an antigen, wherein the first pair of light chains comprises a VL domain and the first pair of heavy chains comprises a VH domain, wherein the VL domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 19, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 20, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 21, and / or the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24.

[0031] In some of the optional embodiments, the VL domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 25, and the VH domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 26, or the VL domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 30, and the VH domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 31.

[0032] Provided herein is a bispecific antibody or antigen-binding fragment thereof, comprising a first pair of light and heavy chains that specifically binds to CTLA4 and a second pair of light and heavy chains that specifically binds to an antigen, wherein the first pair of light chains comprises a VL domain and the first pair of heavy chains comprises a VH domain, wherein the VL domain comprises the amino acid sequence of SEQ ID NO: 25 and / or the VH domain comprises the amino acid sequence of SEQ ID NO: 26, or wherein the VL domain comprises the amino acid sequence of SEQ ID NO: 30 and / or the VH domain comprises the amino acid sequence of SEQ ID NO: 31.

[0033] In some of the optional embodiments, the VL domain comprises the amino acid sequence of SEQ ID NO: 25 and the VH domain comprises the amino acid sequence of SEQ ID NO: 26, or the VL domain comprises the amino acid sequence of SEQ ID NO: 30 and the VH domain comprises the amino acid sequence of SEQ ID NO: 31.

[0034] In some of any of the embodiments, the bispecific antibody or antigen-binding fragment thereof comprises a heavy chain constant domain (CH). In some of any of the embodiments, the bispecific antibody or antigen-binding fragment thereof comprises a CH sequence selected from the group consisting of SEQ ID NOs: 35-38. In some of any of the embodiments, the CH comprises the amino acid substitution S239D or I332E or both, wherein the amino acid residues are numbered according to the EU index of Kabat. In some of any of the embodiments, the bispecific antibody or antigen-binding fragment thereof comprises the CH sequence of SEQ ID NO: 38.

[0035] In some of any of the embodiments, the bispecific antibody or antigen-binding fragment thereof comprises a light chain constant domain (CL). In some of any of the embodiments, the bispecific antibody or antigen-binding fragment thereof comprises the CL sequence of SEQ ID NO: 39.

[0036] In some of the optional embodiments, the light chain comprises the amino acid sequence of SEQ ID NO: 27 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 29, or the light chain comprises the amino acid sequence of SEQ ID NO: 32 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 34.

[0037] Provided herein is a bispecific antibody or antigen-binding fragment thereof, comprising a first pair of light and heavy chains that specifically binds to CTLA4 and a second pair of light and heavy chains that specifically binds to an antigen, wherein the first pair comprises a light chain comprising the amino acid sequence of SEQ ID NO:27 and a heavy chain comprising the amino acid sequence of SEQ ID NO:29.

[0038] Provided herein is a bispecific antibody or antigen-binding fragment thereof, comprising a first pair of light and heavy chains that specifically binds to CTLA4 and a second pair of light and heavy chains that specifically binds to an antigen, wherein the first pair comprises a light chain comprising the amino acid sequence of SEQ ID NO: 32 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 34.

[0039] In some of any of the embodiments, the bispecific antibody or antigen-binding fragment thereof is non-fucosylated or fucose-deficient.

[0040] In some of the embodiments, the bispecific antibody or antigen-binding fragment thereof is conjugated to a drug. In some of the embodiments, the drug is an inhibitor of tubulin polymerization, a DNA damaging agent, or a DNA synthesis inhibitor. In some of the embodiments, the drug is a maytansinoid, an auristatin, a pyrrolobenzodiazepine (PBD) dimer, a calicheamicin, a duocarmycin, an indolinobenzodiazepine dimer, or an exatecan derivative Dxd.

[0041] Also provided herein are nucleic acids encoding any of the provided anti-CTLA4 antibodies or antigen-binding fragments thereof, or any of the provided bispecific antibodies or antigen-binding fragments thereof.

[0042] Also provided are vectors comprising any of the nucleic acids provided herein.

[0043] Also provided are host cells comprising any of the provided anti-CTLA4 antibodies or antigen-binding fragments thereof, any of the provided bispecific antibodies or antigen-binding fragments thereof, or any of the nucleic acids provided herein.

[0044] In some of any such embodiments, the host cells are capable of producing non-fucosylated or fucose-deficient antibodies or antigen-binding fragments thereof. In some of any such embodiments, the host cells have an alpha 1,6-fucosyltransferase (Fut8) knockout. In some of any such embodiments, the host cells overexpress β1,4-N-acetylglucosaminyltransferase III (GnT-III). In some of any such embodiments, the host cells overexpress Golgi μ-mannosidase I (ManII).

[0045] Also provided is a method of producing an antibody or antigen-binding fragment thereof, comprising culturing any of the provided host cells under conditions that produce the antibody or antigen-binding fragment thereof.

[0046] Also provided is a method for producing a nonfucosylated or fucose-deficient antibody or antigen-binding fragment thereof, comprising culturing any of the provided host cells under conditions that produce the antibody or antigen-binding fragment thereof.

[0047] In some of any such embodiments, the method also includes recovering the antibody or antigen-binding fragment thereof produced by the host cell.

[0048] Also provided is an antibody or antigen-binding fragment thereof produced by any of the methods of producing an antibody or antigen-binding fragment thereof provided herein.

[0049] Also provided are compositions comprising any of the provided anti-CTLA4 antibodies or antigen-binding fragments thereof, any of the provided bispecific antibodies or antigen-binding fragments thereof, or any of the provided antibodies or antigen-binding fragments thereof.

[0050] Also provided are pharmaceutical compositions comprising any of the provided anti-CTLA4 antibodies or antigen-binding fragments thereof, any of the provided bispecific antibodies or antigen-binding fragments thereof, or any of the provided antibodies or antigen-binding fragments thereof, and a pharmaceutically acceptable carrier.

[0051] Also provided are pharmaceutical compositions comprising any of the provided anti-CTLA4 antibodies or antigen-binding fragments thereof, any of the provided bispecific antibodies or antigen-binding fragments thereof, or any of the provided antibodies or antigen-binding fragments thereof, and a pharmaceutically acceptable carrier for use in treating or preventing a neoplastic disease in a subject.

[0052] Also provided is the use of any of the provided anti-CTLA4 antibodies or antigen-binding fragments thereof, any of the provided bispecific antibodies or antigen-binding fragments thereof, or any of the provided antibodies or antigen-binding fragments thereof, and a pharmaceutical composition comprising a pharmaceutically acceptable carrier in the manufacture of a medicament for treating or preventing a neoplastic disease in a subject.

[0053] Kits including any of the provided anti-CTLA4 antibodies or antigen-binding fragments thereof, any of the provided bispecific antibodies or antigen-binding fragments thereof, or any of the provided antibodies or antigen-binding fragments thereof are also provided.

[0054] Also provided are methods for treating or preventing a neoplastic disease in a subject, the methods comprising administering to the subject an effective amount of any of the provided anti-CTLA4 antibodies or antigen-binding fragments thereof, any of the provided bispecific antibodies or antigen-binding fragments thereof, any of the provided antibodies or antigen-binding fragments thereof, or any of the provided compositions.

[0055] It should be understood that one, some, or all of the features of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the present invention will be apparent to those skilled in the art. These and other embodiments of the present invention are further described in the detailed description that follows. [Brief explanation of the drawings]

[0056] [Figure 1A] Figures 1A and 1B are graphs showing binding of anti-CTLA4 antibodies to human CTLA4-Fc over a range of antibody concentrations. Figure 1A shows binding of forms of Antibody 1 (Antibody 1-1 and Antibody 1-2) to human CTLA4-Fc over a range of antibody concentrations, showing similar binding between the antibodies shown. Figure 1B shows binding of forms of Antibody 2 (Antibody 2-1, Antibody 2-2, Antibody 2-3, Antibody 2-4, and Antibody 2-5) to human CTLA4-Fc over a range of antibody concentrations, showing similar binding between the antibodies shown. [Figure 1B] See legend to Figure 1A. [Figure 1C] Figures 1C-1E show the binding results of human CTLA-Fc and humanized anti-CTLA4 antibodies, as well as variants (e.g., containing mutations in the Fc region or defucosylated versions) of Antibody 1 (Figure 1C), Antibody 2 (Figure 1D), or ipilimumab (Figure 1E), as assessed by ELISA. As shown, all variants of each of the antibodies bound similarly to human CTLA4-Fc. [Figure 1D] See legend to Figure 1C. [Figure 1E] See legend to Figure 1C. [Figure 2A] 1 shows the results of SPR analysis demonstrating binding between antibody 2-6 (a version of antibody 2 with S239D mutation, I332E mutation in the Fc region) or ipilimumab and human CTLA4 (huCTLA4) at 32 nM, 16 nM, 8 nM, 4 nM, and 2 nM. [Figure 2B]The association rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (KD) for the results shown in Figure 2A are provided, as well as the fold difference between antibody 2-6 and ipilimumab. [Figure 2C] 1 shows the results of SPR analysis demonstrating binding between the Fab of ipilimumab (ipilimumab-Fab) or the Fab of Antibody 2 (Antibody 2-Fab) and rhCTLA4-Fc at 32 nM, 16 nM, 8 nM, 4 nM, and 2 nM. [Figure 2D] The association rate constant (ka), dissociation rate constant (kd), equilibrium dissociation constant (KD), and χ2 values ​​are provided for the results shown in Figure 2C. [Figure 2E] 1 shows the results of SPR analysis demonstrating binding between ipilimumab Fab (ipilimumab-Fab) or Antibody 2 Fab (Antibody 2-Fab) and cynoCTLA4-Fc at 32 nM, 16 nM, 8 nM, 4 nM, and 2 nM. [Figure 2F] The association rate constant (ka), dissociation rate constant (kd), equilibrium dissociation constant (KD), and χ2 values ​​are provided for the results shown in Figure 2E. [Figure 3] 1 shows the results of SPR analysis demonstrating binding between antibody 2-1 (a version of antibody 2 with a wild-type Fc region), antibody 2-6 (a version of antibody 2 with S239D and I332E mutations in the Fc region), or ipilimumab at various concentrations and human FcγRIIIa (CD16a). [Figure 4A] Figures 4A-4D are graphs showing IL-2 levels (pg / mL) (Figures 4A and 4C) or fold change in IL-2 levels (Figures 4B and 4D) for Antibody 1 and Antibody 2, as determined using a Staphylococcal Enterotoxin B (SEB) assay. Antibody 1 (Antibody 1-1 and 1-2) (Figures 4A and 4B) and Antibody 2 (Antibody 2-1, Antibody 2-2, Antibody 2-3, Antibody 2-4, and Antibody 2-5) (Figures 4C and 4D) were tested for their ability to promote IL-2 production from peripheral mononuclear cells using the SEB assay. All tested forms of Antibody 1 and Antibody 2 demonstrated the ability to increase IL-2 levels compared to non-antibody controls (Figures 4A and 4C). [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 4D] See legend to Figure 4A. [Figure 4E] 1 shows the results of an SEB assay using various versions of anti-CTLA4 antibody 1 [having a wild-type Fc region (antibody 1-1), having S239D and I332E mutations in the Fc region (antibody 1-2), and a non-fucosylated version of antibody 1 (antibody 1-aFuc)], antibody 2 [having a wild-type Fc region (antibody 2-1), having S239D and I332E mutations in the Fc region (antibody 2-6), and a non-fucosylated version of antibody 2 (antibody 2-aFuc)], or ipilimumab [having a wild-type Fc (ipilimumab), having S239D and I332E mutations in the Fc region (ipilimumab-m), and an afucosylated version of ipilimumab (ipilimumab-aFuc)]. [Figure 5A] Figures 5A-5D show the ADCC, reporter activation curves and EC50 values ​​from the FcγRIIIa reporter bioassay for the Antibody 1 variant (Figure 5A), the Antibody 2 variant (Figure 5B), the ipilimumab variant (Figure 5C), and the non-fucosylated variants of all three antibodies (Figure 5D). [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 5D] See legend to Figure 5A. [Figure 6A] Figures 6A-6D show the results of immunophenotyping studies evaluating the percentage of CD45+ cells expressing markers including: CD3+ / ICOS+, CD3+ T cells, CD4+ / Ki67+, CD3+ / Ki67+, CD4+ / ICOS+, CD4+ T cells, CD8+ / ICOS+, CD8+ T cells, Tregs+ / ICOS+, CD8+ / Ki67+, Tregs, and Tregs+ / Ki67+. The antibodies tested were as follows: Group 1: IgG, Group 2: Antibody 2-1, Group 4: ipilimumab, and Group 5: ipilimumab-aFuc. [Figure 6B] See legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 6D] See legend to Figure 6A. [Figure 7A] Figures 7A-7D show graphs depicting tumor volume over time following administration of a single injection of 20 μg, 7 μg, or 2 μg of test antibody, as follows: Figure 7A (group mean) and Figure 7B (individual mice) show tumor volume (mm3) over time for mice injected with antibody 2-6, ipilimumab, or the non-fucosylated form of ipilimumab (ipilimumab-aFuc), while Figures 7C (20 μg), 7D (7 μg), and 7E (2 μg) show a comparison of tumor volume (mm3) over time for each dose of different antibodies. [Figure 7B] See legend to Figure 7A. [Figure 7C] See legend to Figure 7A. [Figure 7D] See legend to Figure 7A. [Figure 7E] See legend to Figure 7A. [Figure 8A] Graphs showing the results of efficacy studies of the percentage of CD4+Ki67+ cells (left) and CD4+ICOS+ cells (right) in peripheral blood 5 days after administration, which represents the level of T cell activation. [Figure 8B] Graphs showing tumor weight (left) and CD8 / Treg ratio (right) assessed at day 7 in mice treated with 10 mg / kg (RSV-m control) or 3 mg / kg (ipilimumab or antibody 2-6). [Figure 8C] Graphs showing regulatory T cells in the tumor microenvironment (left) and CD8+ T cells in the tumor microenvironment (right) are shown after administration of RSV-m control, ipilimumab, or antibody 2-6 in mice. [Figure 8D] 1 shows a graph depicting tumor volume (mm3) over time in mice following administration of 0.3 mg / kg of RSV-m control, ipilimumab, or antibodies, including antibodies 2-6. [Figure 9]Graphs depicting the results of two sets of experiments (Experiment 1 and Experiment 2) evaluating pharmacodynamic effects in cynomolgus monkeys by assessing the percentage of Ki67+ cells among total CD4+ cells following administration of antibody 2-6, ipilimumab, or an isotype control. DETAILED DESCRIPTION OF THE INVENTION

[0057] Detailed Description Treatments such as checkpoint inhibitors have shown unprecedented responses in cancer, but their use is limited by immune-related adverse events (irAEs) and other toxicities (e.g., hypophysitis).Protein therapeutics are provided herein to bind to CTLA4 in, for example, the tumor microenvironment, achieving increased sustained response rates and improved safety profiles.Improved binding affinity, increased functional activity such as ADCC, and other advantages described herein of the provided CTLA4-binding proteins, such as antibodies, bispecific antibodies, or antigen-binding fragments thereof, can result in improved response to treatment and improved safety profiles, such as reduced or minimized adverse events that may be associated with certain immunotherapies.

[0058] I. Definition. Before describing the present invention in detail, it is to be understood that this invention is not limited to particular compositions or biological systems, which can, of course, vary. Also, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to an antibody optionally includes a combination of two or more such antibodies, and the like.

[0059] As used herein, the term "about" refers to a normal range of error for the respective value, readily known to one of ordinary skill in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments that are inherently directed to that value or parameter.

[0060] It is understood that aspects and embodiments of the invention described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0061] The term "antibody" includes polyclonal antibodies, monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), and antibody fragments (e.g., Fab, F(ab'), and Fv). The term "immunoglobulin (Ig)" is used interchangeably with "antibody" herein.

[0062] The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of five basic heterotetrameric units, along with an additional polypeptide called the J chain, and contain ten antigen-binding sites, whereas IgA antibodies consist of two to five basic four-chain units that can polymerize to form multivalent aggregates with the J chain. In the case of IgG, the four-chain unit is generally approximately 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds, depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for each α and γ chain, and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at its N-terminus followed by a constant domain at its other end. The VL aligns with the VH, and the CL aligns with the first constant domain of the heavy chain (CH1). Certain amino acid residues are thought to form an interface between the light and heavy chain variable domains. The pairing of the VH and VL together forms a single antigen-binding site. For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

[0063] Light chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the constant domain (CH) of their heavy chains, immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, each with a heavy chain designated α, δ, ε, γ, and μ. The γ and α classes are further divided into subclasses based on relatively minor differences in CH sequence and function; for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. IgG1 antibodies may exist in multiple polymorphic variants, called allotypes (Jefferis and Lefranc 2009. mAbs Vol 1 Issue 4 1-7), any of which are suitable for use in the present invention. Common allotypic variants in the human population are designated by the letters a, f, n, and z.

[0064] An "isolated" antibody is an antibody that has been identified, separated, and / or recovered from a component of its production environment (e.g., natural or recombinant). In some embodiments, an isolated polypeptide is free from association with all other components from its production environment. Contaminating components of its production environment, such as those resulting from recombinantly transfected cells, are materials that typically interfere with research, diagnostic, or therapeutic uses of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the polypeptide is purified to (1) greater than 95% by weight, and in some embodiments, greater than 99% by weight, of the antibody as determined, for example, by the Lowry method; (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a rolling cup sequencer; or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or silver staining, since at least one component of the antibody's natural environment will not be present. An isolated antibody includes the antibody in situ within recombinant cells. Ordinarily, however, isolated polypeptide or antibody will be prepared by at least one purification step.

[0065] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation), which may be present in minor amounts. In some embodiments, the monoclonal antibody has a C-terminal truncation in the heavy and / or light chain. For example, 1, 2, 3, 4, or 5 amino acid residues are truncated at the C-terminus of the heavy and / or light chain. In some embodiments, the C-terminal truncation removes the C-terminal lysine from the heavy chain. In some embodiments, the monoclonal antibody has an N-terminal truncation in the heavy and / or light chain. For example, 1, 2, 3, 4, or 5 amino acid residues are truncated at the N-terminus of the heavy and / or light chain. In some embodiments, truncated forms of monoclonal antibodies may be produced by recombinant techniques. In some embodiments, monoclonal antibodies are highly specific and directed against a single antigenic site. In some embodiments, monoclonal antibodies are highly specific and directed against multiple antigenic sites (e.g., bispecific or multispecific antibodies, etc.). The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, but is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be produced by a variety of techniques, including, for example, hybridoma methods, recombinant DNA methods, phage display techniques, and techniques for producing human or human-like antibodies in animals having some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences.

[0066] The term "naked antibody" refers to an antibody that is not conjugated to a cytotoxic moiety or radiolabel.

[0067] The term "parent antibody" refers to the antibody before modification.

[0068] "Antibody-drug conjugate" or "ADC" refers to an antibody conjugated to one or more heterologous molecules, including, but not limited to, cytotoxic agents.

[0069] The terms "full-length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Specifically, whole antibodies include those having heavy and light chains, including the Fc region. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In some cases, an intact antibody may have one or more effector functions.

[0070] "Antibody fragments" include portions of an intact antibody, the antigen-binding and / or variable regions of the intact antibody. Examples of antigen-binding antibody fragments include domain antibodies (dAbs), Fab, Fab', F(ab')2, and Fv fragments; antibodies; linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062

[1995] ); single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Single heavy-chain or single light-chain antibodies can be engineered or, in the case of heavy chains, isolated from camels, sharks, libraries, or mice engineered to produce single heavy-chain molecules.

[0071] Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments and a residual "Fc" fragment, a name reflecting their ability to readily crystallize. Fab fragments consist of the entire L chain along with the variable region domain of the H chain (VH) and the first constant domain of one heavy chain (CH1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of antibodies produces a single large F(ab')2 fragment, roughly corresponding to two disulfide-linked Fab fragments with different antigen-binding activities and still capable of cross-linking antigen. Fab' fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residues in the constant domain bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0072] The Fc fragment contains the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of the antibody are determined by sequences and glycans within the Fc region, a region also recognized by Fc receptors (FcRs) found on certain cell types.

[0073] "Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. This fragment consists of a dimer of one heavy chain variable region domain and one light chain variable region domain in tight, non-covalent association. The folding of these two domains generates six hypervariable loops (three loops from each H chain and L chain) that contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific HVRs) has the ability to recognize and bind to an antigen, although with lower affinity than the entire binding site.

[0074] A "single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment comprising the VH and VL antibody domains linked in a single polypeptide chain. In some embodiments, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains, enabling the sFv to form the desired structure for antigen binding. For a review of sFvs, see Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0075] "Functional fragments" of antibodies of the present invention comprise a portion of an intact antibody, generally comprising the antigen-binding or variable region of the intact antibody, or the Fv region of the antibody that retains or has modified FcR binding ability. Examples of antibody fragments include linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0076] The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chains are identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remaining chains are identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include PRIMATIZED® antibodies, in which the antigen-binding region of the antibody is derived, for example, from an antibody generated by immunizing macaque monkeys with the antigen of interest. As used herein, "humanized antibodies" is used as a subset of "chimeric antibodies."

[0077] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's HVR are replaced by residues from an HVR of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, possessing the desired specificity, affinity, and / or capacity. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications may be made to further refine antibody performance, such as binding affinity. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of a non-human immunoglobulin, although the FR regions may comprise one or more individual FR residue substitutions that improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc. In some embodiments, the number of these amino acid substitutions in the FRs will be six or fewer in the H chain and three or fewer in the L chain. Humanized antibodies also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998), Harris, Biochem. Soc. Transactions 23:1035-1038 (1995), Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994), and U.S. Patent Nos. 6,982,321 and 7,087,409.In some embodiments, the humanized antibody is directed against a single antigenic site. In some embodiments, the humanized antibody is directed against multiple antigenic sites. Alternative humanization methods are described in U.S. Patent No. 7,981,843 and U.S. Patent Application Publication No. 2006 / 0134098.

[0078] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. Thus, as used herein, "variable region" and "variable domain" may be used interchangeably. The heavy and light chain variable domains may be referred to as "VH" and "VL," respectively. These domains are generally the most variable parts of an antibody (compared to other antibodies of the same class) and contain the antigen-binding site. The heavy and light chain variable domains may be determined using any available method or numbering scheme and may, for example, include the variable domains described in WO2018 / 207701, the contents of which are incorporated herein by reference. In some embodiments, the heavy and / or light chain variable domains may lack one or more amino acid residues on the carboxyl terminus of the variable domain (i.e., at the carboxyl terminus of the fourth framework domain) or may otherwise be included in the description of the variable domain based on a particular numbering scheme. In some embodiments, the heavy and / or light chain variable domains may comprise one or more amino acid residues on the carboxyl terminus of the variable domain (i.e., at the carboxyl terminus of the fourth framework domain) that may not otherwise be included in the description of the variable domain based on a particular numbering scheme.

[0079] As used herein, "hypervariable region," "HVR," or "HV" refers to a region of an antibody variable domain that is hypervariable in sequence and / or forms a structurally defined loop. Generally, antibodies contain six HVRs: three in the VH (H1, H2, and H3) and three in the VL (L1, L2, and L3). In natural antibodies, H3 and L3 are the most diverse of the six HVRs, and H3 in particular is thought to play a unique role in conferring fine specificity to antibodies. See, for example, Xu et al., Immunity 13:37-45 (2000); Johnson and Wu in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003). In fact, naturally occurring camelid antibodies consisting only of heavy chains are functional and stable in the absence of light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993) and Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0080] Several HVR descriptions are in use and are encompassed herein. Kabat complementarity-determining regions (CDRs), HVRs, are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). Chothia HVRs instead refer to the location of structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). "Contact" HVRs are based on analysis of available complex crystal structures. Residues from each of these HVRs are listed below. TIFF2025163156000002.tif53128

[0081] Unless otherwise indicated, variable domain residues (HVR and framework region residues) are numbered according to Kabat et al., supra.

[0082] "Framework" or "FR" residues are those variable domain residues other than the HVR residues as herein defined.

[0083] The phrases "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat," or variations thereof, refer to the numbering system used for the heavy or light chain variable domains of the above-mentioned compilation of antibodies of Kabat et al. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the FR or HVR of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat, etc.). The Kabat numbering of residues may be determined for a given antibody by alignment of the antibody's sequence with the "standard" Kabat numbered sequence at the region of homology.

[0084] For purposes of this specification, an "acceptor human framework" is a framework that includes the amino acid sequence of a VL or VH framework derived from a human immunoglobulin framework or a human consensus framework. An acceptor human framework that is "derived" from a human immunoglobulin framework or a human consensus framework may include the same amino acid sequence or may include pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.

[0085] "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and does not take into account any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be achieved by various methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, and Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment across the full length of the sequences being compared. For example, the percent amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (which may alternatively be referred to as a given amino acid sequence A having or containing a certain percent amino acid sequence identity to a given amino acid sequence B, to a given amino acid sequence B, or to a given amino acid sequence B) is calculated as follows: 100 x fraction X / Y where X is the number of amino acid residues scored as identical matches by the program's sequences in the alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A.

[0086] An antibody that "binds," "specifically binds," or is "specific for" a particular polypeptide or epitope on a particular polypeptide is an antibody that binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptides or polypeptide epitopes. In some embodiments, the binding of an anti-CTLA4 binding protein (e.g., an anti-CTLA4 antibody or antigen-binding fragment thereof) described herein to an unrelated anti-CTLA4 polypeptide is less than about 10% of the antibody binding to CTLA4, as measured by methods known in the art (e.g., enzyme-linked immunosorbent assay (ELISA)). In some embodiments, a binding protein (e.g., an antibody) that binds to CTLA4 (e.g., murine CTLA4 and / or human CTLA4) has a binding affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦2 nM, ≦1 nM, ≦0.7 nM, ≦0.6 nM, ≦0.5 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., ≦10 -8 M or less, e.g., 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13 Equilibrium dissociation constant (K D )

[0087] The term "CTLA4" or "CTLA4 protein" as provided herein includes any recombinant or naturally occurring form of cytotoxic T-lymphocyte-associated protein 4 (CTLA4) or a variant or homolog thereof that maintains CTLA4 protein activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to CTLA4). In some aspects, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to a naturally occurring CTLA4 polypeptide. In some embodiments, the CTLA4 is a protein identified by NCBI sequence reference GI:83700231, a homolog, or a functional fragment thereof. In some embodiments, the CTLA4 is human CTLA4. In some embodiments, the CTLA4 is mouse CTLA4.

[0088] Antibody "effector functions" refer to the biological activities attributable to the Fc region of an antibody (either a native sequence Fc region or an amino acid sequence variant Fc region) and vary with the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0089] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcR) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. Antibodies are required to "arm" the cytotoxic cells and kill the target cells by this mechanism. The primary cells for mediating ADCC, NK cells, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. Fc expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). In some embodiments, the anti-CTLA4 binding protein described herein (e.g., an anti-CTLA4 antibody or antigen-binding fragment thereof) is engineered or expressed in cells that lack the ability to fucosylate Fc glycans to enhance ADCC. To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or 5,821,337, can be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or in addition, the ADCC activity of a molecule of interest can be assessed in vivo in an animal model, such as that disclosed in Clynes et al., PNAS USA 95:652-656 (1998). Other Fc variants that alter ADCC activity and other antibody properties include those described by Ghetie et al., Nat Biotech. 15:637-40, 1997; Duncan et al., Nature 332:563-564, 1988; Lund et al., J. Immunol 147:2657-2662, 1991; Lund et al., Mol Immunol 29:53-59, 1992; Alegre et al., Transplantation 57:1537-1543, 1994; Hutchins et al., Proc Natl.Acad Sci USA 92:11980-11984,1995, Jefferis et al,Immunol Lett.44:111-117,1995, Lund et al.,FASEB J9:115-119,1995, Jefferis et al,Immunol Lett 54:101-104,1996, Lund et al,J Immunol 157:4963-4969,1996, Armor et al.,Eur J Immunol 29:2613-2624,1999, Idusogie et al,J Immunol 164:4178-4184,200, Reddy et al,J Immunol 164:1925-1933,2000, Xu et al.,Cell Immunol 200:16-26,2000, Idusogie et al,J Immunol 166:2571-2575,2001, Shields et al.,J Biol Chem 276:6591-6604,2001, Jefferis et al,Immunol Lett 82:57-65.2002, Presta et al. al.,Biochem Soc Trans 30:487-490,2002, Lazar et al.,Proc.Natl.Acad.Sci.USA 103:4005-4010,2006, and U.S. Patent Nos. 5,624,821, 5,885,573, 5,677,425, 6,165,745, 6,277,375, 5,869,046, 6,121,022, 5,624,821, 5,648,260, 6,194,551, 6,737,056, 6,821,505, 6,277,375, 7,335,742, and 7,317,091.

[0090] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the Fc region of a human IgG heavy chain is usually defined to stretch from the amino acid residue at position Cys226, or from the amino acid residue at position Pro230, to the carboxyl-terminus thereof. Suitable native-sequence Fc regions for use in the antibodies of the invention include human IgG1, IgG2, IgG3, and IgG4.

[0091] As used herein, "binding affinity" refers to the strength of a non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). In some embodiments, the affinity of a binding protein (e.g., an antibody) for CTLA4 is generally determined by the equilibrium dissociation constant (K D Affinity can be measured by common methods known in the art, including those described herein.

[0092] As used herein, "binding affinity" refers to the strength of binding between multiple binding sites of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen).

[0093] An "isolated" nucleic acid molecule encoding an antibody of the present invention is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is normally associated in the environment in which it is produced. In some embodiments, an isolated nucleic acid is free from all components associated with the environment in which it is produced. An isolated nucleic acid molecule encoding a polypeptide or antibody of the present invention is in a form other than the form or environment in which it is found in nature. Thus, an isolated nucleic acid molecule is distinguished from the nucleic acid encoding the polypeptide or antibody of the present invention that naturally exists in a cell.

[0094] The term "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered. Such formulations are sterile.

[0095] As used herein, "carrier" refers to a pharmaceutically acceptable carrier, excipient, or stabilizer that is non-toxic to cells or mammals exposed to it at the dosage and concentration used. Physiologically acceptable carriers are often aqueous pH-buffered solutions. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

[0096] As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable effects of treatment include slowing the rate of disease progression, improving or alleviating the disease state, and remission or improved prognosis. An individual is successfully "treated," for example, if one or more symptoms associated with a disorder (e.g., a neoplastic disease) are alleviated or eliminated. For example, an individual is successfully "treated" if the treatment results in an improvement in the quality of life of the individual suffering from the disease, a reduction in the dose of other drugs required to treat the disease, a reduction in the frequency of disease recurrence, a reduction in the severity of the disease, a delay in the onset or progression of the disease, and / or an extension of the individual's survival.

[0097] As used herein, "in conjunction with" or "in combination with" refers to the administration of one therapeutic modality in addition to another therapeutic modality. Thus, "in conjunction with" or "in combination with" refers to the administration of one therapeutic modality before, during, or after the administration of another therapeutic modality to an individual.

[0098] As used herein, the term "prevention" includes providing prevention with respect to the occurrence or recurrence of a disease in an individual. The individual may be predisposed to, susceptible to, or at risk of developing a disorder, but has not been diagnosed with the disorder. In some embodiments, the anti-CTLA4 binding proteins (e.g., anti-CTLA4 antibodies) described herein are used to delay the onset of a disorder.

[0099] As used herein, an individual "at risk" of developing a disorder may or may not have detectable disease or disease symptoms, and may or may not exhibit detectable disease or disease symptoms prior to the treatment methods described herein. "At risk," as known in the art, indicates that an individual has one or more risk factors, which are measurable parameters that correlate with the development of a disease. Individuals who have one or more of these risk factors have a higher probability of developing a disorder than individuals who do not have one or more of these risk factors.

[0100] An "effective amount" refers to an amount effective, at a dosage and for a period of time necessary, to achieve at least the desired or indicated effect, including a therapeutic or preventative result. An effective amount may be provided in one or more administrations. A "therapeutically effective amount" is at least the minimum concentration necessary to affect a measurable improvement in a particular disorder. The therapeutically effective amount herein may vary depending on factors such as the patient's disease state, age, sex, and weight, as well as the ability of the antibody to elicit a desired response in an individual. A therapeutically effective amount may also be an amount in which the therapeutically beneficial effects outweigh any toxic or harmful effects of the antibody. A "prophylactically effective amount" refers to an amount effective, at a dosage and for a period of time necessary, to achieve the desired preventative result. Typically, but not necessarily, a prophylactic dose is used in subjects before or at an early stage of disease, so the prophylactically effective amount may be less than the therapeutically effective amount.

[0101] "Chronic" administration refers to the administration of a medication continuously, as opposed to acutely, so as to predominate the initial therapeutic effect (activity) over an extended period of time. "Intermittent" administration is treatment that is not continuous without interruption, but rather is cyclic in nature.

[0102] As used herein, an "individual" or "subject" is a mammal. For purposes of treatment, "mammals" include humans, domestic and farm animals, and zoo, sport, or pet animals such as dogs, horses, rabbits, cows, pigs, hamsters, gerbils, mice, ferrets, rats, cats, etc. In some embodiments, the individual or subject is a human.

[0103] II. Anti-CTLA4 Binding Protein Provided herein are proteins that bind to cytotoxic T-lymphocyte-associated protein 4 (CTLA4). In some embodiments, the provided CTLA4-binding proteins are antibodies or antigen-binding fragments thereof, or proteins comprising a CTLA4-binding domain. In some embodiments, the CTLA4-binding protein is an anti-CTLA4 antibody or antigen-binding fragment thereof. In some embodiments, the CTLA4-binding protein is a bispecific antibody or antigen-binding fragment thereof that binds to CTLA4. For example, a bispecific antibody comprising a CTLA-binding domain. In one aspect, a CTLA4-binding protein comprising a CTLA4-binding domain, such as a fusion protein comprising a CTLA4-binding domain described herein, is provided. In some embodiments, the CTLA4-binding protein is a chimeric receptor that binds to CTLA4, for example, via an antigen-binding domain capable of binding to CTLA4.

[0104] The CTLA4 binding proteins provided herein can bind to CTLA4 from various species, for example, some bind to human CTLA4 and / or mouse CTLA4, or cynomolgus monkey CTLA4. In some embodiments, the anti-CTLA4 binding proteins described herein have one or more of the following characteristics: (1) bind to CTLA4 (e.g., human CTLA4), and (2) bind to CTLA4 in vivo at the tumor site.

[0105] In one aspect, provided herein are CTLA4 binding proteins that are particularly useful for treating neoplastic diseases in which CTLA4 plays a role. The CTLA4 binding proteins provided herein include a binding domain that can interact with (e.g., bind to) a CTLA4 protein expressed on the surface of a cell (e.g., a cancer cell or a T cell).

[0106] Also provided herein are CTLA4 binding proteins (e.g., anti-CTLA4 antibodies or antigen-binding fragments thereof), which in some embodiments comprise a CTLA4 binding protein (e.g., an anti-CTLA4 antibody or antigen-binding fragment thereof comprising a first chain and a second chain). In some embodiments, the CTLA4 binding protein is a dimer. In some embodiments, the CTLA4 binding protein is a homodimer. In some embodiments, the CTLA4 binding protein is a heterodimer. In some embodiments, the CTLA4 binding protein is a heterodimer comprising a first chain and a second chain, such as a heterodimer comprising a heavy chain and a light chain. In some embodiments, the CTLA4 binding protein is an anti-CTLA4 antibody or antigen-binding fragment thereof comprising a first chain and a second chain. In some embodiments, the first chain is or comprises a heavy chain and the second chain is or comprises a light chain, or the first chain is or comprises a light chain and the second chain is or comprises a heavy chain. In some embodiments, the first chain is or comprises a heavy chain variable region and the second chain is or comprises a light chain variable region, or the first chain is or comprises a light chain variable region and the second chain is or comprises a heavy chain variable region. In some embodiments, the CTLA4 binding protein is a single-chain protein, such as a single-chain protein comprising both a heavy chain and a light chain.

[0107] Nucleic acids encoding the antibody, bispecific antibody, or any antigen-binding fragment thereof, or chimeric receptor of any one of the foregoing embodiments are also provided. Vectors comprising the nucleic acids of the foregoing embodiments are also provided. In some embodiments, the vector is an expression vector. Host cells comprising the foregoing nucleic acid embodiments are also provided.

[0108] Also provided are methods for producing an antibody, bispecific antibody, or any antigen-binding fragment thereof, or chimeric receptor, comprising culturing the aforementioned host cell under conditions for producing the antibody, bispecific antibody, or chimeric receptor. In some embodiments, the host cell has an alpha 1,6-fucosyltransferase (Fut8) knockout. In some embodiments, the host cell overexpresses β1,4-N-acetylglucosaminyltransferase III (GnT-III). In some embodiments, the host cell further overexpresses Golgi μ-mannosidase II (ManII). Some of any such embodiments further comprise recovering the antibody, bispecific antibody, or any antigen-binding fragment thereof, or chimeric receptor produced by the host cell. In some embodiments, the bispecific antibody or chimeric receptor produced by the aforementioned method.

[0109] Also provided is a composition comprising the antibody, bispecific antibody, or any antigen-binding fragment thereof, or chimeric receptor of any one of the preceding embodiments. Some embodiments include a composition comprising the antibody, bispecific antibody, or any antigen-binding fragment thereof, or chimeric receptor of the preceding embodiments. In some embodiments, the composition is a pharmaceutical composition.

[0110] Kits comprising the antibody, bispecific antibody, or any antigen-binding fragment thereof, chimeric receptor, or composition of any one of the foregoing embodiments are also provided.

[0111] Also provided are methods of treating or preventing a neoplastic disease in a subject, the method comprising administering to the subject an effective amount of the antibody, bispecific antibody, or any antigen-binding fragment thereof, chimeric receptor, or composition of any one of the preceding embodiments. In one embodiment, the neoplastic disease is cancer. In some embodiments, the cancer is leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, spleen cancer, melanoma, breast cancer, neuroblastoma, lung cancer, ovarian cancer, osteosarcoma, bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer, or testicular cancer.

[0112] CTLA4-binding protein The term "CTLA4 binding protein" as provided herein refers to a polypeptide comprising a CTLA4 binding domain capable of binding to or otherwise exhibiting affinity for CTLA4 protein. In some embodiments, the CTLA4 binding protein is an anti-CTLA4 antibody or antigen-binding fragment thereof, a bispecific antibody, an antigen-binding fragment, a single-chain antibody, or the like. In some embodiments, the CTLA4 binding protein is an antibody or antigen-binding fragment thereof that binds to CTLA4. In some embodiments, the antibody or antigen-binding fragment thereof that binds to CTLA4 is an anti-CTLA4 antibody or antigen-binding fragment thereof. Thus, in some embodiments, the CTLA4 binding protein is an anti-CTLA4 antibody or antigen-binding fragment thereof. In some embodiments, the CTLA4 binding protein is a component of a chimeric antigen receptor that binds to CTLA4.

[0113] The term "CTLA4 binding domain" refers to a recombinantly expressed polypeptide domain capable of binding to or otherwise exhibiting affinity for CTLA4 protein found in or on a cell. Methods for determining the degree of binding of a CTLA4 binding domain to CTLA4 are well known in the art.

[0114] In some embodiments, the CTLA4 binding domain provided herein is an antibody capable of binding to CTLA4. In some embodiments, the CTLA4 is human CTLA4. In some embodiments, the CTLA4 is mouse CTLA4. In some embodiments, the antibody is a mouse antibody.

[0115] In some embodiments, the antibody is a humanized antibody, a chimeric antibody, or a human antibody. In some embodiments, the anti-CTLA4 antibody or antigen-binding fragment thereof described herein is a monoclonal antibody. In some embodiments, the anti-CTLA4 antibody or antigen-binding fragment thereof described herein is an antibody fragment (including an antigen-binding fragment), such as a dAb, Fab'-SH, Fv, scFv, or (Fab')2 fragment. In some embodiments, the antibody or antigen-binding fragment thereof is a dimer. In some embodiments, the antibody or antigen-binding fragment thereof is a homodimer. In some embodiments, the antibody or antigen-binding fragment thereof is a heterodimer. In some embodiments, the antibody or antigen-binding fragment thereof is a heterodimer comprising a first chain and a second chain, such as a heterodimer comprising a heavy chain and a light chain. In some embodiments, the antibody or antigen-binding fragment thereof comprises a first chain and a second chain. In some embodiments, the first chain is or comprises a heavy chain, and the second chain is or comprises a light chain, or the first chain is or comprises a light chain and the second chain is or comprises a heavy chain. In some embodiments, the first chain is or comprises a heavy chain variable region, and the second chain is or comprises a light chain variable region, or the first chain is or comprises a light chain variable region, and the second chain is or comprises a heavy chain variable region. In some embodiments, the antibody or antigen-binding fragment thereof comprises a first chain and a second chain (e.g., a light chain and a heavy chain). In some embodiments, the antibody or antigen-binding fragment thereof comprises two first chains and two second chains (e.g., two light chains and two heavy chains).In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1 or 13, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2 or 14, and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3 or 15, and / or the heavy chain variable region comprises (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4 or 16, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5 or 17, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6 or 18.

[0116] In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3, and / or the heavy chain variable region comprises (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3, and the heavy chain variable region comprises (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6.

[0117] In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO:9, and / or the heavy chain variable region comprises (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:10, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:11, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:12. In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO:9, and the heavy chain variable region comprises (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:10, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:11, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:12.

[0118] In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15, and / or the heavy chain variable region comprises (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 16, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15, and the heavy chain variable region comprises (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 16, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 18.

[0119] In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 19, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 20, and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 21, and / or the heavy chain variable region comprises (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 19, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 20, and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 21, and the heavy chain variable region comprises (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24.

[0120] In some embodiments, the VL domain comprises CDR-L1, CDR-L2, and CDR-L3 contained within the amino acid sequence of SEQ ID NO: 25, and the VH domain comprises CDR-H1, CDR-H2, and CDR-H3 contained within the amino acid sequence of SEQ ID NO: 26. In some embodiments, the VL domain comprises CDR-L1, CDR-L2, and CDR-L3 contained within the amino acid sequence of SEQ ID NO: 30, and the VH domain comprises CDR-H1, CDR-H2, and CDR-H3 contained within the amino acid sequence of SEQ ID NO: 31.

[0121] In some embodiments, the first chain is a light chain and the second chain is a heavy chain. In some embodiments, the antibody or antigen-binding fragment thereof comprises two first chains and two second chains. In some embodiments, the first chain is a light chain variable domain and the second chain is a heavy chain variable domain. In some of any such embodiments, the antigen-binding fragment is a dAb, Fab, Fab'-SH, Fv, scFv, or (Fab')2 fragment.

[0122] In some of any such embodiments, the antibody is a murine antibody.

[0123] In some of any such embodiments, the antibody is a humanized antibody, a chimeric antibody, or a human antibody. In some of any such embodiments, the antibody has an IgG1, IgG2, IgG3, or IgG4 isotype. In some of any such embodiments, the IgG1 has the amino acid substitutions S298A, E333A, and K334A; S239D and I332E; S239D, A330L, and I332E; P247I and A339D or A339Q; D280H, K290S with or without S298D, or S298V; F243L, R292P, and Y300L; F243L, R292P , Y300L, and P396L; F243L, R292P, Y300L, V305I, and P396L; G236A, S239D, and I332E; K326A and E333A; K326W and E333S; or K290E or K290N, S298G, T299A, and / or K326E, wherein amino acid residues are numbered according to the EU index of Kabat.

[0124] In some of any such embodiments, the antibody or antigen-binding fragment comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 25 or 30, and / or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26 or 31.

[0125] In some of any of the embodiments, the VL domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 25, and the VH domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 26. In some of any of such embodiments, the antibody or antigen-binding fragment comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 25, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26.

[0126] In some of any of the embodiments, the VL domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 30, and the VH domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 31. In some of any of such embodiments, the antibody or antigen-binding fragment comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31.

[0127] In some embodiments, the antibody or antigen-binding fragment thereof has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO:27 or an amino acid sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology. and / or a heavy chain comprising an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO:28. In some embodiments, the antibody or antigen-binding fragment thereof has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO:27. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 27 and / or a heavy chain comprising the amino acid sequence of SEQ ID NO: 28. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 27 and / or a heavy chain comprising the amino acid sequence of SEQ ID NO: 28. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 27 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 28.

[0128] In some embodiments, the antibody or antigen-binding fragment thereof has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO:27 or an amino acid sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology. and / or a heavy chain comprising an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO:29. In some embodiments, the antibody or antigen-binding fragment thereof has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO:27. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 27 and / or a heavy chain comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 27 and / or a heavy chain comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 27 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 29.

[0129] In some embodiments, the antibody or antigen-binding fragment thereof has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology. and / or a heavy chain comprising an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:33 or that is about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the antibody or antigen-binding fragment thereof has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO: 32. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 32 and / or a heavy chain comprising the amino acid sequence of SEQ ID NO: 33. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 32 and / or a heavy chain comprising the amino acid sequence of SEQ ID NO: 33. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 32 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 33.

[0130] In some embodiments, the antibody or antigen-binding fragment thereof has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology. and / or a heavy chain comprising an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO:34. In some embodiments, the antibody or antigen-binding fragment thereof has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO: 32. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 32 and / or a heavy chain comprising the amino acid sequence of SEQ ID NO: 34. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 32 and / or a heavy chain comprising the amino acid sequence of SEQ ID NO: 34. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 32 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 34.

[0131] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising an amino acid sequence selected from SEQ ID NOs: 27 and 32, and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 28, 29, 33, and 34.

[0132] In some of any such embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 27 and / or a heavy chain comprising the amino acid sequence of SEQ ID NO: 28. In some of any such embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 27 and / or a heavy chain comprising the amino acid sequence of SEQ ID NO: 29. In some of any such embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 27 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 28. In some of any such embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 27 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 29.

[0133] In some of any such embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 32 and / or a heavy chain comprising the amino acid sequence of SEQ ID NO: 33. In some of any such embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 32 and / or a heavy chain comprising the amino acid sequence of SEQ ID NO: 34. In some of any such embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 32 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 33. In some of any such embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 32 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 34.

[0134] Also provided herein are bispecific antibodies comprising a first pair of light and heavy chains that specifically binds to CTLA4 and a second pair of light and heavy chains that specifically binds to an antigen, e.g., an antigen that is a tumor antigen.

[0135] In some of any such embodiments, the bispecific antibody is a murine antibody.

[0136] In some of any such embodiments, the bispecific antibody is a humanized antibody, a chimeric antibody, or a human antibody. In some of any such embodiments, the bispecific antibody has an IgG1, IgG2, IgG3, or IgG4 isotype. In some of any such embodiments, the IgG1 has the following amino acids: S298A, E333A, and K334A; S239D and I332E; S239D, A330L, and I332E; P247I and A339D or A339Q; D280H, K290S with or without S298D, or S298V; F243L, R292P, and Y300L; F243L, R292P, Y300L, and and P396L; F243L, R292P, Y300L, V305I, and P396L; G236A, S239D, and I332E; K326A and E333A; K326W and E333S; or K290E or K290N, S298G, T299A, and / or K326E, where amino acid residues are numbered according to the EU index of Kabat.

[0137] In some of any such embodiments, the first pair comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1 or 13, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2 or 14, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3 or 15, and / or the heavy chain variable region comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4 or 16, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5 or 17, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6 or 18.

[0138] In some of any such embodiments, the first pair comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 25 or 30, and / or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26 or 31.

[0139] In some of any of the embodiments, the first pair comprises a VL domain comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 25, and a VH domain comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 26. In some of any of such embodiments, the first pair comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 25, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26.

[0140] In some of any of the embodiments, the first pair comprises a VL domain comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 30, and a VH domain comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 31. In some of any of such embodiments, the first pair comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31.

[0141] In some of any such embodiments, the first pair comprises a light chain comprising an amino acid sequence selected from SEQ ID NOs: 27 and 32, and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 28, 29, 33, and 34.

[0142] In some of any such embodiments, the first pair comprises a light chain comprising the amino acid sequence of SEQ ID NO: 27 and / or a heavy chain comprising the amino acid sequence of SEQ ID NO: 28. In some of any such embodiments, the first pair comprises a light chain comprising the amino acid sequence of SEQ ID NO: 27 and / or a heavy chain comprising the amino acid sequence of SEQ ID NO: 29. In some of any such embodiments, the first pair comprises a light chain comprising the amino acid sequence of SEQ ID NO: 27 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 28. In some of any such embodiments, the first pair comprises a light chain comprising the amino acid sequence of SEQ ID NO: 27 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 29.

[0143] In some of any such embodiments, the first pair comprises a light chain comprising the amino acid sequence of SEQ ID NO: 32 and / or a heavy chain comprising the amino acid sequence of SEQ ID NO: 33. In some of any such embodiments, the first pair comprises a light chain comprising the amino acid sequence of SEQ ID NO: 32 and / or a heavy chain comprising the amino acid sequence of SEQ ID NO: 34. In some of any such embodiments, the first pair comprises a light chain comprising the amino acid sequence of SEQ ID NO: 32 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 33. In some of any such embodiments, the first pair comprises a light chain comprising the amino acid sequence of SEQ ID NO: 32 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 34.

[0144] In some of any such embodiments, the bispecific antibody comprises a first chain and a second chain, wherein the first chain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1 or 13, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2 or 14, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3 or 15; and / or the second chain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4 or 16, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5 or 17, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6 or 18.

[0145] In some of any such embodiments, the first chain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 25 or 30, and / or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26 or 31.

[0146] In some of any of the embodiments, the first chain comprises a VL domain comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 25, and a VH domain comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 26. In some of any of such embodiments, the first chain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 25, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26.

[0147] In some of any of the embodiments, the first chain comprises a VL domain comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 30, and a VH domain comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 31. In some of any of such embodiments, the first chain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31.

[0148] In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 25 or 30, and / or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26 or 31. In some embodiments, the antibody or antigen-binding fragment comprises an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 25 or that is about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence of SEQ ID NO: 25. The present invention also includes a light chain variable region and / or a heavy chain variable region comprising an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO:26, or about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology. In some embodiments, the antibody or antigen-binding fragment has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO: 25 or an amino acid sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology. and a heavy chain variable region comprising an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO:26 or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO:26.In some embodiments, the antibody or antigen-binding fragment has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO: 30 or comprises an amino acid sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology. The present invention also includes a light chain variable region and / or a heavy chain variable region comprising an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO: 31, or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO: 31. In some embodiments, the antibody or antigen-binding fragment has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO: 30 or an amino acid sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology. and a heavy chain variable region comprising an amino acid sequence with 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 31. In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30 and / or comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31.In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:30 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:31.

[0149] In some embodiments, the antibody or antigen-binding fragment thereof has an IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the antibody or antigen-binding fragment thereof has an IgG1 isotype containing amino acid substitutions that enhance effector function as described herein.

[0150] In some embodiments, the CTLA4-binding domain comprises the light and heavy chains of the antigen-binding arms of a bispecific antibody. In some embodiments of the bispecific antibody, the light chain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1 or 13, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2 or 14, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3 or 15, and / or the heavy chain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4 or 16, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5 or 17, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6 or 18. In some embodiments of the bispecific antibody, the light chain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3, and the heavy chain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments of the bispecific antibody, the light chain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, and the heavy chain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 10, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 11, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments of the bispecific antibody, the light chain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15, and the heavy chain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 16, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 18.In some embodiments of the bispecific antibody, the light chain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 19, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 20, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 21, and the heavy chain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24.

[0151] In some embodiments of the bispecific antibody, the VL domain comprises CDR-L1, CDR-L2, and CDR-L3 comprised within the amino acid sequence of SEQ ID NO: 25, and the VH domain comprises CDR-H1, CDR-H2, and CDR-H3 comprised within the amino acid sequence of SEQ ID NO: 26. In some embodiments of the bispecific antibody, the VL domain comprises CDR-L1, CDR-L2, and CDR-L3 comprised within the amino acid sequence of SEQ ID NO: 30, and the VH domain comprises CDR-H1, CDR-H2, and CDR-H3 comprised within the amino acid sequence of SEQ ID NO: 31.

[0152] In some embodiments of the bispecific antibody, the light chain has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO:25. and / or the heavy chain comprises an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence of SEQ ID NO:26. In some embodiments of the bispecific antibody, the light chain has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO: 30. and / or the heavy chain comprises an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence of SEQ ID NO:31.

[0153] In some embodiments of the bispecific antibody, the light chain comprises the amino acid sequence of SEQ ID NO: 25 or 30 and / or the heavy chain comprises the amino acid sequence of SEQ ID NO: 26 or 31.

[0154] In some of any of the bispecific antibody embodiments, the VL domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 25, and the VH domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 26. In some of any of such bispecific antibody embodiments, the antibody or antigen-binding fragment comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 25, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26.

[0155] In some of any of the bispecific antibody embodiments, the VL domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 30, and the VH domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 31. In some of any of such bispecific antibody embodiments, the antibody or antigen-binding fragment comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30, and a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 31.

[0156] In some embodiments of the bispecific antibody, the light chain has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27 and 32, or an amino acid sequence having about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to an amino acid sequence selected from the group consisting of SEQ ID NOs: 27 and 32. and / or the heavy chain comprises an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous to or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 29, 33, and 34. In some embodiments of the bispecific antibody, the light chain comprises an amino acid sequence selected from SEQ ID NOs: 27 and 32, and / or the heavy chain comprises an amino acid sequence selected from SEQ ID NOs: 28, 29, 33, and 34.

[0157] In some embodiments of the bispecific antibody, the light chain has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO:27. and / or the heavy chain comprises an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous to or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous to the amino acid sequence of SEQ ID NO: 28. In some embodiments of the bispecific antibody, the light chain comprises the amino acid sequence of SEQ ID NO: 27 and the heavy chain comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 28.

[0158] In some embodiments of the bispecific antibody, the light chain has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO:27. and / or the heavy chain comprises an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous to or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous to the amino acid sequence of SEQ ID NO: 29. In some embodiments of the bispecific antibody, the light chain comprises the amino acid sequence of SEQ ID NO: 27 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 29.

[0159] In some embodiments of the bispecific antibody, the light chain has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO: 32. and / or the heavy chain comprises an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence of SEQ ID NO: 33. In some embodiments of the bispecific antibody, the light chain comprises the amino acid sequence of SEQ ID NO: 32 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 33.

[0160] In some embodiments of the bispecific antibody, the light chain has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO: 32. and / or the heavy chain comprises an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous to or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous to the amino acid sequence of SEQ ID NO: 34. In some embodiments of the bispecific antibody, the light chain comprises the amino acid sequence of SEQ ID NO: 32 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 34.

[0161] In some embodiments of the bispecific antibody, the CTLA4 is human CTLA4. In some embodiments of the bispecific antibody, the CTLA4 is mouse CTLA4. In some embodiments, the bispecific antibody is a mouse antibody.

[0162] In some embodiments, the bispecific antibody is a humanized antibody, a chimeric antibody, or a human antibody. In some embodiments, the bispecific antibody has an IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the bispecific antibody has an IgG1 isotype containing amino acid substitutions that enhance effector function as described herein.

[0163] In some embodiments, the bispecific antibody or antigen-binding fragment thereof is a dimer. In some embodiments, the bispecific antibody or antigen-binding fragment thereof is a homodimer. In some embodiments, the bispecific antibody or antigen-binding fragment thereof is a heterodimer. In some embodiments, the bispecific antibody or antigen-binding fragment thereof is a heterodimer comprising a first chain and a second chain, such as a heterodimer comprising a heavy chain and a light chain. In some embodiments, the bispecific antibody or antigen-binding fragment comprises a first chain and a second chain. In some embodiments, the first chain is or comprises a heavy chain and the second chain is or comprises a light chain, or the first chain is or comprises a light chain and the second chain is or comprises a heavy chain. In some embodiments, the first chain is or comprises a heavy chain variable region and the second chain is or comprises a light chain variable region, or the first chain is or comprises a light chain variable region and the second chain is or comprises a heavy chain variable region.

[0164] In some embodiments, the CTLA4-binding domain comprises a first chain and a second chain that bind to CTLA4, such as a portion of a ligand-binding domain for use in a chimeric receptor. In some embodiments of the chimeric receptor, the first chain is a light chain variable domain. In some embodiments, the second chain is a heavy chain variable domain. In some embodiments of the chimeric receptor, the first chain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1 or 13, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2 or 14, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3 or 15, and / or the second chain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4 or 16, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5 or 17, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6 or 18. In some embodiments of the chimeric receptor, the first chain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3, and the second chain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments of the chimeric receptor, the first chain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, and the second chain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 10, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 11, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments of the chimeric receptor, the first chain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15, and the second chain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 16, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 18.In some embodiments of the chimeric receptor, the first chain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 19, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 20, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 21, and the second chain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24.

[0165] In some embodiments of the chimeric receptor, the first chain has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the amino acid sequence of SEQ ID NO:30. and / or the second chain comprises an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to or about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 31. In some embodiments of the chimeric receptor, the first chain comprises the amino acid sequence of SEQ ID NO: 25 or 30 and / or the second chain comprises the amino acid sequence of SEQ ID NO: 26 or 31. In some embodiments of the chimeric receptor, the first chain comprises the amino acid sequence of SEQ ID NO:30 and the second chain comprises the amino acid sequence of SEQ ID NO:31.

[0166] CTLA4-binding proteins can be conjugated to additional molecules by a variety of methods well known in the art. The terms "conjugate" and "conjugation chemistry" refer to reactions with known reactive groups that proceed under relatively mild conditions. These include, but are not limited to, nucleophilic substitutions (e.g., reactions of amines and alcohols with acyl halides, activated esters), electrophilic substitutions (e.g., enamine reactions), and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reactions, Diels-Alder additions). These and other useful reactions are discussed, for example, in March, Advanced Organic Chemistry, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson, Bioconjugate Techniques, Academic Press, San Diego, 1996; and Feeney et al., Modification of Proteins; Advances in Chemistry Series, Vol. 198, American Chemical Society, Washington, DC, 1982.

[0167] Useful reactive functional groups for use in the conjugation chemistry herein include, for example, (a) carboxyl groups and various derivatives thereof, including, but not limited to, N-hydroxysuccinimide esters, N-hydroxybenztriazole esters, acid halides, acylimidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl, and aromatic esters; (b) hydroxyl groups that can be converted to esters, ethers, aldehydes, and the like; (c) haloalkyl groups, where the halide can be subsequently displaced with a nucleophilic group, such as an amine, a carboxylate anion, a thiol anion, a carbanion, or an alkoxide ion, thereby resulting in the covalent attachment of a new group at the site of the halogen atom; (d) dienophile groups, such as, for example, a maleimide group, that can participate in Diels-Alder reactions; and (e) functional groups that can undergo reactions such as Grignard addition or alkyllithium addition. (f) sulfonylhalogen groups for subsequent reaction with amines, e.g., to form sulfonamides; (g) thiol groups, which can be converted to disulfides, reacted with acyl halides, or attached to metals such as gold; (h) amine or sulfhydryl groups, which can be acylated, alkylated, or oxidized; (i) alkenes, which can undergo, e.g., cycloaddition, acylation, Michael addition, etc.; (j) epoxides, which can react with, e.g., amines and hydroxyl compounds; (k) phosphoramidites and other standard functional groups useful in nucleic acid synthesis; (i) metal-silicon oxide linkages; and (l) metal linkages to reactive phosphorus groups (e.g., phosphines), e.g., to form phosphodiester linkages.

[0168] The reactive functional groups can be selected so that they do not participate in or interfere with the chemical stability of the compositions described herein, or alternatively, the reactive functional groups can be protected from participating in the crosslinking reaction by the presence of a protecting group.

[0169] In some embodiments, the linker can be engineered to be fused to an additional molecule and / or a CTLA4 binding protein by various methods well known in the art. For example, a nucleic acid can be engineered to encode a linker that includes an additional molecule and / or a CTLA4 binding protein to generate a fusion protein when recombinantly expressed from a host cell.

[0170] Exemplary CTLA4 Binding Proteins Certain exemplary embodiments of CTLA4 binding proteins incorporating certain features described above are described below. These embodiments are merely exemplary and should not be construed as limiting.

[0171] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that binds to CTLA4 (e.g., human CTLA4), wherein the antibody or antigen-binding fragment thereof comprises a first chain and a second chain. In some embodiments, the antibody or antigen-binding fragment thereof that binds to CTLA4 is any anti-CTLA4 antibody or antigen-binding fragment thereof described herein. In some embodiments, the antibody or antigen-binding fragment thereof comprises two first chains and two second chains. In some embodiments, the first chain is a light chain and the second chain is a heavy chain. In some embodiments, the first chain is a light chain variable domain and the second chain is a heavy chain variable domain. In some embodiments, a) the first chain of the antibody is a light chain and the second chain of the antibody is a light chain, b) the first chain of the antibody is a heavy chain and the second chain of the antibody is a heavy chain, or c) the first chain of the antibody is a light chain and the second chain of the antibody is a heavy chain.

[0172] Also provided herein in some embodiments is an anti-CTLA4 antibody or antigen-binding fragment thereof, comprising a light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 19, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 20, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 21, and a heavy chain variable region comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24.

[0173] In some embodiments, the light chain variable region comprises the amino acid sequence of SEQ ID NO: 30, and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 31. In some embodiments, the anti-CTLA4 antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 34, and a light chain comprising the amino acid sequence of SEQ ID NO: 32.

[0174] In one aspect, provided herein is an anti-CTLA4 antibody, or antigen-binding fragment thereof, comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 25 or 30, and / or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26 or 31. In some embodiments, provided herein is an anti-CTLA4 antibody, or antigen-binding fragment thereof, comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30, and / or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31.

[0175] In a further aspect, provided herein is an anti-CTLA4 antibody or antigen-binding fragment thereof comprising a light chain comprising an amino acid sequence selected from SEQ ID NOs: 27 and 32, and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 28, 29, 33, and 34. In some embodiments, provided herein is an anti-CTLA4 antibody or antigen-binding fragment thereof comprising a light chain comprising the amino acid sequence of SEQ ID NO: 27 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, provided herein is an anti-CTLA4 antibody or antigen-binding fragment thereof comprising a light chain comprising the amino acid sequence of SEQ ID NO: 27 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 28. In some embodiments, provided herein is an anti-CTLA4 antibody or antigen-binding fragment thereof comprising a light chain comprising the amino acid sequence of SEQ ID NO: 32 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 33. In some embodiments, provided herein is an anti-CTLA4 antibody or antigen-binding fragment thereof comprising a light chain comprising the amino acid sequence of SEQ ID NO: 32 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 34.

[0176] In some embodiments, provided herein is an anti-CTLA4 antibody or antigen-binding fragment thereof, comprising a heavy chain variable domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from SEQ ID NOs: 26 and 31. In some embodiments, provided herein is an anti-CTLA4 antibody or antigen-binding fragment thereof, comprising a heavy chain variable domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 26. In some embodiments, provided herein is an anti-CTLA4 antibody or antigen-binding fragment thereof, comprising a heavy chain variable domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 31. In some embodiments, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity contains substitutions, insertions, or deletions relative to the reference sequence, but an antibody comprising the amino acid sequence retains the ability to bind to CTLA4 (e.g., human CTLA4). In some embodiments, the substitutions, insertions, or deletions (e.g., 1, 2, 3, 4, or 5 amino acids) occur in a region outside the HVR (i.e., within the FR). In some embodiments, an anti-CTLA4 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 26 or 31. In some embodiments, an anti-CTLA4 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, an anti-CTLA4 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 31.

[0177] In some embodiments, provided herein is an anti-CTLA4 antibody or antigen-binding fragment thereof comprising a heavy chain variable domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 25 or 30. In some embodiments, provided herein is an anti-CTLA4 antibody or antigen-binding fragment thereof comprising a heavy chain variable domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 25. In some embodiments, provided herein is an anti-CTLA4 antibody or antigen-binding fragment thereof comprising a heavy chain variable domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 30. In some embodiments, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity contains substitutions, insertions, or deletions relative to the reference sequence, but an antibody comprising the amino acid sequence retains the ability to bind to CTLA4 (e.g., human CTLA4). In some embodiments, the substitutions, insertions, or deletions (e.g., 1, 2, 3, 4, or 5 amino acids) occur in a region outside the HVR (i.e., within the FR). In some embodiments, an anti-CTLA4 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 25 or 30. In some embodiments, an anti-CTLA4 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, an anti-CTLA4 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 30.

[0178] There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, each with a heavy chain designated α, δ, ε, γ, and μ. The γ and α classes are further divided into subclasses; for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. IgG1 antibodies may exist in multiple polymorphic variants called allotypes (Jefferis and Lefranc 2009. mAbs Vol 1 Issue 4 1-7), any of which are suitable for use in some of the embodiments herein. Common allotypic variants in the human population are those designated by the letters a, f, n, z, or combinations thereof. In some of the embodiments herein, the antibody has an IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the anti-CTLA4 antibody or antigen-binding fragment thereof provided herein has an IgG1 isotype (e.g., a human IgG1 isotype). In some embodiments, the antibodies provided herein comprise a heavy chain constant domain comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the antibodies provided herein comprise a heavy chain constant domain comprising the amino acid sequence of SEQ ID NO: 38.

[0179] In one aspect of the present invention, a polynucleotide encoding an anti-CTLA4 antibody or an antigen-binding fragment thereof is provided. In certain embodiments, a vector comprising a polynucleotide encoding an anti-CTLA4 antibody or an antigen-binding fragment thereof is provided. In certain embodiments, a host cell comprising such a vector is provided. In another aspect of the present invention, a composition comprising an anti-CTLA4 antibody described herein or a polynucleotide encoding an anti-CTLA4 antibody described herein is provided. In certain embodiments, the composition of the present invention is a pharmaceutical preparation for the treatment of neoplastic diseases in which CTLA4 plays a role, such as those listed herein.

[0180] In some embodiments, the CTLA4 binding protein provided herein is a bispecific antibody that can bind to CTLA4. A bispecific antibody is a monoclonal antibody that has binding specificities for at least two different antigens. In some embodiments, one of the binding specificities is for CTLA4, and the other is for any other antigen. In certain embodiments, a bispecific antibody can bind to two different epitopes of CTLA4.

[0181] In some aspects, provided herein are bispecific antibodies comprising a) a first pair of light chains and heavy chains that specifically bind to CTLA4, and b) a second pair of light chains and heavy chains that specifically bind to an antigen. In some aspects, provided herein are bispecific antibodies comprising a) a first pair of light chains and heavy chains that specifically bind to CTLA4, and b) a second pair of light chains and heavy chains that specifically bind to an antigen. In some embodiments, the antigen is an antigen different from CTLA4. In some embodiments, the light chain of the first pair or the second pair is any light chain described herein. In some embodiments, the heavy chain of the first pair or the second pair is any light chain described herein. In some embodiments, a first pair of light chains comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 19, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 20, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 21, and a first pair of heavy chains comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments, a second pair of light chains comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 19, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 20, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 21, and a second pair of heavy chains comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments, the antigens are directed against different epitopes of CTLA4.

[0182] Bispecific antibodies contemplated herein for use in bispecific antibodies include murine bispecific antibodies, humanized bispecific antibodies, chimeric bispecific antibodies, and human bispecific antibodies. In some of the embodiments herein, the bispecific antibodies have an IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the bispecific antibodies provided herein have an IgG1 isotype (e.g., a human IgG1 isotype). In some embodiments, the antibodies have an IgG1 isotype that includes amino acid substitutions or are expressed by cells that do not have a reduced ability to fucosylate Fc glycans that enhance the effector functions described herein. In some embodiments, the bispecific antibodies provided herein comprise a heavy chain constant domain comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the bispecific antibodies provided herein comprise a heavy chain constant domain comprising the amino acid sequence of SEQ ID NO: 38.

[0183] In one aspect, provided herein is an anti-CTLA4 bispecific antibody comprising a light chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 25 and 30, and / or a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 26 and 31. In a further aspect, provided herein is an anti-CTLA4 bispecific antibody comprising a light chain comprising an amino acid sequence selected from SEQ ID NOs: 27 and 32, and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 28, 29, 33, and 34. In some embodiments, provided herein is an anti-CTLA4 bispecific antibody, or antigen-binding fragment thereof, comprising a light chain comprising the amino acid sequence of SEQ ID NO: 27, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, provided herein is an anti-CTLA4 bispecific antibody, or antigen-binding fragment thereof, comprising a light chain comprising the amino acid sequence of SEQ ID NO: 27, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 28. In some embodiments, provided herein is an anti-CTLA4 bispecific antibody, or antigen-binding fragment thereof, comprising a light chain comprising the amino acid sequence of SEQ ID NO: 32, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 33. In some embodiments, provided herein is an anti-CTLA4 bispecific antibody or antigen-binding fragment thereof, comprising a light chain comprising the amino acid sequence of SEQ ID NO: 32, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 34.

[0184] In some embodiments, provided herein is an anti-CTLA4 bispecific antibody or antigen-binding fragment thereof, comprising a heavy chain variable domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from SEQ ID NOs: 26 and 31. In some embodiments, provided herein is an anti-CTLA4 bispecific antibody or antigen-binding fragment thereof, comprising a heavy chain variable domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 26. In some embodiments, provided herein is an anti-CTLA4 bispecific antibody or antigen-binding fragment thereof, comprising a heavy chain variable domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 31. In some embodiments, the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity contains substitutions, insertions, or deletions relative to the reference sequence, but the antibody comprising the amino acid sequence retains the ability to bind to CTLA4 (e.g., human CTLA4). In some embodiments, the substitutions, insertions, or deletions (e.g., 1, 2, 3, 4, or 5 amino acids) occur in a region outside the HVR (i.e., within the FR). In some embodiments, the anti-CTLA4 bispecific antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising an amino acid sequence selected from SEQ ID NOs: 26 and 31. In some embodiments, the anti-CTLA4 bispecific antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the anti-CTLA4 bispecific antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 31.

[0185] In some embodiments, provided herein is an anti-CTLA4 bispecific antibody or antigen-binding fragment thereof, comprising a heavy chain variable domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from SEQ ID NOs: 25 and 30. In some embodiments, provided herein is an anti-CTLA4 bispecific antibody or antigen-binding fragment thereof, comprising a heavy chain variable domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 25. In some embodiments, provided herein is an anti-CTLA4 bispecific antibody or antigen-binding fragment thereof comprising a heavy chain variable domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 30. In some embodiments, the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity contains substitutions, insertions, or deletions relative to the reference sequence, but the antibody comprising the amino acid sequence retains the ability to bind to CTLA4 (e.g., human CTLA4). In some embodiments, the substitutions, insertions, or deletions (e.g., 1, 2, 3, 4, or 5 amino acids) occur in a region outside the HVR (i.e., within the FR). In some embodiments, the anti-CTLA4 bispecific antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising an amino acid sequence selected from SEQ ID NOs: 25 and 30. In some embodiments, the anti-CTLA4 bispecific antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the anti-CTLA4 bispecific antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 30.

[0186] In some embodiments, the CTLA4 binding proteins provided herein are chimeric receptors (e.g., chimeric antigen receptors (CARs)) capable of binding to CTLA4. CARs are molecules that combine antibody-based specificity for a desired antigen (e.g., CTLA4) with a T cell receptor activation intracellular domain to generate a chimeric protein that exhibits specific anti-tumor cell activity. In one embodiment, provided herein are chimeric receptors engineered to include an extracellular domain having a CTLA4 binding domain described herein fused to the intracellular signaling domain of the T cell antigen receptor complex zeta chain (e.g., CD3 zeta). When expressed in T cells, the chimeric receptors provided herein can redirect antigen recognition based on the antigen binding specificity via the extracellular domain. In some embodiments, the CTLA4 binding domain is preferably fused to an intracellular domain from one or more of a costimulatory molecule and a zeta chain. In some embodiments, the CTLA4 binding domain is fused to one or more intracellular domains selected from the group consisting of a CD137 (4-1BB) signaling domain, a CD28 signaling domain, a CD3 zeta signaling domain, and any combination thereof.

[0187] In some aspects, provided herein is a chimeric receptor comprising: a) a ligand-binding domain comprising a first chain and a second chain that binds to CTLA4; b) a transmembrane domain; and c) an intracellular signaling domain comprising a signaling domain. In some embodiments, the first chain is a light chain variable domain and the second chain is a heavy chain variable domain. In some embodiments, the first chain comprises an amino acid sequence selected from SEQ ID NOs: 25 and 30, and / or the second chain comprises an amino acid sequence selected from SEQ ID NOs: 26 and 31.

[0188] In some aspects, provided herein is a chimeric receptor comprising: a) a ligand-binding domain comprising a first chain and a second chain that binds to CTLA4; b) a transmembrane domain; and d) an intracellular signaling domain comprising a signaling domain. In some embodiments, the first chain is a light chain variable domain and the second chain is a heavy chain variable domain. In some embodiments, the first chain comprises an amino acid sequence selected from SEQ ID NOs: 25 and 30, and / or the second chain comprises an amino acid sequence selected from SEQ ID NOs: 26 and 31.

[0189] In some aspects, provided herein is a chimeric receptor comprising: 1) a ligand binding domain comprising a VL domain and a VH domain that binds to CTLA4, wherein a) the VL domain comprises CDR-L1, CDR-L2, and CDR-L3 comprised within the amino acid sequence of SEQ ID NO: 25, and the VH domain comprises CDR-H1, CDR-H2, and CDR-H3 comprised within the amino acid sequence of SEQ ID NO: 26, or b) the VL domain comprises CDR-L1, CDR-L2, and CDR-L3 comprised within the amino acid sequence of SEQ ID NO: 30, and the VH domain comprises CDR-H1, CDR-H2, and CDR-H3 comprised within the amino acid sequence of SEQ ID NO: 31; 2) a transmembrane domain; and 3) an intracellular signaling domain comprising a signaling domain.

[0190] In some embodiments, the present invention provides a ligand-binding domain that binds to CTLA4, comprising: 1) a VL domain and a VH domain, wherein a) the VL domain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3; and / or the VH domain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6. or b) the VL domain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15, and / or the VH domain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 16, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 18; or c) the VL domain comprises , (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, and / or the VH domain comprises (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 10, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 11, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12, or d) the VL domain comprises (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 19, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21. Provided herein is a chimeric receptor comprising: 1) a ligand-binding domain comprising: (i) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 20; and (ii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 21; and / or a VH domain comprising: (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24; 2) a transmembrane domain; and 3) an intracellular signaling domain comprising a signaling domain.

[0191] In some embodiments, in any of the provided chimeric receptors, a) the VL domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 25, and the VH domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 26, or b) the VL domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 30, and the VH domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 31.

[0192] In some aspects, in any of the provided chimeric receptors, a) the VL domain comprises the amino acid sequence of SEQ ID NO: 25 and the VH domain comprises the amino acid sequence of SEQ ID NO: 26, or b) the VL domain comprises the amino acid sequence of SEQ ID NO: 30 and the VH domain comprises the amino acid sequence of SEQ ID NO: 31.

[0193] 1.Binding affinity The strength, or affinity, of an immunological binding interaction, such as that between an antibody and the antigen for which the antibody is specific, is determined by the equilibrium dissociation constant (K D ), with a smaller Kd representing a higher affinity. The immunological binding properties of a protein can be quantified using methods well known in the art. For example, one method involves measuring the rate of antigen-binding protein (e.g., antibody) / antigen complex formation and dissociation, which depends on the concentration of the complex partners, the affinity of the interaction, and geometric parameters that affect the rate equally in both directions. Both the "on rate constant" (Kon) and the "off rate constant" (Koff) can be determined by calculation of the concentration and the actual rates of association and dissociation. The rate Koff / Kon allows for the elimination of all parameters not related to affinity, resulting in the equilibrium dissociation constant K D See Davies et al., Annual Rev Biochem. 59:439-473, (1990).

[0194] In some aspects, the anti-CTLA4 binding proteins (e.g., anti-CTLA4 antibodies or antigen-binding fragments thereof) described herein bind to CTLA4 with about the same or higher affinity compared to the anti-CTLA4 binding proteins. In certain embodiments, the anti-CTLA4 binding proteins provided herein have an affinity of ≦1 μM, ≦150 nM, ≦100 nM, ≦50 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., ≦10 -8 M or less, e.g., 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13 Equilibrium dissociation constant (K D In some embodiments, the anti-CTLA4 binding proteins (e.g., anti-CTLA4 antibodies or antigen-binding fragments thereof) provided herein have an equilibrium dissociation constant (K) of about 50 pM to about 5 nM. D ) binds to a target protein (e.g., CTLA4 protein). Assays for assessing binding affinity are well known in the art, such as the assay described in Example 1 herein.

[0195] 2. Biological Activity Assay In some embodiments, the anti-CTLA4 binding proteins described herein reduce tumor volume in an in vivo mouse tumor model. Assays for assessing tumor volume reduction are well known in the art, such as the assay described in Example 1 herein.

[0196] III. Preparation of anti-CTLA4 binding protein The anti-CTLA4 binding proteins described herein are prepared using techniques available in the art, exemplary methods of which are described in more detail in the following sections.

[0197] 1. Anti-CTLA4 binding protein: antibody fragment The present invention encompasses antibody fragments as anti-CTLA4 binding proteins.Antibody fragments can be produced by conventional means such as enzyme digestion or by recombinant technology.In certain circumstances, there are advantages to using antibody fragments rather than whole antibodies.For a review of certain antibody fragments, see Hudson et al.(2003)Nat.Med.9:129-134.

[0198] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv, and ScFv antibody fragments can all be expressed in and secreted from E. coli and other cell types, thus allowing the facile production of large amounts of these fragments. Alternatively, Fab'-SH fragments can be directly recovered from the culture medium and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology 10:163-167 (1992)). According to another approach, F(ab')2 fragments can be directly isolated from recombinant host cell culture. Fab and F(ab')2 fragments with increased in vivo half-life containing FcRN / salvage receptor binding epitope residues are described in U.S. Patent No. 5,869,046. Other techniques for generating antibody fragments will be apparent to those skilled in the art. In certain embodiments, the antibody is a single-chain Fv fragment (scFv). See WO 93 / 16185, U.S. Patent Nos. 5,571,894, and 5,587,458. Fvs and scFvs are the only species with intact binding sites lacking constant regions; therefore, they may be suitable for reduced nonspecific binding during in vivo use. scFv fusion proteins can be constructed to result in fusion of an effector protein at either the amino or carboxy terminus of the scFv. See Antibody Engineering, ed. Borrebaeck, supra. Bi-scFvs, comprising two scFvs linked via a polypeptide linker, can also be used as bispecific antibodies. Alternatively, a multi-scFv comprising three or more scFvs may be used as a multispecific antibody.

[0199] The present invention includes linear antibodies (e.g., as described in U.S. Pat. No. 5,641,870) or single-chain immunoglobulins comprising antibody heavy and light chain sequences linked via a suitable linker. Such linear antibodies or immunoglobulins may be monospecific or bispecific. Such single-chain immunoglobulins can be dimerized, thereby maintaining a structure and activity similar to that of an originally tetrameric antibody. The antibody of the present invention may also be an antibody having a single heavy chain variable region and no light chain sequence. Such antibodies are called single-domain antibodies (sdAbs) or nanobodies. These antibodies are also encompassed within the meaning of functional fragments of antibodies according to the present invention.

[0200] 2. Anti-CTLA4 binding protein: humanized antibody The present invention encompasses humanized antibodies. Humanized antibodies are produced according to the guidance provided herein. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues, typically taken from an "import" variable domain. Humanization can be performed essentially following the method of Winter (Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536) by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) in which substantially less of an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0201] 3. Anti-CTLA4 binding protein: human antibody The human anti-CTLA4 antibodies of the present invention can be constructed by combining Fv clone variable domain sequences selected from a human-derived phage display library with known human constant domain sequences. Alternatively, the human monoclonal anti-CTLA4 antibodies of the present invention can be produced by hybridoma technology. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described, for example, by Kozbor J. Immunol., 133:3001 (1984), Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987), and Boerner et al., J. Immunol., 147:86 (1991). Human antibodies are produced according to the guidance provided herein.

[0202] 4. Anti-CTLA4 Binding Protein: Bispecific Antibody A bispecific antibody is a monoclonal antibody that has binding specificities for at least two different antigens. In certain embodiments, the bispecific antibody is a human or humanized antibody. In certain embodiments, one of the binding specificities is for CTLA4 and the other is for any other antigen. In certain embodiments, the bispecific antibody can bind to two different epitopes of CTLA4. Bispecific antibodies may be used to localize cytotoxic agents to cells expressing CTLA4. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). Bispecific antibodies are generated according to the guidance provided herein.

[0203] Methods for producing bispecific antibodies are known in the art. See Milstein and Cuello, Nature, 305:537 (1983); WO93 / 08829 published May 13, 1993; Traunecker et al., EMBO J., 10:3655 (1991); Kontermann and Brinkmann, Drug Discovery Today, 20(7):838-847. For further details on the production of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986). Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be bound to avidin, and the other to biotin. Heteroconjugate antibodies can be produced using any convenient cross-linking method. Suitable cross-linking agents, along with several cross-linking techniques, are well known in the art and are disclosed in US Pat. No. 4,676,980.

[0204] 5. Anti-CTLA4 binding proteins: single domain antibodies In some embodiments, single-domain antibodies are produced according to the guidance provided herein. Single-domain antibodies are single polypeptide chains that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, single-domain antibodies are human single-domain antibodies (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Patent No. 6,248,516 B1). In one embodiment, a single-domain antibody consists of all or part of the heavy chain variable domain of an antibody.

[0205] 6. Anti-CTLA4 Binding Protein: Antibody Variants In some embodiments, amino acid sequence modifications of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of antibodies can be prepared by introducing appropriate changes into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from and / or insertions into and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct retains the desired characteristics. Amino acid changes can be introduced into the subject antibody amino acid sequence at the time the sequence is generated.

[0206] A useful method for identifying specific residues or regions of an antibody that are preferred locations for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a target residue or group of residues is identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to affect the interaction of the amino acid with the antigen. Those amino acid positions that demonstrate functional sensitivity to the substitution are then refined by introducing additional or other variants at or for the substitution site. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation itself need not be. For example, to analyze the performance of a mutation at a given site, Ala scanning or random mutagenesis is performed at the target codon or region, and the expressed immunoglobulin is screened for the desired activity.

[0207] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing several hundred or more residues, as well as intersequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the fusion to the N- or C-terminus of the antibody to an enzyme or a polypeptide to extend the serum half-life of the antibody.

[0208] In some embodiments, FcRn mutations that improve pharmacokinetics include, but are not limited to, M428L, T250Q / M428L, M252Y / S254T / T256E, P257I / N434H, D376V / N434H, P257I / Q3111, N434A, N434W, M428L / N434S, V259I / V308F, M252Y / S254T / T256E, V259I / V308F / M428L, T307Q / N434A, T307Q / N434S, T307Q / E380A / N434A, V308P / N434A, N434H, V308P. In some embodiments, such mutations enhance antibody binding to FcRn at low pH but do not alter antibody affinity at neutral pH.

[0209] In certain embodiments, antibodies of the present invention are modified to increase or decrease the extent of glycosylation of the antibody. Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0210] Addition or deletion of glycosylation sites to an antibody is conveniently accomplished by altering the amino acid sequence to create or remove one or more of the above-mentioned tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by adding, deleting, or substituting one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

[0211] If an antibody contains an Fc region, the carbohydrate attached thereto can be modified. For example, an antibody having a mature carbohydrate structure lacking fucose attached to its Fc region is described in U.S. Patent Application No. US2003 / 0157108 (Presta, L.). See also U.S. Patent Application No. US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). An antibody having a bisecting N-acetylglucosamine (GlcNAc) in the carbohydrate attached to its Fc region is referenced in WO2003 / 011878 (Jean-Mairet et al.) and U.S. Patent No. 6,602,684 (Umana et al.). An antibody having at least one galactose residue in the oligosaccharide attached to its Fc region is reported in WO1997 / 30087 (Patel et al.). See also WO1998 / 58964 (Raju, S.) and WO1999 / 22764 (Raju, S.) concerning antibodies with modified carbohydrates attached to the Fc region thereof. See also US2005 / 0123546 (Umana et al.) concerning antigen-binding molecules with modified glycosylation.

[0212] In certain embodiments, the glycosylation variant comprises an Fc region, and the carbohydrate structure attached to the Fc region lacks or has reduced fucose. Such variants have improved ADCC function. Optionally, the Fc region further comprises one or more amino acid substitutions therein that further improve ADCC, for example, substitutions at positions 298, 333, and / or 334 of the Fc region (EU numbering of residues). Examples of publications related to "non-fucosylated," "defucosylated," or "fucose-deficient" antibodies include US2003 / 0157108, WO2000 / 61739, WO2001 / 29246, US2003 / 0115614, US2002 / 0164328, US2004 / 0093621, US2004 / 0132140, US2004 / 0110704, US2004 / 0110282, US2004 / 0109865, WO2003 / 085119, WO2003 / 084570, WO2005 / 035586, WO2005 / 035778, WO2005 / 053742, Okazaki et al. et al. J. Mol. Biol. 336: 1239-1249 (2004), Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines that produce defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US2003 / 0157108A1; Presta, L; and WO2004 / 056312A1; Adams et al., especially Example 11), and knockout cell lines such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004)), and cells overexpressing β1,4-N-acetylglucosaminyltransferase III (GnT-III) and Golgi μ-mannosidase II (ManII).

[0213] In any of the embodiments herein, the anti-CTLA4 binding protein may be engineered to improve antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In some embodiments, the anti-CTLA4 binding protein may be produced in a cell line with an alpha 1,6-fucosyltransferase (Fut8) knockout. In some further embodiments, the anti-CTLA4 binding protein may be produced in a cell line overexpressing β1,4-N-acetylglucosaminyltransferase III (GnT-III). In further embodiments, the cell line further overexpresses Golgi μ-mannosidase II (ManII). In some of the embodiments herein, the anti-CTLA4 binding protein may include at least one amino acid substitution in the Fc region that improves ADCC activity.

[0214] In one embodiment, an antibody is modified to improve its serum half-life. To increase the serum half-life of an antibody, one can incorporate an FcRN / salvage receptor binding epitope into the antibody (especially an antibody fragment), for example, as described in U.S. Pat. No. 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an epitope in the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that increases the in vivo serum half-life of the IgG molecule (US2003 / 0190311, U.S. Pat. No. 6,821,505, U.S. Pat. No. 6,165,745, U.S. Pat. No. 5,624,821, U.S. Pat. No. 5,648,260, U.S. Pat. No. 6,165,745, U.S. Pat. No. 5,834,597).

[0215] Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule replaced with a different residue. Targeted sites for substitution mutagenesis include hypervariable regions, although modifications of FRs are also contemplated. Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions." If such substitutions result in a desirable change in biological activity, larger substitution changes can be introduced and the products screened, as indicated in Table 1 under "exemplary substitutions," or as detailed below for classes of amino acids.

[0216] [Table 1]

[0217] Substitutional modifications in the biological properties of antibodies are achieved by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the region of the substitution, e.g., as a sheet or helical conformation, (b) the charge or hydrophobicity of the target site molecule, or (c) the bulk of the side chain. Amino acids can be grouped according to the similarity of their side chain properties (A.L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)). (1) Non-polar: Ala(A), Val(V), Leu(L), Ile(I), Pro(P), Phe(F), Trp(W), Met(M) (2) Uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q) (3) Acidic: Asp(D), Glu(E) (4) Basic: Lys(K), Arg(R), His(H)

[0218] Alternatively, the naturally occurring residues can be divided into groups based on the following general side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain directionality: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0219] Non-conservative substitutions involve exchanging a member of one of these classes for another. Such substituted residues also may be introduced into the conservative substitution sites or into the remaining (non-conserved) sites.

[0220] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further development have modified (e.g., improved) biological properties relative to the parent antibody from which they were generated. A convenient method for generating such substitutional variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6–7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibodies thus generated are displayed from filamentous phage particles as fusions to at least a portion of a phage coat protein (e.g., the gene III product of M13) packaged within each particle. Phage-displayed variants are then screened for their biological activity (e.g., binding affinity). To identify potential hypervariable region sites for modification, scanning mutagenesis (e.g., alanine scanning) can be performed to identify hypervariable region residues that significantly contribute to antigen binding. Alternatively, or in addition, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and the antigen. Such contact residues and adjacent residues are candidates for substitution using techniques known in the art, including those described herein. Once such variants are generated, a panel of variants can be screened using techniques known in the art, including those described herein, and antibodies with superior properties in one or more relevant assays can be selected for further development.

[0221] Nucleic acid molecules encoding amino acid sequence variants of antibodies are prepared by a variety of methods known in the art, including, but not limited to, isolation from natural sources (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of variant or non-variant versions of previously prepared antibodies.

[0222] It may be desirable to introduce one or more amino acid modifications into the Fc region of an antibody of the invention, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions, including the hinge cysteine ​​amino acid positions.

[0223] In some embodiments, the anti-CTLA4 binding proteins (e.g., anti-CTLA4 antibodies or antigen-binding fragments thereof, or anti-CTLA4 bispecific antibodies) provided herein have an IgG1 isotype with enhanced effector function. In some embodiments, the anti-CTLA4 antibodies or antigen-binding fragments thereof are non-fucosylated. In some embodiments, the anti-CTLA4 bispecific antibodies are non-fucosylated. In some embodiments, the anti-CTLA4 antibodies or antigen-binding fragments thereof have increased levels of mannose moieties. In some embodiments, the anti-CTLA4 antibodies or antigen-binding fragments thereof have increased levels of bisecting glycan moieties. In some embodiments, the anti-CTLA4 bispecific antibodies have increased levels of mannose moieties. In some embodiments, the IgG1 comprises amino acid mutations.

[0224] In some embodiments, the anti-CTLA4 antibody or antigen-binding fragment thereof, or anti-CTLA4 bispecific antibody provided herein has an IgG1 isotype (e.g., a human IgG1 isotype). In one embodiment, the IgG1 comprises amino acid substitutions S298A, E333A, and K334A, where the amino acid residues are numbered according to the EU index of Kabat. In one embodiment, the IgG1 comprises amino acid substitutions S239D and I332E, where the amino acid residues are numbered according to the EU index of Kabat. In one embodiment, the IgG1 comprises amino acid substitutions S239D, A330L, and I332E, where the amino acid residues are numbered according to the EU index of Kabat. In one embodiment, the IgG1 comprises amino acid substitutions P247I and A339D or A339Q, where the amino acid residues are numbered according to the EU index of Kabat. In one embodiment, IgG1 comprises the amino acid substitutions D280H, K290S with or without S298D, or S298V, where the amino acid residues are numbered according to the EU index of Kabat. In one embodiment, IgG1 comprises the amino acid substitutions F243L, R292P, and Y300L, where the amino acid residues are numbered according to the EU index of Kabat. In one embodiment, IgG1 comprises the amino acid substitutions F243L, R292P, Y300L, and P396L, where the amino acid residues are numbered according to the EU index of Kabat. In one embodiment, IgG1 comprises the amino acid substitutions F243L, R292P, Y300L, V305I, and P396L, where the amino acid residues are numbered according to the EU index of Kabat. In one embodiment, IgG1 comprises the amino acid substitutions G236A, S239D, and I332E, where the amino acid residues are numbered according to the EU index of Kabat. In one embodiment, the IgG1 comprises the amino acid substitutions K326A and E333A, where the amino acid residues are numbered according to the EU index of Kabat. In one embodiment, the IgG1 comprises the amino acid substitutions K326W and E333S, where the amino acid residues are numbered according to the EU index of Kabat.In one embodiment, the IgG1 comprises the amino acid substitutions K290E or K290N, S298G, T299A, and / or K326E, where amino acid residues are numbered according to the EU index of Kabat. In some aspects, exemplary heavy chain constant regions comprised within provided CTLA4 binding proteins, such as antibodies or antigen-binding fragments thereof, comprise the heavy chain constant region sequences set forth in SEQ ID NOs: 35-38. In some aspects, exemplary light chain constant regions comprised within provided CTLA4 binding proteins, such as antibodies or antigen-binding fragments thereof, comprise the light chain constant region sequence set forth in SEQ ID NO: 39.

[0225] In some embodiments, a provided CTLA4 binding protein, such as an antibody or antigen-binding fragment thereof, comprises a heavy chain constant region comprising the sequence set forth in SEQ ID NO: 38. In some embodiments, a provided CTLA4 binding protein, such as an antibody or antigen-binding fragment thereof, comprises a heavy chain constant region comprising the sequence set forth in SEQ ID NO: 35. In some embodiments, a provided CTLA4 binding protein, such as an antibody or antigen-binding fragment thereof, comprises a light chain constant region comprising the sequence set forth in SEQ ID NO: 39.

[0226] In some embodiments, the provided CTLA4 binding proteins, such as antibodies or antigen-binding fragments thereof, comprise a heavy chain constant region comprising the sequence set forth in SEQ ID NO: 38, and a light chain constant region comprising the sequence set forth in SEQ ID NO: 39. In some embodiments, the provided CTLA4 binding proteins, such as antibodies or antigen-binding fragments thereof, comprise a heavy chain constant region comprising the sequence set forth in SEQ ID NO: 35, and a light chain constant region comprising the sequence set forth in SEQ ID NO: 39.

[0227] In accordance with this description and teachings of the art, it is contemplated that in some embodiments, the antibodies of the present invention may contain one or more modifications, for example, in the Fc region, compared to their wild-type counterparts. However, these antibodies will retain substantially the same characteristics required for therapeutic utility compared to their wild-type counterparts. For example, certain modifications may be made in the Fc region that will result in altered (i.e., either improved or reduced) C1q binding and / or complement-dependent cytotoxicity (CDC), as described, for example, in WO 99 / 51642. See also Duncan & Winter Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO 94 / 29351 for other examples of Fc region variants. WO 00 / 42072 (Presta) and WO 2004 / 056312 (Lowman) describe antibody variants with improved or reduced binding to FcRs. The contents of these patent publications are specifically incorporated herein by reference. See also Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001). Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005 / 0014934A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Polypeptide variants with altered Fc region amino acid sequences and increased or decreased C1q binding ability are described in U.S. Patent No. 6,194,551 B1 and WO 99 / 51642. The contents of these patent publications are specifically incorporated herein by reference. See also Idusogie et al. J Immunol. 164:4178-4184 (2000).

[0228] 7. Antibody-drug conjugates The present invention also provides antibody-drug conjugates (ADCs) comprising the anti-CTLA4 binding proteins provided herein conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioactive isotope.

[0229] In one embodiment, the one or more drugs conjugated to the antibody-drug conjugate include, but are not limited to, maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064, and European Patent EP 0425235 B1); auristatins, such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483, 5,780,588, and 7,498,298); dolastatins; calicheamicin or a derivative thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993), and Lode et al., Cancer Res. 58:2925-2928 (1998); anthracyclines such as daunomycin or doxorubicin (Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002), King et al., J. Med. Chem. 45:4336-4343 (2002), and U.S. Patent No. 6,630,579); methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065.

[0230] In another embodiment, the one or more drugs conjugated to the antibody-drug conjugate include, but are not limited to, inhibitors of tubulin polymerization (e.g., maytansinoids and auristatins), DNA damaging agents (e.g., pyrrolobenzodiazepine (PBD) dimers, calicheamicins, duocarmycins, and indolinobenzodiazepine dimers), and DNA synthesis inhibitors (e.g., exatecan derivatives Dxd).

[0231] In another embodiment, the antibody-drug conjugate comprises an antibody described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and the trichothecenes.

[0232] In another embodiment, the antibody-drug conjugate comprises an antibody described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for generating radioconjugates. Examples include At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 When a radioactive complex is used for detection, the radioactive complex may be, for example, tc 99m Or I 123or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0233] Conjugates of an anti-CTLA4 binding protein (e.g., an anti-CTLA4 antibody or antigen-binding fragment thereof) and a cytotoxic agent can be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO 94 / 11026. The linker may also be a "cleavable linker" that facilitates the release of the cytotoxic drug in cells. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker, or disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992), U.S. Patent No. 5,208,020) may be used.

[0234] The ADCs herein expressly contemplate such conjugates prepared with cross-linking reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology Inc., Rockford, Ill., USA).

[0235] 8. Vectors, Host Cells, and Recombinant Methods For recombinant production of the anti-CTLA4 binding protein of the present invention, the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning (DNA amplification) or expression. The DNA encoding the antibody can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody). Many vectors are available. The choice of vector depends in part on the host cell used. Generally, the host cell is either prokaryotic or eukaryotic (generally mammalian) in origin. It will be understood that constant regions of any isotype, including IgG, IgM, IgA, IgD, and IgE constant regions, can be used for this purpose, and that such constant regions can be obtained from any human or animal species.

[0236] 9. Production of Binding Proteins Using Prokaryotic Host Cells a) Vector construction Polynucleotide sequences encoding polypeptide components of the anti-CTLA4 binding proteins of the present invention can be obtained using standard recombinant techniques. The desired polynucleotide sequence may be isolated and sequenced from antibody-producing cells, such as hybridoma cells. Alternatively, polynucleotides can be synthesized using a nucleotide synthesizer or PCR technology. Once obtained, the polypeptide-encoding sequence is inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in a prokaryotic host. Many vectors available and known in the art can be used for the purposes of the present invention. The selection of an appropriate vector depends primarily on the size of the nucleic acid to be inserted into the vector and the specific host cell to be transformed with the vector. Each vector contains various components depending on its function (amplification or expression of the heterologous polynucleotide, or both) and its compatibility with the specific host cell in which it resides. Vector components generally include, but are not limited to, an origin of replication, a selectable marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, the heterologous nucleic acid insert, and a transcription termination sequence.

[0237] Generally, plasmid vectors containing replicons and control sequences derived from species compatible with the host cell are used in connection with these hosts. The vectors usually carry a replication site and marking sequences capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from the E. coli species. pBR322 contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance, thus providing an easy means for identifying transformed cells. pBR322, its derivatives, or other microbial plasmids or bacteriophages may also contain, or be modified to contain, promoters that can be used by the microorganism for expression of endogenous proteins. Examples of pBR322 derivatives used to express specific antibodies are described in Carter et al., U.S. Patent No. 5,648,237.

[0238] Additionally, phage vectors containing replicons and control sequences compatible with host microorganisms can be used as transformation vectors in connection with these hosts. For example, bacteriophages such as λGEM.TM.-11 can be utilized to generate recombinant vectors that can be used to transform susceptible host cells such as E. coli LE392.

[0239] The expression vector of the present invention may contain two or more promoter-cistron pairs, each encoding a polypeptide component. A promoter is a non-translated regulatory sequence located upstream (5') of a cistron that controls its expression. Prokaryotic promoters are typically classified into two classes: inducible and constitutive. An inducible promoter is a promoter that initiates an increase in the level of transcription of the cistron under its control in response to changes in culture conditions, such as the presence or absence of nutrients or a change in temperature.

[0240] Numerous promoters recognized by various potential host cells are well known. The selected promoter can be operably linked to the cistron DNA encoding the light or heavy chain by removing the promoter from the source DNA via restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the present invention. Both the native promoter sequence and many heterologous promoters can be used to direct the amplification and / or expression of the target gene. In some embodiments, heterologous promoters are utilized because they generally allow for greater transcription and higher yields of the expressed target gene compared to the native target polypeptide promoter.

[0241] Suitable promoters for use with prokaryotic hosts include the PhoA promoter, the β-galactamase and lactose promoter system, the tryptophan (trp) promoter system, and hybrid promoters such as the tac or trc promoter. However, other promoters that function in bacteria (such as other known bacterial or phage promoters) are also suitable. Their nucleotide sequences have been published, allowing those skilled in the art to operably ligate them into the cistrons encoding the target light and heavy chains using linkers or adapters to provide any necessary restriction sites (Siebenlist et al. (1980) Cell 20:269).

[0242] In one aspect of the present invention, each cistron in a recombinant vector contains a secretory signal sequence component that directs translocation of the expressed polypeptide across a membrane. Generally, the signal sequence may be a component of the vector or may be part of the target polypeptide DNA inserted into the vector. The signal sequence selected for purposes of the present invention should be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the native signal sequence for a heterologous polypeptide, the signal sequence is replaced by a prokaryotic signal sequence selected from the group consisting of, for example, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STII) leaders, LamB, PhoE, PelB, OmpA, and MBP. In one embodiment of the present invention, the signal sequences used in both cistrons of the expression system are STII signal sequences or variants thereof.

[0243] In another embodiment, production of immunoglobulins according to the present invention can occur within the cytoplasm of the host cell, and therefore does not require the presence of a secretory signal sequence within each cistron. In this regard, immunoglobulin light and heavy chains can be expressed, folded, and assembled to form functional immunoglobulins within the cytoplasm, with or without additional sequences, such as additional molecules. Certain host strains (e.g., E. coli trxB strains) provide cytoplasmic conditions favorable for disulfide bond formation, thereby allowing proper folding and assembly of the expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

[0244] The anti-CTLA4 binding proteins of the invention can also be produced by using an expression system that allows for the quantitative ratio of expressed polypeptide components to be adjusted to maximize the yield of secreted and properly assembled antibodies of the invention, such adjustment being achieved at least in part by simultaneously adjusting the translational strength of the polypeptide components.

[0245] Prokaryotic host cells suitable for expressing the anti-CTLA4 binding proteins of the present invention include, for example, archaebacteria and eubacteria, such as gram-negative or gram-positive organisms. Examples of useful bacteria include Escherichia (e.g., E. coli), Bacilli (e.g., B. subtilis), Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment, gram-negative cells are used. In one embodiment, E. coli cells are used as hosts in the present invention. Examples of E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, DC: American Society for Microbiology, 1987), pp. 1190-1219, ATCC deposit number 27,325) and its derivatives, including strain 33D3 having the genotype W3110 ΔfhuA(ΔtonA)ptr3 lac Iq lacL8 ΔompT Δ(nmpc-fepE)degP41 kanR (U.S. Pat. No. 5,639,635). Other strains such as E. coli 294 (ATCC 31,446) and its derivatives, E. coli B, E. coli λ 1776 (ATCC 31,537), and E. coli RV308 (ATCC 31,608) are also suitable. These examples are illustrative and not limiting. Methods for constructing derivatives of any of the above-mentioned bacteria with defined genotypes are known in the art and are described, for example, in Bass et al., Proteins, 8:309-314 (1990). It is generally necessary to select an appropriate bacterium taking into account the replicability of the replicon within the bacterial cell.For example, species of Escherichia coli, Serratia, or Salmonella can be suitably used as hosts when well-known plasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supply the replicon. Typically, the host cell should secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors may desirably be incorporated into the cell culture.

[0246] b) Binding protein production Host cells are transformed with the above-described expression vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[0247] Transformation refers to the introduction of DNA into a prokaryotic host so that the DNA is replicable either as an extrachromosomal element or by chromosomal integration. Depending on the host cell used, transformation is carried out using standard techniques appropriate for such cells. Calcium treatment using calcium chloride is commonly used for bacterial cells that contain substantial cell wall barriers. Another method for transformation uses polyethylene glycol / DMSO. Yet another technique that can be used is electroporation.

[0248] Prokaryotic cells used to produce the anti-CTLA4 binding proteins of the present invention are grown in media known in the art and suitable for culturing the selected host cells. Examples of suitable media include Luria Broth (LB) plus necessary nutritional supplements. In some embodiments, the media also contains a selection agent selected based on the construction of the expression vector, which selectively allows the growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the medium for the growth of cells expressing an ampicillin resistance gene.

[0249] Any necessary supplements other than carbon, nitrogen, and inorganic phosphate sources may also be introduced at appropriate concentrations, either alone or in admixture with the medium, such as another supplement or a complex nitrogen source. Optionally, the culture medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, dithioerythritol, and dithiothreitol.

[0250] Prokaryotic host cells are cultured at a suitable temperature. In certain embodiments, for growth of E. coli, the growth temperature ranges from about 20°C to about 39°C, from about 25°C to about 37°C, or about 30°C. The pH of the medium can be any pH ranging from about 5 to about 9, depending primarily on the host organism. In certain embodiments, for E. coli, the pH is about 6.8 to about 7.4, or about 7.0.

[0251] When an inducible promoter is used in the expression vector of the present invention, protein expression is induced under the conditions suitable for promoter activation.In one aspect of the present invention, PhoA promoter is used to control the transcription of polypeptide.Therefore, transformed host cells are cultured in phosphate-limited medium for induction.In certain embodiments, phosphate-limited medium is CRAP medium (see, for example, Simmons et al., J.Immunol.Methods (2002), 263:133-147).As known in the art, various other inducers can also be used according to the vector construction used.

[0252] In one embodiment, the expressed anti-CTLA4 binding protein of the present invention is secreted into the periplasm of the host cell and recovered from the periplasm of the host cell. Protein recovery typically involves disrupting the microorganism, generally by such means as osmotic shock, sonication, or lysis. Once the cells are disrupted, cell debris or whole cells can be removed by centrifugation or filtration. The protein can be further purified, for example, by affinity resin chromatography. Alternatively, the protein can be transported into the culture medium and isolated therein. The cells can be removed from the culture, and the culture supernatant can be filtered and concentrated for further purification of the produced protein. The expressed polypeptide can be further isolated and characterized using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assay.

[0253] In one aspect of the present invention, anti-CTLA4 binding protein production is carried out in large quantities by a fermentation process. A variety of large-scale fed-batch fermentation procedures are available for recombinant protein production. Large-scale fermentations have a volume of at least 1000 liters, and in certain embodiments, a volume of about 1,000 to 100,000 liters. These fermenters use agitator impellers to distribute oxygen and nutrients, particularly glucose. Small-scale fermentation generally refers to fermentation in fermentors with a volumetric capacity of about 100 liters or less, and can range from about 1 liter to about 100 liters.

[0254] In fermentation processes, induction of protein expression typically begins after cells have grown under suitable conditions to a desired density, approximately 180-220 OD550, at which point the cells are in early stationary phase. Various inducers are known in the art and may be used according to the vector construct used, as described above. Cells may be grown for a shorter period before induction. Cells are usually induced for approximately 12-50 hours, although longer or shorter induction times may be used.

[0255] Various fermentation conditions can be modified to improve the production yield and quality of the polypeptides of the present invention. For example, to improve the proper assembly and folding of secreted antibody polypeptides, prokaryotic host cells can be co-transformed with an additional vector overexpressing a chaperone protein, such as a Dsb protein (DsbA, DsbB, DsbC, DsbD, and / or DsbG) or FkpA (a peptidyl prolyl cis, trans-isomerase with chaperone activity). Chaperone proteins have been demonstrated to promote the proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al. (1999) J. Biol. Chem. 274:19601-19605; Georgiou et al., U.S. Patent No. 6,083,715; Georgiou et al., U.S. Patent No. 6,027,888; Bothmann and Pluckthun(2000) J.Biol.Chem.275:17100-17105;Ramm and Pluckthun(2000)J.Biol.Chem.275:17106-17113;Arie et al.(2001)Mol.Microbiol.39:199-210.

[0256] To minimize proteolysis of expressed heterologous proteins (especially proteolytically sensitive proteins), certain host strains deficient in proteolytic enzymes can be used in the present invention. For example, host cell strains can be modified to affect genetic mutations in genes encoding known bacterial proteases, such as protease III, OmpT, DegP, Tsp, protease I, protease Mi, protease V, protease VI, and combinations thereof. Several E. coli protease-deficient strains are described, for example, in Joly et al. (1998) supra; Georgiou et al., U.S. Pat. No. 5,264,365; Georgiou et al., U.S. Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72 (1996).

[0257] In one embodiment, an E. coli strain that is deficient in a proteolytic enzyme and transformed with a plasmid that overexpresses one or more chaperone proteins is used as a host cell in the expression system of the present invention.

[0258] c) Binding protein purification In one embodiment, the antibody protein produced herein is further purified to obtain a substantially homogeneous preparation for further assays and uses. Standard protein purification methods known in the art can be used. The following procedures are examples of suitable purification procedures: fractionation on an immunoaffinity column or an ion exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica or a cation exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration using, for example, Sephadex G-75.

[0259] In one embodiment, Protein A immobilized on a solid phase is used for immunoaffinity purification of antibody products of the invention. Protein A is a 41 kD cell wall protein from Staphylococcus aureus that binds with high affinity to the Fc region of antibodies. Lindmark et al. (1983) J. Immunol. Meth. 62:1-13. The solid phase to which Protein A is immobilized can be a glass or silica surface, or a column, including a controlled pore glass column or a silicic acid column. In some applications, the column may be coated with a reagent such as glycerol to prevent nonspecific adhesion of contaminants.

[0260] As a first step in purification, the preparation derived from the cell culture described above can be applied to a Protein A-immobilized solid phase to allow specific binding of the antibody of interest to Protein A. The solid phase is then washed to remove contaminants nonspecifically bound to the solid phase. Finally, the antibody of interest is recovered from the solid phase by elution.

[0261] 10. Production of Binding Proteins Using Eukaryotic Host Cells Vectors for use in eukaryotic host cells generally include one or more of the following components, but are not limited to: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0262] a) Signal Sequence Component Vectors for use in eukaryotic host cells may also contain a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide of interest. The heterologous signal sequence selected may be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In mammalian cell expression, mammalian signal sequences as well as viral secretory leaders, such as the herpes simplex gD signal, are available. The DNA of such a precursor region is ligated in reading frame to the DNA encoding the antibody.

[0263] b) Origin of replication Generally, the origin of replication component is not needed for mammalian expression vectors. For example, the SV40 origin may typically be used only because it contains the early promoter.

[0264] c) Selective Gene Components Expression and cloning vectors may contain a selection gene, also referred to as a selection marker. Typical selection genes encode (a) a protein that confers resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline, (b) a protein that complements an auxotrophic deficiency, if relevant, or (c) a protein that supplies a critical nutrient not available from complex media.

[0265] One example of a selection scheme utilizes drugs to arrest the growth of host cells. Those cells that are successfully transformed with a heterologous gene produce proteins that confer drug resistance and therefore survive the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolic acid, and hygromycin.

[0266] Other examples of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to produce anti-CTLA4 binding protein encoding nucleic acids such as DHFR, thymidine kinase, metallothionein-I and -II, primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, and the like.

[0267] For example, in some embodiments, cells transformed with the DHFR selection gene are first identified by culturing all transformants in a culture medium containing methotrexate (Mtx), a competitive antagonist of DHFR. In some embodiments, when wild-type DHFR is used, a suitable host cell is a Chinese hamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCC CRL-9096).

[0268] Alternatively, host cells transformed or co-transformed with DNA sequences encoding an anti-CTLA4 binding protein, wild-type DHFR protein, and another selectable marker, such as aminoglycoside 3'-phosphotransferase (APH), particularly wild-type hosts containing endogenous DHFR, can be selected by growing the cells in medium containing a selection agent for the selectable marker, such as an aminoglycoside antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Patent No. 4,965,199. Host cells may also include NS0, including cell lines deficient in glutamine synthetase (GS). Methods for using GS as a selectable marker for mammalian cells are described in U.S. Patent Nos. 5,122,464 and 5,891,693.

[0269] d) Promoter components Expression and cloning vectors usually contain a promoter that is recognized by the host organism and operably linked to a nucleic acid encoding the anti-CTLA4 binding protein of interest. Promoter sequences are known for eukaryotes. For example, nearly all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region, where N can be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence, which may signal addition of a polyA tail to the 3' end of the coding sequence. In certain embodiments, any or all of these sequences may be suitably inserted into a eukaryotic expression vector.

[0270] Transcription from vectors in mammalian host cells is controlled by promoters derived from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, and simian virus 40 (SV40), from heterologous mammalian promoters such as the actin promoter or immunoglobulin promoters, and from heat shock promoters, provided that such promoters are compatible with the host cell system.

[0271] The early and late promoters of SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate early promoter of human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment. A system for expressing DNA in mammalian hosts using bovine papillomavirus as a vector is disclosed in U.S. Pat. No. 4,419,446. A modification of this system is described in U.S. Pat. No. 4,601,978. See also Reyes et al., Nature 297:598-601 (1982), which describes the expression of human β-interferon cDNA in mouse cells under the control of the thymidine kinase promoter from herpes simplex virus. Alternatively, the Rous sarcoma virus long terminal repeat can be used as a promoter.

[0272] e) Enhancer element component Transcription of DNA encoding the antibody of the present invention by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). However, typically, one uses an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the human cytomegalovirus early promoter enhancer, the mouse cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18 (1982), which describes enhancer elements for the activation of eukaryotic promoters. Enhancers may be spliced ​​into the vector at either a 5' or 3' position to the antibody polypeptide-coding sequence, but are generally located 5' from the promoter.

[0273] f) transcription termination component Expression vectors used in eukaryotic host cells may also contain sequences necessary for the termination of transcription and stabilization of mRNA. Such sequences are commonly available from the 5' and, occasionally, 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94 / 11026 and the expression vector disclosed therein.

[0274] g) Selection and transformation of host cells Suitable host cells for cloning or expressing the DNA in the vectors herein include the higher eukaryotic cells described herein, including vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines include monkey kidney cell line CV1 transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary carcinoma tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); MRC5 cells; FS4 cells; and a human hepatoma line (Hep G2).

[0275] Host cells are transformed with the above-described expression or cloning vectors for production of anti-CTLA4 binding proteins and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[0276] h) Cultivation of host cells The host cells used to produce the anti-CTLA4 binding proteins of the present invention may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), minimal essential medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's modified Eagle's medium (DMEM, Sigma) are suitable for culturing host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469, WO90 / 03430, WO87 / 00195, or U.S. Pat. Reissue 30,985 may be used as a culture medium for host cells. Any of these media may be supplemented, if necessary, with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the drug GENTAMYCIN™), trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other supplements may also be included at appropriate concentrations that would be known to those of skill in the art. Culture conditions, such as temperature, pH, etc., will be those previously used with the host cell selected for expression and will be apparent to those of ordinary skill in the art.

[0277] i) Purification of binding proteins When using recombinant technology, the anti-CTLA4 binding protein can be produced intracellularly or directly secreted into the culture medium. If the antibody is produced intracellularly as a first step, particulate debris, either host cells or lysed fragments, can be removed, for example, by centrifugation or ultrafiltration. If the anti-CTLA4 binding protein is secreted into the culture medium, the supernatant from such an expression system can first be concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF can be included in any of the above steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of adventitious contaminants.

[0278] Antibody compositions prepared from cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a convenient technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Methods 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand is attached can be agarose, although other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. If the antibody contains a CH3 domain, Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification, such as fractionation on an ion exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, are also available, depending on the antibody to be recovered.

[0279] Following any preliminary purification steps, the mixture containing the binding protein of interest and contaminants may be subjected to further purification, for example, by low pH hydrophobic interaction chromatography performed at a low salt concentration (e.g., about 0-0.25 M salt) and using an elution buffer at a pH of about 2.5-4.5.

[0280] Generally, a variety of methodologies for preparing antibodies for use in research, testing, and clinical use are established in the art, consistent with those described above and / or as deemed appropriate by those skilled in the art for a particular antibody of interest.

[0281] IV. Composition Also provided herein, in some embodiments, are compositions (eg, pharmaceutical compositions) comprising any of the anti-CTLA4 binding proteins described herein.

[0282] Therapeutic formulations are prepared for storage by mixing the active ingredient having the desired purity with any pharmaceutically acceptable carrier, excipient, or stabilizer (Remington: The Science and Practice of Pharmacy, 20th Ed., Lippincott Williams & Wiklins, Pub., Gennaro Ed., Philadelphia, Pa. 2000). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed and include buffers; antioxidants including ascorbic acid, methionine, vitamin E, and sodium metabisulfite; preservatives, isotonicity agents, stabilizers, metal complexes (e.g., Zn-protein complexes); and chelating agents such as EDTA and / or nonionic surfactants.

[0283] Buffers can be used to adjust the pH within a range that optimizes therapeutic efficacy, particularly when stability is pH-dependent. Buffers can be present at concentrations ranging from about 20 mM to about 250 mM. Suitable buffers for use with the present invention include both organic and inorganic acids and their salts, such as citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, and acetate. Additionally, buffers may be comprised of histidine and trimethylamine salts, such as Tris.

[0284] Preservatives can be added to prevent microbial growth and are typically present in the range of about 0.2% to 1.0% (w / v). Suitable preservatives for use with the present invention include octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol.

[0285] Isotonicity agents, sometimes known as "stabilizers," may be present to adjust or maintain the isotonicity of the liquid in the composition. When used with large, charged biomolecules, such as proteins and antibodies, they are often referred to as "stabilizers" because they can interact with the charged groups on amino acid side chains, thereby reducing the likelihood of intermolecular and intramolecular interactions. Taking into account the relative amounts of other components, isotonicity agents may be present in any amount from about 0.1% to about 25% by weight, or from about 1% to about 5% by weight. In some embodiments, isotonicity agents include polyhydric sugar alcohols, trihydric or higher hydric sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0286] Additional excipients include agents that can act as one or more of: (1) bulking agents, (2) solubility enhancers, (3) stabilizers, and (4) agents that prevent denaturation or adhesion to the container wall. Such excipients include polyhydric sugar alcohols (listed above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myo-inisitose, myo-inisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), and polyethylene glycol. sulfur-containing reducing agents such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose); disaccharides (e.g., lactose, maltose, sucrose); trisaccharides such as raffinose; and polysaccharides such as dextrin or dextran.

[0287] A non-ionic surfactant or detergent (also known as a "wetting agent") can be present to help solubilize the therapeutic agent and protect the therapeutic protein from agitation-induced aggregation, which also allows the formulation to be exposed to shear surface stresses without causing denaturation of the active therapeutic protein or antibody. The non-ionic surfactant is present in a range of about 0.05 mg / ml to about 1.0 mg / ml, or about 0.07 mg / ml to about 0.2 mg / ml. In some embodiments, the non-ionic surfactant is present in a range of about 0.001% to about 0.1% w / v, or about 0.01% to about 0.1% w / v, or about 0.01% to about 0.025% w / v.

[0288] Suitable nonionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), poloxamers (184, 188, etc.), PLURONIC® polyol, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50, and 60, glycerol monostearate, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. Anionic detergents that can be used include sodium lauryl sulfate, sodium dioctyl sulfosuccinate, and sodium dioctyl sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0289] For the preparation to be used for in vivo administration, the preparation must be sterile.The preparation can be sterilized by filtration through a sterile filtration membrane.The therapeutic composition herein is generally placed into a container with a sterile access port, for example, an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle.

[0290] The route of administration will be in accordance with known and accepted methods, for example, by subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional, or intraarticular route, topical administration, by inhalation, or by slow or sustained release means, in a suitable manner such as injection or infusion, as single or multiple boluses, or as infusion over an extended period of time.

[0291] The anti-CTLA4 binding proteins described herein (e.g., anti-CTLA4 antibodies or antigen-binding fragments thereof) can be used alone or in combination with other therapeutic agents, as in the methods described herein. The term "in combination with" encompasses two or more therapeutic agents (e.g., anti-CTLA4 binding proteins and therapeutic agents) contained in the same or separate formulations. In some embodiments, "in combination with" refers to "simultaneous" administration, where administration of an anti-CTLA4 binding protein of the invention occurs simultaneously with administration of one or more additional therapeutic agents (e.g., simultaneously with or within one hour between administration of the anti-CTLA4 binding protein and administration of one or more additional therapeutic agents). In some embodiments, "in combination with" refers to sequential administration, where administration of an anti-CTLA4 binding protein of the invention occurs before and / or after administration of one or more additional therapeutic agents (e.g., more than one hour between administration of the anti-CTLA4 binding protein and administration of one or more additional therapeutic agents). Agents contemplated herein include, but are not limited to, a cytotoxic agent, a cytokine, an agent that targets an immune checkpoint molecule, an agent that targets an immunostimulatory molecule, or a growth inhibitory agent.

[0292] The formulations herein may also contain multiple active compounds as needed for the particular indication being treated, preferably with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may include a cytotoxic agent, a cytokine, an agent that targets an immune checkpoint or stimulatory molecule, or a growth inhibitory agent. Such molecules are suitably present in combination in amounts effective for the intended purpose.

[0293] V. Treatment method Provided herein are methods for treating or preventing a disease in a subject, comprising administering to the subject an effective amount of an anti-CTLA4 binding protein (e.g., an anti-CTLA4 antibody or antigen-binding fragment thereof) or composition thereof described herein. In some embodiments, the subject (e.g., a human patient) has been diagnosed with or is at risk of developing a neoplastic disorder (e.g., cancer).

[0294] For disease prevention or treatment, the appropriate dosage of the active agent depends on the type of disease being treated (as defined above), the severity and course of the disease, whether the agent is being administered for prophylactic or therapeutic purposes, previous treatments, the subject's clinical history and response to the agent, and the discretion of the treating physician. The agent is suitably administered to the subject at one time or over a series of treatments. In some embodiments of the methods described herein, the interval between administrations of the anti-CTLA4 binding protein (e.g., an anti-CTLA4 antibody or antigen-binding fragment thereof) is about one month or more. In some embodiments, the interval between administrations is about two months, about three months, about four months, about five months, about six months, or more. As used herein, the interval between administrations refers to the period between one administration of the antibody and the next administration of the antibody. As used herein, an interval of about one month includes four weeks. In some embodiments, the interval between administrations is about one week, about two weeks, about three weeks, about four weeks, about eight weeks, about 12 weeks, about 16 weeks, about 20 weeks, about 24 weeks, or more. In some embodiments, the treatment involves multiple administrations of the antibody, and the interval between administrations can vary. For example, the interval between the first and second administrations is about one month, and the interval between subsequent administrations is about three months. In some embodiments, the interval between the first and second administrations is about one month, the interval between the second and third administrations is about two months, and the interval between subsequent administrations is about three months. In some embodiments, the anti-CTLA4 binding proteins (e.g., anti-CTLA4 antibodies or antigen-binding fragments thereof) described herein are administered in a flat dose. Depending on the type and severity of the disease, an antibody dose of about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) can be an initial candidate dose for administration to a patient, whether by one or more separate administrations or by continuous infusion, for example. One typical daily dosage can range from about 1 μg / kg to about 100 mg / kg or more, depending on the factors described above. For repeated administrations over several days or longer, depending on the condition, treatment would generally be maintained until a desired suppression of disease symptoms occurs. One exemplary dosage of antibody would be in the range of about 0.05 mg / kg to about 10 mg / kg.Thus, one or more doses of about 0.5 mg / kg, about 2.0 mg / kg, about 4.0 mg / kg, or about 10 mg / kg (or any combination thereof) may be administered to a patient. In some embodiments, an anti-CTLA4 binding protein described herein (e.g., an anti-CTLA4 antibody or antigen-binding fragment thereof) is administered to a subject at a dosage of about 25 mg to about 500 mg per dose. In some embodiments, an anti-CTLA4 binding protein described herein (e.g., an anti-CTLA4 antibody or antigen-binding fragment thereof) is administered to a subject at a dosage of about 0.1 mg / kg to about 10 mg / kg or about 1.0 mg / kg to about 10 mg / kg. In some embodiments, an anti-CTLA4 binding protein (e.g., an anti-CTLA4 antibody or antigen-binding fragment thereof) described herein is administered to a subject at a dose of any of about 0.1 mg / kg, about 0.5 mg / kg, about 1.0 mg / kg, about 1.5 mg / kg, about 2.0 mg / kg, about 2.5 mg / kg, about 3.0 mg / kg, about 3.5 mg / kg, about 4.0 mg / kg, about 4.5 mg / kg, about 5.0 mg / kg, about 5.5 mg / kg, about 6.0 mg / kg, about 6.5 mg / kg, about 7.0 mg / kg, about 7.5 mg / kg, about 8.0 mg / kg, about 8.5 mg / kg, about 9.0 mg / kg, about 9.5 mg / kg, or about 10.0 mg / kg. Any of the above-mentioned dosing frequencies may be used.

[0295] Therapeutic methods contemplated herein are treatment of disorders or diseases with the anti-CTLA4 binding proteins described herein (e.g., anti-CTLA4 antibodies or antigen-binding fragments thereof). Disorders or diseases treatable with the formulations of the invention include leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, splenic cancer, melanoma, breast cancer, neuroblastoma, lung cancer, ovarian cancer, osteosarcoma, bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer (e.g., Merkel cell carcinoma), or testicular cancer.

[0296] In some embodiments, provided herein are methods of treating or preventing cancer by administering an anti-CTLA4 binding protein described herein (e.g., an anti-CTLA4 antibody or antigen-binding fragment thereof). As used herein, the term "cancer" refers to all types of cancer, neoplasm, or malignant tumors found in mammals, including leukemia, lymphoma, melanoma, neuroendocrine tumors, carcinoma, and sarcoma. Exemplary cancers that may be treated by an anti-CTLA4 binding protein (e.g., an anti-CTLA4 antibody or antigen-binding fragment thereof), pharmaceutical composition, or method provided herein include lymphoma, sarcoma, bladder cancer, bone cancer, brain cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, myeloma, thyroid cancer, leukemia, prostate cancer, breast cancer (e.g., triple negative, ER positive, ER negative, chemotherapy resistant, Herceptin resistant, HER2 positive, doxorubicin resistant, tamoxifen resistant, ductal cancer, thyroid cancer ...thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer cancer, lobular carcinoma, primary, metastatic), ovarian cancer, splenic cancer, liver cancer (e.g., hepatocellular carcinoma), lung cancer (e.g., non-small cell lung cancer, squamous cell lung carcinoma, adenocarcinoma, large cell lung carcinoma, small cell lung carcinoma, carcinoid, sarcoma), glioblastoma multiforme, glioma, melanoma, prostate cancer, castration-resistant prostate cancer, breast cancer, triple-negative breast cancer, glioblastoma, ovarian cancer, lung cancer, squamous cell carcinoma (e.g., of the head, neck, or esophagus), colorectal cancer, leukemia, acute myeloid leukemia, lymphoma, B-cell lymphoma, or multiple myeloma. Additional examples include cancer of the thyroid gland, cancer of the endocrine system, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, esophageal cancer, liver cancer, kidney cancer, lung cancer, non-small cell lung cancer, melanoma, mesothelioma, ovarian cancer, sarcoma, stomach cancer, uterine cancer, or medulloblastoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocythemia, primary macroglobulinemia, primary brain tumors, cancer, malignant pancreatic insulinoma, malignant carcinoid, bladder cancer, precancerous skin lesion, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer, malignant hypercalcemia, endometrial cancer, adrenocortical carcinoma, neoplasm of the endocrine or exocrine spleen, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid carcinoma, hepatocellular carcinoma, Paget's disease of the nipple, phyllodes tumor, lobular carcinoma, ductal carcinoma, carcinoma of the pancreatic stellate cells, carcinoma of the hepatic stellate cells, or prostate cancer.

[0297] In some embodiments, provided herein are methods for treating or preventing leukemia by administering an anti-CTLA4 binding protein (e.g., an anti-CTLA4 antibody or antigen-binding fragment thereof) described herein. The term "leukemia" broadly refers to a progressive, malignant disease of the blood-forming organs, generally characterized by distorted proliferation and development of white blood cells and their precursors in the blood and bone marrow. Leukemias are generally classified clinically based on (1) the acute or chronic duration and characteristics of the disease, (2) the type of cell involved, myeloid (myelogenous), lymphocytic (lymphatic), or monocytic, and (3) the increased or absent number of abnormal cells in blood leukemia or leukemia (subleukemia). Exemplary leukemias that may be treated by a compound, pharmaceutical composition, or method provided herein include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, leukocytic leukemia, and leukocytic leukemia. leukemia), basophilic leukemia, blastic leukemia, bovine leukemia, chronic myeloid leukemia, leukemia cutis, fetal leukemia, eosinophilic leukemia, Gross' leukemia, hairy cell leukemia, hemoblastic leukemia, hemoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphoblastic leukemia, lymphoid leukemia, lymphocytic (lymphocytic) leukemia phoid) leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myelogranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, multiple myeloma, plasma cell leukemia, promyelocytic leukemia, Leder cell leukemia, Schilling leukemia, stem cell leukemia, subleukemic leukemia, or anaplastic cell leukemia.

[0298] In some embodiments, provided herein are methods for treating or preventing sarcoma by administering an anti-CTLA4 binding protein (e.g., an anti-CTLA4 antibody or antigen-binding fragment thereof) described herein. The term "sarcoma" generally refers to a tumor that is made up of a substance like embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrous or homogeneous substance. Sarcomas that may be treated by the compounds, pharmaceutical compositions, or methods provided herein include chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, and leukocyte sarcoma. sarcoma, botryoid sarcoma, chloromatous sarcoma, chorioepithelioma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, B-cell immunoblastic sarcoma, lymphoma, T-cell immunoblastic sarcoma, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymal sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, or telangiectatic sarcoma sarcoma).

[0299] In some embodiments, provided herein are methods for treating or preventing melanoma by administering an anti-CTLA4 binding protein (e.g., an anti-CTLA4 antibody or antigen-binding fragment thereof) described herein. The term "melanoma" is taken to mean a tumor arising from the melanocytic system of the skin and other organs. Melanomas that can be treated by the compounds, pharmaceutical compositions, or methods provided herein include, for example, acral lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungual melanoma, or superficial spreading melanoma.

[0300] In some embodiments, provided herein are methods for treating or preventing carcinoma by administering an anti-CTLA4 binding protein described herein (e.g., an anti-CTLA4 antibody or antigen-binding fragment thereof). The term "carcinoma" refers to a malignant new growth composed of epithelial cells that tend to infiltrate surrounding tissues and give rise to metastases. Exemplary carcinomas that may be treated by a compound, pharmaceutical composition, or method provided herein include, for example, medullary thyroid carcinoma, familial medullary thyroid carcinoma, acinar carcinoma, acinous carcinoma, adenoid cystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatous, adrenocortical carcinoma, alveolar cell carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchoalveolar carcinoma, bronchogenic carcinoma, cerebrocellular carcinoma, cholangiocarcinoma, choriocarcinoma, colloid carcinoma, comedocarcinoma, corpus carcinoma, cribriform carcinoma, armorial carcinoma, carcinoma of the skin, cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, ductal carcinoma, ductal carcinoma, scirrhous carcinoma, embryonal carcinoma, encephalomyocarcinoma, epidermoid carcinoma, adenoid carcinoma (carcinoma epitheliale adenoides), exophytic carcinoma, ulcerative carcinoma (carcinoma ex ulcere), fibrous carcinoma (carcinoma fibrosum), gelatinous carcinoma (gelatinifonni carcinoma), gelatinous carcinoma, giant cell carcinoma, giant cell carcinoma (carcinoma gigantocellulare), adenocarcinoma, granulosa cell carcinoma, hair matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hürthle cell carcinoma, hyaline carcinoma, hypernephroid carcinoma, infantile embryonal carcinoma, carcinoma in situIn situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kurticaysky cell carcinoma, large cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipocarcinoma, lobular carcinoma, lymphoepithelial carcinoma, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, myxomatous carcinoma Myxomatodes, nasopharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, squamous cell carcinoma, atheromatous carcinoma, renal cell carcinoma of the kidney, reserve cell carcinoma, sarcomatoid carcinoma, Schneiderian carcinoma, scirrhous carcinoma, scrotal carcinoma, signet ring cell carcinoma, simplex carcinoma, small cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, cavernous carcinoma, squamous cell carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, nodular carcinoma tuberosum, tubular carcinoma, nodular carcinoma, verrucous carcinoma, or choriocarcinoma.

[0301] In some embodiments, provided herein are methods for treating or preventing metastatic cancer by administering an anti-CTLA4 binding protein (e.g., an anti-CTLA4 antibody or antigen-binding fragment thereof) described herein. As used herein, the terms "metastasis," "metastatic," and "metastatic cancer" are used interchangeably and refer to the spread of a neoplastic disease or disorder, e.g., cancer, from one organ or another non-adjacent organ or body part. Cancer begins at a site of origin, such as the breast, referred to as a primary tumor, e.g., primary breast cancer. Some cancer cells from the primary tumor or site of origin acquire the ability to penetrate and infiltrate surrounding normal tissue in the local area and / or penetrate the walls of the lymphatic or vascular system circulating through the system to other sites and tissues in the body. A second, clinically detectable tumor formed from cancer cells of the primary tumor is referred to as a metastatic or secondary tumor. When cancer cells metastasize, the metastatic tumor and its cells are assumed to be similar to those of the original tumor. Therefore, when lung cancer metastasizes to the breast, the secondary tumor in the breast is made up of abnormal lung cells, not abnormal breast cells.The secondary tumor in the breast is referred to as metastatic lung cancer.Therefore, the term metastatic cancer refers to a disease in which a subject has or has had a primary tumor, and has one or more secondary tumors.The term non-metastatic cancer or a subject with non-metastatic cancer refers to a disease in which a subject has a primary tumor but does not have one or more secondary tumors.For example, metastatic lung cancer refers to a disease in a subject who has a primary lung tumor or has a history of a primary lung tumor, and has one or more secondary tumors, for example, in a second location or multiple locations in the breast.

[0302] In some embodiments, diseases or disorders that may be beneficial by the CTLA4 binding proteins described herein include diseases caused (in whole or in part) by CTLA4 or CTLA4 activity or function (e.g., diabetes, cancer (e.g., prostate cancer, kidney cancer, metastatic cancer, melanoma, castration-resistant prostate cancer, breast cancer, triple-negative breast cancer, glioblastoma, ovarian cancer, lung cancer, squamous cell carcinoma (e.g., of the head, neck, or esophagus), colorectal cancer, leukemia, acute myeloid leukemia, lymphoma, B-cell lymphoma, or multiple myeloma)), or the symptoms of the disease are caused (in whole or in part) by CTLA4 or CTLA4 activity or function.

[0303] VI. Manufactured Articles or Kits In another aspect, an article of manufacture or kit is provided that includes an anti-CTLA4 binding protein (e.g., an anti-CTLA4 antibody or antigen-binding fragment thereof) described herein. The article of manufacture or kit may further include instructions for use of the binding protein in a method of the invention. Thus, in certain embodiments, the article of manufacture or kit includes instructions for use of the anti-CTLA4 binding protein (e.g., an anti-CTLA4 antibody or antigen-binding fragment thereof) in a method for treating or preventing a disorder (e.g., cancer) in an individual, comprising administering to the individual an effective amount of the anti-CTLA4 binding protein (e.g., an anti-CTLA4 antibody or antigen-binding fragment thereof). In certain embodiments, the individual is a human. In some embodiments, the individual has a disease selected from the group consisting of leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, spleen cancer, melanoma, breast cancer, neuroblastoma, lung cancer, ovarian cancer, osteosarcoma, bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer, or testicular cancer.

[0304] The article of manufacture or kit may further comprise a container. Suitable containers include, for example, bottles, vials (e.g., dual-chamber vials), syringes (such as single-chamber or dual-chamber syringes), and test tubes. The container may be made of various materials, such as glass or plastic. The container holds the formulation. In some embodiments, the formulation is a lyophilized formulation.

[0305] The article of manufacture or kit may further include a label or package insert on or associated with the container, which may indicate instructions for reconstitution and / or use of the formulation. The label or package insert may further indicate that the formulation is useful or intended for subcutaneous, intravenous, or other administration for treating or preventing a disorder (e.g., cancer) in an individual. The container holding the formulation may be a single-use or multi-use vial, which allows for repeated administration of the reconstituted formulation. The article of manufacture or kit may further include a second container containing a suitable diluent. The article of manufacture or kit may further include other materials desirable from commercial, therapeutic, and user standpoints, including other buffers, diluents, filters, needles, syringes, and package inserts containing instructions for use.

[0306] In certain embodiments, the present invention provides a kit for single-dose administration units. Such kit comprises a container of aqueous formulation of therapeutic antibody, including both single-chamber and multi-chamber pre-filled syringes. Exemplary pre-filled syringes are available from Vetter GmbH (Ravensburg, Germany).

[0307] The article of manufacture or kit herein optionally further comprises a container containing a second medicament, wherein the anti-CTLA4 binding protein (e.g., an anti-CTLA4 antibody or antigen-binding fragment thereof) is the first medicament, and the article or kit further comprises instructions on a label or package insert for treating a subject with the second medicament in an effective amount.

[0308] In another embodiment, provided herein is an article of manufacture or kit comprising the formulations described herein for administration in an automatic injector. Automatic injectors can be described as injection devices that, upon activation, deliver their contents without any additional action required from the patient or the administering person. They are particularly suitable for self-medication of therapeutic formulations when the delivery rate must be constant and the delivery time is longer than a few moments.

[0309] Illustrative Embodiments Among the exemplary embodiments provided are the following: 1. An anti-CTLA4 antibody or antigen-binding fragment thereof comprising a light chain variable (VL) domain and a heavy chain variable (VH) domain, a) the VL domain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3, and / or the VH domain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6, or b) the VL domain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15, and / or the VH domain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 16, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 18, or c) the VL domain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, and / or the VH domain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 10, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 11, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12, or d) the VL domain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 19, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 20, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 21, and / or the VH domain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24; An anti-CTLA4 antibody or antigen-binding fragment thereof. 2.a) the VL domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 25, and the VH domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 26, or b) the VL domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 30, and the VH domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 31; 2. The anti-CTLA4 antibody or antigen-binding fragment thereof of embodiment 1. 3.a) the VL domain comprises the amino acid sequence of SEQ ID NO: 25 and the VH domain comprises the amino acid sequence of SEQ ID NO: 26, or b) the VL domain comprises the amino acid sequence of SEQ ID NO: 30, and the VH domain comprises the amino acid sequence of SEQ ID NO: 31; The anti-CTLA4 antibody or antigen-binding fragment thereof of embodiment 1 or 2. 4. An anti-CTLA4 antibody or antigen-binding fragment thereof, comprising a light chain variable (VL) domain and a heavy chain variable (VH) domain, a) the VL domain comprises the amino acid sequence of SEQ ID NO: 25 and / or the VH domain comprises the amino acid sequence of SEQ ID NO: 26, or b) the VL domain comprises the amino acid sequence of SEQ ID NO: 30 and / or the VH domain comprises the amino acid sequence of SEQ ID NO: 31; An anti-CTLA4 antibody or antigen-binding fragment thereof. 5. An anti-CTLA4 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4, wherein the VL domain comprises the amino acid sequence of SEQ ID NO: 25 and the VH domain comprises the amino acid sequence of SEQ ID NO: 26. 6. An anti-CTLA4 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4, wherein the VL domain comprises the amino acid sequence of SEQ ID NO: 30 and the VH domain comprises the amino acid sequence of SEQ ID NO: 31. 7. An anti-CTLA4 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 6, comprising a heavy chain constant domain (CH). 8. An anti-CTLA4 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 7, comprising a CH sequence selected from the group consisting of SEQ ID NOs: 35 to 38. 9. The anti-CTLA4 antibody or antigen-binding fragment thereof of embodiment 7 or 8, wherein the CH comprises the amino acid substitution S239D or I332E, or both, and the amino acid residues are numbered according to the EU index of Kabat. 10. An anti-CTLA4 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 9, comprising the CH sequence of SEQ ID NO: 38. 11. An anti-CTLA4 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 10, comprising a light chain constant domain (CL). 12. An anti-CTLA4 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 11, comprising the CL sequence of SEQ ID NO: 39. 13.a) the light chain comprises the amino acid sequence of SEQ ID NO: 27 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 29, or b) the light chain comprises the amino acid sequence of SEQ ID NO: 32 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 34; 13. An anti-CTLA4 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 12. 14. An anti-CTLA4 antibody or antigen-binding fragment thereof, comprising a light chain comprising the amino acid sequence of SEQ ID NO: 27 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 29. 15. An anti-CTLA4 antibody or antigen-binding fragment thereof, comprising a light chain comprising the amino acid sequence of SEQ ID NO: 32 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 34. 16. The anti-CTLA4 antibody or antigen-binding fragment thereof of any one of embodiments 1 to 15, which is nonfucosylated or fucose-deficient. 17. An anti-CTLA4 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 16, which is conjugated to an agent. 18. The anti-CTLA4 antibody or antigen-binding fragment thereof of embodiment 17, wherein the agent is an inhibitor of tubulin polymerization, a DNA damaging agent, or a DNA synthesis inhibitor. 19. The anti-CTLA4 antibody or antigen-binding fragment thereof of embodiment 17, wherein the agent is a maytansinoid, an auristatin, a pyrrolobenzodiazepine (PBD) dimer, a calicheamicin, a duocarmycin, an indolinobenzodiazepine dimer, or an exatecan derivative Dxd. 20.1) A first pair of light and heavy chains that specifically binds to CTLA4; 2) a second pair of light and heavy chains that specifically bind to the antigen; A bispecific antibody or antigen-binding fragment thereof comprising: the light chains of the first pair comprise a VL domain and the heavy chains of the first pair comprise a VH domain; a) the VL domain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3, and / or the VH domain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6, or b) the VL domain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15, and / or the VH domain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 16, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 18, or c) the VL domain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, and / or the VH domain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 10, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 11, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12, or d) the VL domain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 19, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 20, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 21, and / or the VH domain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24; A bispecific antibody or antigen-binding fragment thereof. 21.a) the VL domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 25, and the VH domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 26, or b) the VL domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 30, and the VH domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 31; 21. The bispecific antibody or antigen-binding fragment thereof of embodiment 20. 22.a) the VL domain comprises the amino acid sequence of SEQ ID NO: 25 and the VH domain comprises the amino acid sequence of SEQ ID NO: 26, or b) the VL domain comprises the amino acid sequence of SEQ ID NO: 30, and the VH domain comprises the amino acid sequence of SEQ ID NO: 31; 22. The bispecific antibody or antigen-binding fragment thereof of embodiment 20 or 21. 23.1) A first pair of light and heavy chains that specifically binds to CTLA4; 2) a second pair of light and heavy chains that specifically bind to the antigen; A bispecific antibody or antigen-binding fragment thereof comprising: the light chains of the first pair comprise a VL domain and the heavy chains of the first pair comprise a VH domain; a) the VL domain comprises the amino acid sequence of SEQ ID NO: 25 and / or the VH domain comprises the amino acid sequence of SEQ ID NO: 26, or b) the VL domain comprises the amino acid sequence of SEQ ID NO: 30 and / or the VH domain comprises the amino acid sequence of SEQ ID NO: 31; A bispecific antibody or antigen-binding fragment thereof. 24. The bispecific antibody or antigen-binding fragment thereof according to any one of embodiments 20 to 23, wherein the VL domain comprises the amino acid sequence of SEQ ID NO: 25 and the VH domain comprises the amino acid sequence of SEQ ID NO: 26. 25. The bispecific antibody or antigen-binding fragment thereof according to any one of embodiments 20 to 23, wherein the VL domain comprises the amino acid sequence of SEQ ID NO: 30 and the VH domain comprises the amino acid sequence of SEQ ID NO: 31. 26. The bispecific antibody or antigen-binding fragment thereof according to any one of embodiments 20 to 25, comprising a heavy chain constant domain (CH). 27. The bispecific antibody or antigen-binding fragment thereof according to any one of embodiments 20 to 26, comprising a CH sequence selected from the group consisting of SEQ ID NOs: 35 to 38. 28. The bispecific antibody or antigen-binding fragment thereof according to embodiment 26 or 27, wherein said CH comprises the amino acid substitution S239D or I332E or both, and wherein the amino acid residues are numbered according to the EU index of Kabat. 29. The bispecific antibody or antigen-binding fragment thereof according to any one of embodiments 20 to 28, comprising the CH sequence of SEQ ID NO: 38. 30. The bispecific antibody or antigen-binding fragment thereof according to any one of embodiments 20 to 29, comprising a light chain constant domain (CL). 31. The bispecific antibody or antigen-binding fragment thereof according to any one of embodiments 20 to 30, comprising the CL sequence of SEQ ID NO: 39. 32. a) the light chain comprises the amino acid sequence of SEQ ID NO: 27 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 29, or b) the light chain comprises the amino acid sequence of SEQ ID NO: 32 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 34; 32. The bispecific antibody or antigen-binding fragment thereof according to any one of embodiments 20 to 31. 33.1) A first pair of light and heavy chains that specifically binds to CTLA4; 2) a second pair of light and heavy chains that specifically bind to the antigen; A bispecific antibody or antigen-binding fragment thereof comprising: the first pair comprising a light chain comprising the amino acid sequence of SEQ ID NO: 27 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 29; A bispecific antibody or antigen-binding fragment thereof. 34.1) A first pair of light and heavy chains that specifically binds to CTLA4; 2) a second pair of light and heavy chains that specifically bind to the antigen; A bispecific antibody or antigen-binding fragment thereof comprising: the first pair comprising a light chain comprising the amino acid sequence of SEQ ID NO: 32 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 34; A bispecific antibody or antigen-binding fragment thereof. 35. The bispecific antibody or antigen-binding fragment thereof according to any one of embodiments 20 to 34, which is nonfucosylated or fucose-deficient. 36. The bispecific antibody or antigen-binding fragment thereof according to any one of embodiments 20 to 35, which is conjugated to an agent. 37. The bispecific antibody or antigen-binding fragment thereof of embodiment 36, wherein the agent is an inhibitor of tubulin polymerization, a DNA damaging agent, or a DNA synthesis inhibitor. 38. The bispecific antibody or antigen-binding fragment thereof of embodiment 36, wherein the agent is a maytansinoid, an auristatin, a pyrrolobenzodiazepine (PBD) dimer, a calicheamicin, a duocarmycin, an indolinobenzodiazepine dimer, or an exatecan derivative Dxd. 39. A nucleic acid encoding an anti-CTLA4 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 19, or a bispecific antibody or antigen-binding fragment thereof according to any one of embodiments 20 to 38. 40. A vector comprising the nucleic acid of embodiment 39. 41. A host cell comprising an anti-CTLA4 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 19, a bispecific antibody or antigen-binding fragment thereof according to any one of embodiments 20 to 38, or a nucleic acid according to embodiment 39. 42. The host cell of embodiment 41, capable of producing an antibody or antigen-binding fragment thereof that is nonfucosylated or fucose-deficient. 43. The host cell of embodiment 41 or 42, having an alpha 1,6-fucosyltransferase (Fut8) knockout. 44. A host cell according to any one of embodiments 41 to 43, which overexpresses β1,4-N-acetylglucosaminyltransferase III (GnT-III). 45. A host cell according to any one of embodiments 41 to 44, which overexpresses Golgi μ-mannosidase I (ManII). 46. ​​A method for producing an antibody or antigen-binding fragment thereof, comprising culturing a host cell according to any one of embodiments 41 to 45 under conditions that produce the antibody or antigen-binding fragment thereof. 47. A method for producing a nonfucosylated or fucose-deficient antibody or antigen-binding fragment thereof, comprising culturing a host cell according to any one of embodiments 42 to 45 under conditions for producing said antibody or antigen-binding fragment thereof. 48. The method of any one of embodiments 43 to 45, further comprising recovering the antibody or antigen-binding fragment thereof produced by the host cell. 49. An antibody or antigen-binding fragment thereof produced by a method according to any one of embodiments 46 to 48. 50. A composition comprising an anti-CTLA4 antibody or antigen-binding fragment thereof described in any one of embodiments 1 to 19, a bispecific antibody or antigen-binding fragment thereof described in any one of embodiments 20 to 38, or an antibody or antigen-binding fragment thereof described in embodiment 49. 51. A pharmaceutical composition comprising an anti-CTLA4 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 19, a bispecific antibody or antigen-binding fragment thereof according to any one of embodiments 20 to 38, or an antibody or antigen-binding fragment thereof according to embodiment 49, and a pharmaceutically acceptable carrier. 52. For use in treating or preventing a neoplastic disease in a subject: A pharmaceutical composition comprising an anti-CTLA4 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 19, a bispecific antibody or antigen-binding fragment thereof according to any one of embodiments 20 to 38, or an antibody or antigen-binding fragment thereof according to embodiment 49, and a pharmaceutically acceptable carrier. 53. In the manufacture of a medicament for treating or preventing a neoplastic disease in a subject, A pharmaceutical composition comprising an anti-CTLA4 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 19, a bispecific antibody or antigen-binding fragment thereof according to any one of embodiments 20 to 38, or an antibody or antigen-binding fragment thereof according to embodiment 49, and a pharmaceutically acceptable carrier. Use of. 54. A kit comprising an anti-CTLA4 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 19, a bispecific antibody or antigen-binding fragment thereof according to any one of embodiments 20 to 38, or an antibody or antigen-binding fragment thereof according to embodiment 49. 55. A method for treating or preventing a neoplastic disease in a subject, comprising administering to the subject an effective amount of an anti-CTLA4 antibody or antigen-binding fragment thereof described in any one of embodiments 1 to 19, a bispecific antibody or antigen-binding fragment thereof described in any one of embodiments 20 to 38, an antibody or antigen-binding fragment thereof described in embodiment 49, or a composition described in any one of embodiments 50 to 52. [Example]

[0310] The present invention will be more fully understood by reference to the following examples. However, they should not be construed as limiting the scope of the present invention. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or variations thereof in light of this will be suggested to those skilled in the art and are to be included within the spirit and purpose of this application and the scope of the appended claims.

[0311] Example 1: In vitro characterization of anti-CTLA4 antibody binding to CTLA4: low-density antigen-binding ELISA and SPR Binding between exemplary anti-CTLA4 antibodies and variants to CTLA4 or FcγRIIIa (CD16a) was assessed by enzyme-linked immunosorbent assay (ELISA) or surface plasmon resonance (SPR).

[0312] method antibody The antibodies tested include humanized anti-CTLA4 antibodies designated Antibody 1 and Antibody 2, as well as variants / forms / versions thereof. According to a specific numbering scheme, Antibody 1 comprises a light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3, and a heavy chain variable region comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6. According to the Kabat numbering scheme, Antibody 1 comprises a light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, and a heavy chain variable region comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 10, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 11, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12. According to a specific numbering scheme, antibody 2 comprises a light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15, and a heavy chain variable region comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 16, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 18. According to the Kabat numbering scheme, antibody 2 comprises a light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 19, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 20, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 21, and a heavy chain variable region comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24. For example, various forms of Antibody 1 were generated and evaluated, each containing specific mutations in the Fc domain or a non-fucosylated version, and each was designated by the nomenclature "Antibody 1-#." For example, various forms of Antibody 2 were generated and evaluated, each containing specific mutations in the Fc domain or a non-fucosylated version, and each was designated by the nomenclature "Antibody 2-#."

[0313] ELISA All ELISAs were performed in a manner substantially similar to that described below or generally consistent with methods known in the art. Polystyrene 96-well microplates (Fisher #07-200-591) were coated with 1 μg / mL of human CTLA4-Fc (R&D #7268-CT) per well and stored at 4°C overnight. Plates were washed with 0.05% Tween in TBS (TBS-T), blocked with 1% BSA (Sigma Aldrich #B4287-25G), and washed with TBS-T. Serial dilutions of samples containing the anti-CTLA4 antibodies described below were made in assay buffer (PBS + 0.05% Tween + 1% BSA), added to the plates, and orbitally shaken at 100 RPM for 1 hour at room temperature. After washing with TBS-T, anti-human kappa light chain [clone: ​​SB81a]-HRP (Abcam #ab79115) diluted 1:8000 in assay buffer was applied to the wells and shaken for 1 hour at room temperature. The plate was washed with TBS-T, and HRP substrate (Super Signal Pico Chemiluminescent Substrate, Thermo #37069) was applied to the plate, and luminescence was recorded using a spectrophotometer (BioTek). Data were analyzed using GraphPad Prism.

[0314] SPR All surface plasmon resonance (SPR) analyses were performed in a manner substantially similar to that described below or generally consistent with methods known in the art. SPR was performed on certain antibodies to evaluate binding between the antibody or its fragment and a target protein, such as human CTLA4 (huCTLA4), cynomolgus monkey CTLA4 (cynoCTLA4), or FcγRIIIa (CD16a). In some studies, SPR analysis was performed to test the binding of certain antibodies, such as antibody 2-6 (a version of antibody 2 with S239D and I332E mutations in the Fc region) and the anti-CTLA4 antibody ipilimumab, to human CTLA4. In some studies, SPR analysis was performed to test the binding of certain antigen-binding fragments, such as the Fabs of antibodies such as antibody 2 and ipilimumab, to recombinant human CTLA4 (rhCTLA4). In some studies, SPR analysis was performed to test the binding of certain antigen-binding fragments, such as the Fab of antibodies like Antibody 2 and ipilimumab, to cynoCTLA4-Fc. SPR analysis was performed on a GE Biacore T200 instrument at 37°C. Antibodies and isotype controls (reference channels) were coupled to a CM5 chip (GE Healthcare) at an immobilization level of 100 RU. Peptides were synthesized and diluted in HBS-EP+ running buffer (GE Healthcare). Binding data were analyzed using Biacore evaluation software, version 3.0.

[0315] result ELISA Figures 1A and 1B show the results of binding studies, assessed by ELISA, to human CTLA-Fc using the above-described humanized anti-CTLA4 antibodies, Antibody 1 and Antibody 2, or variants thereof. Figure 1A shows that there is no detectable difference between the binding to human CTLA4-Fc of a version of Antibody 1 with a wild-type Fc region (Antibody 1-1) and a version of Antibody 1 with S239D and I332E mutations in the Fc region (Antibody 1-2). Figure 1B compares the CTLA4 binding of various forms of antibody 2, including a version of antibody 2 with a wild-type Fc region (antibody 2-1), a version of antibody 2 with S239D and I332E mutations in the Fc region (antibody 2-2), a version of antibody 2 with S239D, I332E, and two hinge region mutations in the Fc region (antibody 2-3), a version of antibody 2 with S239D, I332E, and A330L mutations in the Fc region (antibody 2-4), and a version of antibody 2 with S239D, I332E, and A330L mutations in the Fc region and two hinge region mutations (antibody 2-5). As shown in Figure 1B, all forms of the antibody bound similarly to human CTLA4-Fc. The mean EC 50 The values ​​are provided in Table 2 below.

[0316] [Table 2]

[0317] Figures 1C-1E show the results of binding studies of humanized CTLA4 antibodies, Antibody 1, Antibody 2, or ipilimumab, or variants thereof, containing mutations in the Fc region or non-fucosylated versions, to human CTLA-Fc as assessed by ELISA. The mean EC values ​​of the tested antibodies were 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 50Values ​​are provided in the respective figures. Figure 1C shows that there is no detectable difference between the binding to human CTLA4-Fc of a version of Antibody 1 with a wild-type Fc region (Antibody 1-1), a version of Antibody 1 with S239D and I332E mutations in the Fc region (Antibody 1-2), and a non-fucosylated version of Antibody 1 (Antibody 1-aFuc). Figure 1D shows that there is no detectable difference between the binding to human CTLA4-Fc of a version of Antibody 2 with a wild-type Fc region (Antibody 2-1), a version of Antibody 2 with S239D and I332E mutations in the Fc region (Antibody 2-6), and a non-fucosylated version of Antibody 2 (Antibody 2-aFuc). Figure 1E shows that there was no detectable difference between the binding of a version of ipilimumab with a wild-type Fc (ipilimumab), a version of ipilimumab with S239D and I332E mutations in the Fc region (ipilimumab-m), and a non-fucosylated version of ipilimumab (ipilimumab-aFuc) to human CTLA4-Fc. As shown in Figures 1C-1E, all variants of each antibody bound similarly to human CTLA4-Fc.

[0318] SPR Figure 2A shows the results of SPR analysis demonstrating binding between antibody 2-6 (a version of antibody 2 with the S239D mutation and the I332E mutation in the Fc region) or ipilimumab and human CTLA4 (huCTLA4) at 32 nM, 16 nM, 8 nM, 4 nM, and 2 nM. Figure 2B shows the association rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (K). D ), as well as the fold difference between antibody 2-6 and ipilimumab. As shown in Figure 2B, antibody 2-6 has a K of 91 pM. D whereas ipilimumab has a K of 1640 pM D This indicates that antibodies 2-6 have D SPR analysis of the binding between antibody 2-6 and cynoCTLA4 showed a K of 131 pM. D showed.

[0319] Figure 2C shows the results of SPR analysis demonstrating binding between ipilimumab Fab (ipilimumab-Fab) or Antibody 2 Fab (Antibody 2-Fab) and rhCTLA4-Fc at 32 nM, 16 nM, 8 nM, 4 nM, and 2 nM. Figure 2D shows the association rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (K D ), and χ 2 The binding results shown in Figure 2C are summarized in terms of K values. As shown in Figure 2D, ipilimumab exhibited a K of 4.74 nM. D value, while antibody 2-Fab has a K of 4.53 pM D value, which indicates that antibody 2-Fab has a K D Additional binding affinity data between rhCTLA4-Fc and either ipilimumab-Fab or Antibody 2-Fab is provided in Table 3 below, which shows that rhCTLA4-Fc has a much higher binding affinity to ipilimumab-Fab than ipilimumab-Fab, as measured by the average K D was 5.71 ± 0.87 nM, and the mean K of antibody 2-Fab D is 3.86±2.1 pM.

[0320] [Table 3]

[0321] Figure 2E shows the results of SPR analysis demonstrating binding between ipilimumab Fab (ipilimumab-Fab) or Antibody 2 Fab (Antibody 2-Fab) and cynoCTLA4-Fc at 32 nM, 16 nM, 8 nM, 4 nM, and 2 nM. Figure 2F shows the association rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (K D ), and χ 2 The binding results shown in Figure 2E are summarized in terms of K values. As shown in Figure 2F, ipilimumab exhibited a K of 28.6 nM. D value, while antibody 2-Fab has a K of 0.623 nM D value, which indicates that antibody 2-Fab has a K DAdditional binding affinity data between cynoCTLA4-Fc and either ipilimumab-Fab or antibody 2-Fab is provided in Table 4 below, which shows that ipilimumab-Fab has a much higher binding affinity for cynoCTLA4-Fc than ipilimumab-Fab. D was 23.1 ± 6.3 nM, and the mean K of antibody 2-Fab D is 0.779±0.15nM.

[0322] [Table 4]

[0323] Figure 3 shows the results of SPR analysis demonstrating binding between antibody 2-1 (a version of antibody 2 with a wild-type Fc region), antibody 2-6 (a version of antibody 2 with S239D and I332E mutations in the Fc region), or ipilimumab at various concentrations and human FcγRIIIa (CD16a). Kinetics and affinity could not be calculated based on the results.

[0324] Example 2: Enterotoxin analysis The ability of various antibodies to stimulate IL-2 production from peripheral blood mononuclear cells (PBMCs) was examined using a staphylococcal enterotoxin B (SEB) assay.

[0325] method SEB assay For the SEB assay, dilutions of the selected antibodies were prepared in pre-warmed medium (RPMI + 10% heat-inactivated FBS + 1% HEPES + 1% MEM NEAA + 1% Na-pyruvate). Antibody solutions were plated in triplicate, and medium alone was added to the "PBMC + SEB" wells, the "PBMC only" wells, and the outer perimeter wall. SEB solution was prepared in pre-warmed medium and added to all experimental wells except the "PBMC only" wells. PBMCs (BioIVT) were thawed in a 37°C water bath. Cells were transferred dropwise to a conical tube, and 20 volumes of pre-warmed medium were added to wash the cells. The cells were centrifuged, and the medium was aspirated. The cells were resuspended in 20 mL of pre-warmed medium, and an aliquot was taken for counting. The remaining cells were centrifuged and resuspended in a large volume to obtain a cell count of 1 × 10 cells. 6 Cells were seeded at 100 μL per well and the plates were incubated in a 37°C, 5% CO2 incubator for 5 days. On day 5, the plates were spun at 1000 RPM for 5 minutes. From each well, 250 μL of cells were transferred to a new 96-well plate. The plate was spun again and 225 μL of cells were transferred to a PCR strip. Samples were stored at -80°C until analyzed by ELISA.

[0326] IL-2 ELISA IL-2 levels in cell supernatants produced using the protocol described above were determined by analysis with the Human IL-2 ELISA MAX Deluxe Set (BioLegend, Catalog No. #431806). Cell supernatant samples were diluted with assay buffer to fall within the standard curve. Samples were analyzed using GraphPad Prism and with Tukey's multiple comparison test (one-way ANOVA) to determine statistical significance between treatment groups.

[0327] result Various forms of Antibody 1 and Antibody 2 were tested in the SEB assay. Data showing the levels of IL-2 produced in the presence of Antibody 1-1 or Antibody 1-2, or without any antibody, as described in Example 1, are shown in Figure 4A. Data showing the levels of IL-2 produced in the presence of Antibody 2-1, Antibody 2-2, Antibody 2-3, Antibody 2-4, and Antibody 2-5, or without any antibody, as described in Example 1, are shown in Figure 4C. All tested antibodies demonstrated the ability to increase IL-2 levels compared to the no-antibody control. The fold increase in IL-2 levels for each antibody over the no-antibody control, along with a comparison to the isotype control, are shown in Figure 4B (Antibody 1 and variants) and Figure 4D (Antibody 2 and variants).

[0328] The results of a similar evaluation by SEB assay using various versions of anti-CTLA4 antibody 1 [having a wild-type Fc region (antibody 1-1), having S239D and I332E mutations in the Fc region (antibody 1-2), and a non-fucosylated version of antibody 1 (antibody 1-aFuc)], antibody 2 [having a wild-type Fc region (antibody 2-1), having S239D and I332E mutations in the Fc region (antibody 2-6), and a non-fucosylated version of antibody 2 (antibody 2-aFuc)], or ipilimumab [having a wild-type Fc (ipilimumab), having S239D and I332E mutations in the Fc region (ipilimumab-m), and an afucosylated version of ipilimumab (ipilimumab-aFuc)] are shown in Figure 4E. The results showed that antibodies with S239D and I332E mutations in the Fc region resulted in higher IL-2 production.

[0329] Example 3: ADCC activity by anti-CTLA4 antibody The ability of anti-CTLA4 antibodies to effect antibody-dependent cellular cytotoxicity (ADCC) was assessed using an FcγRIIIa reporter bioassay.

[0330] method FcγRIIIa reporter bioassay FcγRIIIa reporter bioassays were performed using various anti-CTLA4 antibodies. Antibodies were diluted in pre-warmed complete medium (RPMI1640 with FBS). CTLA4 effector cells (target cells: Promega J158A) were thawed and transferred to a conical tube containing complete medium. Cells were mixed and counted to a cell density of 1 × 10 6 The concentration was adjusted to 3x10 cells / mL. Target cells were added to each well of a 96-well plate (Corning, Cat. #3917). Diluted antibodies were added to the appropriate wells and tested in duplicate. The contents of each well were gently mixed and the plate was incubated at 37°C for 15 minutes. Effector cells (FcγRIIIa expressing Jurkat cells) were thawed and transferred to a conical tube containing complete medium. The cells were mixed and counted to a cell density of 3x10 6 The concentration was adjusted to cells / mL. Effector cells were immediately dispensed into each well and gently mixed. The plate was covered with a lid and maintained at 37°C for 6 hours. One hour before measurement, Bio-Glo substrate and Bio-Glo buffer were removed from 4°C. Bio-Glo buffer was transferred to the Bio-Glo substrate bottle to create Bio-Glo reagent and gently mixed by inversion. The bottle was maintained at room temperature. After incubation, the assay plate was removed from the incubator and maintained at room temperature for 10 minutes. Bio-Glo reagent was added to each well, and the plate was incubated at room temperature for 5-15 minutes. The plate was then read in a luminometer.

[0331] The antibodies tested included anti-CTLA4 antibody 1 [having a wild-type Fc region (antibody 1-1), having S239D and I332E mutations in the Fc region (antibody 1-2), and a non-fucosylated version of antibody 1 (antibody 1-aFuc)], antibody 2 [having a wild-type Fc region (antibody 2-1), having S239D and I332E mutations in the Fc region (antibody 2-6), and a non-fucosylated version of antibody 2 (antibody 2-aFuc)], or ipilimumab [having a wild-type Fc (ipilimumab), having S239D and I332E mutations in the Fc region (ipilimumab-m), and an afucosylated version of ipilimumab (ipilimumab-aFuc)].

[0332] result Figures 5A-5D show reporter activation curves and EC values ​​from ADCC reporter bioassays for the Antibody 1 variant (Figure 5A), the Antibody 2 variant (Figure 5B), the ipilimumab variant (Figure 5C), and the nonfucosylated variants of all three antibodies (Figure 5D). 50 The results showed that antibodies with the S239D and I332E mutations in the Fc region and the non-fucosylated antibody resulted in higher activation compared to the respective versions with wild-type Fc regions for each antibody.

[0333] Example 4: In vivo immunophenotyping of immune cells in a tumor-bearing mouse model expressing human CTLA4 after administration of anti-CTLA4 antibodies The immunophenotype of immune cells was determined in a genetically engineered mouse model bearing MC38 tumors with a human CTLA4 knock-in that was administered various anti-CTLA4 antibodies.

[0334] method MC38 cells (1 x 10 6) were subcutaneously injected into mouse models with a knock-in of human CTLA4. On day 0, mice were randomized based on tumor volume measurements and injected intraperitoneally with a single injection of 200 μg of test antibody (Antibody 2-1, ipilimumab, the nonfucosylated form of ipilimumab (ipilimumab-aFuc), or an IgG control. Immunophenotyping was performed on CD45+ splenocytes and CD45+ intratumoral cells 5 days after injection. CD45+ cells were assessed for expression of markers including CD3+ / ICOS+, CD3+ T cells, CD4+ / Ki67+, CD3+ / Ki67+, CD4+ / ICOS+, CD4+ T cells, CD8+ / ICOS+, CD8+ T cells, Treg+ / ICOS+, CD8+ / Ki67+, Treg, and Treg+ / Ki67+, with the relative percentage of CD45+ cells expressing those markers assessed.

[0335] result Results from immunophenotyping of MC38 tumor-bearing human CTLA4 knock-in mice are shown in Figures 6A-6D. The relative percentages of CD45+ spleen (Figures 6A and 6B) or CD45+ intratumor (Figures 6C and 6D) are provided for markers CD3+ / ICOS+, CD3+ T cells, CD4+ / Ki67+, CD3+ / Ki67+, CD4+ / ICOS+, CD4+ T cells, CD8+ / ICOS+, CD8+ T cells, Treg+ / ICOS+, CD8+ / Ki67+, Treg, and Treg+ / Ki67+. Statistics are provided in Table 5 (spleen cells) and Table 6 (intratumoral cells). Group 1: IgG, Group 2: Antibody 2-1, Group 4: ipilimumab, Group 5: ipilimumab-aFuc.

[0336] [Table 5]

[0337] [Table 6]

[0338] Example 5: In vivo tumor growth inhibition of anti-CTLA4 antibodies in a tumor-bearing mouse model expressing human CTLA4 The tumor growth inhibitory activity of various anti-CTLA4 antibodies was evaluated after administration in a genetically engineered mouse model bearing MC38 tumors with human CTLA4 knock-in.

[0339] MC38 cells (1 x 10 6 (1000 cells / ml) were subcutaneously injected into a mouse model containing a knock-in of the human CTLA4 extracellular domain coding region. Mice were intraperitoneally injected with a single injection of 20 μg, 7 μg, or 2 μg of test antibody (antibody 2-6, ipilimumab, the non-fucosylated form of ipilimumab (ipilimumab-aFuc), or an RSV antibody with S239D and I332E mutations in the Fc domain (RSV-m)). Tumor volume was measured over time after the single injection of test antibody.

[0340] The results are shown in Figures 7A-7D. Figure 7A (group means) and Figure 7B (individual mice) show tumor volume (mm) over time in mice injected with antibody 2-6, ipilimumab, or the nonfucosylated form of ipilimumab (ipilimumab-aFuc). 3 ), and the results showed that all of the antibodies tested exhibited tumor growth inhibition. Figure 7C (20 μg), Figure 7D (7 μg), and Figure 7E (2 μg) show the tumor volume (mm ) over time at each dose of the different antibodies. 3 ) shows a comparison of the results. The results showed that antibodies 2-6 showed the best tumor growth inhibition at the 2 μg and 7 μg doses, and similar tumor growth inhibition to the ipilimumab antibody at the 20 μg dose.

[0341] Example 6: Efficacy and Pharmacodynamics of Anti-CTLA4 Antibodies in the MB49 Murine Bladder Tumor Model method The efficacy and pharmacodynamics (PD) of anti-CTLA4 antibodies were evaluated using the MB49 mouse bladder tumor model. Each test antibody (RSV-m control antibody, ipilimumab, and antibodies 2-6) was administered, followed by evaluation of tumor volume and body weight to assess efficacy. The anti-RSV control antibody contained the S239D and I332E mutations (RSV-m control antibody). Peripheral immunophenotyping was also performed on day 5 to assess the percentage of CD4+Ki67+ cells and CD4+ICOS+ cells in peripheral blood on day 5 post-administration. For efficacy studies, mice were evaluated using doses in mg / kg, as shown in Table 7. As used herein, doses in mg / kg are also referred to as "mpk." MB49 cells were subcutaneously inoculated into C57 / BL6-huCTLA4 mice. Treatment was initiated when tumors reached approximately 350 mm 3 One-way ANOVA with Dunnett's post-hoc test was performed to determine statistical significance of treatment vs. control (RSV-m control). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0342] [Table 7]

[0343] Pharmacodynamics were assessed by administering antibody to each of five cohorts of mice using the doses (mg / kg) shown in Table 8. Immunophenotyping is performed in tumors, liver, spleen, and blood, among other readouts. T cells are assessed by measuring Foxp3+CD25+ cells as a percentage of CD4+ cells, and CD8+ cells as a percentage of CD45+ cells.

[0344] [Table 8]

[0345] result FIG. 8A shows the results of the efficacy study for the percentage of CD4+Ki67+ cells (left) and CD4+ICOS+ cells (right) in peripheral blood 5 days after administration, which represents the level of T cell activation.

[0346] As shown in Figure 8B, tumor weight and CD8 / Treg ratio were assessed on day 7 in mice treated with 10 mg / kg (RSV-m control) or 3 mg / kg (ipilimumab or antibody 2-6). Mice treated with antibody 2-6 exhibited the lowest tumor weight and highest CD8 / Treg ratio in tumors at 3 mg / kg on day 7. No changes in body weight, spleen weight, kidney weight, or liver weight were observed in any of the treatment groups (data not shown).

[0347] As shown in Figure 8C, regulatory T cell depletion and CD8+ T cell activation is observed with antibody 2-6 but not with ipilimumab.

[0348] As shown in Figure 8D, potent anti-tumor activity was observed after treatment with anti-CTLA4 antibodies. At a dose of 0.3 mg / kg, antibodies 2-6 exhibited superior anti-tumor activity compared to ipilimumab.

[0349] Example 7: In vivo evaluation of anti-CTLA4 antibodies in cynomolgus monkeys The pharmacodynamic (PD) effects of anti-CTLA4 antibodies were evaluated in cynomolgus monkeys. In two separate sets of experiments, cynomolgus monkeys were administered ipilimumab, antibody 2-6, or an isotype control, and the PD effects were assessed by measuring the percentage of Ki67+ cells among CD4+ cells.

[0350] Results from the first and second sets of PD experiments are shown in Figure 9. Antibodies 2-6 are more potent than ipilimumab in inducing peripheral PD effects, as indicated by an increased percentage of Ki67+ cells in CD4+ cells compared to ipilimumab.

[0351] Pharmacokinetics was also evaluated using cynomolgus monkeys after a 10 mg / kg intravenous dose of the test antibodies (RSV-m control, ipilimumab, or antibody 2-6). Plasma levels of each antibody were measured, and the half-life (days), Cmax (μg / mL), and area under the curve (AUC) (days * μg / mL) were determined in two studies. The half-life (days), Cmax (μg / mL), and area under the curve (AUC) (days * μg / mL) results from the pharmacokinetic studies for each tested antibody were calculated. Ipilimumab had the longest half-life and maximum AUC. Antibody 2-6 had a Cmax similar to ipilimumab.

[0352] Sequence information SEQUENCE LISTING <110> XILIO DEVELOPMENT, INC. <120> ANTI-CTLA4 ANTIBODIES AND METHODS OF USE THEREOF <150> US 62 / 785,111 <151> 2018-12-26 <160> 39 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR-L1 Antibody 1 <400> 1 Gln Ser Leu Leu Asn Ser Asp Gly Asn Thr Tyr 1 5 10 <210> 2 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> CDR-L2 Antibody 1 <400> 2 Leu Val Ser 1 <210> 3 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR-L3 Antibody 1 <400> 3 Val Gln Gly Thr His Asp Pro 1 5 <210> 4 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR-H1 Antibody 1 <400> 4 Tyr His Thr Ile Thr Ser Gly Tyr 1 5 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR-H2 Antibody 1 <400> 5 Ile Ser Tyr Ser Gly Asn Thr 1 5 <210> 6 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDR-H3 Antibody 1 <400> 6 Ala Ser Met Met Val Pro His Tyr Tyr Val Met Asp Ala 1 5 10 <210> 7 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDR-L1 (Kabat) Antibody 1 <400> 7 Arg Ser Ser Gln Ser Leu Leu Asn Ser Asp Gly Asn Thr Tyr Leu Tyr 1 5 10 15 <210> 8 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR-L2 (Kabat) Antibody 1 <400> 8 Leu Val Ser Lys Leu Gly Ser 1 5 <210> 9 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR-L3 (Kabat) Antibody 1 <400> 9 Val Gln Gly Thr His Asp Pro Trp Thr 1 5 <210> 10 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> CDR-H1 (Kabat) Antibody 1 <400> 10 Ser Gly Tyr Asp Trp Thr 1 5 <210> 11 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDR-H2 (Kabat) Antibody 1 <400> 11 Tyr Ile Ser Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 12 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR-H3(Kabat) Antibody 1 <400> 12 Met Met Val Pro His Tyr Tyr Val Met Asp Ala 1 5 10 <210> 13 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> CDR-L1 Antibody 2 <400> 13 Ser Ala Leu Ser Tyr Met 1 5 <210> 14 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> CDR-L2 Antibody 2 <400> 14 Gly Thr Ser 1 <210> 15 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR-L3 Antibody 2 <400> 15 His His Trp Ser Asn Thr Gln 1 5 <210> 16 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR-H1 Antibody 2 <400> 16 Gly Tyr Thr Phe Thr Asn Tyr Phe 1 5 <210> 17 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR-H2 Antibody 2 <400> 17 Val Asp Pro Glu Gln Gly Arg Ala Asp 1 5 <210> 18 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR-H3 Antibody 2 <400> 18 Arg Arg Ala Met Asp Asn Tyr Gly Phe Ala Tyr 1 5 10 <210> 19 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR-L1 (Kabat) Antibody 2 <400> 19 Ser Ala Asn Ser Ala Leu Ser Tyr Met Tyr 1 5 10 <210> 20 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR-L2 (Kabat) Antibody 2 <400> 20 Gly Thr Ser Asn Leu Ala Ser 1 5 <210> 21 <211> 9 <212> PRT <213> Artificial Sequence <220> &l...

Claims

1. An anti-CTLA4 antibody or antigen-binding fragment thereof, comprising a light chain variable (VL) domain and a heavy chain variable (VH) domain, a) the VL domain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3, and / or the VH domain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6, or b) the VL domain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15, and / or the VH domain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 16, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 18; or c) the VL domain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, and / or the VH domain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 10, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 11, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12; or d) the VL domain comprises (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 19, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 20, and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 21, and / or the VH domain comprises (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24; An anti-CTLA4 antibody or antigen-binding fragment thereof.

2. a) the VL domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 25, and the VH domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 26, or b) the VL domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 30, and the VH domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 31; The anti-CTLA4 antibody or antigen-binding fragment thereof according to claim 1.

3. a) the VL domain comprises the amino acid sequence of SEQ ID NO: 25 and the VH domain comprises the amino acid sequence of SEQ ID NO: 26, or b) the VL domain comprises the amino acid sequence of SEQ ID NO: 30, and the VH domain comprises the amino acid sequence of SEQ ID NO: 31; The anti-CTLA4 antibody or antigen-binding fragment thereof according to claim 1 or 2.

4. An anti-CTLA4 antibody or antigen-binding fragment thereof, comprising a light chain variable (VL) domain and a heavy chain variable (VH) domain, a) the VL domain comprises the amino acid sequence of SEQ ID NO: 25 and / or the VH domain comprises the amino acid sequence of SEQ ID NO: 26, or b) the VL domain comprises the amino acid sequence of SEQ ID NO: 30 and / or the VH domain comprises the amino acid sequence of SEQ ID NO: 31; An anti-CTLA4 antibody or antigen-binding fragment thereof.

5. The anti-CTLA4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the VL domain comprises the amino acid sequence of SEQ ID NO: 25 and the VH domain comprises the amino acid sequence of SEQ ID NO:

26.

6. The anti-CTLA4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the VL domain comprises the amino acid sequence of SEQ ID NO: 30 and the VH domain comprises the amino acid sequence of SEQ ID NO:

31.

7. The anti-CTLA4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, comprising a heavy chain constant domain (CH).

8. The anti-CTLA4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, comprising a CH sequence selected from the group consisting of SEQ ID NOs: 35 to 38.

9. 9. The anti-CTLA4 antibody or antigen-binding fragment thereof of claim 7 or 8, wherein the CH comprises the amino acid substitution S239D or I332E or both, and the amino acid residues are numbered according to the EU index of Kabat.

10. 10. The anti-CTLA4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, comprising the CH sequence of SEQ ID NO:

38.

11. The anti-CTLA4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, comprising a light chain constant domain (CL).

12. 12. The anti-CTLA4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, comprising the CL sequence of SEQ ID NO:

39.

13. a) the light chain comprises the amino acid sequence of SEQ ID NO: 27 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 29, or b) the light chain comprises the amino acid sequence of SEQ ID NO: 32 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 34; An anti-CTLA4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12.

14. An anti-CTLA4 antibody or antigen-binding fragment thereof, comprising a light chain comprising the amino acid sequence of SEQ ID NO:27 and a heavy chain comprising the amino acid sequence of SEQ ID NO:

29.

15. An anti-CTLA4 antibody or antigen-binding fragment thereof, comprising a light chain comprising the amino acid sequence of SEQ ID NO: 32 and a heavy chain comprising the amino acid sequence of SEQ ID NO:

34.

16. 16. The anti-CTLA4 antibody or antigen-binding fragment thereof of any one of claims 1 to 15, which is nonfucosylated or fucose-deficient.

17. The anti-CTLA4 antibody or antigen-binding fragment thereof of any one of claims 1 to 16, which is conjugated to a drug.

18. 18. The anti-CTLA4 antibody or antigen-binding fragment thereof of claim 17, wherein the agent is an inhibitor of tubulin polymerization, a DNA damaging agent, or a DNA synthesis inhibitor.

19. 18. The anti-CTLA4 antibody or antigen-binding fragment thereof of claim 17, wherein the drug is a maytansinoid, an auristatin, a pyrrolobenzodiazepine (PBD) dimer, a calicheamicin, a duocarmycin, an indolinobenzodiazepine dimer, or an exatecan derivative Dxd.

20. 1) a first pair of light and heavy chains that specifically binds to CTLA4; 2) a second pair of light and heavy chains that specifically bind to the antigen; A bispecific antibody or antigen-binding fragment thereof comprising: the light chains of the first pair comprise a VL domain and the heavy chains of the first pair comprise a VH domain; a) the VL domain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3, and / or the VH domain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6, or b) the VL domain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15, and / or the VH domain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 16, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 18; or c) the VL domain comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, and / or the VH domain comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 10, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 11, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12; or d) the VL domain comprises (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 19, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 20, and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 21, and / or the VH domain comprises (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24; A bispecific antibody or antigen-binding fragment thereof.

21. a) the VL domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 25, and the VH domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 26, or b) the VL domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 30, and the VH domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 31; 21. The bispecific antibody or antigen-binding fragment thereof of claim 20.

22. a) the VL domain comprises the amino acid sequence of SEQ ID NO: 25 and the VH domain comprises the amino acid sequence of SEQ ID NO: 26, or b) the VL domain comprises the amino acid sequence of SEQ ID NO: 30, and the VH domain comprises the amino acid sequence of SEQ ID NO: 31; A bispecific antibody or antigen-binding fragment thereof according to claim 20 or 21.

23. 1) a first pair of light and heavy chains that specifically binds to CTLA4; 2) a second pair of light and heavy chains that specifically bind to the antigen; A bispecific antibody or antigen-binding fragment thereof comprising: the light chains of the first pair comprise a VL domain and the heavy chains of the first pair comprise a VH domain; a) the VL domain comprises the amino acid sequence of SEQ ID NO: 25 and / or the VH domain comprises the amino acid sequence of SEQ ID NO: 26, or b) the VL domain comprises the amino acid sequence of SEQ ID NO: 30 and / or the VH domain comprises the amino acid sequence of SEQ ID NO: 31; A bispecific antibody or antigen-binding fragment thereof.

24. 24. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 20 to 23, wherein the VL domain comprises the amino acid sequence of SEQ ID NO: 25 and the VH domain comprises the amino acid sequence of SEQ ID NO:

26.

25. 24. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 20 to 23, wherein the VL domain comprises the amino acid sequence of SEQ ID NO: 30 and the VH domain comprises the amino acid sequence of SEQ ID NO:

31.

26. 26. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 20 to 25, comprising a heavy chain constant domain (CH).

27. 27. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 20 to 26, comprising a CH sequence selected from the group consisting of SEQ ID NOs: 35 to 38.

28. 28. The bispecific antibody or antigen-binding fragment thereof of claim 26 or 27, wherein the CH comprises the amino acid substitution S239D or I332E or both, wherein the amino acid residues are numbered according to the EU index of Kabat.

29. 29. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 20 to 28, comprising the CH sequence of SEQ ID NO:

38.

30. 30. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 20 to 29, comprising a light chain constant domain (CL).

31. 31. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 20 to 30, comprising the CL sequence of SEQ ID NO:

39.

32. a) the light chain comprises the amino acid sequence of SEQ ID NO: 27 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 29, or b) the light chain comprises the amino acid sequence of SEQ ID NO: 32 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 34; A bispecific antibody or antigen-binding fragment thereof according to any one of claims 20 to 31.

33. 1) a first pair of light and heavy chains that specifically binds to CTLA4; 2) a second pair of light and heavy chains that specifically bind to the antigen; A bispecific antibody or antigen-binding fragment thereof comprising: the first pair comprising a light chain comprising the amino acid sequence of SEQ ID NO: 27 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 29; A bispecific antibody or antigen-binding fragment thereof.

34. 1) a first pair of light and heavy chains that specifically binds to CTLA4; 2) a second pair of light and heavy chains that specifically bind to the antigen; A bispecific antibody or antigen-binding fragment thereof comprising: the first pair comprising a light chain comprising the amino acid sequence of SEQ ID NO: 32 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 34; A bispecific antibody or antigen-binding fragment thereof.

35. 35. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 20 to 34, which is nonfucosylated or fucose-deficient.

36. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 20 to 35, which is conjugated to a drug.

37. 37. The bispecific antibody or antigen-binding fragment thereof of claim 36, wherein the agent is an inhibitor of tubulin polymerization, a DNA damaging agent, or a DNA synthesis inhibitor.

38. 37. The bispecific antibody or antigen-binding fragment thereof of claim 36, wherein the drug is a maytansinoid, an auristatin, a pyrrolobenzodiazepine (PBD) dimer, a calicheamicin, a duocarmycin, an indolinobenzodiazepine dimer, or an exatecan derivative Dxd.

39. A nucleic acid encoding the anti-CTLA4 antibody or antigen-binding fragment thereof of any one of claims 1 to 19, or the bispecific antibody or antigen-binding fragment thereof of any one of claims 20 to 38.

40. A vector comprising the nucleic acid of claim 39.

41. A host cell comprising the anti-CTLA4 antibody or antigen-binding fragment thereof of any one of claims 1 to 19, the bispecific antibody or antigen-binding fragment thereof of any one of claims 20 to 38, or the nucleic acid of claim 39.

42. 42. The host cell of claim 41, capable of producing an antibody or antigen-binding fragment thereof that is nonfucosylated or fucose-deficient.

43. 43. The host cell of claim 41 or 42, having an alpha 1,6-fucosyltransferase (Fut8) knockout.

44. The host cell according to any one of claims 41 to 43, which overexpresses β1,4-N-acetylglucosaminyltransferase III (GnT-III).

45. 45. The host cell of any one of claims 41 to 44, which overexpresses Golgi μ-mannosidase I (ManII).

46. 46. ​​A method for producing an antibody or antigen-binding fragment thereof, comprising culturing the host cell of any one of claims 41 to 45 under conditions to produce the antibody or antigen-binding fragment thereof.

47. 46. ​​A method for producing a nonfucosylated or fucose-deficient antibody or antigen-binding fragment thereof, comprising culturing the host cell of any one of claims 42 to 45 under conditions to produce the antibody or antigen-binding fragment thereof.

48. 46. ​​The method of any one of claims 43 to 45, further comprising recovering the antibody or antigen-binding fragment thereof produced by the host cell.

49. An antibody or antigen-binding fragment thereof produced by the method of any one of claims 46 to 48.

50. A composition comprising an anti-CTLA4 antibody or antigen-binding fragment thereof of any one of claims 1 to 19, a bispecific antibody or antigen-binding fragment thereof of any one of claims 20 to 38, or an antibody or antigen-binding fragment thereof of claim 49.

51. A pharmaceutical composition comprising an anti-CTLA4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 19, a bispecific antibody or antigen-binding fragment thereof according to any one of claims 20 to 38, or an antibody or antigen-binding fragment thereof according to claim 49, and a pharmaceutically acceptable carrier.

52. For use in treating or preventing a neoplastic disease in a subject, A pharmaceutical composition comprising an anti-CTLA4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 19, a bispecific antibody or antigen-binding fragment thereof according to any one of claims 20 to 38, or an antibody or antigen-binding fragment thereof according to claim 49, and a pharmaceutically acceptable carrier.

53. In the manufacture of a medicament for treating or preventing a neoplastic disease in a subject, A pharmaceutical composition comprising an anti-CTLA4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 19, a bispecific antibody or antigen-binding fragment thereof according to any one of claims 20 to 38, or an antibody or antigen-binding fragment thereof according to claim 49, and a pharmaceutically acceptable carrier. Use of.

54. A kit comprising an anti-CTLA4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 19, a bispecific antibody or antigen-binding fragment thereof according to any one of claims 20 to 38, or an antibody or antigen-binding fragment thereof according to claim 49.

55. 52. A method for treating or preventing a neoplastic disease in a subject, comprising administering to the subject an effective amount of an anti-CTLA4 antibody or antigen-binding fragment thereof of any one of claims 1 to 19, a bispecific antibody or antigen-binding fragment thereof of any one of claims 20 to 38, an antibody or antigen-binding fragment thereof of claim 49, or a composition of any one of claims 50 to 52.

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