Anti-CTLA-4 binding proteins and methods of use thereof

Abstract

Provided herein are antigen binding proteins (ABPs) having binding specificity for CTLA-4, and compositions, including pharmaceutical compositions, diagnostic compositions, and kits, comprising such ABPs. Also provided are methods of making CTLA-4 ABPs, and methods of using CTLA-4 ABPs for therapeutic, e.g., cancer treatment, diagnostic, and research purposes.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to and benefit of U.S. Provisional Patent Application No. 63 / 218,198, filed July 2, 2021, the entire contents of which are incorporated herein by reference.

[0002] (Sequence Listing) This application contains a Sequence Listing with 12,088 sequences submitted via EFS-Web, which is incorporated herein by reference in its entirety. The ASCII copy, created on June 29, 2022, is named "49446_WO_Sequence_Listing_Final" and is 1.86 megabytes in size.

[0003] FIELD OF THE INVENTION Provided herein are antigen binding proteins (ABPs) that have binding specificity for CTLA-4, and compositions, including pharmaceutical compositions, diagnostic compositions, and kits, comprising such ABPs. Also provided are methods of making CTLA-4 ABPs and methods of using CTLA-4 ABPs, e.g., for therapeutic, diagnostic, and research purposes. [Background technology]

[0004] CTLA-4, also known as cytotoxic T lymphocyte-associated protein 4 and CD152 (cluster of differentiation 152), is a cell surface receptor that suppresses T cell inflammatory activity, T cell costimulation, activation, and proliferation. CTLA-4 is constitutively expressed by regulatory T cells (Tregs) and is upregulated in stimulated T cells. CD80 and CD86, which are also expressed on antigen-presenting cells (APCs) such as dendritic cells (DCs), are the primary ligands for CTLA-4. The interaction between CTLA-4 and its ligands is crucial for downregulating immune responses and promoting self-tolerance by suppressing T cell inflammatory activity. This activity prevents autoimmune diseases and prevents the immune system from killing cancer cells.

[0005] CTLA-4 is a member of the immunoglobulin superfamily that is expressed by activated T cells and transmits inhibitory signals to T cells. CTLA-4 binds to CD80 and CD86 with higher affinity and avidity than CD28, thereby preventing CTLA-4 from competing with CD28 for its ligand. CTLA-4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. CTLA-4 is also found on regulatory T cells (Tregs) and contributes to their inhibitory function. T cell activation via the T cell receptor and CD28 results in increased expression of CTLA-4. The mechanism of action of CTLA-4 in T cells remains somewhat controversial. Biochemical evidence suggests that CTLA-4 recruits phosphatases to the T cell receptor (TCR), thereby attenuating the signal. This study remains unconfirmed in the literature since its initial report. More recent studies have suggested that CTLA-4 may function in vivo by capturing and removing B7-1 and B7-2 from the membrane of antigen-presenting cells, thereby making them unavailable for CD28 induction.

[0006] CTLA-4 variants have been associated with insulin-dependent diabetes mellitus, Graves' disease, Hashimoto's thyroiditis, celiac disease, systemic lupus erythematosus, thyroid eye disease, primary biliary cirrhosis, and other autoimmune diseases. The relatively high binding affinity of CTLA-4 to CD80 and CD86 makes it a potential therapeutic target for autoimmune diseases. A soluble fusion protein of CTLA-4 and an antibody (CTLA-4-Ig) has been developed for clinical use.

[0007] Recently, CTLA-4 antibodies have been used to treat some types of cancer, with varying degrees of efficacy. CTLA-4 inhibitors have been shown to antagonize the binding of CTLA-4 to its ligand, thereby activating the immune system to attack tumors. The current mechanism of action of known anti-CTLA-4 therapies is to block the interaction between CTLA-4 and its ligand for checkpoint inhibition. For example, CTLA-4 monoclonal antibodies (mAbs), such as ipilimumab, were originally intended to block the binding of CTLA-4 to its ligands, the B7 proteins CD80 and CD86, i.e., "checkpoint inhibition." Blocking CTLA-4 binding to B7 proteins frees them to bind to CD28, inducing T cell costimulation and activation. CTLA-4 antibodies have also been used to induce antibody-dependent cell-mediated cytotoxicity (ADCC) of Tregs specific to the tumor microenvironment, thereby reducing immune tolerance to tumors. Thus, in addition to blocking the interaction of CTLA-4 with its B7 ligand, anti-CTLA-4 mAbs also inhibit intratumoral FOXP3 expression in tumors that express relatively high levels of surface CTLA-4. + It can induce antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) of regulatory T cells (Treg).

[0008] Therefore, inhibition of CTLA-4 function is currently one of the most promising systemic therapeutic approaches for various diseases. There is a need to develop CTLA-4 ABPs that can be used in the treatment, diagnosis, and research of various diseases, including cancer and autoimmune diseases. International Application No. PCT / US2019 / 068820, filed December 27, 2019, and published as WO 2020140084(A1), describes CTLA-4 ABPs, and is incorporated herein by reference in its entirety. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Publication No. 2020140084 Summary of the Invention

[0010] Provided herein are ABPs (e.g., GIGA-564, GIGA-2328) that have binding specificity for CTLA-4 and methods of using the ABPs, which specifically bind to human CTLA-4 (SEQ ID NO: 7001) or a fragment of human CTLA-4.

[0011] In particular, in one aspect, the present disclosure provides GIGA-564, a CTLA-4 monoclonal antibody that has minimal ability to block CTLA-4 binding to its CD80 / CD86 ligands, but has excellent anti-tumor activity with reduced toxicity. Anti-CTLA-4 antibodies have been demonstrated to induce lower peripheral Treg proliferation and more efficient intratumoral Treg depletion in mouse models expressing human CTLA-4. The anti-tumor activity of the anti-CTLA-4 antibody was further enhanced when it was further defucosylated (GIGA-2328).

[0012] The present disclosure also provides that the anti-CTLA-4 monoclonal antibodies bind to CTLA-4 at a different epitope from other known anti-CTLA-4 antibodies (e.g., ipilimumab) and have limited checkpoint inhibitor activity, making them weak checkpoint inhibitors. Surprisingly, the efficacy of the anti-CTLA-4 antibodies described herein was found to be associated with reduced FcR-mediated Treg depletion and proliferation of remaining Tregs in the tumor microenvironment. The anti-CTLA-4 antibodies also induced lower Treg proliferation and had enhanced ability to induce in vitro FcR signaling and in vivo intratumoral Treg depletion. The experimental results described herein suggest that the enhanced FcR activity of weak checkpoint inhibitors likely contributes to their enhanced antitumor activity. They also show that weak checkpoint inhibition was associated with lower toxicity in mouse models.

[0013] Furthermore, it has been demonstrated that the anti-CTLA-4 monoclonal antibodies provided herein can enhance the anti-tumor effect in combination with anti-PD-1 antibodies, suggesting that anti-CTLA-4 antibodies may be effective against tumors resistant to anti-PD-1 antibodies. Based on these studies, the present disclosure provides methods for treating cancers resistant to anti-PD-1 or anti-PD-L1 therapy. Also provided are dosing regimens and pharmaceutical formulations that can be used in the treatment methods.

[0014] Accordingly, the present disclosure provides a method of treating cancer, comprising administering to a cancer patient an effective amount of an antigen binding protein that specifically binds to human cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA-4 ABP), wherein the anti-CTLA-4 ABP comprises a CDR1-L consisting of SEQ ID NO: 12078, a CDR2-L consisting of SEQ ID NO: 12079, a CDR3-L consisting of SEQ ID NO: 12080, a CDR1-H consisting of SEQ ID NO: 12075, a CDR2-H consisting of SEQ ID NO: 12076, and a CDR3-H consisting of SEQ ID NO: 12077.

[0015] In some embodiments, the cancer is resistant to anti-PD-1 or anti-PD-L1 therapy. In some embodiments, the cancer is resistant to anti-PD-1 antibody or anti-PD-L1 antibody therapy. In some embodiments, the cancer patient has progressed or relapsed after anti-PD-1 or anti-PD-L1 therapy. In some embodiments, the method further comprises determining whether the cancer is resistant to anti-PD-1 therapy or anti-PD-L1 therapy.

[0016] In some embodiments, the cancer patient has melanoma, RCC (renal cell carcinoma), NSCLC (non-small cell lung cancer), Merkel cell carcinoma, cSCC, mesothelioma, hepatocellular carcinoma, esophageal cancer, breast cancer, sarcoma, MSI-Hi / dMMR colorectal cancer, ovarian cancer, or cervical cancer, bladder cancer, prostate cancer, TMB-HI tumors of any origin, tumors that are MSI, tumors that are dMMR, T-cell leukemia / lymphoma, NHL, tumors in which the cancer cells express CTLA-4.

[0017] In some embodiments, the method further comprises administering an antigen binding protein that specifically binds to human PD-1 or anti-PD-L1 (anti-PD-1 ABP or anti-PD-L1 ABP). In some embodiments, the anti-PD-1 ABP is pembrolizumab. In some embodiments, the anti-CTLA-4 ABP and anti-PD-1 ABP are administered in a weight ratio selected from 3:1, 3:10, 1:3, 1:10, 10:1, 10:3, 9:1, and 1:1. In some embodiments, the anti-CTLA-4 ABP and anti-PD-1 ABP are administered in a weight ratio of 2:1 to 10:1. In some embodiments, the anti-CTLA-4 ABP and anti-PD-1 ABP are administered in a weight ratio of 1:1 to 1:10. In some embodiments, the anti-CTLA-4 ABP and anti-PD-1 ABP are administered in a weight ratio of 3:1.

[0018] In some embodiments, the anti-CTLA-4 ABP and the anti-PD-1 ABP are administered on the same day. In some embodiments, the anti-CTLA-4 ABP and the anti-PD-1 ABP are administered on different days. In some embodiments, the anti-CTLA-4 ABP and the anti-PD-L1 ABP are administered on the same day. In some embodiments, the anti-CTLA-4 ABP and the anti-PD-L1 ABP are administered on different days.

[0019] In some embodiments, the effective amount of anti-CTLA-4 ABP is less than 30 mg / kg. In some embodiments, the effective amount of anti-CTLA-4 ABP is at least 0.01 mg / kg, 0.03 mg / kg, 0.1 mg / kg, 0.3 mg / kg, or 1 mg / kg. In some embodiments, the effective amount of anti-CTLA-4 ABP is 0.5 mg / kg to 30 mg / kg. In some embodiments, the effective amount of anti-CTLA-4 ABP is 1 mg / kg to 18 mg / kg. In some embodiments, the effective amount of anti-CTLA-4 ABP is 1 mg / kg to 10 mg / kg. In some embodiments, the effective amount of anti-CTLA-4 ABP is 1 mg / kg, 3 mg / kg, or 30 mg / kg. In some embodiments, the effective amount of anti-CTLA-4 ABP is 9 mg / kg or 27 mg / kg. In some embodiments, the effective amount of anti-CTLA-4 ABP is 50 mg to 2500 mg. In some embodiments, the effective amount of anti-CTLA-4 ABP is 50 mg to 1000 mg. In some embodiments, the effective amount of anti-CTLA-4 ABP is 70 mg to 150 mg, 150 mg to 500 mg, 500 mg to 800 mg, 700 mg to 900 mg, 800 mg to 1200 mg, 1200 mg to 1500 mg, or 1500 mg to 2500 mg. In some embodiments, the effective amount of anti-CTLA-4 ABP is 80 mg, 240 mg, 720 mg, 800 mg, 1440 mg, or 2160 mg. In some embodiments, the anti-CTLA-4 ABP is administered at a dose of 50 mg, 100 mg, 150 mg, 250 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1500 mg, 2000 mg, or 2500 mg per administration.

[0020] In some embodiments, the anti-CTLA-4 ABP comprises a variable light chain (VL) comprising a sequence at least 97% identical to SEQ ID NO: 14, and a variable heavy chain (VH) comprising a sequence at least 97% identical to SEQ ID NO: 114. In some embodiments, the anti-CTLA-4 ABP comprises a variable light chain (VL) comprising the sequence of SEQ ID NO: 14, and a variable heavy chain (VH) comprising the sequence of SEQ ID NO: 114.

[0021] In some embodiments, the anti-CTLA-4 ABP comprises an scFv or a full-length monoclonal antibody. In some embodiments, the anti-CTLA-4 ABP comprises an immunoglobulin constant region. In some embodiments, the anti-CTLA-4 ABP is an IgG1 ABP. In some embodiments, the anti-CTLA-4 ABP is an IGHG1 ABP. * 01 human heavy chain constant region gene segment. In some embodiments, the anti-CTLA-4 ABP comprises a lysine at amino acid position 97 (R97) according to the IMGT exon numbering. In some embodiments, the anti-CTLA-4 ABP comprises a lysine at amino acid position 97 (R214) according to the EU numbering.

[0022] In some embodiments, the anti-CTLA-4 ABP comprises a defucosylated Fc region. In some embodiments, the anti-CTLA-4 ABP is produced from cells comprising the bacterial protein RMD (GDP-6-deoxy-D-lyxo-4-hexulose reductase) or a variant thereof. In some embodiments, the cells are cultured in the absence of fucose. In some embodiments, the anti-CTLA-4 ABP is produced from cells lacking or having reduced expression of Fut8. In some embodiments, the anti-CTLA-4 ABP is produced from cells cultured in the presence of the fucosylation inhibitor 2-Fluorfucose (2FF). In some embodiments, the anti-CTLA-4 ABP is produced from cells overexpressing glycosyltransferase (GnTIII). In some embodiments, the anti-CTLA-4 ABP is isolated based on its fucosylation status. In some embodiments, the anti-CTLA-4 ABP comprises an Fc region lacking core fucosylation of N-glycans in the Fc portion. In some embodiments, the ABP is a defucosylated monoclonal antibody.

[0023] In some embodiments, the anti-CTLA-4 ABP is administered in a pharmaceutical composition. In some embodiments, the pharmaceutical composition has a pH of 5.0 to 6.5. In some embodiments, the pharmaceutical composition has a pH of 6.0 to 6.5. In some embodiments, the pharmaceutical composition comprises 20 mM histidine or citrate buffer. In some embodiments, the pharmaceutical composition comprises 20 mM histidine. In some embodiments, the pharmaceutical composition comprises 50 mM NaCl. In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of 170 mM to 270 mM. In some embodiments, the pharmaceutical composition comprises 0.1 to 1 mg / mL polysorbate 20. In some embodiments, the pharmaceutical composition comprises 0.2 mg / mL polysorbate 20. In some embodiments, the pharmaceutical composition comprises 20 mM histidine, 270 mM sucrose, and 0.2 mg / mL polysorbate 20, and has a pH of 6.2.

[0024] In some embodiments, the pharmaceutical composition comprises 5 mg / mL to 20 mg / mL of anti-CTLA-4 ABP. In some embodiments, the pharmaceutical composition comprises 20 mg / mL of anti-CTLA-4 ABP. In some embodiments, the pharmaceutical composition comprises 10 mg / mL of anti-CTLA-4 ABP. In some embodiments, the pharmaceutical composition comprises 5 mg / mL of anti-CTLA-4 ABP.

[0025] In some embodiments, the step of administering the anti-CTLA-4 ABP is repeated. In some embodiments, the step of administering the anti-CTLA-4 ABP is repeated at least two, three, four, or more times. In some embodiments, the step of administering the anti-CTLA-4 ABP is repeated every day, every two, three, four, five, or six days. In some embodiments, the step of administering the anti-CTLA-4 ABP is repeated every week, every two, three, four, five, six, or seven weeks. In some embodiments, the step of administering the anti-CTLA-4 ABP is repeated every 1-2 weeks, every 2-3 weeks, every 3-4 weeks, every 4-5 weeks, every 5-6 weeks, every 6-7 weeks, every 7-8 weeks, every 8-9 weeks, every 9-10 weeks, every 10-11 weeks, every 11-12 weeks, every 12-13 weeks, every 13-14 weeks, or every 14-15 weeks. In some embodiments, the step of administering the anti-CTLA-4 ABP is repeated monthly, every 2 months, every 3 months, every 4 months, every 5 months, or less frequently. In some embodiments, the step of administering the anti-CTLA-4 ABP is repeated every 1-2 months, every 2-3 months, every 3-4 months, every 4-5 months, or every 5-6 months. In some embodiments, an anti-PD-1 antibody or anti-PD-L1 antibody is administered in combination with an anti-CTLA-4 ABP in each of the repeated administrations. In some embodiments, the anti-PD-1 antibody or anti-PD-L1 antibody is administered in combination with an anti-CTLA-4 ABP in some but not all of the repeated doses.

[0026] In one aspect, the disclosure provides a pharmaceutical composition comprising an anti-CTLA-4 ABP and a pharmaceutically acceptable excipient, wherein the anti-CTLA-4 ABP is an isolated antigen binding protein (ABP) that specifically binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and comprises a CDR1-L consisting of SEQ ID NO: 12078, a CDR2-L consisting of SEQ ID NO: 12079, a CDR3-L consisting of SEQ ID NO: 12080, a CDR1-H consisting of SEQ ID NO: 12075, a CDR2-H consisting of SEQ ID NO: 12076, and a CDR3-H consisting of SEQ ID NO: 12077.

[0027] In some embodiments, the anti-CTLA-4 ABP comprises a variable light chain (VL) comprising the sequence of SEQ ID NO:14 and a variable heavy chain (VH) comprising the sequence of SEQ ID NO:114.

[0028] In some embodiments, the pharmaceutical composition has a pH of 5.0 to 6.5. In some embodiments, the pharmaceutical composition has a pH of 6.0 to 6.5. In some embodiments, the pharmaceutical composition has a pH of 6.2.

[0029] In some embodiments, the pharmaceutical composition comprises 20 mM histidine or citrate buffer. In some embodiments, the pharmaceutical composition comprises 20 mM histidine. In some embodiments, the pharmaceutical composition comprises 50 mM NaCl. In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of 170 mM to 270 mM. In some embodiments, the pharmaceutical composition comprises 0.1 to 1 mg / mL polysorbate 20. In some embodiments, the pharmaceutical composition comprises 0.2 mg / mL polysorbate 20. In some embodiments, the pharmaceutical composition comprises 20 mM histidine, 270 mM sucrose, and 0.02% PS-20, and has a pH of 6.2.

[0030] In some embodiments, the pharmaceutical composition comprises 5 mg / mL to 20 mg / mL of anti-CTLA-4 ABP. In some embodiments, the pharmaceutical composition comprises 20 mg / mL of anti-CTLA-4 ABP. In some embodiments, the pharmaceutical composition comprises 10 mg / mL of anti-CTLA-4 ABP. In some embodiments, the pharmaceutical composition comprises 5 mg / mL of anti-CTLA-4 ABP.

[0031] In some embodiments, less than 50% of the anti-CTLA-4 ABPs are fucosylated. In some embodiments, less than 40% of the anti-CTLA-4 ABPs are fucosylated. In some embodiments, less than 30% of the anti-CTLA-4 ABPs are fucosylated. In some embodiments, less than 20% of the anti-CTLA-4 ABPs are fucosylated. In some embodiments, less than 10% of the anti-CTLA-4 ABPs are fucosylated. In some embodiments, between 3% and 30% of the anti-CTLA-4 ABPs are fucosylated. In some embodiments, between 10% and 30% of the anti-CTLA-4 ABPs are fucosylated. In some embodiments, between 15% and 25% of the anti-CTLA-4 ABPs are fucosylated.

[0032] In some embodiments, the pharmaceutical composition is formulated for injection, hi some embodiments, the pharmaceutical composition is formulated for iv infusion.

[0033] The present disclosure further provides unit dose forms of pharmaceutical compositions. In some embodiments, the unit dose comprises 50 mg to 5000 mg of anti-CTLA-4 ABP. In some embodiments, the unit dose comprises 50 mg to 2500 mg of anti-CTLA-4 ABP. In some embodiments, the unit dose comprises 10 mg to 2000 mg of anti-CTLA-4 ABP. In some embodiments, the unit dose comprises anti-CTLA-4 ABP in an amount of 70 mg to 150 mg, 150 mg to 500 mg, 500 mg to 800 mg, 700 mg to 900 mg, 800 mg to 1200 mg, 1200 mg to 1500 mg, or 1500 mg to 2500 mg. In some embodiments, the unit dose comprises anti-CTLA-4 ABP in an amount of 80 mg, 240 mg, 720 mg, 800 mg, 1440 mg, or 2160 mg. In some embodiments, the unit dose comprises an anti-CTLA-4 ABP in an amount of 50 mg, 100 mg, 150 mg, 250 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1500 mg, 2000 mg, or 2500 mg in each administration.

[0034] In some embodiments, when bound to CTLA-4, the ABP contacts amino acids K130, Y139, L141, and I143 of CTLA-4, but not amino acid R70, or R70 is not a major energetic contributor to the interaction between CTLA-4 and the ABP; and / or CTLA-4, when bound to the ABP, can associate with CD80 / CD86; and / or the interaction between the ABP and amino acid L74A and / or E68 of CTLA-4 is greater than the interaction between ipilimumab and amino acid L74A of CTLA-4.

[0035] In some embodiments, the ABP comprises a CDR1-L consisting of SEQ ID NO: 12078 or SEQ ID NO: 1014, a CDR2-L consisting of SEQ ID NO: 12079 or SEQ ID NO: 2014, a CDR3-L consisting of SEQ ID NO: 12080 or SEQ ID NO: 3014, a CDR1-H consisting of SEQ ID NO: 12075 or SEQ ID NO: 4014, a CDR2-H consisting of SEQ ID NO: 12076 or SEQ ID NO: 5014, and a CDR3-H consisting of SEQ ID NO: 12077 or SEQ ID NO: 6014. In some embodiments, the ABP comprises a CDR1-L consisting of SEQ ID NO: 12004, a CDR2-L consisting of SEQ ID NO: 12014, a CDR3-L consisting of SEQ ID NO: 12024, a CDR1-H consisting of SEQ ID NO: 12039, a CDR2-H consisting of SEQ ID NO: 12049, and a CDR3-H consisting of SEQ ID NO: 12059. In some embodiments, the ABP comprises a CDR1-L consisting of SEQ ID NO: 12005, a CDR2-L consisting of SEQ ID NO: 12015, a CDR3-L consisting of SEQ ID NO: 12025, a CDR1-H consisting of SEQ ID NO: 12040, a CDR2-H consisting of SEQ ID NO: 12050, and a CDR3-H consisting of SEQ ID NO: 12060. In some embodiments, the ABP comprises a CDR1-L consisting of SEQ ID NO: 12006, a CDR2-L consisting of SEQ ID NO: 12016, a CDR3-L consisting of SEQ ID NO: 12026, a CDR1-H consisting of SEQ ID NO: 12041, a CDR2-H consisting of SEQ ID NO: 12051, and a CDR3-H consisting of SEQ ID NO: 12061. In some embodiments, the ABP comprises a CDR1-L consisting of SEQ ID NO: 12007, a CDR2-L consisting of SEQ ID NO: 12017, a CDR3-L consisting of SEQ ID NO: 12027, a CDR1-H consisting of SEQ ID NO: 12042, a CDR2-H consisting of SEQ ID NO: 12052, and a CDR3-H consisting of SEQ ID NO: 12062. In some embodiments, the ABP comprises a CDR1-L consisting of SEQ ID NO: 12008, a CDR2-L consisting of SEQ ID NO: 12018, a CDR3-L consisting of SEQ ID NO: 12028, a CDR1-H consisting of SEQ ID NO: 12043, a CDR2-H consisting of SEQ ID NO: 12053, and a CDR3-H consisting of SEQ ID NO: 12063.

[0036] In some embodiments, the ABP comprises a variable light chain (V) comprising a sequence that is at least 97% identical to SEQ ID NO: 14.L ) and a variable heavy chain (V) comprising a sequence at least 97% identical to SEQ ID NO: 114. H ) and

[0037] In some embodiments, the ABP comprises an scFv or a full-length monoclonal antibody. In some embodiments, the ABP comprises an immunoglobulin constant region.

[0038] In some embodiments, the ABP has a K of less than 500 nM as measured by surface plasmon resonance. D ABP binds to human CTLA-4 with a K of less than 200 nM as measured by surface plasmon resonance. D ABP binds to human CTLA-4 with a K of less than 25 nM as measured by surface plasmon resonance. D ABP binds to human CTLA-4 with a K of less than 25 nM. D It binds to human CTLA-4 on the cell surface.

[0039] In some embodiments, the ABP is an IgG1 ABP. In some embodiments, the ABP is an IGHG1 ABP. * 01 human heavy chain constant region gene segment. In some embodiments, the ABP comprises a lysine at amino acid position 97 (R97) according to the IMGT exon numbering. In some embodiments, the ABP comprises a lysine at amino acid position 97 (R214) according to the EU numbering.

[0040] In some embodiments, the ABP comprises a defucosylated Fc region.

[0041] In some embodiments, the ABP is produced from cells containing the bacterial protein RMD (GDP-6-deoxy-D-lyxo-4-hexulose reductase) or a variant thereof, and in some embodiments, the cells are cultured in the absence of fucose.

[0042] In some embodiments, the ABP is produced from cells lacking or having reduced expression of Fut8. In some embodiments, the ABP is produced from cells cultured in the presence of 2-Fluorfucose (2FF), a fucosylation inhibitor. In some embodiments, the ABP is produced from cells overexpressing glycosyltransferase (GnTIII). In some embodiments, the ABP is isolated based on its fucosylation status.

[0043] In some embodiments, the ABP comprises an Fc region that lacks core fucosylation of N-glycans in the Fc portion, hi some embodiments, the ABP is a defucosylated monoclonal antibody.

[0044] Aspects of the present disclosure also include pharmaceutical compositions comprising an ABP of the present disclosure and a pharmaceutically acceptable excipient.

[0045] In some embodiments, less than 50% of the ABPs are fucosylated. In some embodiments, less than 40% of the ABPs are fucosylated. In some embodiments, less than 30% of the ABPs are fucosylated. In some embodiments, less than 20% of the ABPs are fucosylated. In some embodiments, less than 10% of the ABPs are fucosylated. In some embodiments, more than 30% of the ABPs are fucosylated. In some embodiments, more than 40% of the ABPs are fucosylated. In some embodiments, more than 50% of the ABPs are fucosylated. In some embodiments, more than 60% of the ABPs are fucosylated. In some embodiments, more than 70% of the ABPs are fucosylated. In some embodiments, more than 80% of the ABPs are fucosylated. In some embodiments, more than 90% of the ABPs are fucosylated.

[0046] In some embodiments, the pharmaceutical composition has a pH of 5.0 to 6.5. In some embodiments, the pharmaceutical composition comprises 20 mM histidine or citrate buffer. In some embodiments, the pharmaceutical composition comprises 50 mM NaCl. In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of 170 mM to 270 mM. In some embodiments, the pharmaceutical composition comprises 170 mM or 270 mM sucrose. In some embodiments, the pharmaceutical composition comprises 5 mg / mL to 20 mg / mL ABP. In some embodiments, the pharmaceutical composition comprises 20 mg / mL ABP. In some embodiments, the pharmaceutical composition comprises 5 mg / mL ABP.

[0047] An aspect of the present disclosure provides a method of treating a disease, comprising administering an effective amount of any of the ABPs disclosed herein or a pharmaceutical composition thereof to a subject in need thereof.

[0048] In some embodiments, the disease is selected from the group consisting of cancer, AIDS, Alzheimer's disease, and a viral or bacterial infection, hi some embodiments, the disease is selected from the group consisting of an autoimmune disease, an autoinflammatory disease, and inflammation.

[0049] In some embodiments, the method further comprises administering to the subject one or more additional therapeutic agents. In some embodiments, the additional therapeutic agents are selected from anti-PD-L1, anti-PD1, a LAG-3 inhibitor, a CD47 inhibitor, a TIGIT inhibitor, a chemotherapeutic agent, an immunostimulant, radiation, a BRAF inhibitor, a MEK inhibitor, a PI3K inhibitor, a cytokine, a polynucleotide encoding a cytokine, an oncolytic virus encoding a cytokine, and combinations thereof. In some embodiments, the method further comprises adoptive cell therapy or treatment with a cancer vaccine, an oncolytic virus, or an anti-CD40 inhibitor.

[0050] Aspects of the present disclosure include an isolated polynucleotide encoding an ABP. Aspects of the present disclosure include a vector comprising the isolated polynucleotide. Aspects of the present disclosure provide a host cell comprising an isolated polynucleotide or vector of the present disclosure. In some embodiments, the host cell further comprises the bacterial protein RMD (GDP-6-deoxy-D-lyxo-4-hexulose reductase). In some embodiments, the host cell is cultured in the absence of fucose. In some embodiments, the host cell lacks or has reduced expression of Fut8. In some embodiments, the host cell is cultured in the presence of 2-Fluorfucose (2FF), a fucosylation inhibitor. In some embodiments, the host cell overexpresses glycosyltransferase (GnTIII). In some embodiments, the host cell is a CHOZN GS-derived cell line that utilizes the 2G UNic membrane translation enhancer element.

[0051] An embodiment of the present disclosure provides a method of producing an isolated antigen binding protein (ABP) that specifically binds to human CTLA-4, the method comprising inducing expression of the ABP in a host cell of the present disclosure and isolating the ABP.

[0052] In some embodiments, the method further comprises isolating the ABP based on its fucosylation state. In some embodiments, the host cell is cultured in a culture medium containing a fucosylation inhibitor. In some embodiments, the fucosylation inhibitor is 2-Fluorfucose (2FF).

[0053] CTLA-4 in subjects with limited residual Treg expansion HI A method for reducing Tregs, comprising administering an effective amount of an ABP or pharmaceutical composition described herein.

[0054] In some embodiments, the subject is a human subject, optionally a human subject with RCC (renal cell carcinoma), NSCLC (non-small cell lung cancer), Merkel cell carcinoma, cSCC, mesothelioma, MSI colorectal cancer, ovarian cancer, or cervical cancer.

[0055] In some embodiments, the method further comprises administering to the subject one or more additional therapeutic agents, hi some embodiments, the additional therapeutic agents are anti-PD-L1 or anti-PD1, or a combination thereof.

[0056] In another aspect, the present disclosure provides an isolated antigen binding protein (ABP) that specifically binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the ABP comprising: (a) a CDR1-L consisting of SEQ ID NO: 12078, a CDR2-L consisting of SEQ ID NO: 12079, a CDR3-L consisting of SEQ ID NO: 12080, a CDR1-H consisting of SEQ ID NO: 12075, a CDR2-H consisting of SEQ ID NO: 12076, and a CDR3-H consisting of SEQ ID NO: 12077; (b) a CDR1-L consisting of SEQ ID NO: 1014, a CDR2-L consisting of SEQ ID NO: 12014, a CDR3-H consisting of SEQ ID NO: 12016, and a CDR3-H consisting of SEQ ID NO: 12017; (c) CDR1-L consisting of SEQ ID NO: 12004, CDR2-L consisting of SEQ ID NO: 12014, CDR3-L consisting of SEQ ID NO: 12024, CDR1-H consisting of SEQ ID NO: 12039, CDR2-H consisting of SEQ ID NO: 12049, and CDR3-H consisting of SEQ ID NO: 12059; (d) CDR1-L consisting of SEQ ID NO: 12005, CDR2-L consisting of SEQ ID NO: 1201 (e) CDR1-L consisting of SEQ ID NO: 12006, CDR2-L consisting of SEQ ID NO: 12016, CDR3-L consisting of SEQ ID NO: 12026, CDR1-H consisting of SEQ ID NO: 12041, CDR2-H consisting of SEQ ID NO: 12051, and CDR3-H consisting of SEQ ID NO: 12061; (f) CDR consisting of SEQ ID NO: 12007 1-L, CDR2-L consisting of SEQ ID NO: 12017, CDR3-L consisting of SEQ ID NO: 12027, CDR1-H consisting of SEQ ID NO: 12042, CDR2-H consisting of SEQ ID NO: 12052, and CDR3-H consisting of SEQ ID NO: 12062, or (g) CDR1-L consisting of SEQ ID NO: 12008, CDR2-L consisting of SEQ ID NO: 12018, CDR3-L consisting of SEQ ID NO: 12028, CDR1-H consisting of SEQ ID NO: 12043, CDR2-H consisting of SEQ ID NO: 12053, and CDR3-H consisting of SEQ ID NO: 12063.

[0057] In some embodiments, the ABP comprises a variable light chain (V) comprising a sequence that is at least 97% identical to SEQ ID NO: 14. L ) and a variable heavy chain (V) comprising a sequence at least 97% identical to SEQ ID NO: 114. H ) and

[0058] In some embodiments, the ABP comprises an scFv or a full-length monoclonal antibody. In some embodiments, the ABP comprises an immunoglobulin constant region.

[0059] In some embodiments, the ABP has a K of less than 500 nM as measured by surface plasmon resonance. D ABP binds to human CTLA-4 with a K of less than 200 nM as measured by surface plasmon resonance. D ABP binds to human CTLA-4 with a K of less than 25 nM as measured by surface plasmon resonance. D ABP binds to human CTLA-4 with a K of less than 25 nM. D It binds to human CTLA-4 on the cell surface.

[0060] In some embodiments, the ABP is an IgG1 ABP. In some embodiments, the ABP is an IGHG1 ABP. * 01 human heavy chain constant region gene segment. In some embodiments, the ABP comprises a lysine at amino acid position 97 (R97) according to the IMGT exon numbering. In some embodiments, the ABP comprises a lysine at amino acid position 97 (R214) according to the EU numbering.

[0061] In some embodiments, the ABP comprises a defucosylated Fc region.

[0062] In some embodiments, the ABP is produced from cells containing the bacterial protein RMD (GDP-6-deoxy-D-lyxo-4-hexulose reductase) or a variant thereof. In some embodiments, the cells are cultured in the absence of fucose. In some embodiments, the ABP is produced from cells lacking or having reduced expression of Fut8. In some embodiments, the ABP is produced from cells cultured in the presence of the fucosylation inhibitor 2-Fluorfucose (2FF). In some embodiments, the ABP is produced from cells overexpressing glycosyltransferase (GnTIII). In some embodiments, the ABP is isolated based on its fucosylation status.

[0063] In some embodiments, the ABP comprises an Fc region lacking core fucosylation of N-glycans in the Fc portion. In some embodiments, the ABP is a defucosylated monoclonal antibody. In some embodiments, the defucosylated Fc region has less than 30% fucosylation, and less than 30% fucosylation enhances FcgRIII (Fc gamma receptor III) signaling. In some embodiments, the defucosylated Fc region has less than 30% fucosylation, and less than 30% fucosylation enhances FcgRIIIa (Fc gamma receptor IIIa) signaling.

[0064] An aspect of the present disclosure includes a pharmaceutical composition comprising an ABP of the present disclosure and a pharmaceutically acceptable excipient.

[0065] In some embodiments, less than 50% of the ABPs are fucosylated. In some embodiments, less than 40% of the ABPs are fucosylated. In some embodiments, less than 30% of the ABPs are fucosylated. In some embodiments, less than 20% of the ABPs are fucosylated. In some embodiments, less than 10% of the ABPs are fucosylated. In some embodiments, more than 30% of the ABPs are fucosylated. In some embodiments, more than 40% of the ABPs are fucosylated. In some embodiments, more than 50% of the ABPs are fucosylated. In some embodiments, more than 60% of the ABPs are fucosylated. In some embodiments, more than 70% of the ABPs are fucosylated. In some embodiments, more than 80% of the ABPs are fucosylated. In some embodiments, more than 90% of the ABPs are fucosylated.

[0066] In some embodiments, the pharmaceutical composition has a pH of 5.0 to 6.5. In some embodiments, the pharmaceutical composition comprises 20 mM histidine or citrate buffer. In some embodiments, the pharmaceutical composition comprises 50 mM NaCl. In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of 170 mM to 270 mM. In some embodiments, the pharmaceutical composition comprises 170 mM or 270 mM sucrose. In some embodiments, the pharmaceutical composition comprises 5 mg / mL to 20 mg / mL ABP. In some embodiments, the pharmaceutical composition comprises 20 mg / mL ABP. In some embodiments, the pharmaceutical composition comprises 5 mg / mL ABP.

[0067] An aspect of the present disclosure provides a method of treating a disease, comprising administering an effective amount of any of the ABPs disclosed herein or a pharmaceutical composition thereof to a subject in need thereof.

[0068] In some embodiments, the disease is selected from the group consisting of cancer, AIDS, Alzheimer's disease, and a viral or bacterial infection, hi some embodiments, the disease is selected from the group consisting of an autoimmune disease, an autoinflammatory disease, and inflammation.

[0069] In some embodiments, the method further comprises administering to the subject one or more additional therapeutic agents. In some embodiments, the additional therapeutic agents are selected from anti-PD-L1, anti-PD1, a LAG-3 inhibitor, a CD47 inhibitor, a TIGIT inhibitor, a chemotherapeutic agent, an immunostimulant, radiation, a BRAF inhibitor, a MEK inhibitor, a PI3K inhibitor, a cytokine, a polynucleotide encoding a cytokine, an oncolytic virus encoding a cytokine, and combinations thereof. In some embodiments, the method further comprises adoptive cell therapy or treatment with a cancer vaccine, an oncolytic virus, or an anti-CD40 inhibitor.

[0070] Aspects of the present disclosure include an isolated polynucleotide encoding an ABP. Aspects of the present disclosure include a vector comprising the isolated polynucleotide. Aspects of the present disclosure provide a host cell comprising an isolated polynucleotide or vector of the present disclosure. In some embodiments, the host cell further comprises the bacterial protein RMD (GDP-6-deoxy-D-lyxo-4-hexulose reductase). In some embodiments, the host cell is cultured in the absence of fucose. In some embodiments, the host cell lacks or has reduced expression of Fut8. In some embodiments, the host cell is cultured in the presence of 2-Fluorfucose (2FF), a fucosylation inhibitor. In some embodiments, the host cell overexpresses glycosyltransferase (GnTIII).

[0071] Aspects of the present disclosure provide methods of treating cancer, comprising administering an effective amount of an ABP or pharmaceutical composition of the present disclosure to a subject in need thereof. In some embodiments, the subject has a malignant tumor. In some embodiments, the ABP, when administered, induces increased Fc receptor (FcR) signaling compared to ipilimumab, and the administration increases CTLA-4 expression in the subject. HIIn some embodiments, the administration reduces the amount of Tregs in the subject compared to ipilimumab. In some embodiments, the administration reduces tumors more effectively than ipilimumab.

[0072] In some embodiments, the method further comprises administering to the subject one or more additional therapeutic agents. In some embodiments, the additional therapeutic agents are selected from anti-PD-L1, anti-PD1, a TIGIT inhibitor, a LAG-3 inhibitor, a CD47 inhibitor, a BRAF inhibitor, a MEK inhibitor, a PI3K inhibitor, a chemotherapeutic agent, an immunostimulant, radiation, a cytokine, a polynucleotide encoding a cytokine, an oncolytic virus encoding a cytokine, and combinations thereof. In some embodiments, the method further comprises adoptive cell therapy or treatment with a cancer vaccine, an oncolytic virus, or an anti-CD40 inhibitor.

[0073] In some embodiments, the ABP comprises a defucosylated Fc region with less than 30% fucosylation, wherein less than 30% fucosylation enhances FcgRIII signaling. In some embodiments, the ABP comprises a defucosylated Fc region with less than 30% fucosylation, wherein less than 30% fucosylation enhances FcgRIIIa signaling. In some embodiments, the ABP comprises a fucosylated Fc region with more than 70% fucosylation.

[0074] Aspects of the present disclosure include an isolated polynucleotide encoding an ABP. Aspects of the present disclosure include a vector comprising the isolated polynucleotide. Aspects of the present disclosure provide a host cell comprising an isolated polynucleotide or vector of the present disclosure. In some embodiments, the host cell further comprises the bacterial protein RMD (GDP-6-deoxy-D-lyxo-4-hexulose reductase). In some embodiments, the host cell is cultured in the absence of fucose. In some embodiments, the host cell lacks or has reduced expression of Fut8. In some embodiments, the host cell is cultured in the presence of 2-Fluorfucose (2FF), a fucosylation inhibitor. In some embodiments, the host cell overexpresses glycosyltransferase (GnTIII).

[0075] An embodiment of the present disclosure provides a method of producing an isolated antigen binding protein (ABP) that specifically binds to human CTLA-4, the method comprising inducing expression of the ABP in a host cell of the present disclosure and isolating the ABP, wherein the ABP comprises a defucosylated Fc.

[0076] In some embodiments, the method further comprises isolating the ABP based on its fucosylation state. In some embodiments, the host cell is cultured in a culture medium containing a fucosylation inhibitor. In some embodiments, the fucosylation inhibitor is 2-Fluorfucose (2FF).

[0077] In another aspect, the present disclosure provides an isolated antigen binding protein (ABP) that specifically binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), comprising: * 01 human heavy chain constant region gene segment.

[0078] In some embodiments, the ABP comprises (a) a CDR1-L consisting of SEQ ID NO: 12078, a CDR2-L consisting of SEQ ID NO: 12079, a CDR3-L consisting of SEQ ID NO: 12080, a CDR1-H consisting of SEQ ID NO: 12075, a CDR2-H consisting of SEQ ID NO: 12076, and a CDR3-H consisting of SEQ ID NO: 12077; (b) a CDR1-L consisting of SEQ ID NO: 1014, a CDR2-L consisting of SEQ ID NO: 2014, a CDR3-L consisting of SEQ ID NO: 3014, a CDR1-H consisting of SEQ ID NO: 4014, and a CDR3-H consisting of SEQ ID NO: 4015; (c) CDR1-L consisting of SEQ ID NO: 12004, CDR2-L consisting of SEQ ID NO: 12014, CDR3-L consisting of SEQ ID NO: 12024, CDR1-H consisting of SEQ ID NO: 12039, CDR2-H consisting of SEQ ID NO: 12049, and CDR3-H consisting of SEQ ID NO: 12059; (d) CDR1-L consisting of SEQ ID NO: 12005, CDR2-L consisting of SEQ ID NO: 12015, and CDR3-H consisting of SEQ ID NO: 12025 (e) CDR1-L consisting of SEQ ID NO: 12006, CDR2-L consisting of SEQ ID NO: 12016, CDR3-L consisting of SEQ ID NO: 12026, CDR1-H consisting of SEQ ID NO: 12041, CDR2-H consisting of SEQ ID NO: 12051, and CDR3-H consisting of SEQ ID NO: 12061; (f) CDR1-L consisting of SEQ ID NO: 12007, CDR2-L consisting of SEQ ID NO: 12017 (g) a CDR1-L consisting of SEQ ID NO: 12008, a CDR2-L consisting of SEQ ID NO: 12018, a CDR3-L consisting of SEQ ID NO: 12028, a CDR1-H consisting of SEQ ID NO: 12043, a CDR2-H consisting of SEQ ID NO: 12053, and a CDR3-H consisting of SEQ ID NO: 12063. In some embodiments, the ABP comprises a variable light chain (V) comprising a sequence at least 97% identical to SEQ ID NO: 14. L ) and a variable heavy chain (V) comprising a sequence at least 97% identical to SEQ ID NO: 114. H) and

[0079] In some embodiments, the ABP comprises (a) a CDR1-L consisting of any one of SEQ ID NOs: 1001-1028, a CDR2-L consisting of any one of SEQ ID NOs: 1001-1028, a CDR3-L consisting of any one of SEQ ID NOs: 2001-2028, a CDR1-H consisting of any one of SEQ ID NOs: 3001-3028, and a CDR2-H consisting of any one of SEQ ID NOs: 3001-3028. In some embodiments, the ABP comprises a variable light chain (V) comprising a sequence at least 97% identical to any one of SEQ ID NOs: 1-28. L ) and a variable heavy chain (V) comprising a sequence at least 97% identical to SEQ ID NOs: 1 to 128. H ) and

[0080] In some embodiments, the ABP comprises a lysine at amino acid position 97 (R97) according to the IMGT exon numbering. In some embodiments, the ABP comprises a lysine at amino acid position 97 (R214) according to the EU numbering. In some embodiments, the ABP comprises a defucosylated Fc region. In some embodiments, the ABP is produced from cells comprising the bacterial protein RMD (GDP-6-deoxy-D-lyxo-4-hexulose reductase) or a variant thereof. In some embodiments, the cells are cultured in the absence of fucose. In some embodiments, the ABP is produced from cells lacking or having reduced expression of Fut8. In some embodiments, the ABP is produced from cells cultured in the presence of the fucosylation inhibitor 2-Fluorfucose (2FF). In some embodiments, the ABP is produced from cells overexpressing glycosyltransferase (GnTIII). In some embodiments, the ABP is isolated based on its fucosylation status. In some embodiments, the ABP comprises an Fc region lacking core fucosylation of N-glycans in the Fc moiety. In some embodiments, the ABP is a defucosylated monoclonal antibody.

[0081] An aspect of the present disclosure includes a pharmaceutical composition comprising an ABP of the present disclosure and a pharmaceutically acceptable excipient.

[0082] In some embodiments, less than 50% of the ABPs are fucosylated. In some embodiments, less than 40% of the ABPs are fucosylated. In some embodiments, less than 30% of the ABPs are fucosylated. In some embodiments, less than 20% of the ABPs are fucosylated. In some embodiments, less than 10% of the ABPs are fucosylated. In some embodiments, more than 30% of the ABPs are fucosylated. In some embodiments, more than 40% of the ABPs are fucosylated. In some embodiments, more than 50% of the ABPs are fucosylated. In some embodiments, more than 60% of the ABPs are fucosylated. In some embodiments, more than 70% of the ABPs are fucosylated. In some embodiments, more than 80% of the ABPs are fucosylated. In some embodiments, more than 90% of the ABPs are fucosylated.

[0083] In some embodiments, the pharmaceutical composition has a pH of 5.0 to 6.5. In some embodiments, the pharmaceutical composition comprises 20 mM histidine or citrate buffer. In some embodiments, the pharmaceutical composition comprises 50 mM NaCl. In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of 170 mM to 270 mM. In some embodiments, the pharmaceutical composition comprises 170 mM or 270 mM sucrose. In some embodiments, the pharmaceutical composition comprises 5 mg / mL to 20 mg / mL ABP. In some embodiments, the pharmaceutical composition comprises 20 mg / mL ABP. In some embodiments, the pharmaceutical composition comprises 5 mg / mL ABP.

[0084] Aspects of the present disclosure provide a method of treating a disease, comprising administering an effective amount of an ABP or a pharmaceutical composition to a subject in need thereof.

[0085] In some embodiments, the disease is selected from the group consisting of cancer, AIDS, Alzheimer's disease, and a viral or bacterial infection, hi some embodiments, the disease is selected from the group consisting of an autoimmune disease, an autoinflammatory disease, and inflammation.

[0086] In some embodiments, the method further comprises administering to the subject one or more additional therapeutic agents. In some embodiments, the additional therapeutic agents are selected from anti-PD-L1, anti-PD1, a TIGIT inhibitor, a LAG-3 inhibitor, a CD47 inhibitor, a BRAF inhibitor, a MEK inhibitor, a PI3K inhibitor, a chemotherapeutic agent, an immunostimulant, radiation, a cytokine, a polynucleotide encoding a cytokine, an oncolytic virus encoding a cytokine, and combinations thereof. In some embodiments, the method further comprises adoptive cell therapy or treatment with a cancer vaccine, an oncolytic virus, or an anti-CD40 inhibitor.

[0087] Aspects of the present disclosure include an isolated polynucleotide encoding an ABP. Aspects of the present disclosure include a vector comprising the isolated polynucleotide. Aspects of the present disclosure provide a host cell comprising an isolated polynucleotide or vector of the present disclosure. In some embodiments, the host cell further comprises the bacterial protein RMD (GDP-6-deoxy-D-lyxo-4-hexulose reductase). In some embodiments, the host cell is cultured in the absence of fucose. In some embodiments, the host cell lacks or has reduced expression of Fut8. In some embodiments, the host cell is cultured in the presence of 2-Fluorfucose (2FF), a fucosylation inhibitor. In some embodiments, the host cell overexpresses glycosyltransferase (GnTIII).

[0088] Aspects of the present disclosure provide a method for producing an isolated antigen binding protein (ABP) that specifically binds to human CTLA-4, the method comprising inducing expression of the ABP in a host cell and isolating the ABP.

[0089] In some embodiments, the method further comprises isolating the ABP based on its fucosylation state. In some embodiments, the host cell is cultured in a culture medium containing a fucosylation inhibitor. In some embodiments, the fucosylation inhibitor is 2-Fluorfucose (2FF).

[0090] Embodiments of the present disclosure provide a method for the treatment of CTLA-4 in subjects with limited residual Treg expansion. HI A method for reducing Tregs is provided, comprising administering an effective amount of an ABP or a pharmaceutical composition.

[0091] In some embodiments, the subject is a human subject, optionally with melanoma, RCC (renal cell carcinoma), NSCLC (non-small cell lung cancer), Merkel cell carcinoma, cSCC, mesothelioma, MSI colorectal cancer, ovarian cancer, or cervical cancer. In some embodiments, the method further comprises administering one or more additional therapeutic agents to the subject. In some embodiments, the additional therapeutic agent is anti-PD-L1 or anti-PD1, or a combination thereof. In some embodiments, the subject has a tumor with high levels of Tregs, high levels of CTLA-4, high levels of NK cells, or high levels of activating FcR.

[0092] In another aspect, the present disclosure provides IGHG1 * An isolated antigen binding protein (ABP) that specifically binds to an antigen is provided, comprising a human heavy chain constant region gene segment.

[0093] In some embodiments, the ABP comprises a lysine at amino acid position 97 (R97) according to the IMGT exon numbering. In some embodiments, the ABP comprises a lysine at amino acid position 97 (R214) according to the EU numbering.

[0094] In some embodiments, the ABP comprises a defucosylated Fc region. In some embodiments, the ABP is produced from cells comprising the bacterial protein RMD (GDP-6-deoxy-D-lyxo-4-hexulose reductase) or a variant thereof. In some embodiments, the cells are cultured in the absence of fucose. In some embodiments, the ABP is produced from cells lacking or having reduced expression of Fut8. In some embodiments, the ABP is produced from cells cultured in the presence of 2-Fluorfucose (2FF), a fucosylation inhibitor.

[0095] In some embodiments, the ABP is produced from cells overexpressing glycosyltransferase (GnTIII). In some embodiments, the ABP is isolated based on its fucosylation status. In some embodiments, the ABP comprises an Fc region lacking core fucosylation of N-glycans in the Fc portion. In some embodiments, the ABP is a defucosylated monoclonal antibody.

[0096] In some embodiments, the ABP is selected from an anti-CTLA-4 antibody or antigen-binding fragment thereof, an anti-PD-L1 antibody or antigen-binding fragment thereof, an anti-PD1 antibody or antigen-binding fragment thereof, a TIGIT antibody or antigen-binding fragment thereof, a LAG-3 antibody or antigen-binding fragment thereof, a CD47 antibody or antigen-binding fragment thereof, a BRAF antibody or antigen-binding fragment thereof, a MEK antibody or antigen-binding fragment thereof, an OX40 antibody or antigen-binding fragment thereof, a 41BB antibody or antigen-binding fragment thereof, and a PI3K antibody or antigen-binding fragment thereof.

[0097] In some embodiments, the ABP comprises (a) a CDR1-L consisting of SEQ ID NO: 12078, a CDR2-L consisting of SEQ ID NO: 12079, a CDR3-L consisting of SEQ ID NO: 12080, a CDR1-H consisting of SEQ ID NO: 12075, a CDR2-H consisting of SEQ ID NO: 12076, and a CDR3-H consisting of SEQ ID NO: 12077; (b) a CDR1-L consisting of SEQ ID NO: 1014, a CDR2-L consisting of SEQ ID NO: 2014, a CDR3-L consisting of SEQ ID NO: 3014, a CDR1-H consisting of SEQ ID NO: 4014, and a CDR3-H consisting of SEQ ID NO: 4015; (c) CDR1-L consisting of SEQ ID NO: 12004, CDR2-L consisting of SEQ ID NO: 12014, CDR3-L consisting of SEQ ID NO: 12024, CDR1-H consisting of SEQ ID NO: 12039, CDR2-H consisting of SEQ ID NO: 12049, and CDR3-H consisting of SEQ ID NO: 12059; (d) CDR1-L consisting of SEQ ID NO: 12005, CDR2-L consisting of SEQ ID NO: 12015, and CDR3-H consisting of SEQ ID NO: 12025 (e) CDR1-L consisting of SEQ ID NO: 12006, CDR2-L consisting of SEQ ID NO: 12016, CDR3-L consisting of SEQ ID NO: 12026, CDR1-H consisting of SEQ ID NO: 12041, CDR2-H consisting of SEQ ID NO: 12051, and CDR3-H consisting of SEQ ID NO: 12061; (f) CDR1-L consisting of SEQ ID NO: 12007, CDR2-L consisting of SEQ ID NO: 12017 (g) a CDR1-L consisting of SEQ ID NO: 12008, a CDR2-L consisting of SEQ ID NO: 12018, a CDR3-L consisting of SEQ ID NO: 12028, a CDR1-H consisting of SEQ ID NO: 12043, a CDR2-H consisting of SEQ ID NO: 12053, and a CDR3-H consisting of SEQ ID NO: 12063.

[0098] In some embodiments, the ABP comprises a variable light chain (V) comprising a sequence that is at least 97% identical to SEQ ID NO: 14. L) and a variable heavy chain (V) comprising a sequence at least 97% identical to SEQ ID NO: 114. H ) and

[0099] In some embodiments, the ABP comprises (a) a CDR1-L consisting of any one of SEQ ID NO: 12081, a CDR2-L consisting of SEQ ID NO: 12082, a CDR3-L consisting of SEQ ID NO: 12083, a CDR1-H consisting of SEQ ID NO: 12084, a CDR2-H consisting of SEQ ID NO: 12085, and a CDR3-H consisting of SEQ ID NO: 12086.

[0100] In some embodiments, the ABP comprises a variable light chain (V) comprising a sequence that is at least 97% identical to SEQ ID NO: 12088. L ) and a variable heavy chain (V) comprising a sequence at least 97% identical to SEQ ID NO: 12087. H ) and

[0101] In some embodiments, the pharmaceutical composition has a pH of 5.0 to 6.5. In some embodiments, the pharmaceutical composition comprises 20 mM histidine or citrate buffer. In some embodiments, the pharmaceutical composition comprises 50 mM NaCl. In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of 170 mM to 270 mM. In some embodiments, the pharmaceutical composition comprises 170 mM or 270 mM sucrose. In some embodiments, the pharmaceutical composition comprises 5 mg / mL to 20 mg / mL ABP. In some embodiments, the pharmaceutical composition comprises 20 mg / mL ABP. In some embodiments, the pharmaceutical composition comprises 5 mg / mL ABP.

[0102] Aspects of the present disclosure include methods of treating a disease, comprising administering an effective amount of an ABP or a pharmaceutical composition to a subject in need thereof.

[0103] In some embodiments, the disease is selected from the group consisting of cancer, AIDS, Alzheimer's disease, and a viral or bacterial infection. In some embodiments, the disease is selected from the group consisting of an autoimmune disease, an autoinflammatory disease, and inflammation. In some embodiments, the method further comprises administering one or more additional therapeutic agents to the subject.

[0104] In some embodiments, the additional therapeutic agent is selected from a chemotherapeutic agent, an immunostimulant, radiation, a cytokine, a polynucleotide encoding a cytokine, an oncolytic virus encoding a cytokine, and combinations thereof.

[0105] Aspects of the present disclosure include an isolated polynucleotide encoding an ABP. Aspects of the present disclosure include a vector comprising the isolated polynucleotide. Aspects of the present disclosure provide a host cell comprising an isolated polynucleotide or vector of the present disclosure. In some embodiments, the host cell further comprises the bacterial protein RMD (GDP-6-deoxy-D-lyxo-4-hexulose reductase). In some embodiments, the host cell is cultured in the absence of fucose. In some embodiments, the host cell lacks or has reduced expression of Fut8. In some embodiments, the host cell is cultured in the presence of 2-Fluorfucose (2FF), a fucosylation inhibitor. In some embodiments, the host cell overexpresses glycosyltransferase (GnTIII).

[0106] Aspects of the present disclosure provide a method for producing an isolated antigen binding protein (ABP) that specifically binds to human CTLA-4, the method comprising inducing expression of the ABP in a host cell and isolating the ABP.

[0107] In some embodiments, the method further comprises isolating the ABP based on its fucosylation state. In some embodiments, the host cell is cultured in a culture medium containing a fucosylation inhibitor. In some embodiments, the fucosylation inhibitor is 2-Fluorfucose (2FF).

[0108] Embodiments of the present disclosure provide a method for the treatment of CTLA-4 in subjects with limited residual Treg expansion. HI A method for reducing Tregs is provided, comprising administering an effective amount of an ABP or a pharmaceutical composition.

[0109] In some embodiments, the subject is a human subject, optionally a human subject with melanoma, RCC (renal cell carcinoma), NSCLC (non-small cell lung cancer), Merkel cell carcinoma, cSCC, mesothelioma, MSI colorectal cancer, ovarian cancer, or cervical cancer.

[0110] In some embodiments, the method further comprises administering one or more additional therapeutic agents to the subject. In some embodiments, the subject has a tumor with high levels of Tregs, high levels of CTLA-4, high levels of NK cells, or high levels of activating FcR.

[0111] Embodiments of the present disclosure provide a method for the treatment of CTLA-4 in subjects with limited residual Treg expansion. HI Provided is a method for reducing Tregs, comprising administering to a subject an effective amount of an antigen binding protein (ABP) that specifically binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).

[0112] In some embodiments, the subject is a human subject, optionally a human subject with melanoma, RCC (renal cell carcinoma), NSCLC (non-small cell lung cancer), Merkel cell carcinoma, cSCC, mesothelioma, MSI colorectal cancer, ovarian cancer, or cervical cancer. In some embodiments, the cancer patient has melanoma, RCC (renal cell carcinoma), NSCLC (non-small cell lung cancer), Merkel cell carcinoma, cSCC, mesothelioma, hepatocellular carcinoma, esophageal cancer, breast cancer, sarcoma, MSI-Hi / dMMR colorectal cancer, ovarian cancer, or cervical cancer, bladder cancer, prostate cancer, TMB-HI tumors of any origin, tumors that are MSI, tumors that are dMMR, T-cell leukemia / lymphoma, NHL, tumors in which the cancer cells express CTLA-4.

[0113] In some embodiments, the method further comprises administering one or more additional therapeutic agents to the subject.

[0114] In some embodiments, the ABP comprises a variable light chain (V) comprising a sequence that is at least 97% identical to SEQ ID NO: 14. L ) and a variable heavy chain (V) comprising a sequence at least 97% identical to SEQ ID NO: 114. H ) and

[0115] In some embodiments, the ABP comprises (a) a CDR1-L consisting of any one of SEQ ID NOs: 1001 to 1028, a CDR2-L consisting of any one of SEQ ID NOs: 3001 to 3028, a CDR1-H consisting of any one of SEQ ID NOs: 3001 to 3028, and a CDR2-H consisting of any one of SEQ ID NOs: 3001 to 3028.

[0116] In some embodiments, the ABP comprises a variable light chain (V) comprising a sequence at least 97% identical to any one of SEQ ID NOs: 1-28. L ) and a variable heavy chain (V) comprising a sequence that is at least 97% identical to SEQ ID NOs: 101 to 128. H ) and [Brief explanation of the drawings]

[0117] [Figure 1]This method summarizes the steps of generating an scFv library from B cells isolated from mice with fully human antibody variable regions, and selecting yeast cells expressing scFvs with affinity for an antigen derived from B cells expressing antibodies with affinity for the antigen. Figure 1 discloses SEQ ID NOs: 11971 to 11998 in order of appearance.

[0118] [Figure 2] The scFv amplification procedure is shown below. First, IgK and IgH are amplified separately using a mixture of primers for the IgK C region, IgG C region, and all V regions. Second, the VH and CK primers contain complementary regions that result in the formation of an overlap-extension amplicon, which is the fusion product between IgK and IgH. The complementary region includes a DNA sequence encoding a Gly-Ser-rich scFv linker sequence. Third, semi-nested PCR is performed to add adapters for Illumina sequencing or yeast display.

[0119] [Figure 3] Includes a schematic diagram of monoclonal antibodies grouped into epitope bins as determined by high-throughput Array SPR.

[0120] [Figure 4-1] Figure 4 includes plots from histopathological staining of hCTLA-4 KI mice bearing MC38 tumors treated with PBS or anti-CTLA-4 ABP. Plots show H&E (Figure 4A), immunoglobulin (Ig) (Figures 4B and 4C), and C3 staining (Figures 4D and 4E) scoring from the right kidney. ipi is ipilimumab, and CTLA4.A14.2a is antibody A14 cloned onto a murine IgG2a backbone. [Figure 4-2]Figure 4 includes plots from histopathological staining of hCTLA-4 KI mice bearing MC38 tumors treated with PBS or anti-CTLA-4 ABP. Plots show H&E (Figure 4A), immunoglobulin (Ig) (Figures 4B and 4C), and C3 staining (Figures 4D and 4E) scoring from the right kidney. ipi is ipilimumab, and CTLA4.A14.2a is antibody A14 cloned onto a murine IgG2a backbone. [Figure 4-3] Figure 4 includes plots from histopathological staining of hCTLA-4 KI mice bearing MC38 tumors treated with PBS or anti-CTLA-4 ABP. Plots show H&E (Figure 4A), immunoglobulin (Ig) (Figures 4B and 4C), and C3 staining (Figures 4D and 4E) scoring from the right kidney. ipi is ipilimumab, and CTLA4.A14.2a is antibody A14 cloned onto a murine IgG2a backbone. [Figure 4-4] Figure 4 includes plots from histopathological staining of hCTLA-4 KI mice bearing MC38 tumors treated with PBS or anti-CTLA-4 ABP. Plots show H&E (Figure 4A), immunoglobulin (Ig) (Figures 4B and 4C), and C3 staining (Figures 4D and 4E) scoring from the right kidney. ipi is ipilimumab, and CTLA4.A14.2a is antibody A14 cloned onto a murine IgG2a backbone. [Figure 4-5] Figure 4 includes plots from histopathological staining of hCTLA-4 KI mice bearing MC38 tumors treated with PBS or anti-CTLA-4 ABP. Plots show H&E (Figure 4A), immunoglobulin (Ig) (Figures 4B and 4C), and C3 staining (Figures 4D and 4E) scoring from the right kidney. ipi is ipilimumab, and CTLA4.A14.2a is antibody A14 cloned onto a murine IgG2a backbone.

[0121] [Figure 5]Included are plots showing alkaline phosphatase levels in treated hCTLA4 KI mice bearing MC38 tumors. IPI is ipilimumab, and CTLA4.A14.2a is antibody A14 cloned onto a murine IgG2a backbone. U / L is units per liter.

[0122] [Figure 6] Plots of the percentage of intratumoral regulatory T cells (Treg) and intratumoral natural killer (NK) cells after the indicated treatments are included.

[0123] [Figure 7] Included are plots showing weight change in hCTLA4 mice receiving the indicated treatments. Ipi is ipilimumab, and CTLA4.A14.2a is antibody A14 cloned onto a murine IgG2a backbone. Error bars represent + / - standard error of the mean.

[0124] [Figure 8-1] Figure 8 includes plots showing the effect of control, Ipi, and anti-CTLA4 (CTLA4.A2, CTLA4.A14, CTLA4.A14.2a) treatment on the percentage of indicated cell populations, including CD3+ cells (Figure 8A), CD4+ cells (Figure 8B), CD69+ cells (Figure 8C), ICOS+ cells (Figure 8D), PD1+ cells (Figure 8E), and FOXP3+ cells (Figure 8F), in hCTLA-4 KI mice implanted with MC38 tumor cells. Ipi is ipilimumab, and CTLA4.A14.2a is antibody A14 cloned on a murine IgG2a backbone. [Figure 8-2]Figure 8 includes plots showing the effect of control, Ipi, and anti-CTLA4 (CTLA4.A2, CTLA4.A14, CTLA4.A14.2a) treatment on the percentage of indicated cell populations, including CD3+ cells (Figure 8A), CD4+ cells (Figure 8B), CD69+ cells (Figure 8C), ICOS+ cells (Figure 8D), PD1+ cells (Figure 8E), and FOXP3+ cells (Figure 8F), in hCTLA-4 KI mice implanted with MC38 tumor cells. Ipi is ipilimumab, and CTLA4.A14.2a is antibody A14 cloned on a murine IgG2a backbone. [Figure 8-3] Figure 8 includes plots showing the effect of control, Ipi, and anti-CTLA4 (CTLA4.A2, CTLA4.A14, CTLA4.A14.2a) treatment on the percentage of indicated cell populations, including CD3+ cells (Figure 8A), CD4+ cells (Figure 8B), CD69+ cells (Figure 8C), ICOS+ cells (Figure 8D), PD1+ cells (Figure 8E), and FOXP3+ cells (Figure 8F), in hCTLA-4 KI mice implanted with MC38 tumor cells. Ipi is ipilimumab, and CTLA4.A14.2a is antibody A14 cloned on a murine IgG2a backbone. [Figure 8-4] Figure 8 includes plots showing the effect of control, Ipi, and anti-CTLA4 (CTLA4.A2, CTLA4.A14, CTLA4.A14.2a) treatment on the percentage of indicated cell populations, including CD3+ cells (Figure 8A), CD4+ cells (Figure 8B), CD69+ cells (Figure 8C), ICOS+ cells (Figure 8D), PD1+ cells (Figure 8E), and FOXP3+ cells (Figure 8F), in hCTLA-4 KI mice implanted with MC38 tumor cells. Ipi is ipilimumab, and CTLA4.A14.2a is antibody A14 cloned on a murine IgG2a backbone. [Figure 8-5]Figure 8 includes plots showing the effect of control, Ipi, and anti-CTLA4 (CTLA4.A2, CTLA4.A14, CTLA4.A14.2a) treatment on the percentage of indicated cell populations, including CD3+ cells (Figure 8A), CD4+ cells (Figure 8B), CD69+ cells (Figure 8C), ICOS+ cells (Figure 8D), PD1+ cells (Figure 8E), and FOXP3+ cells (Figure 8F), in hCTLA-4 KI mice implanted with MC38 tumor cells. Ipi is ipilimumab, and CTLA4.A14.2a is antibody A14 cloned on a murine IgG2a backbone. [Figure 8-6] Figure 8 includes plots showing the effect of control, Ipi, and anti-CTLA4 (CTLA4.A2, CTLA4.A14, CTLA4.A14.2a) treatment on the percentage of indicated cell populations, including CD3+ cells (Figure 8A), CD4+ cells (Figure 8B), CD69+ cells (Figure 8C), ICOS+ cells (Figure 8D), PD1+ cells (Figure 8E), and FOXP3+ cells (Figure 8F), in hCTLA-4 KI mice implanted with MC38 tumor cells. Ipi is ipilimumab, and CTLA4.A14.2a is antibody A14 cloned on a murine IgG2a backbone.

[0125] [Figure 9-1] Figure 9 includes plots showing the effect of control, Ipi, and anti-CTLA-4 (CTLA4.A2, CTLA4.A14, CTLA4.A14.2a) treatment on the percentage of indicated cell populations, including CD8+ cells (Figure 9A), CD69+ cells (Figure 9B), ICOS+ cells (Figure 9C), and PD1+ cells (Figure 9D), in hCTLA-4 KI mice implanted with MC38 tumor cells. Ipi is ipilimumab, and CTLA4.A14.2a is antibody A14 cloned on a murine IgG2a backbone. [Figure 9-2]Figure 9 includes plots showing the effect of control, Ipi, and anti-CTLA-4 (CTLA4.A2, CTLA4.A14, CTLA4.A14.2a) treatment on the percentage of indicated cell populations, including CD8+ cells (Figure 9A), CD69+ cells (Figure 9B), ICOS+ cells (Figure 9C), and PD1+ cells (Figure 9D), in hCTLA-4 KI mice implanted with MC38 tumor cells. Ipi is ipilimumab, and CTLA4.A14.2a is antibody A14 cloned on a murine IgG2a backbone. [Figure 9-3] Figure 9 includes plots showing the effect of control, Ipi, and anti-CTLA-4 (CTLA4.A2, CTLA4.A14, CTLA4.A14.2a) treatment on the percentage of indicated cell populations, including CD8+ cells (Figure 9A), CD69+ cells (Figure 9B), ICOS+ cells (Figure 9C), and PD1+ cells (Figure 9D), in hCTLA-4 KI mice implanted with MC38 tumor cells. Ipi is ipilimumab, and CTLA4.A14.2a is antibody A14 cloned on a murine IgG2a backbone. [Figure 9-4] Figure 9 includes plots showing the effect of control, Ipi, and anti-CTLA-4 (CTLA4.A2, CTLA4.A14, CTLA4.A14.2a) treatment on the percentage of indicated cell populations, including CD8+ cells (Figure 9A), CD69+ cells (Figure 9B), ICOS+ cells (Figure 9C), and PD1+ cells (Figure 9D), in hCTLA-4 KI mice implanted with MC38 tumor cells. Ipi is ipilimumab, and CTLA4.A14.2a is antibody A14 cloned on a murine IgG2a backbone.

[0126] [Figure 10] Included are plots showing the effect of control, Ipi, and anti-CTLA-4 treatment on the percentage of dendritic cells (DCs) and activated dendritic cells (CD86+). Ipi is ipilimumab, and CTLA4.A14.2a is antibody A14 cloned onto a murine IgG2a backbone.

[0127] [Figure 11]Included are plots showing mean tumor volumes after treatment with 0.3 mg / kg of the indicated anti-CTLA-4.

[0128] [Figure 12-1] Figure 12A shows that FcR effector function is required for the antitumor effect of ipilimumab in a mouse model. Figure 12A shows the frequency of CD4+FOXP3- T cells from the LNs of individual control (C57BL / 6) or hCTLA-4 KI mice that were CD44LoCD62L+, as determined by flow cytometry (mean + / - SEM). Figure 12B shows hCTLA-4 KI mice bearing MC38 tumors were randomized (day 0) when tumors reached 50–151 mm3 and treated biweekly for five doses with the indicated antibody at 5 mg / kg. The plot shows tumor volume (mean ± SEM) over time for MC38 tumors treated with the indicated antibody. Tumor volumes of mice euthanized due to tumor burden exceeding 3000 mm3 were advanced. Thin vertical lines indicate censored data. Thin gray vertical lines indicate animals likely lost after dosing due to anti-drug antibody (ADA)-induced hypersensitivity, and thin black vertical lines indicate animals euthanized due to tumor burden, whose data were carried forward. Ipi analog refers to ipilimumab mAb produced and purified by the applicant, and N297Q indicates that the antibody contains the N297Q mutation in the Fc domain of the antibody to abolish Fc effector function. n=8 (isotype, aCTLA-4.28, Ipi-N297Q), n=7 (ipi analog and aCTLA-4.28). For the change in tumor volume between groups, p=0.0004 when comparing Ipi analog to isotype and p=0.0006 when comparing aCTLA-4.28 to isotype (linear mixed-effects model). [Figure 12-2]Figure 12A shows that FcR effector function is required for the antitumor effect of ipilimumab in a mouse model. Figure 12A shows the frequency of CD4+FOXP3- T cells from the LNs of individual control (C57BL / 6) or hCTLA-4 KI mice that were CD44LoCD62L+, as determined by flow cytometry (mean + / - SEM). Figure 12B shows hCTLA-4 KI mice bearing MC38 tumors were randomized (day 0) when tumors reached 50–151 mm3 and treated biweekly for five doses with the indicated antibody at 5 mg / kg. The plot shows tumor volume (mean ± SEM) over time for MC38 tumors treated with the indicated antibody. Tumor volumes of mice euthanized due to tumor burden exceeding 3000 mm3 were advanced. Thin vertical lines indicate censored data. Thin gray vertical lines indicate animals likely lost after dosing due to anti-drug antibody (ADA)-induced hypersensitivity, and thin black vertical lines indicate animals euthanized due to tumor burden, whose data were carried forward. Ipi analog refers to ipilimumab mAb produced and purified by the applicant, and N297Q indicates that the antibody contains the N297Q mutation in the Fc domain of the antibody to abolish Fc effector function. n=8 (isotype, aCTLA-4.28, Ipi-N297Q), n=7 (ipi analog and aCTLA-4.28). For the change in tumor volume between groups, p=0.0004 when comparing Ipi analog to isotype and p=0.0006 when comparing aCTLA-4.28 to isotype (linear mixed-effects model).

[0129] [Figure 13-1]Figure 13A shows that GIGA-564 has little ability to block the interaction between CTLA-4 and CD80 / CD86 in vitro. Figure 13B shows that GIGA-564 has little ability to block the interaction between CTLA-4 and CD80 / CD86 in vitro. The blocking ability of CTLA-4 mAbs against CD80 or CD86 binding was measured using a plate-based ELISA. CTLA-4 was used to coat plates, and antibody samples were then incubated. His-tagged CD80 or CD86 was then added, and the amount of ligand available for binding to CTLA-4 was measured. Blocking mAbs prevent CD80 or CD86 from binding to CTLA-4, resulting in a reduced absorbance signal due to the lack of CD80 or CD86 binding to CTLA-4. Weak blocking mAbs still allow CD80 or CD86 to bind, preventing the loss of all absorbance signal. Figure 13B. Plots show the ability of GIGA-564 to block the interaction between CTLA-4 and the B7 ligands CD80 and CD86, as assessed by ELISA described in (Figure 13A), compared to ipilimumab and CTLA-4.28. Absorbance values ​​were corrected for the anti-PD-1 control (pembrolizumab) and displayed as the average of two technical replicates. Figures 13C-13D. Key residues mediating CTLA-4 binding were identified for GIGA-564 and ipilimumab by shotgun mutagenesis of CTLA-4 followed by staining and flow cytometry assessment of binding. Figure 13C shows the crystal structure of the complex between CD80 and CTLA-4 (Protein Database [PDB] 1I8L), which highlights the key differentiation residue R70 (visualized in Pymol) and shows that CTLA-4 epitope residues are shared between ipilimumab and GIGA-564. Furthermore, G142 was identified as a secondary residue in the epitope of ipilimumab but not GIGA-564. Figure 13D shows the key amino acids on CTLA-4 that are of interest for these epitopes; those found by mutational analysis to be important for CTLA-4 binding to CD80 or CD86 in cell-based assays are marked in gray to indicate the epitope residues in these proteins. [Figure 13-2]Figure 13A shows that GIGA-564 has little ability to block the interaction between CTLA-4 and CD80 / CD86 in vitro. Figure 13B shows that GIGA-564 has little ability to block the interaction between CTLA-4 and CD80 / CD86 in vitro. The blocking ability of CTLA-4 mAbs against CD80 or CD86 binding was measured using a plate-based ELISA. CTLA-4 was used to coat plates, and antibody samples were then incubated. His-tagged CD80 or CD86 was then added, and the amount of ligand available for binding to CTLA-4 was measured. Blocking mAbs prevent CD80 or CD86 from binding to CTLA-4, resulting in a reduced absorbance signal due to the lack of CD80 or CD86 binding to CTLA-4. Weak blocking mAbs still allow CD80 or CD86 to bind, preventing the loss of all absorbance signal. Figure 13B. Plots show the ability of GIGA-564 to block the interaction between CTLA-4 and the B7 ligands CD80 and CD86, as assessed by ELISA described in (Figure 13A), compared to ipilimumab and CTLA-4.28. Absorbance values ​​were corrected for the anti-PD-1 control (pembrolizumab) and displayed as the average of two technical replicates. Figures 13C-13D. Key residues mediating CTLA-4 binding were identified for GIGA-564 and ipilimumab by shotgun mutagenesis of CTLA-4 followed by staining and flow cytometry assessment of binding. Figure 13C shows the crystal structure of the complex between CD80 and CTLA-4 (Protein Database [PDB] 1I8L), which highlights the key differentiation residue R70 (visualized in Pymol) and shows that CTLA-4 epitope residues are shared between ipilimumab and GIGA-564. Furthermore, G142 was identified as a secondary residue in the epitope of ipilimumab but not GIGA-564. Figure 13D shows the key amino acids on CTLA-4 that are of interest for these epitopes; those found by mutational analysis to be important for CTLA-4 binding to CD80 or CD86 in cell-based assays are marked in gray to indicate the epitope residues in these proteins. [Figure 13-3]Figure 13A shows that GIGA-564 has little ability to block the interaction between CTLA-4 and CD80 / CD86 in vitro. Figure 13B shows that GIGA-564 has little ability to block the interaction between CTLA-4 and CD80 / CD86 in vitro. The blocking ability of CTLA-4 mAbs against CD80 or CD86 binding was measured using a plate-based ELISA. CTLA-4 was used to coat plates, and antibody samples were then incubated. His-tagged CD80 or CD86 was then added, and the amount of ligand available for binding to CTLA-4 was measured. Blocking mAbs prevent CD80 or CD86 from binding to CTLA-4, resulting in a reduced absorbance signal due to the lack of CD80 or CD86 binding to CTLA-4. Weak blocking mAbs still allow CD80 or CD86 to bind, preventing the loss of all absorbance signal. Figure 13B. Plots show the ability of GIGA-564 to block the interaction between CTLA-4 and the B7 ligands CD80 and CD86, as assessed by ELISA described in (Figure 13A), compared to ipilimumab and CTLA-4.28. Absorbance values ​​were corrected for the anti-PD-1 control (pembrolizumab) and displayed as the average of two technical replicates. Figures 13C-13D. Key residues mediating CTLA-4 binding were identified for GIGA-564 and ipilimumab by shotgun mutagenesis of CTLA-4 followed by staining and flow cytometry assessment of binding. Figure 13C shows the crystal structure of the complex between CD80 and CTLA-4 (Protein Database [PDB] 1I8L), which highlights the key differentiation residue R70 (visualized in Pymol) and shows that CTLA-4 epitope residues are shared between ipilimumab and GIGA-564. Furthermore, G142 was identified as a secondary residue in the epitope of ipilimumab but not GIGA-564. Figure 13D shows the key amino acids on CTLA-4 that are of interest for these epitopes; those found by mutational analysis to be important for CTLA-4 binding to CD80 or CD86 in cell-based assays are marked in gray to indicate the epitope residues in these proteins. [Figure 13-4]Figure 13A shows that GIGA-564 has little ability to block the interaction between CTLA-4 and CD80 / CD86 in vitro. Figure 13B shows that GIGA-564 has little ability to block the interaction between CTLA-4 and CD80 / CD86 in vitro. The blocking ability of CTLA-4 mAbs against CD80 or CD86 binding was measured using a plate-based ELISA. CTLA-4 was used to coat plates, and antibody samples were then incubated. His-tagged CD80 or CD86 was then added, and the amount of ligand available for binding to CTLA-4 was measured. Blocking mAbs prevent CD80 or CD86 from binding to CTLA-4, resulting in a reduced absorbance signal due to the lack of CD80 or CD86 binding to CTLA-4. Weak blocking mAbs still allow CD80 or CD86 to bind, preventing the loss of all absorbance signal. Figure 13B. Plots show the ability of GIGA-564 to block the interaction between CTLA-4 and the B7 ligands CD80 and CD86, as assessed by ELISA described in (Figure 13A), compared to ipilimumab and CTLA-4.28. Absorbance values ​​were corrected for the anti-PD-1 control (pembrolizumab) and displayed as the average of two technical replicates. Figures 13C-13D. Key residues mediating CTLA-4 binding were identified for GIGA-564 and ipilimumab by shotgun mutagenesis of CTLA-4 followed by staining and flow cytometry assessment of binding. Figure 13C shows the crystal structure of the complex between CD80 and CTLA-4 (Protein Database [PDB] 1I8L), which highlights the key differentiation residue R70 (visualized in Pymol) and shows that CTLA-4 epitope residues are shared between ipilimumab and GIGA-564. Furthermore, G142 was identified as a secondary residue in the epitope of ipilimumab but not GIGA-564. Figure 13D shows the key amino acids on CTLA-4 that are of interest for these epitopes; those found by mutational analysis to be important for CTLA-4 binding to CD80 or CD86 in cell-based assays are marked in gray to indicate the epitope residues in these proteins.

[0130] [Figure 14-1] Figure 14A shows that GIGA-564 inhibits tumor growth in a mouse model. hCTLA-4 KI mice bearing MC38 tumors were randomized (day 0) when tumors reached 50-150 mm3 and treated biweekly for five doses with the indicated antibody at 5 mg / kg. The plot shows tumor volume (mean ± SEM) over time for MC38 tumors treated with the indicated antibody. Tumor volumes of mice euthanized due to tumor burden exceeding 3000 mm3 were advanced. Thin gray vertical lines indicate censored data (animals likely lost after dosing due to ADA-induced hypersensitivity). This experiment is also described in Figure 12B. n = 7 (GIGA-564), n = 6 (vehicle and commercial ipilimumab). Figure 14B. hCTLA-4 KI mice bearing RM-1 tumors were randomized (day 0) when tumors reached 40-125 mm3 and treated with the indicated antibodies at 5 mg / kg on days 0, 3, and 6. The plot shows tumor volume (mean ± SEM) over time for RM-1 tumors treated with the indicated antibodies. Tumor volumes from mice euthanized due to tumor burden exceeding 3000 mm3 were tracked until all mice in that group died (thin black vertical line). The thin vertical line indicates censored data. The thin gray vertical line indicates one mouse in the ipilimumab-treated group was euthanized due to tumor ulceration. n = 11 (ipilimumab and GIGA-564-treated), n = 7 (isotype-treated). Figure 14C (also shown in Figure 11). hCTLA-4 KI mice bearing MC38 tumors were randomized (day 0) when tumors reached 65–125 mm3 and treated with 0.3 mg / kg of the indicated antibody on days 0, 3, and 6. The plot shows tumor volume (mean ± SEM) over time for MC38 tumors treated with the indicated antibody. Tumor volume from mice euthanized due to tumor burden exceeding 3000 mm3 was tracked until all mice in that group died (thin black vertical line). n = 13 (ipilimumab and GIGA-564), n = 8 (isotype-treated animals). P values ​​are shown for statistical significance in longitudinally measured tumor volume changes (linear mixed-effects model). [Figure 14-2]Figure 14A shows that GIGA-564 inhibits tumor growth in a mouse model. hCTLA-4 KI mice bearing MC38 tumors were randomized (day 0) when tumors reached 50-150 mm3 and treated biweekly for five doses with the indicated antibody at 5 mg / kg. The plot shows tumor volume (mean ± SEM) over time for MC38 tumors treated with the indicated antibody. Tumor volumes of mice euthanized due to tumor burden exceeding 3000 mm3 were advanced. Thin gray vertical lines indicate censored data (animals likely lost after dosing due to ADA-induced hypersensitivity). This experiment is also described in Figure 12B. n = 7 (GIGA-564), n = 6 (vehicle and commercial ipilimumab). Figure 14B. hCTLA-4 KI mice bearing RM-1 tumors were randomized (day 0) when tumors reached 40-125 mm3 and treated with the indicated antibodies at 5 mg / kg on days 0, 3, and 6. The plot shows tumor volume (mean ± SEM) over time for RM-1 tumors treated with the indicated antibodies. Tumor volumes from mice euthanized due to tumor burden exceeding 3000 mm3 were tracked until all mice in that group died (thin black vertical line). The thin vertical line indicates censored data. The thin gray vertical line indicates one mouse in the ipilimumab-treated group was euthanized due to tumor ulceration. n = 11 (ipilimumab and GIGA-564-treated), n = 7 (isotype-treated). Figure 14C (also shown in Figure 11). hCTLA-4 KI mice bearing MC38 tumors were randomized (day 0) when tumors reached 65–125 mm3 and treated with 0.3 mg / kg of the indicated antibody on days 0, 3, and 6. The plot shows tumor volume (mean ± SEM) over time for MC38 tumors treated with the indicated antibody. Tumor volume from mice euthanized due to tumor burden exceeding 3000 mm3 was tracked until all mice in that group died (thin black vertical line). n = 13 (ipilimumab and GIGA-564), n = 8 (isotype-treated animals). P values ​​are shown for statistical significance in longitudinally measured tumor volume changes (linear mixed-effects model). [Figure 14-3]Figure 14A shows that GIGA-564 inhibits tumor growth in a mouse model. hCTLA-4 KI mice bearing MC38 tumors were randomized (day 0) when tumors reached 50-150 mm3 and treated biweekly for five doses with the indicated antibody at 5 mg / kg. The plot shows tumor volume (mean ± SEM) over time for MC38 tumors treated with the indicated antibody. Tumor volumes of mice euthanized due to tumor burden exceeding 3000 mm3 were advanced. Thin gray vertical lines indicate censored data (animals likely lost after dosing due to ADA-induced hypersensitivity). This experiment is also described in Figure 12B. n = 7 (GIGA-564), n = 6 (vehicle and commercial ipilimumab). Figure 14B. hCTLA-4 KI mice bearing RM-1 tumors were randomized (day 0) when tumors reached 40-125 mm3 and treated with the indicated antibodies at 5 mg / kg on days 0, 3, and 6. The plot shows tumor volume (mean ± SEM) over time for RM-1 tumors treated with the indicated antibodies. Tumor volumes from mice euthanized due to tumor burden exceeding 3000 mm3 were tracked until all mice in that group died (thin black vertical line). The thin vertical line indicates censored data. The thin gray vertical line indicates one mouse in the ipilimumab-treated group was euthanized due to tumor ulceration. n = 11 (ipilimumab and GIGA-564-treated), n = 7 (isotype-treated). Figure 14C (also shown in Figure 11). hCTLA-4 KI mice bearing MC38 tumors were randomized (day 0) when tumors reached 65–125 mm3 and treated with 0.3 mg / kg of the indicated antibody on days 0, 3, and 6. The plot shows tumor volume (mean ± SEM) over time for MC38 tumors treated with the indicated antibody. Tumor volume from mice euthanized due to tumor burden exceeding 3000 mm3 was tracked until all mice in that group died (thin black vertical line). n = 13 (ipilimumab and GIGA-564), n = 8 (isotype-treated animals). P values ​​are shown for statistical significance in longitudinally measured tumor volume changes (linear mixed-effects model).

[0131] [Figure 15-1]Figure 15A shows that GIGA-564 induces lower peripheral Treg proliferation but potently mediates intratumoral Treg depletion. Figure 15A. hCTLA-4 KI mice (n=12) were treated with 5 mg / kg hIgG1 isotype control, ipilimumab, or GIGA-564 on days 0, 3, and 6 and euthanized on day 7 for flow cytometry analysis. Two samples from the GIGA-564 group were excluded from analysis due to low cell counts. Ki67-expressing CD8 T cells (Live, CD45+TCRβ+CD8+, CD4 T cells (Live, CD45+TCRβ+CD4+FOXP3-), and T cells (Live, CD45+TCRβ+CD4+FOXP3+) in non-draining (left anterior axillary) lymph nodes (LNs) were determined by flow cytometry (first three panels). The right panel shows the fold change in the percentage of cells of each subtype expressing Ki67 in ipilimumab- or GIGA-564-treated mice relative to the mean frequency of that cell type expressed in the isotype control-treated group. In this right panel, p values ​​were calculated using the Mann-Whitney (Wilcoxon) test without adjustment for multiple pairwise comparisons. Fewer proliferating T cells may further enhance efficacy compared to ipilimumab in patients. Figures 15B-15E. hCTLA-4 expression in established MC38 tumor-bearing mice. KI mice were randomized (n=6) and treated once with 5 mg / kg of hIgG1 isotype control, ipilimumab, or GIGA-564. The following day, cells from non-draining LNs and tumors were analyzed by flow cytometry. Figure 15B. Treg frequencies (as a percentage of CD45+ cells) in LNs (left) and tumors (right) in each treatment group. Figure 15C. CTLA-4 geometric mean fluorescence intensity (MFI) of Tregs in LNs (left) and tumors (right). Figure 15D. Representative contour plots of CD4 T cells in each treatment group. X- and Y-axes correspond to FOXP3 and CTLA-4, respectively. Figure 15E. CD8 versus Tregs in LNs (left) and tumors (right). T cell ratios. Unless otherwise indicated, p values ​​were calculated using the Wilcoxon rank-sum test, and horizontal lines indicate means. Flow cytometry demonstrated that GIGA-564 was more effective than ipilimumab in depleting CTLA-4+ Tregs in the tumor microenvironment.The data from FIG. 15D are shown again in FIG. [Figure 15-2]Figure 15A shows that GIGA-564 induces lower peripheral Treg proliferation but potently mediates intratumoral Treg depletion. Figure 15A. hCTLA-4 KI mice (n=12) were treated with 5 mg / kg hIgG1 isotype control, ipilimumab, or GIGA-564 on days 0, 3, and 6 and euthanized on day 7 for flow cytometry analysis. Two samples from the GIGA-564 group were excluded from analysis due to low cell counts. Ki67-expressing CD8 T cells (Live, CD45+TCRβ+CD8+, CD4 T cells (Live, CD45+TCRβ+CD4+FOXP3-), and T cells (Live, CD45+TCRβ+CD4+FOXP3+) in non-draining (left anterior axillary) lymph nodes (LNs) were determined by flow cytometry (first three panels). The right panel shows the fold change in the percentage of cells of each subtype expressing Ki67 in ipilimumab- or GIGA-564-treated mice relative to the mean frequency of that cell type expressed in the isotype control-treated group. In this right panel, p values ​​were calculated using the Mann-Whitney (Wilcoxon) test without adjustment for multiple pairwise comparisons. Fewer proliferating T cells may further enhance efficacy compared to ipilimumab in patients. Figures 15B-15E. hCTLA-4 expression in established MC38 tumor-bearing mice. KI mice were randomized (n=6) and treated once with 5 mg / kg of hIgG1 isotype control, ipilimumab, or GIGA-564. The following day, cells from non-draining LNs and tumors were analyzed by flow cytometry. Figure 15B. Treg frequencies (as a percentage of CD45+ cells) in LNs (left) and tumors (right) in each treatment group. Figure 15C. CTLA-4 geometric mean fluorescence intensity (MFI) of Tregs in LNs (left) and tumors (right). Figure 15D. Representative contour plots of CD4 T cells in each treatment group. X- and Y-axes correspond to FOXP3 and CTLA-4, respectively. Figure 15E. CD8 versus Tregs in LNs (left) and tumors (right). T cell ratios. Unless otherwise indicated, p values ​​were calculated using the Wilcoxon rank-sum test, and horizontal lines indicate means. Flow cytometry demonstrated that GIGA-564 was more effective than ipilimumab in depleting CTLA-4+ Tregs in the tumor microenvironment.The data from FIG. 15D are shown again in FIG. [Figure 15-3]Figure 15A shows that GIGA-564 induces lower peripheral Treg proliferation but potently mediates intratumoral Treg depletion. Figure 15A. hCTLA-4 KI mice (n=12) were treated with 5 mg / kg hIgG1 isotype control, ipilimumab, or GIGA-564 on days 0, 3, and 6 and euthanized on day 7 for flow cytometry analysis. Two samples from the GIGA-564 group were excluded from analysis due to low cell counts. Ki67-expressing CD8 T cells (Live, CD45+TCRβ+CD8+, CD4 T cells (Live, CD45+TCRβ+CD4+FOXP3-), and T cells (Live, CD45+TCRβ+CD4+FOXP3+) in non-draining (left anterior axillary) lymph nodes (LNs) were determined by flow cytometry (first three panels). The right panel shows the fold change in the percentage of cells of each subtype expressing Ki67 in ipilimumab- or GIGA-564-treated mice relative to the mean frequency of that cell type expressed in the isotype control-treated group. In this right panel, p values ​​were calculated using the Mann-Whitney (Wilcoxon) test without adjustment for multiple pairwise comparisons. Fewer proliferating T cells may further enhance efficacy compared to ipilimumab in patients. Figures 15B-15E. hCTLA-4 expression in established MC38 tumor-bearing mice. KI mice were randomized (n=6) and treated once with 5 mg / kg of hIgG1 isotype control, ipilimumab, or GIGA-564. The following day, cells from non-draining LNs and tumors were analyzed by flow cytometry. Figure 15B. Treg frequencies (as a percentage of CD45+ cells) in LNs (left) and tumors (right) in each treatment group. Figure 15C. CTLA-4 geometric mean fluorescence intensity (MFI) of Tregs in LNs (left) and tumors (right). Figure 15D. Representative contour plots of CD4 T cells in each treatment group. X- and Y-axes correspond to FOXP3 and CTLA-4, respectively. Figure 15E. CD8 versus Tregs in LNs (left) and tumors (right). T cell ratios. Unless otherwise indicated, p values ​​were calculated using the Wilcoxon rank-sum test, and horizontal lines indicate means. Flow cytometry demonstrated that GIGA-564 was more effective than ipilimumab in depleting CTLA-4+ Tregs in the tumor microenvironment.The data from FIG. 15D are shown again in FIG. [Figure 15-4]Figure 15A shows that GIGA-564 induces lower peripheral Treg proliferation but potently mediates intratumoral Treg depletion. Figure 15A. hCTLA-4 KI mice (n=12) were treated with 5 mg / kg hIgG1 isotype control, ipilimumab, or GIGA-564 on days 0, 3, and 6 and euthanized on day 7 for flow cytometry analysis. Two samples from the GIGA-564 group were excluded from analysis due to low cell counts. Ki67-expressing CD8 T cells (Live, CD45+TCRβ+CD8+, CD4 T cells (Live, CD45+TCRβ+CD4+FOXP3-), and T cells (Live, CD45+TCRβ+CD4+FOXP3+) in non-draining (left anterior axillary) lymph nodes (LNs) were determined by flow cytometry (first three panels). The right panel shows the fold change in the percentage of cells of each subtype expressing Ki67 in ipilimumab- or GIGA-564-treated mice relative to the mean frequency of that cell type expressed in the isotype control-treated group. In this right panel, p values ​​were calculated using the Mann-Whitney (Wilcoxon) test without adjustment for multiple pairwise comparisons. Fewer proliferating T cells may further enhance efficacy compared to ipilimumab in patients. Figures 15B-15E. hCTLA-4 expression in established MC38 tumor-bearing mice. KI mice were randomized (n=6) and treated once with 5 mg / kg of hIgG1 isotype control, ipilimumab, or GIGA-564. The following day, cells from non-draining LNs and tumors were analyzed by flow cytometry. Figure 15B. Treg frequencies (as a percentage of CD45+ cells) in LNs (left) and tumors (right) in each treatment group. Figure 15C. CTLA-4 geometric mean fluorescence intensity (MFI) of Tregs in LNs (left) and tumors (right). Figure 15D. Representative contour plots of CD4 T cells in each treatment group. X- and Y-axes correspond to FOXP3 and CTLA-4, respectively. Figure 15E. CD8 versus Tregs in LNs (left) and tumors (right). T cell ratios. Unless otherwise indicated, p values ​​were calculated using the Wilcoxon rank-sum test, and horizontal lines indicate means. Flow cytometry demonstrated that GIGA-564 was more effective than ipilimumab in depleting CTLA-4+ Tregs in the tumor microenvironment.The data from FIG. 15D are shown again in FIG. [Figure 15-5]Figure 15A shows that GIGA-564 induces lower peripheral Treg proliferation but potently mediates intratumoral Treg depletion. Figure 15A. hCTLA-4 KI mice (n=12) were treated with 5 mg / kg hIgG1 isotype control, ipilimumab, or GIGA-564 on days 0, 3, and 6 and euthanized on day 7 for flow cytometry analysis. Two samples from the GIGA-564 group were excluded from analysis due to low cell counts. Ki67-expressing CD8 T cells (Live, CD45+TCRβ+CD8+, CD4 T cells (Live, CD45+TCRβ+CD4+FOXP3-), and T cells (Live, CD45+TCRβ+CD4+FOXP3+) in non-draining (left anterior axillary) lymph nodes (LNs) were determined by flow cytometry (first three panels). The right panel shows the fold change in the percentage of cells of each subtype expressing Ki67 in ipilimumab- or GIGA-564-treated mice relative to the mean frequency of that cell type expressed in the isotype control-treated group. In this right panel, p values ​​were calculated using the Mann-Whitney (Wilcoxon) test without adjustment for multiple pairwise comparisons. Fewer proliferating T cells may further enhance efficacy compared to ipilimumab in patients. Figures 15B-15E. hCTLA-4 expression in established MC38 tumor-bearing mice. KI mice were randomized (n=6) and treated once with 5 mg / kg of hIgG1 isotype control, ipilimumab, or GIGA-564. The following day, cells from non-draining LNs and tumors were analyzed by flow cytometry. Figure 15B. Treg frequencies (as a percentage of CD45+ cells) in LNs (left) and tumors (right) in each treatment group. Figure 15C. CTLA-4 geometric mean fluorescence intensity (MFI) of Tregs in LNs (left) and tumors (right). Figure 15D. Representative contour plots of CD4 T cells in each treatment group. X- and Y-axes correspond to FOXP3 and CTLA-4, respectively. Figure 15E. CD8 versus Tregs in LNs (left) and tumors (right). T cell ratios. Unless otherwise indicated, p values ​​were calculated using the Wilcoxon rank-sum test, and horizontal lines indicate means. Flow cytometry demonstrated that GIGA-564 was more effective than ipilimumab in depleting CTLA-4+ Tregs in the tumor microenvironment.The data from FIG. 15D are shown again in FIG.

[0132] [Figure 16-1] Figures 16A-D show that GIGA-564 induces more FcR signaling than ipilimumab. Target CHO cells expressing human CTLA-4 with the Y201G mutation to enhance surface expression were incubated with a titration series of ipilimumab (black squares), GIGA-564 (black stars), or a variant of GIGA-564 with the LALA-PG mutation to disrupt FcγR binding (GIGA-564_LALA-PG, gray circles). Jurkat / NFAT-Luc effector cells bearing (Figure 16A) mouse FcγRIV or FcγRIII, (Figure 16B) human FcγRIIIa (high-affinity V158 or low-affinity F158 variant), (Figure 16C) human FcγRIIa (high-affinity H131 or low-affinity R131 variant), or (Figure 16D) human FcγRIIb were subsequently added. Cells were incubated at 37°C for 6 hours, after which luciferase activity was measured. Data shown are relative light units (RLU) emitted from effector cells and are plotted as the average of technical duplicates. [Figure 16-2]Figures 16A-D show that GIGA-564 induces more FcR signaling than ipilimumab. Target CHO cells expressing human CTLA-4 with the Y201G mutation to enhance surface expression were incubated with a titration series of ipilimumab (black squares), GIGA-564 (black stars), or a variant of GIGA-564 with the LALA-PG mutation to disrupt FcγR binding (GIGA-564_LALA-PG, gray circles). Jurkat / NFAT-Luc effector cells bearing (Figure 16A) mouse FcγRIV or FcγRIII, (Figure 16B) human FcγRIIIa (high-affinity V158 or low-affinity F158 variant), (Figure 16C) human FcγRIIa (high-affinity H131 or low-affinity R131 variant), or (Figure 16D) human FcγRIIb were subsequently added. Cells were incubated at 37°C for 6 hours, after which luciferase activity was measured. Data shown are relative light units (RLU) emitted from effector cells and are plotted as the average of technical duplicates. [Figure 16-3] Figures 16A-D show that GIGA-564 induces more FcR signaling than ipilimumab. Target CHO cells expressing human CTLA-4 with the Y201G mutation to enhance surface expression were incubated with a titration series of ipilimumab (black squares), GIGA-564 (black stars), or a variant of GIGA-564 with the LALA-PG mutation to disrupt FcγR binding (GIGA-564_LALA-PG, gray circles). Jurkat / NFAT-Luc effector cells bearing (Figure 16A) mouse FcγRIV or FcγRIII, (Figure 16B) human FcγRIIIa (high-affinity V158 or low-affinity F158 variant), (Figure 16C) human FcγRIIa (high-affinity H131 or low-affinity R131 variant), or (Figure 16D) human FcγRIIb were subsequently added. Cells were incubated at 37°C for 6 hours, after which luciferase activity was measured. Data shown are relative light units (RLU) emitted from effector cells and are plotted as the average of technical duplicates. [Figure 16-4]Figures 16A-D show that GIGA-564 induces more FcR signaling than ipilimumab. Target CHO cells expressing human CTLA-4 with the Y201G mutation to enhance surface expression were incubated with a titration series of ipilimumab (black squares), GIGA-564 (black stars), or a variant of GIGA-564 with the LALA-PG mutation to disrupt FcγR binding (GIGA-564_LALA-PG, gray circles). Jurkat / NFAT-Luc effector cells bearing (Figure 16A) mouse FcγRIV or FcγRIII, (Figure 16B) human FcγRIIIa (high-affinity V158 or low-affinity F158 variant), (Figure 16C) human FcγRIIa (high-affinity H131 or low-affinity R131 variant), or (Figure 16D) human FcγRIIb were subsequently added. Cells were incubated at 37°C for 6 hours, after which luciferase activity was measured. Data shown are relative light units (RLU) emitted from effector cells and are plotted as the average of technical duplicates.

[0133] [Figure 17-1]Figure 17A shows that GIGA-564 confers protection in a mouse tumor reimplantation model. hCTLA-4 KI mice bearing MC38 tumors were randomized (day 8) when tumors reached 60–120 mm3 and then treated with the indicated antibodies at 1 mg / kg every 3 days for three doses. The plot shows tumor volume (mean ± SEM) over time for MC38 tumors treated with the indicated antibodies. Tumor volumes were advanced (thin black vertical lines) from mice euthanized due to tumor burden exceeding 3000 mm3. n = 8 (isotype and commercial ipilimumab), n = 9 (GIGA-564). Figure 17B shows that MC38 cells were implanted into the opposite flank of naive C57BL / 6 mice and mice previously treated with ipilimumab or GIGA-564 that had a durable complete response at day 43 (Figure 17A) (0 mm3 tumor volume). The plot shows tumor volume (mean ± SEM) over time for MC38 tumors in mice pretreated with the indicated antibodies or in naive mice (n = 5). Tumor volume (mm3) was analyzed using a linear mixed-effects model with treatment group and day as fixed effects and animal identifier (ID) as a random effect to account for repeated measurements. Statistical comparisons were performed using the Wald test against the isotype control group in the initial transplant model and against the naive control group in the re-transplant model. Compared to the naive group, pretreatment with ipilimumab (p = 0.0089) or GIGA-564 (p = 0.0088) limited tumor growth. [Figure 17-2]Figure 17A shows that GIGA-564 confers protection in a mouse tumor reimplantation model. hCTLA-4 KI mice bearing MC38 tumors were randomized (day 8) when tumors reached 60–120 mm3 and then treated with the indicated antibodies at 1 mg / kg every 3 days for three doses. The plot shows tumor volume (mean ± SEM) over time for MC38 tumors treated with the indicated antibodies. Tumor volumes were advanced (thin black vertical lines) from mice euthanized due to tumor burden exceeding 3000 mm3. n = 8 (isotype and commercial ipilimumab), n = 9 (GIGA-564). Figure 17B shows that MC38 cells were implanted into the opposite flank of naive C57BL / 6 mice and mice previously treated with ipilimumab or GIGA-564 that had a durable complete response at day 43 (Figure 17A) (0 mm3 tumor volume). The plot shows tumor volume (mean ± SEM) over time for MC38 tumors in mice pretreated with the indicated antibodies or in naive mice (n = 5). Tumor volume (mm3) was analyzed using a linear mixed-effects model with treatment group and day as fixed effects and animal identifier (ID) as a random effect to account for repeated measurements. Statistical comparisons were performed using the Wald test against the isotype control group in the initial transplant model and against the naive control group in the re-transplant model. Compared to the naive group, pretreatment with ipilimumab (p = 0.0089) or GIGA-564 (p = 0.0088) limited tumor growth.

[0134] [Figure 18-1]In a mouse model, GIGA-564 plus pembrolizumab induces lower toxicity than ipilimumab plus pembrolizumab. Four- to five-week-old hCTLA-4 / hPD-1 double KI mice on a BALB / c background were treated every three days for nine doses with vehicle, pembrolizumab, pembrolizumab plus ipilimumab (Ipi), or pembrolizumab plus GIGA-564. One week after the last dose, mice were euthanized, and tissues were collected for pathological analysis. Figure 18A shows the percentage change in body weight over time in mice treated with the indicated treatments (mean ± SEM, n = 10). Four mice in the pembrolizumab + ipilimumab group died on day 12, while three mice in the pembrolizumab + GIGA-564 treatment group died on day 12 and one mouse died on day 15, likely due to ADA-induced hypersensitivity after administration. A mixed-effects model revealed no statistically significant differences in the percentage body weight change between groups. Figures 18B-18C. Plots show the mean + / - SEM for dermatitis (Figure 18B) or colonic epithelial damage (colitis, Figure 18C) scores induced by each treatment regimen. Horizontal lines indicate medians. Adjusted p-values ​​were calculated using the Benjamini-Hochberg step-down method to account for multiple comparisons. [Figure 18-2]In a mouse model, GIGA-564 plus pembrolizumab induces lower toxicity than ipilimumab plus pembrolizumab. Four- to five-week-old hCTLA-4 / hPD-1 double KI mice on a BALB / c background were treated every three days for nine doses with vehicle, pembrolizumab, pembrolizumab plus ipilimumab (Ipi), or pembrolizumab plus GIGA-564. One week after the last dose, mice were euthanized, and tissues were collected for pathological analysis. Figure 18A shows the percentage change in body weight over time in mice treated with the indicated treatments (mean ± SEM, n = 10). Four mice in the pembrolizumab + ipilimumab group died on day 12, while three mice in the pembrolizumab + GIGA-564 treatment group died on day 12 and one mouse died on day 15, likely due to ADA-induced hypersensitivity after administration. A mixed-effects model revealed no statistically significant differences in the percentage body weight change between groups. Figures 18B-18C. Plots show the mean + / - SEM for dermatitis (Figure 18B) or colonic epithelial damage (colitis, Figure 18C) scores induced by each treatment regimen. Horizontal lines indicate medians. Adjusted p-values ​​were calculated using the Benjamini-Hochberg step-down method to account for multiple comparisons. [Figure 18-3]In a mouse model, GIGA-564 plus pembrolizumab induces lower toxicity than ipilimumab plus pembrolizumab. Four- to five-week-old hCTLA-4 / hPD-1 double KI mice on a BALB / c background were treated every three days for nine doses with vehicle, pembrolizumab, pembrolizumab plus ipilimumab (Ipi), or pembrolizumab plus GIGA-564. One week after the last dose, mice were euthanized, and tissues were collected for pathological analysis. Figure 18A shows the percentage change in body weight over time in mice treated with the indicated treatments (mean ± SEM, n = 10). Four mice in the pembrolizumab + ipilimumab group died on day 12, while three mice in the pembrolizumab + GIGA-564 treatment group died on day 12 and one mouse died on day 15, likely due to ADA-induced hypersensitivity after administration. A mixed-effects model revealed no statistically significant differences in the percentage body weight change between groups. Figures 18B-18C. Plots show the mean + / - SEM for dermatitis (Figure 18B) or colonic epithelial damage (colitis, Figure 18C) scores induced by each treatment regimen. Horizontal lines indicate medians. Adjusted p-values ​​were calculated using the Benjamini-Hochberg step-down method to account for multiple comparisons.

[0135] [Figure 19] A model demonstrating the mechanism of action of ipilimumab and GIGA-564 is shown. Upper panel: Ipilimumab blocks the interaction of CTLA-4 with CD80 / CD86, allowing antigen-presenting cells (APCs) to costimulate peripheral Tregs and enhance their proliferation. GIGA-564 weakly blocks the interaction of CTLA-4 with CD80 / CD86, thus inducing lower Treg proliferation. Lower panel: Ipilimumab and GIGA-564 bind to CTLA-4 on intratumoral Tregs and induce Treg death through interaction with Fc receptors (FcR) on effector cells. GIGA-564 induces stronger FcR signaling than ipilimumab and therefore more efficiently depletes intratumoral Tregs.

[0136] [Figure 20-1]In vitro characterization of scFvs reformatted as full-length antibodies is shown. Figure 20A. Clonal cluster analysis of anti-CTLA-4 scFv clones enriched by FACS. Each node represents an scFv clone (full-length IgK + IgH). The total number of amino acid differences was calculated between each pairwise alignment of scFv sequences. Edges indicate pairwise alignments with ≤9 amino acid differences (clustergram modified from Figure 7 in Asensio et al., 2019). The ipilimumab (ipi) scFv sequence was included for comparison. The scFv clones of full-length antibodies described in this study are indicated by ID numbers. Figure 20B. The affinity of the indicated antibodies for soluble CTLA-4 was determined by SPR (Carterra). The plot shows the association and dissociation signals at a 5-fold dilution series of antigen starting at 500 nM. Figure 20C. A 50:50 mixture of CTLA-4+ and CD27+ (CTLA-4-) CHO cells was stained with 10 μg / mL of the indicated antibody. A PE-conjugated anti-human IgG secondary antibody was used to detect cells labeled with the indicated anti-CTLA-4 antibody, while anti-CD27-FITC identified CD27+ CHO cells. Histograms show staining of CTLA-4+ (CD27-FITC-) or CTLA-4- (CD27-FITC+) cells with the indicated antibodies as determined by flow cytometry. Figure 20D. The cell-based CTLA-4 blocking bioassay (Promega) involved co-culturing CTLA-4-expressing Jurkat cells with Raji cells, which naturally express CD80 and CD86, in the presence of the indicated mAb. mAbs that bind to CTLA-4 and block its ability to interact with CD80 / CD86 result in CD28 pathway activation and luciferase expression. The plot shows the amount of luciferase expression (relative luciferase units, RLU) induced when cells were cultured with a titration series of the indicated antibodies. Due to sample size constraints with each Promega bioassay kit, this set of aCTLA-4 mAbs was analyzed using multiple plates.To control for plate-to-plate variation in maximum signal, an ipilimumab analog was run on each plate, and a representative sample was used to calculate the EC50 and maximum signal for ipilimumab in Table 23. Table 23 shows the plates on which each antibody was tested. Plates A and B were run simultaneously, and plates C and D were run separately at a later time. Figure 20E. Correlation between antibody affinity and cell-based assay activity. When antibody affinity (KD) for CTLA-4 was compared to the blocking EC50 and maximum RLU signal, and the data was fitted to a linear regression, no correlation was found for either. Each data point represents a single antibody, and the following antibody distinctions are made: ipilimumab analog (red square), GIGA-564 (inverted triangle), and aCTLA-4.28 (star). Data are from (Figure 20D) and Table 23. [Figure 20-2]In vitro characterization of scFvs reformatted as full-length antibodies is shown. Figure 20A. Clonal cluster analysis of anti-CTLA-4 scFv clones enriched by FACS. Each node represents an scFv clone (full-length IgK + IgH). The total number of amino acid differences was calculated between each pairwise alignment of scFv sequences. Edges indicate pairwise alignments with ≤9 amino acid differences (clustergram modified from Figure 7 in Asensio et al., 2019). The ipilimumab (ipi) scFv sequence was included for comparison. The scFv clones of full-length antibodies described in this study are indicated by ID numbers. Figure 20B. The affinity of the indicated antibodies for soluble CTLA-4 was determined by SPR (Carterra). The plot shows the association and dissociation signals at a 5-fold dilution series of antigen starting at 500 nM. Figure 20C. A 50:50 mixture of CTLA-4+ and CD27+ (CTLA-4-) CHO cells was stained with 10 μg / mL of the indicated antibody. A PE-conjugated anti-human IgG secondary antibody was used to detect cells labeled with the indicated anti-CTLA-4 antibody, while anti-CD27-FITC identified CD27+ CHO cells. Histograms show staining of CTLA-4+ (CD27-FITC-) or CTLA-4- (CD27-FITC+) cells with the indicated antibodies as determined by flow cytometry. Figure 20D. The cell-based CTLA-4 blocking bioassay (Promega) involved co-culturing CTLA-4-expressing Jurkat cells with Raji cells, which naturally express CD80 and CD86, in the presence of the indicated mAb. mAbs that bind to CTLA-4 and block its ability to interact with CD80 / CD86 result in CD28 pathway activation and luciferase expression. The plot shows the amount of luciferase expression (relative luciferase units, RLU) induced when cells were cultured with a titration series of the indicated antibodies. Due to sample size constraints with each Promega bioassay kit, this set of aCTLA-4 mAbs was analyzed using multiple plates.To control for plate-to-plate variation in maximum signal, an ipilimumab analog was run on each plate, and a representative sample was used to calculate the EC50 and maximum signal for ipilimumab in Table 23. Table 23 shows the plates on which each antibody was tested. Plates A and B were run simultaneously, and plates C and D were run separately at a later time. Figure 20E. Correlation between antibody affinity and cell-based assay activity. When antibody affinity (KD) for CTLA-4 was compared to the blocking EC50 and maximum RLU signal, and the data was fitted to a linear regression, no correlation was found for either. Each data point represents a single antibody, and the following antibody distinctions are made: ipilimumab analog (red square), GIGA-564 (inverted triangle), and aCTLA-4.28 (star). Data are from (Figure 20D) and Table 23. [Figure 20-3]In vitro characterization of scFvs reformatted as full-length antibodies is shown. Figure 20A. Clonal cluster analysis of anti-CTLA-4 scFv clones enriched by FACS. Each node represents an scFv clone (full-length IgK + IgH). The total number of amino acid differences was calculated between each pairwise alignment of scFv sequences. Edges indicate pairwise alignments with ≤9 amino acid differences (clustergram modified from Figure 7 in Asensio et al., 2019). The ipilimumab (ipi) scFv sequence was included for comparison. The scFv clones of full-length antibodies described in this study are indicated by ID numbers. Figure 20B. The affinity of the indicated antibodies for soluble CTLA-4 was determined by SPR (Carterra). The plot shows the association and dissociation signals at a 5-fold dilution series of antigen starting at 500 nM. Figure 20C. A 50:50 mixture of CTLA-4+ and CD27+ (CTLA-4-) CHO cells was stained with 10 μg / mL of the indicated antibody. A PE-conjugated anti-human IgG secondary antibody was used to detect cells labeled with the indicated anti-CTLA-4 antibody, while anti-CD27-FITC identified CD27+ CHO cells. Histograms show staining of CTLA-4+ (CD27-FITC-) or CTLA-4- (CD27-FITC+) cells with the indicated antibodies as determined by flow cytometry. Figure 20D. The cell-based CTLA-4 blocking bioassay (Promega) involved co-culturing CTLA-4-expressing Jurkat cells with Raji cells, which naturally express CD80 and CD86, in the presence of the indicated mAb. mAbs that bind to CTLA-4 and block its ability to interact with CD80 / CD86 result in CD28 pathway activation and luciferase expression. The plot shows the amount of luciferase expression (relative luciferase units, RLU) induced when cells were cultured with a titration series of the indicated antibodies. Due to sample size constraints with each Promega bioassay kit, this set of aCTLA-4 mAbs was analyzed using multiple plates.To control for plate-to-plate variation in maximum signal, an ipilimumab analog was run on each plate, and a representative sample was used to calculate the EC50 and maximum signal for ipilimumab in Table 23. Table 23 shows the plates on which each antibody was tested. Plates A and B were run simultaneously, and plates C and D were run separately at a later time. Figure 20E. Correlation between antibody affinity and cell-based assay activity. When antibody affinity (KD) for CTLA-4 was compared to the blocking EC50 and maximum RLU signal, and the data was fitted to a linear regression, no correlation was found for either. Each data point represents a single antibody, and the following antibody distinctions are made: ipilimumab analog (red square), GIGA-564 (inverted triangle), and aCTLA-4.28 (star). Data are from (Figure 20D) and Table 23. [Figure 20-4]In vitro characterization of scFvs reformatted as full-length antibodies is shown. Figure 20A. Clonal cluster analysis of anti-CTLA-4 scFv clones enriched by FACS. Each node represents an scFv clone (full-length IgK + IgH). The total number of amino acid differences was calculated between each pairwise alignment of scFv sequences. Edges indicate pairwise alignments with ≤9 amino acid differences (clustergram modified from Figure 7 in Asensio et al., 2019). The ipilimumab (ipi) scFv sequence was included for comparison. The scFv clones of full-length antibodies described in this study are indicated by ID numbers. Figure 20B. The affinity of the indicated antibodies for soluble CTLA-4 was determined by SPR (Carterra). The plot shows the association and dissociation signals at a 5-fold dilution series of antigen starting at 500 nM. Figure 20C. A 50:50 mixture of CTLA-4+ and CD27+ (CTLA-4-) CHO cells was stained with 10 μg / mL of the indicated antibody. A PE-conjugated anti-human IgG secondary antibody was used to detect cells labeled with the indicated anti-CTLA-4 antibody, while anti-CD27-FITC identified CD27+ CHO cells. Histograms show staining of CTLA-4+ (CD27-FITC-) or CTLA-4- (CD27-FITC+) cells with the indicated antibodies as determined by flow cytometry. Figure 20D. The cell-based CTLA-4 blocking bioassay (Promega) involved co-culturing CTLA-4-expressing Jurkat cells with Raji cells, which naturally express CD80 and CD86, in the presence of the indicated mAb. mAbs that bind to CTLA-4 and block its ability to interact with CD80 / CD86 result in CD28 pathway activation and luciferase expression. The plot shows the amount of luciferase expression (relative luciferase units, RLU) induced when cells were cultured with a titration series of the indicated antibodies. Due to sample size constraints with each Promega bioassay kit, this set of aCTLA-4 mAbs was analyzed using multiple plates.To control for plate-to-plate variation in maximum signal, an ipilimumab analog was run on each plate, and a representative sample was used to calculate the EC50 and maximum signal for ipilimumab in Table 23. Table 23 shows the plates on which each antibody was tested. Plates A and B were run simultaneously, and plates C and D were run separately at a later time. Figure 20E. Correlation between antibody affinity and cell-based assay activity. When antibody affinity (KD) for CTLA-4 was compared to the blocking EC50 and maximum RLU signal, and the data was fitted to a linear regression, no correlation was found for either. Each data point represents a single antibody, and the following antibody distinctions are made: ipilimumab analog (red square), GIGA-564 (inverted triangle), and aCTLA-4.28 (star). Data are from (Figure 20D) and Table 23. [Figure 20-5]In vitro characterization of scFvs reformatted as full-length antibodies is shown. Figure 20A. Clonal cluster analysis of anti-CTLA-4 scFv clones enriched by FACS. Each node represents an scFv clone (full-length IgK + IgH). The total number of amino acid differences was calculated between each pairwise alignment of scFv sequences. Edges indicate pairwise alignments with ≤9 amino acid differences (clustergram modified from Figure 7 in Asensio et al., 2019). The ipilimumab (ipi) scFv sequence was included for comparison. The scFv clones of full-length antibodies described in this study are indicated by ID numbers. Figure 20B. The affinity of the indicated antibodies for soluble CTLA-4 was determined by SPR (Carterra). The plot shows the association and dissociation signals at a 5-fold dilution series of antigen starting at 500 nM. Figure 20C. A 50:50 mixture of CTLA-4+ and CD27+ (CTLA-4-) CHO cells was stained with 10 μg / mL of the indicated antibody. A PE-conjugated anti-human IgG secondary antibody was used to detect cells labeled with the indicated anti-CTLA-4 antibody, while anti-CD27-FITC identified CD27+ CHO cells. Histograms show staining of CTLA-4+ (CD27-FITC-) or CTLA-4- (CD27-FITC+) cells with the indicated antibodies as determined by flow cytometry. Figure 20D. The cell-based CTLA-4 blocking bioassay (Promega) involved co-culturing CTLA-4-expressing Jurkat cells with Raji cells, which naturally express CD80 and CD86, in the presence of the indicated mAb. mAbs that bind to CTLA-4 and block its ability to interact with CD80 / CD86 result in CD28 pathway activation and luciferase expression. The plot shows the amount of luciferase expression (relative luciferase units, RLU) induced when cells were cultured with a titration series of the indicated antibodies. Due to sample size constraints with each Promega bioassay kit, this set of aCTLA-4 mAbs was analyzed using multiple plates.To control for plate-to-plate variation in maximum signal, an ipilimumab analog was run on each plate, and a representative sample was used to calculate the EC50 and maximum signal for ipilimumab in Table 23. Table 23 shows the plates on which each antibody was tested. Plates A and B were run simultaneously, and plates C and D were run separately at a later time. Figure 20E. Correlation between antibody affinity and cell-based assay activity. When antibody affinity (KD) for CTLA-4 was compared to the blocking EC50 and maximum RLU signal, and the data was fitted to a linear regression, no correlation was found for either. Each data point represents a single antibody, and the following antibody distinctions are made: ipilimumab analog (red square), GIGA-564 (inverted triangle), and aCTLA-4.28 (star). Data are from (Figure 20D) and Table 23. [Figure 20-6]In vitro characterization of scFvs reformatted as full-length antibodies is shown. Figure 20A. Clonal cluster analysis of anti-CTLA-4 scFv clones enriched by FACS. Each node represents an scFv clone (full-length IgK + IgH). The total number of amino acid differences was calculated between each pairwise alignment of scFv sequences. Edges indicate pairwise alignments with ≤9 amino acid differences (clustergram modified from Figure 7 in Asensio et al., 2019). The ipilimumab (ipi) scFv sequence was included for comparison. The scFv clones of full-length antibodies described in this study are indicated by ID numbers. Figure 20B. The affinity of the indicated antibodies for soluble CTLA-4 was determined by SPR (Carterra). The plot shows the association and dissociation signals at a 5-fold dilution series of antigen starting at 500 nM. Figure 20C. A 50:50 mixture of CTLA-4+ and CD27+ (CTLA-4-) CHO cells was stained with 10 μg / mL of the indicated antibody. A PE-conjugated anti-human IgG secondary antibody was used to detect cells labeled with the indicated anti-CTLA-4 antibody, while anti-CD27-FITC identified CD27+ CHO cells. Histograms show staining of CTLA-4+ (CD27-FITC-) or CTLA-4- (CD27-FITC+) cells with the indicated antibodies as determined by flow cytometry. Figure 20D. The cell-based CTLA-4 blocking bioassay (Promega) involved co-culturing CTLA-4-expressing Jurkat cells with Raji cells, which naturally express CD80 and CD86, in the presence of the indicated mAb. mAbs that bind to CTLA-4 and block its ability to interact with CD80 / CD86 result in CD28 pathway activation and luciferase expression. The plot shows the amount of luciferase expression (relative luciferase units, RLU) induced when cells were cultured with a titration series of the indicated antibodies. Due to sample size constraints with each Promega bioassay kit, this set of aCTLA-4 mAbs was analyzed using multiple plates.To control for plate-to-plate variation in maximum signal, an ipilimumab analog was run on each plate, and a representative sample was used to calculate the EC50 and maximum signal for ipilimumab in Table 23. Table 23 shows the plates on which each antibody was tested. Plates A and B were run simultaneously, and plates C and D were run separately at a later time. Figure 20E. Correlation between antibody affinity and cell-based assay activity. When antibody affinity (KD) for CTLA-4 was compared to the blocking EC50 and maximum RLU signal, and the data was fitted to a linear regression, no correlation was found for either. Each data point represents a single antibody, and the following antibody distinctions are made: ipilimumab analog (red square), GIGA-564 (inverted triangle), and aCTLA-4.28 (star). Data are from (Figure 20D) and Table 23.

[0137] [Figure 21] This figure provides validation of the binding of the N297Q mutant to cell surface CTLA-4. CHO cells with and without human CTLA-4 expression were incubated with 10 μg / μL of the indicated antibody, and then an anti-human IgG secondary antibody conjugated to FITC was used to detect cells bound by the indicated CTLA-4 antibody. The histograms show staining of CTLA-4+ cells (light gray) or CTLA-4- cells (dark gray) by the indicated antibody as determined by flow cytometry.

[0138] [Figure 22-1]Figure 22 shows that costimulation enhances Treg proliferation. Histograms show CellTrace Violet signals (gated on Live, CD3+CD4+ cells) of Treg cells (Figure 22A) or Tconv cells (Figure 22B) cultured in the presence of anti-CD3 antibody-coated M-450 tosyl activation beads with or without CD80, or Treg cells (Figure 22C) or Tconv cells (Figure 22D) activated with anti-CD3 antibody + CD80-coated M-450 tosyl activation beads in the presence of rhCTLA-4 (abatacept) and / or anti-CTLA-4 mAb (aCTLA-4.28). [Figure 22-2] Figure 22 shows that costimulation enhances Treg proliferation. Histograms show CellTrace Violet signals (gated on Live, CD3+CD4+ cells) of Treg cells (Figure 22A) or Tconv cells (Figure 22B) cultured in the presence of anti-CD3 antibody-coated M-450 tosyl activation beads with or without CD80, or Treg cells (Figure 22C) or Tconv cells (Figure 22D) activated with anti-CD3 antibody + CD80-coated M-450 tosyl activation beads in the presence of rhCTLA-4 (abatacept) and / or anti-CTLA-4 mAb (aCTLA-4.28). [Figure 22-3] Figure 22 shows that costimulation enhances Treg proliferation. Histograms show CellTrace Violet signals (gated on Live, CD3+CD4+ cells) of Treg cells (Figure 22A) or Tconv cells (Figure 22B) cultured in the presence of anti-CD3 antibody-coated M-450 tosyl activation beads with or without CD80, or Treg cells (Figure 22C) or Tconv cells (Figure 22D) activated with anti-CD3 antibody + CD80-coated M-450 tosyl activation beads in the presence of rhCTLA-4 (abatacept) and / or anti-CTLA-4 mAb (aCTLA-4.28). [Figure 22-4]Figure 22 shows that costimulation enhances Treg proliferation. Histograms show CellTrace Violet signals (gated on Live, CD3+CD4+ cells) of Treg cells (Figure 22A) or Tconv cells (Figure 22B) cultured in the presence of anti-CD3 antibody-coated M-450 tosyl activation beads with or without CD80, or Treg cells (Figure 22C) or Tconv cells (Figure 22D) activated with anti-CD3 antibody + CD80-coated M-450 tosyl activation beads in the presence of rhCTLA-4 (abatacept) and / or anti-CTLA-4 mAb (aCTLA-4.28).

[0139] [Figure 23-1] Figures 23A and 23C show that anti-CTLA-4 depletes intratumoral Tregs in hCTLA-4 KI mice. Flow cytometry analysis of cells from hCTLA-4 KI mice bearing MC38 tumors administered with CTLA-4 mAb. Figures 23A and 23C show that six mice per group were treated with 5 mg / kg of hIgG1 isotype control, ipilimumab, or GIGA-564 on days 0 and 3, and cells were analyzed on day 4. Figures 23B and 23D show that twelve mice per group were treated with 5 mg / kg of hIgG1 isotype control, ipilimumab, or GIGA-564 on days 0, 3, and 6, and cells were analyzed on day 7. Two lymph node samples from the GIGA-564 group were excluded from analysis due to low cell counts. Figures 23A-B. Frequency of Tregs (as a percentage of Live, CD45+TCRβ+CD4+FOXP3+, CD45+ cells) in lymph nodes (left) and tumors (right) in each treatment group. Figures 23C-D. Geometric mean fluorescence intensity (MFI) of intracellular CTLA-4 in Tregs in LNs (left) and tumors (right). P values ​​were calculated using the Wilcoxon rank sum test. Lines indicate mean + / - SEM. [Figure 23-2]Figures 23A and 23C show that anti-CTLA-4 depletes intratumoral Tregs in hCTLA-4 KI mice. Flow cytometry analysis of cells from hCTLA-4 KI mice bearing MC38 tumors administered with CTLA-4 mAb. Figures 23A and 23C show that six mice per group were treated with 5 mg / kg of hIgG1 isotype control, ipilimumab, or GIGA-564 on days 0 and 3, and cells were analyzed on day 4. Figures 23B and 23D show that twelve mice per group were treated with 5 mg / kg of hIgG1 isotype control, ipilimumab, or GIGA-564 on days 0, 3, and 6, and cells were analyzed on day 7. Two lymph node samples from the GIGA-564 group were excluded from analysis due to low cell counts. Figures 23A-B. Frequency of Tregs (as a percentage of Live, CD45+TCRβ+CD4+FOXP3+, CD45+ cells) in lymph nodes (left) and tumors (right) in each treatment group. Figures 23C-D. Geometric mean fluorescence intensity (MFI) of intracellular CTLA-4 in Tregs in LNs (left) and tumors (right). P values ​​were calculated using the Wilcoxon rank sum test. Lines indicate mean + / - SEM. [Figure 23-3]Figures 23A and 23C show that anti-CTLA-4 depletes intratumoral Tregs in hCTLA-4 KI mice. Flow cytometry analysis of cells from hCTLA-4 KI mice bearing MC38 tumors administered with CTLA-4 mAb. Figures 23A and 23C show that six mice per group were treated with 5 mg / kg of hIgG1 isotype control, ipilimumab, or GIGA-564 on days 0 and 3, and cells were analyzed on day 4. Figures 23B and 23D show that twelve mice per group were treated with 5 mg / kg of hIgG1 isotype control, ipilimumab, or GIGA-564 on days 0, 3, and 6, and cells were analyzed on day 7. Two lymph node samples from the GIGA-564 group were excluded from analysis due to low cell counts. Figures 23A-B. Frequency of Tregs (as a percentage of Live, CD45+TCRβ+CD4+FOXP3+, CD45+ cells) in lymph nodes (left) and tumors (right) in each treatment group. Figures 23C-D. Geometric mean fluorescence intensity (MFI) of intracellular CTLA-4 in Tregs in LNs (left) and tumors (right). P values ​​were calculated using the Wilcoxon rank sum test. Lines indicate mean + / - SEM. [Figure 23-4]Figures 23A and 23C show that anti-CTLA-4 depletes intratumoral Tregs in hCTLA-4 KI mice. Flow cytometry analysis of cells from hCTLA-4 KI mice bearing MC38 tumors administered with CTLA-4 mAb. Figures 23A and 23C show that six mice per group were treated with 5 mg / kg of hIgG1 isotype control, ipilimumab, or GIGA-564 on days 0 and 3, and cells were analyzed on day 4. Figures 23B and 23D show that twelve mice per group were treated with 5 mg / kg of hIgG1 isotype control, ipilimumab, or GIGA-564 on days 0, 3, and 6, and cells were analyzed on day 7. Two lymph node samples from the GIGA-564 group were excluded from analysis due to low cell counts. Figures 23A-B. Frequency of Tregs (as a percentage of Live, CD45+TCRβ+CD4+FOXP3+, CD45+ cells) in lymph nodes (left) and tumors (right) in each treatment group. Figures 23C-D. Geometric mean fluorescence intensity (MFI) of intracellular CTLA-4 in Tregs in LNs (left) and tumors (right). P values ​​were calculated using the Wilcoxon rank sum test. Lines indicate mean + / - SEM.

[0140] [Figure 24-1]Figure 24A shows that GIGA-564 induces more FcR signaling than ipilimumab. Figure 24A shows that purified CTLA-4 antibody was diluted in an eight-point, five-fold titration series from a starting concentration of 5 μg / mL in different pH buffers and then added to wells coated with rhCTLA-4-Fc. Bound antibody was detected with anti-constant κ-HRP and absorbance at 450 nm was measured. Figure 24B shows that CHO cells expressing wild-type hCTLA-4 were incubated with titrations of ipilimumab or GIGA-564 at 37°C to allow internalization. The amount of antibody remaining on the surface was then determined by staining with anti-human IgG Fc. Figures 24C-D show that GIGA-564 from three different production runs was tested for fucosylation levels (each bar represents a single data point) (Figure 24C) and human FcγRIIIA signaling (Figure 24D). PN-2758.01 and PN-4088.01 were generated from transient transfection of ExpiCHO cells, while PN-4261.01 was derived from a stable expression pool of CHOZN clones (EP-1, enriched pool 1). Figure 24C. Fucosylation levels determined by UPLC analysis are shown for each sample. Figures 24D-24E. Graphs show human FcγRIIIA (V variant) signaling determined in cell-based assays for the three indicated preparations of GIGA-564 (Figure 24D) or GIGA-564 and ipilimumab (Figure 24E) with the indicated amounts of fucosylation. A negative control protein (GIGA-564_LALAPG) with a mutation that disrupts Fc receptor binding was also tested in the reporter bioassay. Data shown are RLU released from effector cells. [Figure 24-2]Figure 24A shows that GIGA-564 induces more FcR signaling than ipilimumab. Figure 24A shows that purified CTLA-4 antibody was diluted in an eight-point, five-fold titration series from a starting concentration of 5 μg / mL in different pH buffers and then added to wells coated with rhCTLA-4-Fc. Bound antibody was detected with anti-constant κ-HRP and absorbance at 450 nm was measured. Figure 24B shows that CHO cells expressing wild-type hCTLA-4 were incubated with titrations of ipilimumab or GIGA-564 at 37°C to allow internalization. The amount of antibody remaining on the surface was then determined by staining with anti-human IgG Fc. Figures 24C-D show that GIGA-564 from three different production runs was tested for fucosylation levels (each bar represents a single data point) (Figure 24C) and human FcγRIIIA signaling (Figure 24D). PN-2758.01 and PN-4088.01 were generated from transient transfection of ExpiCHO cells, while PN-4261.01 was derived from a stable expression pool of CHOZN clones (EP-1, enriched pool 1). Figure 24C. Fucosylation levels determined by UPLC analysis are shown for each sample. Figures 24D-24E. Graphs show human FcγRIIIA (V variant) signaling determined in cell-based assays for the three indicated preparations of GIGA-564 (Figure 24D) or GIGA-564 and ipilimumab (Figure 24E) with the indicated amounts of fucosylation. A negative control protein (GIGA-564_LALAPG) with a mutation that disrupts Fc receptor binding was also tested in the reporter bioassay. Data shown are RLU released from effector cells. [Figure 24-3]Figure 24A shows that GIGA-564 induces more FcR signaling than ipilimumab. Figure 24A shows that purified CTLA-4 antibody was diluted in an eight-point, five-fold titration series from a starting concentration of 5 μg / mL in different pH buffers and then added to wells coated with rhCTLA-4-Fc. Bound antibody was detected with anti-constant κ-HRP and absorbance at 450 nm was measured. Figure 24B shows that CHO cells expressing wild-type hCTLA-4 were incubated with titrations of ipilimumab or GIGA-564 at 37°C to allow internalization. The amount of antibody remaining on the surface was then determined by staining with anti-human IgG Fc. Figures 24C-D show that GIGA-564 from three different production runs was tested for fucosylation levels (each bar represents a single data point) (Figure 24C) and human FcγRIIIA signaling (Figure 24D). PN-2758.01 and PN-4088.01 were generated from transient transfection of ExpiCHO cells, while PN-4261.01 was derived from a stable expression pool of CHOZN clones (EP-1, enriched pool 1). Figure 24C. Fucosylation levels determined by UPLC analysis are shown for each sample. Figures 24D-24E. Graphs show human FcγRIIIA (V variant) signaling determined in cell-based assays for the three indicated preparations of GIGA-564 (Figure 24D) or GIGA-564 and ipilimumab (Figure 24E) with the indicated amounts of fucosylation. A negative control protein (GIGA-564_LALAPG) with a mutation that disrupts Fc receptor binding was also tested in the reporter bioassay. Data shown are RLU released from effector cells. [Figure 24-4]Figure 24A shows that GIGA-564 induces more FcR signaling than ipilimumab. Figure 24A shows that purified CTLA-4 antibody was diluted in an eight-point, five-fold titration series from a starting concentration of 5 μg / mL in different pH buffers and then added to wells coated with rhCTLA-4-Fc. Bound antibody was detected with anti-constant κ-HRP and absorbance at 450 nm was measured. Figure 24B shows that CHO cells expressing wild-type hCTLA-4 were incubated with titrations of ipilimumab or GIGA-564 at 37°C to allow internalization. The amount of antibody remaining on the surface was then determined by staining with anti-human IgG Fc. Figures 24C-D show that GIGA-564 from three different production runs was tested for fucosylation levels (each bar represents a single data point) (Figure 24C) and human FcγRIIIA signaling (Figure 24D). PN-2758.01 and PN-4088.01 were generated from transient transfection of ExpiCHO cells, while PN-4261.01 was derived from a stable expression pool of CHOZN clones (EP-1, enriched pool 1). Figure 24C. Fucosylation levels determined by UPLC analysis are shown for each sample. Figures 24D-24E. Graphs show human FcγRIIIA (V variant) signaling determined in cell-based assays for the three indicated preparations of GIGA-564 (Figure 24D) or GIGA-564 and ipilimumab (Figure 24E) with the indicated amounts of fucosylation. A negative control protein (GIGA-564_LALAPG) with a mutation that disrupts Fc receptor binding was also tested in the reporter bioassay. Data shown are RLU released from effector cells. [Figure 24-5]Figure 24A shows that GIGA-564 induces more FcR signaling than ipilimumab. Figure 24A shows that purified CTLA-4 antibody was diluted in an eight-point, five-fold titration series from a starting concentration of 5 μg / mL in different pH buffers and then added to wells coated with rhCTLA-4-Fc. Bound antibody was detected with anti-constant κ-HRP and absorbance at 450 nm was measured. Figure 24B shows that CHO cells expressing wild-type hCTLA-4 were incubated with titrations of ipilimumab or GIGA-564 at 37°C to allow internalization. The amount of antibody remaining on the surface was then determined by staining with anti-human IgG Fc. Figures 24C-D show that GIGA-564 from three different production runs was tested for fucosylation levels (each bar represents a single data point) (Figure 24C) and human FcγRIIIA signaling (Figure 24D). PN-2758.01 and PN-4088.01 were generated from transient transfection of ExpiCHO cells, while PN-4261.01 was derived from a stable expression pool of CHOZN clones (EP-1, enriched pool 1). Figure 24C. Fucosylation levels determined by UPLC analysis are shown for each sample. Figures 24D-24E. Graphs show human FcγRIIIA (V variant) signaling determined in cell-based assays for the three indicated preparations of GIGA-564 (Figure 24D) or GIGA-564 and ipilimumab (Figure 24E) with the indicated amounts of fucosylation. A negative control protein (GIGA-564_LALAPG) with a mutation that disrupts Fc receptor binding was also tested in the reporter bioassay. Data shown are RLU released from effector cells.

[0141] [Figure 25-1]Figures 25A-25D show that GIGA-564 results in lower toxicity than ipilimumab in a mouse model. Four- to five-week-old hCTLA-4 / hPD-1 double KI mice on a BALB / c background were treated with vehicle, pembrolizumab, pembrolizumab + ipilimumab, or pembrolizumab + GIGA-564 every three days for nine doses. One week after the last dose, mice were euthanized, and tissues were collected for pathological analysis. Graphs show the colon length (Figure 25A) or spleen weight (Figure 25B) of mice from each group at the time of euthanasia. The number of CD45+ cells / mm2 counted in randomly selected CD45-stained cardiac FFPE sections (Figure 25C) or cardiac pathology score determined by a pathologist (Figure 25D) are shown. Figure 25E. hCTLA-4 KI mice bearing MC38 tumors were treated with vehicle (PBS) or 5 mg / kg ipilimumab or GIGA-564 twice weekly for five treatments (Figure 14A). Twenty days after the start of treatment, mice were euthanized, and kidneys were processed into formalin-fixed, paraffin-embedded (FFPE) blocks. FFPE sections were stained for anti-mouse immunoglobulin G3 or C3 and scored by a board-certified veterinary pathologist blinded to the study. Positive staining in glomeruli was along the capillary basement membrane. Graphs show the percentage of glomeruli positive for anti-mouse IgG or C3 and the relative intensity of positive glomeruli; at least five glomeruli were examined in each section at 40x / high magnification. Data from Figure 25E are also shown in Figures 4A-E. Lines indicate mean ± SEM. To account for multiple comparisons, adjusted p-values ​​were calculated using the Benjamini-Hochberg step-down method. [Figure 25-2]Figures 25A-25D show that GIGA-564 results in lower toxicity than ipilimumab in a mouse model. Four- to five-week-old hCTLA-4 / hPD-1 double KI mice on a BALB / c background were treated with vehicle, pembrolizumab, pembrolizumab + ipilimumab, or pembrolizumab + GIGA-564 every three days for nine doses. One week after the last dose, mice were euthanized, and tissues were collected for pathological analysis. Graphs show the colon length (Figure 25A) or spleen weight (Figure 25B) of mice from each group at the time of euthanasia. The number of CD45+ cells / mm2 counted in randomly selected CD45-stained cardiac FFPE sections (Figure 25C) or cardiac pathology score determined by a pathologist (Figure 25D) are shown. Figure 25E. hCTLA-4 KI mice bearing MC38 tumors were treated with vehicle (PBS) or 5 mg / kg ipilimumab or GIGA-564 twice weekly for five treatments (Figure 14A). Twenty days after the start of treatment, mice were euthanized, and kidneys were processed into formalin-fixed, paraffin-embedded (FFPE) blocks. FFPE sections were stained for anti-mouse immunoglobulin G3 or C3 and scored by a board-certified veterinary pathologist blinded to the study. Positive staining in glomeruli was along the capillary basement membrane. Graphs show the percentage of glomeruli positive for anti-mouse IgG or C3 and the relative intensity of positive glomeruli; at least five glomeruli were examined in each section at 40x / high magnification. Data from Figure 25E are also shown in Figures 4A-E. Lines indicate mean ± SEM. To account for multiple comparisons, adjusted p-values ​​were calculated using the Benjamini-Hochberg step-down method. [Figure 25-3]Figures 25A-25D show that GIGA-564 results in lower toxicity than ipilimumab in a mouse model. Four- to five-week-old hCTLA-4 / hPD-1 double KI mice on a BALB / c background were treated with vehicle, pembrolizumab, pembrolizumab + ipilimumab, or pembrolizumab + GIGA-564 every three days for nine doses. One week after the last dose, mice were euthanized, and tissues were collected for pathological analysis. Graphs show the colon length (Figure 25A) or spleen weight (Figure 25B) of mice from each group at the time of euthanasia. The number of CD45+ cells / mm2 counted in randomly selected CD45-stained cardiac FFPE sections (Figure 25C) or cardiac pathology score determined by a pathologist (Figure 25D) are shown. Figure 25E. hCTLA-4 KI mice bearing MC38 tumors were treated with vehicle (PBS) or 5 mg / kg ipilimumab or GIGA-564 twice weekly for five treatments (Figure 14A). Twenty days after the start of treatment, mice were euthanized, and kidneys were processed into formalin-fixed, paraffin-embedded (FFPE) blocks. FFPE sections were stained for anti-mouse immunoglobulin G3 or C3 and scored by a board-certified veterinary pathologist blinded to the study. Positive staining in glomeruli was along the capillary basement membrane. Graphs show the percentage of glomeruli positive for anti-mouse IgG or C3 and the relative intensity of positive glomeruli; at least five glomeruli were examined in each section at 40x / high magnification. Data from Figure 25E are also shown in Figures 4A-E. Lines indicate mean ± SEM. To account for multiple comparisons, adjusted p-values ​​were calculated using the Benjamini-Hochberg step-down method. [Figure 25-4]Figures 25A-25D show that GIGA-564 results in lower toxicity than ipilimumab in a mouse model. Four- to five-week-old hCTLA-4 / hPD-1 double KI mice on a BALB / c background were treated with vehicle, pembrolizumab, pembrolizumab + ipilimumab, or pembrolizumab + GIGA-564 every three days for nine doses. One week after the last dose, mice were euthanized, and tissues were collected for pathological analysis. Graphs show the colon length (Figure 25A) or spleen weight (Figure 25B) of mice from each group at the time of euthanasia. The number of CD45+ cells / mm2 counted in randomly selected CD45-stained cardiac FFPE sections (Figure 25C) or cardiac pathology score determined by a pathologist (Figure 25D) are shown. Figure 25E. hCTLA-4 KI mice bearing MC38 tumors were treated with vehicle (PBS) or 5 mg / kg ipilimumab or GIGA-564 twice weekly for five treatments (Figure 14A). Twenty days after the start of treatment, mice were euthanized, and kidneys were processed into formalin-fixed, paraffin-embedded (FFPE) blocks. FFPE sections were stained for anti-mouse immunoglobulin G3 or C3 and scored by a board-certified veterinary pathologist blinded to the study. Positive staining in glomeruli was along the capillary basement membrane. Graphs show the percentage of glomeruli positive for anti-mouse IgG or C3 and the relative intensity of positive glomeruli; at least five glomeruli were examined in each section at 40x / high magnification. Data from Figure 25E are also shown in Figures 4A-E. Lines indicate mean ± SEM. To account for multiple comparisons, adjusted p-values ​​were calculated using the Benjamini-Hochberg step-down method. [Figure 25-5]Figures 25A-25D show that GIGA-564 results in lower toxicity than ipilimumab in a mouse model. Four- to five-week-old hCTLA-4 / hPD-1 double KI mice on a BALB / c background were treated with vehicle, pembrolizumab, pembrolizumab + ipilimumab, or pembrolizumab + GIGA-564 every three days for nine doses. One week after the last dose, mice were euthanized, and tissues were collected for pathological analysis. Graphs show the colon length (Figure 25A) or spleen weight (Figure 25B) of mice from each group at the time of euthanasia. The number of CD45+ cells / mm2 counted in randomly selected CD45-stained cardiac FFPE sections (Figure 25C) or cardiac pathology score determined by a pathologist (Figure 25D) are shown. Figure 25E. hCTLA-4 KI mice bearing MC38 tumors were treated with vehicle (PBS) or 5 mg / kg ipilimumab or GIGA-564 twice weekly for five treatments (Figure 14A). Twenty days after the start of treatment, mice were euthanized, and kidneys were processed into formalin-fixed, paraffin-embedded (FFPE) blocks. FFPE sections were stained for anti-mouse immunoglobulin G3 or C3 and scored by a board-certified veterinary pathologist blinded to the study. Positive staining in glomeruli was along the capillary basement membrane. Graphs show the percentage of glomeruli positive for anti-mouse IgG or C3 and the relative intensity of positive glomeruli; at least five glomeruli were examined in each section at 40x / high magnification. Data from Figure 25E are also shown in Figures 4A-E. Lines indicate mean ± SEM. To account for multiple comparisons, adjusted p-values ​​were calculated using the Benjamini-Hochberg step-down method.

[0142] [Figure 26] Figure 26 shows that the checkpoint inhibitor ipilimumab increases the proportion of proliferating Tregs in mice expressing humanized CTLA-4. The data in Figure 26 are from the same experiment as Figure 15A.

[0143] [Figure 27]These results demonstrate that intratumor Treg depletion, rather than checkpoint blockade, is the mechanism of action of anti-CTLA-4. Figure 27 (left) shows that Fc effector function in anti-CTLA-4 is required for a robust anti-tumor response. As shown, tumor volume was reduced with ipilimumab and GIGA-577 compared to ADCC-deficient ipilimumab and ADCC-deficient GIGA-577. The data in Figure 27 include data such as those shown in Figure 12B and Figure 14A.

[0144] [Figure 28] This demonstrates that the GIGA-564 anti-CTLA-4 antibody has weak checkpoint inhibitory activity but strong affinity for CTLA-4. A schematic diagram comparing the conventional mechanism with the mechanism of action of the present invention described in this application is shown. The depletion of Tregs in tumors after treatment with GIGA-564 is due to ADCC / ADCP binding, rather than the interaction between CTLA-4 and its ligand.

[0145] [Figure 29] Figure 29 shows that GIGA-564 weakly blocks the interaction of CD80 / CD86 binding to CTLA-4 compared to ipilimumab. Figure 29 can be reproduced from the data shown in Figure 20D (Plate A).

[0146] [Figure 30] These results demonstrate that GIGA-564 has superior antitumor activity compared to ipilimumab. Humanized CTLA-4 knockin mice (n=8-13) bearing MC38 tumors were administered 0.3 mpk of GIGA-564 or ipilimumab on days 0, 3, and 6 of the study. After administration of GIGA-564, human CTLA-4 knockin mice bearing MC38 tumors showed reduced tumor volume and increased survival compared to ipilimumab and isotype. The experiment described in Figure 30 is also described in Figures 11 and 14C. The tumor growth data in Figure 30 includes the data shown in Figures 11 and 14C.

[0147] [Figure 31]This shows that GIGA-564 induces less Treg proliferation than ipilimumab. Human CTLA-4 knock-in mice bearing MC38 tumors were treated with 5 mpk of ipilimumab or GIGA-564 on days 0, 3, and 6 and sacrificed on day 7. GIGA-564 treatment resulted in fewer proliferating Tregs in the periphery. The experiment described here was also depicted in Figure 15A. Figure 31 contains the data previously shown in Figure 15A.

[0148] [Figure 32] Representative contour plots of CD4 T cells in each treatment group are shown. The X and Y axes correspond to FOXP3 and CTLA-4, respectively. Figure 32 repeats the results of Figure 15D.

[0149] [Figure 33] Figure 33 shows that GIGA-564 induces more FcR signaling than ipilimumab when co-cultured with hCTLA-4+ cells. Cell signaling via CTLA-4-Fc-FcR interactions was evaluated in vitro using human CTLA-4+ cells. GIGA-564 demonstrated increased FcR signaling compared to ipilimumab. Further experiments ruled out that this difference was due to Fc-FcR affinity or CTLA-4 cell surface cycling. Figure 33 may re-present some of the data in Figure 16B.

[0150] [Figure 34-1] This paper presents the development and validation of CHO cells expressing cynomolgus monkey (cyno) CTLA-4 on the cell surface. Flow histograms show that cyno CTLA-4 and hCTLA-4 CHO cell lines express the antigen bound by anti-CTLA-4 clones L3D10 or BNI3 on ​​the cell surface, confirming the expression of cyno or human CTLA-4 on the surface of these cell lines, respectively. [Figure 34-2]This paper presents the development and validation of CHO cells expressing cynomolgus monkey (cyno) CTLA-4 on the cell surface. Flow histograms show that cyno CTLA-4 and hCTLA-4 CHO cell lines express the antigen bound by anti-CTLA-4 clones L3D10 or BNI3 on ​​the cell surface, confirming the expression of cyno or human CTLA-4 on the surface of these cell lines, respectively. [Figure 34-3] This paper presents the development and validation of CHO cells expressing cynomolgus monkey (cyno) CTLA-4 on the cell surface. Flow histograms show that cyno CTLA-4 and hCTLA-4 CHO cell lines express the antigen bound by anti-CTLA-4 clones L3D10 or BNI3 on ​​the cell surface, confirming the expression of cyno or human CTLA-4 on the surface of these cell lines, respectively.

[0151] [Figure 35-1] Figure 35 shows that GIGA-564 has a reduced ability to bind to surface-expressed cyno CTLA-4 compared to ipilimumab. Figure 35 shows flow cytometry analysis of mAb binding to cyno or human CTLA-4+ CHO cells. The data show that GIGA-564 and Ipi have relatively similar binding abilities to hCTLA-4, but compared to Ipi, GIGA-564 has significantly reduced binding ability to cell surface-expressed cyno CTLA-4. For reference, the data for Atezo binding (negative control) are shown again on multiple plots. [Figure 35-2] Figure 35 shows that GIGA-564 has a reduced ability to bind to surface-expressed cyno CTLA-4 compared to ipilimumab. Figure 35 shows flow cytometry analysis of mAb binding to cyno or human CTLA-4+ CHO cells. The data show that GIGA-564 and Ipi have relatively similar binding abilities to hCTLA-4, but compared to Ipi, GIGA-564 has significantly reduced binding ability to cell surface-expressed cyno CTLA-4. For reference, the data for Atezo binding (negative control) are shown again on multiple plots. [Figure 35-3] Figure 35 shows that GIGA-564 has a reduced ability to bind to surface-expressed cyno CTLA-4 compared to ipilimumab. Figure 35 shows flow cytometry analysis of mAb binding to cyno or human CTLA-4+ CHO cells. The data show that GIGA-564 and Ipi have relatively similar binding abilities to hCTLA-4, but compared to Ipi, GIGA-564 has significantly reduced binding ability to cell surface-expressed cyno CTLA-4. For reference, the data for Atezo binding (negative control) are shown again on multiple plots. [Figure 35-4] Figure 35 shows that GIGA-564 has a reduced ability to bind to surface-expressed cyno CTLA-4 compared to ipilimumab. Figure 35 shows flow cytometry analysis of mAb binding to cyno or human CTLA-4+ CHO cells. The data show that GIGA-564 and Ipi have relatively similar binding abilities to hCTLA-4, but compared to Ipi, GIGA-564 has significantly reduced binding ability to cell surface-expressed cyno CTLA-4. For reference, the data for Atezo binding (negative control) are shown again on multiple plots.

[0152] [Figure 36-1]These results show that GIGA-564 reduced binding to several presentations of cyno CTLA-4 compared to ipilimumab when tested using an ELISA assay. In these ELISAs, we tested the binding of GIGA-564 to various CTLA-4 proteins supplied by multiple manufacturers, including Fc-chimeric and His-tagged forms. To test the binding of GIGA-564 to Fc-chimeric CTLA-4 proteins, rcmCTLA-4-Fc from R&D systems (9336-CT-200, Figure 36A), Sino Biological (90213-C02H, Figure 36B), or ACROBiosystems (CT4-C5256, Figure 36C) was coated at 1 μg / mL onto half of a separate ELISA plate. Recombinant human CTLA-4-Fc (rhCTLA-4) from R&D Systems (7268-CT-100, Figures 36A-36C) was coated at 1 μg / mL on the other half of each plate. Similarly, to test the binding of GIGA-564 to his-tagged CTLA-4, rcmCTLA-4-His from Sino Biological (90213-C08H, Figure 36D) or ACROBiosystems (CT4-C5227, Figure 36E) was coated at 1 μg / mL on one half of a separate ELISA plate. RhCTLA-4-Fc (ACROBiosystems, CT4-H5229, Figures 36D-36E) was coated at 1 μg / mL on the other half of each of these plates. After coating, the plates were incubated overnight at 4°C. The next day, plates were blocked with 5% milk in PBST for 1 hour at room temperature on a plate shaker. A titration series of ipilimumab, atezolizumab (negative control), and GIGA-564 (GG-564) (starting at 5 μg / mL) was added to the plate and incubated for 1 hour at room temperature on a plate shaker to allow mAb binding. Excess unbound mAb was removed by washing with PBST. Bound mAb was then detected using an HRP-conjugated anti-κ light chain antibody (0.5 μg / mL, Southern Biotech 2060-50). After incubation for 1 hour at room temperature on a plate shaker and washing, the plate was developed with TMB substrate.After a sufficient signal was obtained, color development was stopped by adding 1N hydrochloric acid. Absorbance at 450 nm was read using a Spectramax i3x plate reader (Molecular Devices). EC50 values ​​were calculated by plotting the logarithm of absorbance versus concentration using Prism (GraphPad). Ipi has similar binding to human and cyno CTLA-4, whereas GIGA-564 mostly exhibits a lower ability to bind to cyno CTLA-4 compared to human CTLA-4. These results suggest that the Ipi and GIGA-564 epitopes are indeed different. [Figure 36-2]These results show that GIGA-564 reduced binding to several presentations of cyno CTLA-4 compared to ipilimumab when tested using an ELISA assay. In these ELISAs, we tested the binding of GIGA-564 to various CTLA-4 proteins supplied by multiple manufacturers, including Fc-chimeric and His-tagged forms. To test the binding of GIGA-564 to Fc-chimeric CTLA-4 proteins, rcmCTLA-4-Fc from R&D systems (9336-CT-200, Figure 36A), Sino Biological (90213-C02H, Figure 36B), or ACROBiosystems (CT4-C5256, Figure 36C) was coated at 1 μg / mL onto half of a separate ELISA plate. Recombinant human CTLA-4-Fc (rhCTLA-4) from R&D Systems (7268-CT-100, Figures 36A-36C) was coated at 1 μg / mL on the other half of each plate. Similarly, to test the binding of GIGA-564 to his-tagged CTLA-4, rcmCTLA-4-His from Sino Biological (90213-C08H, Figure 36D) or ACROBiosystems (CT4-C5227, Figure 36E) was coated at 1 μg / mL on one half of a separate ELISA plate. RhCTLA-4-Fc (ACROBiosystems, CT4-H5229, Figures 36D-36E) was coated at 1 μg / mL on the other half of each of these plates. After coating, the plates were incubated overnight at 4°C. The next day, plates were blocked with 5% milk in PBST for 1 hour at room temperature on a plate shaker. A titration series of ipilimumab, atezolizumab (negative control), and GIGA-564 (GG-564) (starting at 5 μg / mL) was added to the plate and incubated for 1 hour at room temperature on a plate shaker to allow mAb binding. Excess unbound mAb was removed by washing with PBST. Bound mAb was then detected using an HRP-conjugated anti-κ light chain antibody (0.5 μg / mL, Southern Biotech 2060-50). After incubation for 1 hour at room temperature on a plate shaker and washing, the plate was developed with TMB substrate.After a sufficient signal was obtained, color development was stopped by adding 1N hydrochloric acid. Absorbance at 450 nm was read using a Spectramax i3x plate reader (Molecular Devices). EC50 values ​​were calculated by plotting the logarithm of absorbance versus concentration using Prism (GraphPad). Ipi has similar binding to human and cyno CTLA-4, whereas GIGA-564 mostly exhibits a lower ability to bind to cyno CTLA-4 compared to human CTLA-4. These results suggest that the Ipi and GIGA-564 epitopes are indeed different. [Figure 36-3]These results show that GIGA-564 reduced binding to several presentations of cyno CTLA-4 compared to ipilimumab when tested using an ELISA assay. In these ELISAs, we tested the binding of GIGA-564 to various CTLA-4 proteins supplied by multiple manufacturers, including Fc-chimeric and His-tagged forms. To test the binding of GIGA-564 to Fc-chimeric CTLA-4 proteins, rcmCTLA-4-Fc from R&D systems (9336-CT-200, Figure 36A), Sino Biological (90213-C02H, Figure 36B), or ACROBiosystems (CT4-C5256, Figure 36C) was coated at 1 μg / mL onto half of a separate ELISA plate. Recombinant human CTLA-4-Fc (rhCTLA-4) from R&D Systems (7268-CT-100, Figures 36A-36C) was coated at 1 μg / mL on the other half of each plate. Similarly, to test the binding of GIGA-564 to his-tagged CTLA-4, rcmCTLA-4-His from Sino Biological (90213-C08H, Figure 36D) or ACROBiosystems (CT4-C5227, Figure 36E) was coated at 1 μg / mL on one half of a separate ELISA plate. RhCTLA-4-Fc (ACROBiosystems, CT4-H5229, Figures 36D-36E) was coated at 1 μg / mL on the other half of each of these plates. After coating, the plates were incubated overnight at 4°C. The next day, plates were blocked with 5% milk in PBST for 1 hour at room temperature on a plate shaker. A titration series of ipilimumab, atezolizumab (negative control), and GIGA-564 (GG-564) (starting at 5 μg / mL) was added to the plate and incubated for 1 hour at room temperature on a plate shaker to allow mAb binding. Excess unbound mAb was removed by washing with PBST. Bound mAb was then detected using an HRP-conjugated anti-κ light chain antibody (0.5 μg / mL, Southern Biotech 2060-50). After incubation for 1 hour at room temperature on a plate shaker and washing, the plate was developed with TMB substrate.After a sufficient signal was obtained, color development was stopped by adding 1N hydrochloric acid. Absorbance at 450 nm was read using a Spectramax i3x plate reader (Molecular Devices). EC50 values ​​were calculated by plotting the logarithm of absorbance versus concentration using Prism (GraphPad). Ipi has similar binding to human and cyno CTLA-4, whereas GIGA-564 mostly exhibits a lower ability to bind to cyno CTLA-4 compared to human CTLA-4. These results suggest that the Ipi and GIGA-564 epitopes are indeed different. [Figure 36-4]These results show that GIGA-564 reduced binding to several presentations of cyno CTLA-4 compared to ipilimumab when tested using an ELISA assay. In these ELISAs, we tested the binding of GIGA-564 to various CTLA-4 proteins supplied by multiple manufacturers, including Fc-chimeric and His-tagged forms. To test the binding of GIGA-564 to Fc-chimeric CTLA-4 proteins, rcmCTLA-4-Fc from R&D systems (9336-CT-200, Figure 36A), Sino Biological (90213-C02H, Figure 36B), or ACROBiosystems (CT4-C5256, Figure 36C) was coated at 1 μg / mL onto half of a separate ELISA plate. Recombinant human CTLA-4-Fc (rhCTLA-4) from R&D Systems (7268-CT-100, Figures 36A-36C) was coated at 1 μg / mL on the other half of each plate. Similarly, to test the binding of GIGA-564 to his-tagged CTLA-4, rcmCTLA-4-His from Sino Biological (90213-C08H, Figure 36D) or ACROBiosystems (CT4-C5227, Figure 36E) was coated at 1 μg / mL on one half of a separate ELISA plate. RhCTLA-4-Fc (ACROBiosystems, CT4-H5229, Figures 36D-36E) was coated at 1 μg / mL on the other half of each of these plates. After coating, the plates were incubated overnight at 4°C. The next day, plates were blocked with 5% milk in PBST for 1 hour at room temperature on a plate shaker. A titration series of ipilimumab, atezolizumab (negative control), and GIGA-564 (GG-564) (starting at 5 μg / mL) was added to the plate and incubated for 1 hour at room temperature on a plate shaker to allow mAb binding. Excess unbound mAb was removed by washing with PBST. Bound mAb was then detected using an HRP-conjugated anti-κ light chain antibody (0.5 μg / mL, Southern Biotech 2060-50). After incubation for 1 hour at room temperature on a plate shaker and washing, the plate was developed with TMB substrate.After a sufficient signal was obtained, color development was stopped by adding 1N hydrochloric acid. Absorbance at 450 nm was read using a Spectramax i3x plate reader (Molecular Devices). EC50 values ​​were calculated by plotting the logarithm of absorbance versus concentration using Prism (GraphPad). Ipi has similar binding to human and cyno CTLA-4, whereas GIGA-564 mostly exhibits a lower ability to bind to cyno CTLA-4 compared to human CTLA-4. These results suggest that the Ipi and GIGA-564 epitopes are indeed different. [Figure 36-5]These results show that GIGA-564 reduced binding to several presentations of cyno CTLA-4 compared to ipilimumab when tested using an ELISA assay. In these ELISAs, we tested the binding of GIGA-564 to various CTLA-4 proteins supplied by multiple manufacturers, including Fc-chimeric and His-tagged forms. To test the binding of GIGA-564 to Fc-chimeric CTLA-4 proteins, rcmCTLA-4-Fc from R&D systems (9336-CT-200, Figure 36A), Sino Biological (90213-C02H, Figure 36B), or ACROBiosystems (CT4-C5256, Figure 36C) was coated at 1 μg / mL onto half of a separate ELISA plate. Recombinant human CTLA-4-Fc (rhCTLA-4) from R&D Systems (7268-CT-100, Figures 36A-36C) was coated at 1 μg / mL on the other half of each plate. Similarly, to test the binding of GIGA-564 to his-tagged CTLA-4, rcmCTLA-4-His from Sino Biological (90213-C08H, Figure 36D) or ACROBiosystems (CT4-C5227, Figure 36E) was coated at 1 μg / mL on one half of a separate ELISA plate. RhCTLA-4-Fc (ACROBiosystems, CT4-H5229, Figures 36D-36E) was coated at 1 μg / mL on the other half of each of these plates. After coating, the plates were incubated overnight at 4°C. The next day, plates were blocked with 5% milk in PBST for 1 hour at room temperature on a plate shaker. A titration series of ipilimumab, atezolizumab (negative control), and GIGA-564 (GG-564) (starting at 5 μg / mL) was added to the plate and incubated for 1 hour at room temperature on a plate shaker to allow mAb binding. Excess unbound mAb was removed by washing with PBST. Bound mAb was then detected using an HRP-conjugated anti-κ light chain antibody (0.5 μg / mL, Southern Biotech 2060-50). After incubation for 1 hour at room temperature on a plate shaker and washing, the plate was developed with TMB substrate.After a sufficient signal was obtained, color development was stopped by adding 1N hydrochloric acid. Absorbance at 450 nm was read using a Spectramax i3x plate reader (Molecular Devices). EC50 values ​​were calculated by plotting the logarithm of absorbance versus concentration using Prism (GraphPad). Ipi has similar binding to human and cyno CTLA-4, whereas GIGA-564 mostly exhibits a lower ability to bind to cyno CTLA-4 compared to human CTLA-4. These results suggest that the Ipi and GIGA-564 epitopes are indeed different.

[0153] [Figure 37] 1 provides response plots generated to determine optimized formulations for GIGA-564. The response plots showed the effect of pH and sucrose concentration when NaCl and buffer concentrations were fixed at 100 mM and 30 mM, respectively.

[0154] [Figure 38] 1 shows theoretical response plots generated to determine the buffer concentration and amount of NaCl predicted to result in the highest Tm for the indicated formulations of GIGA-564 antibody.

[0155] [Figure 39] 1 shows a Pareto analysis showing which variables have the most impact on the formulation of GIGA-564.

[0156] [Figure 40-1] 1 provides a conformational analysis of formulations of GIGA-564. The plot shows that high NaCl is most important for conformation, pH has some effect, with lower pH being better, sucrose has some effect, with higher pH being better. [Figure 40-2]1 provides a conformational analysis of formulations of GIGA-564. The plot shows that high NaCl is most important for conformation, pH has some effect, with lower pH being better, sucrose has some effect, with higher pH being better.

[0157] [Figure 41] This shows that GIGA-2328 is designed to have hypofucosylation for the purpose of enhancing FcγRIIIa signaling. An ADCC reporter bioassay for Promega Corporation's human FcγRIIIa, V variant (G7011) was performed according to the manufacturer's instructions. Briefly, CHO target cells stably expressing human CTLA-4 with the Y201G mutation to enhance cell surface expression were suspended in RPMI 1640 + 4% FBS medium with the indicated antibodies and incubated at 37°C for 30 minutes. Jurkat / NFAT-Luc effector cells expressing human FcγRIIIa, V variant were added to each well at a 5:1 effector:target ratio and incubated at 37°C for 6 hours. Luciferase activity was measured using the provided Bio-Glo luciferase assay reagent with a SpectraMax i3x reader. Measured luciferase activity, expressed in relative luminescence units (RLU), was plotted against antibody concentration.

[0158] [Figure 42-1]This study demonstrates that GIGA-564 induces antibody-dependent cellular cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP) by human PBMCs against target cell lines expressing CTLA-4. Cryopreserved human PBMCs from two donors were thawed and recultured overnight in RMPI medium containing 100 U / mL IL-2 and 10% FBS. CHO target cells stably expressing human CTLA-4 with the Y201G mutation to enhance cell surface expression were stained with CellTrace Violet (Thermo Fisher Scientific) and then incubated with the indicated concentrations of either GIGA-564 or a human IgG1 isotype control for 30 minutes at 37°C. PMBC effector cells were then added at an effector-to-target ratio of 20:1, and the samples were incubated at 37°C for 4 hours to allow ADCC / ADCP. Samples were then washed with MACS buffer, stained with the dead cell dye 7-aminoactinomycin D (7-AAD) to label dead cells, and analyzed by flow cytometry. (Figure 42A) Representative gating strategy for determining ADCC / ADCP. Samples were first gated on target cells (CellTrace Violet+), then single cells, and then dead (7-AAD+) cells. (Figure 42B) Plot of the percentage of dead cells at each concentration of GIGA-564 and human IgG1 isotype. GIGA-564 results in a higher percentage of dead CTLA-4+ target cells than the isotype control. [Figure 42-2]This study demonstrates that GIGA-564 induces antibody-dependent cellular cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP) by human PBMCs against target cell lines expressing CTLA-4. Cryopreserved human PBMCs from two donors were thawed and recultured overnight in RMPI medium containing 100 U / mL IL-2 and 10% FBS. CHO target cells stably expressing human CTLA-4 with the Y201G mutation to enhance cell surface expression were stained with CellTrace Violet (Thermo Fisher Scientific) and then incubated with the indicated concentrations of either GIGA-564 or a human IgG1 isotype control for 30 minutes at 37°C. PMBC effector cells were then added at an effector-to-target ratio of 20:1, and the samples were incubated at 37°C for 4 hours to allow ADCC / ADCP. Samples were then washed with MACS buffer, stained with the dead cell dye 7-aminoactinomycin D (7-AAD) to label dead cells, and analyzed by flow cytometry. (Figure 42A) Representative gating strategy for determining ADCC / ADCP. Samples were first gated on target cells (CellTrace Violet+), then single cells, and then dead (7-AAD+) cells. (Figure 42B) Plot of the percentage of dead cells at each concentration of GIGA-564 and human IgG1 isotype. GIGA-564 results in a higher percentage of dead CTLA-4+ target cells than the isotype control.

[0159] [Figure 43-1]These results suggest that IgG1 allotypes can affect FcγRIIIa receptor-mediated signaling. Figure 43A shows sequence information for the IgG1 allotypes IGHG1*08 and IGHG1*01, which differ by one amino acid in the CH1 domain. The residues that differ between these two allotypes are highlighted, and surrounding residues are provided for reference. The locations are provided in both IMGT and EU numbering. Figure 43B shows that clinical ipilimumab (Yervoy) uses the IGHG1*08 allotype and results in lower FcγRIIIa signaling than ipilimumab and GIGA-564, both produced by GigaGen and using the IGHG1*01 allotype. These results suggest that allotypes can affect FcγRIIIa signaling. An ADCC reporter bioassay for human FcγRIIIa, V variant (G7011), was purchased from Promega Corporation. The assay was performed according to the manufacturer's instructions. Briefly, CHO target cells stably expressing human CTLA-4 with a Y201G mutation to enhance cell surface expression were suspended in RPMI 1640 + 4% FBS medium with the indicated antibodies and incubated for 30 minutes at 37°C. Jurkat / NFAT-Luc effector cells expressing human FcγRIIIa, V variant, were added to each well at a 5:1 effector:target ratio and incubated for 6 hours at 37°C. Luciferase activity was measured using the provided Bio-Glo luciferase assay reagent with a SpectraMax i3x reader. Measured luciferase activity, expressed in relative light units (RLU), was plotted against antibody concentration. [Figure 43-2]These results suggest that IgG1 allotypes can affect FcγRIIIa receptor-mediated signaling. Figure 43A shows sequence information for the IgG1 allotypes IGHG1*08 and IGHG1*01, which differ by one amino acid in the CH1 domain. The residues that differ between these two allotypes are highlighted, and surrounding residues are provided for reference. The locations are provided in both IMGT and EU numbering. Figure 43B shows that clinical ipilimumab (Yervoy) uses the IGHG1*08 allotype and results in lower FcγRIIIa signaling than ipilimumab and GIGA-564, both produced by GigaGen and using the IGHG1*01 allotype. These results suggest that allotypes can affect FcγRIIIa signaling. An ADCC reporter bioassay for human FcγRIIIa, V variant (G7011), was purchased from Promega Corporation. The assay was performed according to the manufacturer's instructions. Briefly, CHO target cells stably expressing human CTLA-4 with a Y201G mutation to enhance cell surface expression were suspended in RPMI 1640 + 4% FBS medium with the indicated antibodies and incubated for 30 minutes at 37°C. Jurkat / NFAT-Luc effector cells expressing human FcγRIIIa, V variant, were added to each well at a 5:1 effector:target ratio and incubated for 6 hours at 37°C. Luciferase activity was measured using the provided Bio-Glo luciferase assay reagent with a SpectraMax i3x reader. Measured luciferase activity, expressed in relative light units (RLU), was plotted against antibody concentration.

[0160] [Figure 44-1] The results of differential scanning fluorimetry (DSF) analysis of nine formulations containing GIGA-564 (G1 to G9) under four different conditions: (A) storage at 5°C, (B) 5xF / T, (C) stirring, and (D) storage for 2 weeks at 40°C are shown. The compositions of G1 to G9 are shown in the table below. [Figure 44-2]The results of differential scanning fluorimetry (DSF) analysis of nine formulations containing GIGA-564 (G1 to G9) under four different conditions: (A) storage at 5°C, (B) 5xF / T, (C) stirring, and (D) storage for 2 weeks at 40°C are shown. The compositions of G1 to G9 are shown in the table below. [Table 42]

[0161] [Figure 45] The results of dynamic light scattering (DLS) analysis of the cumulant diameter of nine formulations containing GIGA-564 (G1 to G9) under four different conditions: (A) storage at 5°C, (B) 5×F / T, (C) stirring, and (D) storage at 40°C for 2 weeks are shown.

[0162] [Figure 46-1] The figures show the results of SE-HPLC analysis of nine formulations containing GIGA-564 (G1 to G9) under four different conditions: (A) storage at 5°C, (B) 5xF / T, (C) stirring, and (D) storage at 40°C for 2 weeks. [Figure 46-2] The figures show the results of SE-HPLC analysis of nine formulations containing GIGA-564 (G1 to G9) under four different conditions: (A) storage at 5°C, (B) 5xF / T, (C) stirring, and (D) storage at 40°C for 2 weeks.

[0163] [Figure 47-1] The BioAnalyzer results for nine formulations containing GIGA-564 (G1 to G9) are shown under four different conditions: (A) storage at 5°C, (B) 5xF / T, (C) stirring, and (D) storage at 40°C for 2 weeks. [Figure 47-2] The BioAnalyzer results for nine formulations containing GIGA-564 (G1 to G9) are shown under four different conditions: (A) storage at 5°C, (B) 5xF / T, (C) stirring, and (D) storage at 40°C for 2 weeks.

[0164] [Figure 48-1]Figure 48 shows the CTLA-4 binding affinity of GIGA-564 in formulation 1 (G1) (Figure 48A) or 4 (G4) (Figure 48B) tested by ELISA. [Figure 48-2] Figure 48 shows the CTLA-4 binding affinity of GIGA-564 in formulation 1 (G1) (Figure 48A) or 4 (G4) (Figure 48B) tested by ELISA.

[0165] [Figure 49-1] Figure 49 shows human FcγRIIIa-V variant signaling induced by GIGA-564 in formulation 1 (G1 or buffer 1) (Figure 49A) or 4 (G4 or buffer 4) (Figure 49B) tested by ELISA, as determined by cell-based assay. [Figure 49-2] Figure 49 shows human FcγRIIIa-V variant signaling induced by GIGA-564 in formulation 1 (G1 or buffer 1) (Figure 49A) or 4 (G4 or buffer 4) (Figure 49B) tested by ELISA, as determined by cell-based assay.

[0166] [Figure 50-1] Figure 1 shows MC38 tumor size in hCTLA-4 KI mice over time after treatment with PBS or the indicated amounts of ipilimumab (commercially available Yervoy), GIGA-564, defucosylated ipilimumab (next generation), or GIGA-2328 (2328, more defucosylated GIGA-564) on days 0, 3, and 6. Data are median + / - 95% CI. [Figure 50-2] Figure 1 shows MC38 tumor size in hCTLA-4 KI mice over time after treatment with PBS or the indicated amounts of ipilimumab (commercially available Yervoy), GIGA-564, defucosylated ipilimumab (next generation), or GIGA-2328 (2328, more defucosylated GIGA-564) on days 0, 3, and 6. Data are median + / - 95% CI.

[0167] [Figure 51]Panels A and B provide the human FcRIIIa-V activity induced by GIGA-564, GIGA-564_XF (defucosylated GIGA-564, also known as GIGA-2328 produced in a stable cell line), transiently produced GIGA-2328, GIGA-564 with the LALA-PG mutation to eliminate Fc function, ipilimumab, ipilimumab_XF (defucosylated ipilimumab), and GIGA-564_AEX (GIGA-564 purified by Protein A and then further purified by anion exchange) as determined by cell-based assays.

[0168] [Figure 52-1] 1 provides serum concentrations of GIGA-564 in NHPs after a single IV bolus administration of two different doses of GIGA-564 (3 mg / kg and 30 mg / kg). [Figure 52-2] 1 provides dose-corrected serum concentrations of GIGA-564 in NHPs after a single IV bolus administration of two different doses of GIGA-564 (3 mg / kg and 30 mg / kg).

[0169] [Figure 53] 1 provides serum concentrations of GIGA-564 in individual NHPs following a single IV bolus administration of two different doses of GIGA-564 (3 mg / kg and 30 mg / kg).

[0170] [Figure 54-1] 1 provides predicted serum concentrations of GIGA-564 in human subjects after administration of two different doses of GIGA-564 (3 mg / kg and 30 mg / kg) based on PK studies in NHPs. [Figure 54-2] 1 provides predicted dose-corrected serum concentrations of GIGA-564 in human subjects after administration of two different doses of GIGA-564 (3 mg / kg and 30 mg / kg) based on PK studies in NHPs.

[0171] [Figure 55-1]1 provides predicted serum concentrations of GIGA-564 in individual human subjects following administration of two different doses of GIGA-564 (3 mg / kg and 30 mg / kg) based on PK studies in NHPs. [Figure 55-2] 1 provides the serum concentration time profile of GIGA-564 following once-monthly dosing of GIGA-564 in human subjects predicted based on PK studies in NHPs.

[0172] [Figure 56-1] Figure 1 provides cytokine / chemokine release from human peripheral blood mononuclear cells (PBMCs) in response to GIGA-564 or control under wet binding conditions. [Figure 56-2] Figure 1 provides cytokine / chemokine release from human peripheral blood mononuclear cells (PBMCs) in response to GIGA-564 or control under wet binding conditions.

[0173] [Figure 57-1] 1 provides cytokine / chemokine release from human peripheral blood mononuclear cells (PBMCs) in response to ipilimumab or control under wet binding conditions. [Figure 57-2] 1 provides cytokine / chemokine release from human peripheral blood mononuclear cells (PBMCs) in response to ipilimumab or control under wet binding conditions.

[0174] [Figure 58-1] Figure 1 provides cytokine / chemokine release from human peripheral blood mononuclear cells (PBMCs) in response to soluble GIGA-564 or control. [Figure 58-2] Figure 1 provides cytokine / chemokine release from human peripheral blood mononuclear cells (PBMCs) in response to soluble GIGA-564 or control.

[0175] [Figure 59-1] 1 provides cytokine / chemokine release from human peripheral blood mononuclear cells (PBMCs) in response to soluble ipilimumab or control. [Figure 59-2]1 provides cytokine / chemokine release from human peripheral blood mononuclear cells (PBMCs) in response to soluble ipilimumab or control.

[0176] [Figure 60] Cytotoxic effects of soluble or wet-bound GIGA-564 (top) and ipilimumab (bottom) in PBMCs are shown.

[0177] [Figure 61] Figure 1 shows the time course of tumor growth in hCTLA-4 KI mice bearing established MC38 tumors treated on days 0, 3, and 6 with PBS or GIGA-564 (1 mg / kg, 0.3 mg / kg, 0.1 mg / kg, 0.03 mg / kg, 0.01 mg / kg). DETAILED DESCRIPTION OF THE INVENTION

[0178] 7.1.Definition Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Generally, the nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification, unless otherwise indicated. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990), which are incorporated herein by reference. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical syntheses, chemical analyses, formulation, formulation, and delivery, and treatment of patients.

[0179] The following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0180] The terms "CTLA-4," "CTLA-4 protein," and "CTLA-4 antigen" are used interchangeably herein and refer to human CTLA-4, or any variants (e.g., splice and allelic variants), isoforms, and species homologs of human CTLA-4, naturally expressed by a cell or expressed by a cell transfected with the ctla4 gene. In some embodiments, the CTLA-4 protein is a CTLA-4 protein naturally expressed by a primate (e.g., monkey or human), rodent (e.g., mouse or rat), dog, camel, cat, cow, goat, horse, or sheep. In some embodiments, the CTLA-4 protein is human CTLA-4 (hCTLA-4, SEQ ID NO: 7001).

[0181] The term "immunoglobulin" refers to a class of structurally related proteins that generally contain two pairs of polypeptide chains: one pair of light (L) chains and one pair of heavy (H) chains. In "intact immunoglobulins," all four of these chains are interconnected by disulfide bonds. The structural features of immunoglobulins are well understood. See, e.g., Paul, Fundamental Immunology 7th ed., Ch. 5 (2013) Lippincott Williams & Wilkins, Philadelphia, PA. Briefly, each heavy chain typically contains a heavy chain variable region (V H ) and heavy chain constant region (C H The heavy chain constant region typically comprises C H1 , C H2 , and C H3 Each light chain typically contains three domains, abbreviated as V L ) and a light chain constant region. The light chain constant region typically comprises C L It contains one domain, abbreviated as .

[0182] The term "antigen binding protein" (ABP) refers to a protein comprising one or more antigen binding domains that specifically bind to an antigen or epitope. In some embodiments, the antigen binding domain binds to an antigen or epitope with specificity and affinity similar to a naturally occurring antibody. In some embodiments, the ABP comprises an antibody. In some embodiments, the ABP consists of an antibody. In some embodiments, the ABP consists essentially of an antibody. In some embodiments, the ABP comprises an surrogate scaffold. In some embodiments, the ABP consists of an surrogate scaffold. In some embodiments, the ABP consists essentially of an surrogate scaffold. In some embodiments, the ABP comprises an antibody fragment. In some embodiments, the ABP consists of an antibody fragment. In some embodiments, the ABP consists essentially of an antibody fragment. A "CTLA-4 ABP," "anti-CTLA-4 ABP," or "CTLA-4-specific ABP," as provided herein, is an ABP that specifically binds to the antigen CTLA-4. In some embodiments, the ABP binds to the extracellular domain of CTLA-4. In certain embodiments, the CTLA-4 ABPs provided herein bind to an epitope of CTLA-4 that is conserved among CTLA-4 proteins from different species.

[0183] The term "antibody" is used herein in its broadest sense and includes a specific type of immunoglobulin molecule that contains one or more antigen-binding domains that specifically bind to an antigen or epitope. Antibodies specifically include intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multispecific antibodies. An example of an antigen-binding domain is a V H -V L The antigen-binding domain is formed by a dimer. Antibodies are a type of ABP.

[0184] The terms "defucosylated" or "defucosylated" in the context of Fc refer to the substantial absence of core fucosylation of N-glycans covalently attached directly or indirectly to N-glycosylation sites, e.g., amino acid residue position 297 of the human IgG1 Fc region, numbered according to the EU index (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)), or to corresponding residues in non-IgG1 or non-human IgG1 immunoglobulins.

[0185] When a fucosylation ratio is indicated in the context of a composition comprising an antibody, the ratio indicates the proportion of fucosylated antibodies among all antibodies in the composition. For example, 70% fucosylation indicates that 70% of the antibodies in the composition are fucosylated and 30% of the antibodies in the composition are defucosylated.

[0186] The term "alternative scaffold" refers to a molecule in which one or more regions can be diversified to generate one or more antigen-binding domains that specifically bind to an antigen or epitope. In some embodiments, the antigen-binding domains bind to an antigen or epitope with similar specificity and affinity as naturally occurring antibodies. Exemplary alternative scaffolds include those derived from fibronectin (e.g., Adnectins™), beta-sandwich (e.g., iMabs), lipocalins (e.g., Anticalins®), EETI-II / AGRP, BPTI / LACI-D1 / ITI-D2 (e.g., Kunitz domains), thioredoxin peptide aptamers, protein A (e.g., Affibody®), ankyrin repeats (e.g., DARPins), gamma-B-crystallin / ubiquitin (e.g., Affilins), CTLD3 (e.g., tetranectin), fynomers, and (LDLR-A modules) (e.g., avimers). Further information regarding alternative scaffolds is provided in Binz et al., Nat. Biotechnol., 2005 23:1257-1268; Skerra, Current Opin. in Biotech., 2007 18:295-304, and Silacci et al., J. Biol. Chem., 2014, 289:14392-14398, each of which is incorporated by reference in its entirety. Alternative scaffolds are a type of ABP.

[0187] The term "antigen-binding domain" refers to a portion of an ABP capable of specifically binding to an antigen or epitope.

[0188] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to that of a naturally occurring antibody and having a heavy chain including an Fc region.

[0189] The term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain that, in naturally occurring antibodies, interacts with Fc receptors and specific proteins of the complement system. The structures of various immunoglobulin Fc regions and the glycosylation sites contained therein are known in the art. See Schroeder and Cavacini, J. Allergy Clin. Immunol., 2010, 125:S41-52, which is incorporated by reference in its entirety. The Fc region may be a naturally occurring Fc region or an altered Fc region as described elsewhere in this disclosure.

[0190] V H and V L The regions can be further subdivided into regions of hypervariability (also called "hypervariable regions (HVRs)" or "complementarity determining regions" (CDRs)), interspersed with more conserved regions. The more conserved regions are called framework regions (FRs). Each V H and V L generally contain three CDRs and four FRs arranged (N-terminus to C-terminus) in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDRs are involved in antigen binding and affect the antigen specificity and binding affinity of the antibody. See Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. (1991) Public Health Service, National Institutes of Health, Bethesda, MD, which is incorporated herein by reference in its entirety.

[0191] Light chains from any vertebrate species can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the sequence of their constant domain.

[0192] Heavy chains from any vertebrate species can be assigned to one of five different classes (or isotypes): IgA, IgD, IgE, IgG, and IgM. These classes are also called α, δ, ε, γ, and μ, respectively. The IgG and IgA classes are further divided into subclasses based on sequence and functional differences. Humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0193] The amino acid sequence boundaries of the CDRs can be determined by one of skill in the art using any of several known numbering schemes, including those described by Kabat et al., supra (the "Kabat" numbering scheme), Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (the "Chothia" numbering scheme), MacCallum et al., 1996, J. Mol. Biol. 262:732-745 (the "Contact" numbering scheme), Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 (the "IMGT" numbering scheme), and Honegge and Pluckthun, J. Mol. Biol., 2001, 309:657-70 (the "AHo" numbering scheme), each of which is incorporated by reference in its entirety.

[0194] Table 1 shows the CDR1-L(V L CDR1), CDR2-L(V L CDR2), CDR3-L(V L CDR3), CDR1-H(V H CDR1), CDR2-H(V H CDR2 of V), and CDR3-H (V H For CDR1-H, residue numbering is provided using both the Kabat and Chothia numbering schemes.

[0195] CDRs can be assigned using antibody numbering software such as Abnum, available at www.bioinf.org.uk / abs / abnum / , and described in Abhinandan and Martin, Immunology, 2008, 45:3832-3839, which is incorporated by reference in its entirety. [Table 1] * The C-terminus of CDR1-H, when numbered using the Kabat numbering convention, varies between 32 and 34 depending on the length of the CDR.

[0196] The "EU numbering scheme" is commonly used when referring to residues in antibody heavy chain constant regions (eg, as reported in Kabat et al., supra).

[0197] "Antibody fragments" include portions of intact antibodies, such as the antigen-binding or variable regions of the intact antibody. Antibody fragments include, for example, Fv fragments, Fab fragments, F(ab')2 fragments, Fab' fragments, scFv (sFv) fragments, and scFv-Fc fragments.

[0198] An "Fv" fragment comprises a non-covalent dimer of one heavy- and one light-chain variable domain.

[0199] A "Fab" fragment contains the variable domains of the heavy and light chains, as well as the constant domain of the light chain and the first constant domain of the heavy chain (C H1 Fab fragments can be produced, for example, by recombinant methods or by papain digestion of a full-length antibody.

[0200] An "F(ab')2" fragment contains two Fab' fragments linked by a disulfide bond near the hinge region. F(ab')2 fragments can be produced, for example, by recombinant methods or by pepsin digestion of an intact antibody. F(ab')2 fragments can be dissociated, for example, by treatment with β-mercaptoethanol.

[0201] "Single-chain Fv" or "sFv" or "scFv" antibody fragments contain V in a single polypeptide chain. H Domain and V L Includes domain. V H and V L are generally linked by a peptide linker. See Pluckthun A. (1994). In some embodiments, the linker is (GGGGS) n (SEQ ID NO: 11968). In some embodiments, n=1, 2, 3, 4, 5, or 6. See Antibodies from Escherichia coli. In Rosenberg M. & Moore GP (Eds.), The Pharmacology of Monoclonal Antibodies vol. 113 (pp. 269-315). Springer-Verlag, New York, incorporated by reference in its entirety.

[0202] An "scFv-Fc" fragment comprises an scFv linked to an Fc domain. For example, the Fc domain can be linked to the C-terminus of the scFv. The Fc domain can be V or V, depending on the orientation of the variable domains in the scFv. H or V L may be followed by (i.e., V H -V L or V L -V H ). Any suitable Fc domain known in the art or described herein can be used. In some cases, the Fc domain comprises an IgG4 Fc domain.

[0203] The term "single domain antibody" refers to a molecule in which one variable domain of an antibody specifically binds to an antigen in the absence of other variable domains. Single domain antibodies and fragments thereof are described in Arabi Ghahroudi et al., FEBS Letters, 1998, 414:521-526 and Muyldermans et al., Trends in Biochem. Sci., 2001, 26:230-245, each of which is incorporated by reference in its entirety.

[0204] A "monospecific ABP" is an ABP that contains a binding site that specifically binds to a single epitope. An example of a monospecific ABP is a naturally occurring IgG molecule that is bivalent but recognizes the same epitope in each antigen-binding domain. The binding specificity can be present in any suitable valency.

[0205] The term "monoclonal antibody" refers to an antibody that is a substantially homogeneous antibody population. A substantially homogeneous antibody population contains antibodies that are substantially similar and bind to the same epitope, excluding variants that may normally arise during monoclonal antibody production. Such variants are generally present only in small amounts. Monoclonal antibodies are typically obtained by a process that includes selecting a single antibody from a plurality of antibodies. For example, the selection process can be the selection of a unique clone from a pool of multiple clones, e.g., hybridoma clones, phage clones, yeast clones, bacterial clones, or other recombinant DNA clones. The selected antibody can be further modified, for example, to improve its affinity for the target ("affinity maturation"), to humanize the antibody, to improve its production in cell culture, and / or to reduce its immunogenicity in a subject.

[0206] The term "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, and the remainder of the heavy and / or light chain is derived from a different source or species.

[0207] "Humanized" forms of non-human antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. Humanized antibodies are generally human antibodies (recipient antibodies) in which residues from one or more CDRs are replaced by residues from one or more CDRs of a non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken, or non-human primate antibody, that has the desired specificity, affinity, or biological effect. In some instances, selected framework region residues of the recipient antibody are replaced by the corresponding framework region residues from the donor antibody. Humanized antibodies can also contain residues that are not found in either the recipient antibody or the donor antibody. Such modifications can be made to further refine antibody function. For further details, see Jones et al., Nature, 1986, 321:522-525; Riechmann et al., Nature, 1988, 332:323-329; and Presta, Curr. Op. Struct. Biol., 1992, 2:593-596, each of which is incorporated by reference in its entirety.

[0208] A "human antibody" is an antibody possessing an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or derived from a non-human source that utilizes the human antibody repertoire or human antibody coding sequences (e.g., obtained from a human source or designed de novo). Human antibodies specifically exclude humanized antibodies. In some embodiments, rodents are genetically engineered to replace their rodent antibody sequences with human antibodies.

[0209] An "isolated ABP" or "isolated nucleic acid" is an ABP or nucleic acid that has been separated and / or recovered from a component of its natural environment. Components of natural environment may include enzymes, hormones, and other proteinaceous or non-proteinaceous substances. In some embodiments, an isolated ABP is purified to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence, for example, by use of a spinning cup sequencer. In some embodiments, an isolated ABP is purified to homogeneity by gel electrophoresis (e.g., SDS-PAGE) under reducing or non-reducing conditions with detection by Coomassie blue or silver staining. Since at least one component of the ABP's natural environment is absent, an isolated ABP includes an ABP in situ within a recombinant cell. In some embodiments, an isolated ABP or isolated nucleic acid is prepared by at least one purification step. In some embodiments, an isolated ABP or isolated nucleic acid is purified to at least 80%, 85%, 90%, 95%, or 99% by weight. In some embodiments, the isolated ABP or isolated nucleic acid is purified to at least 80%, 85%, 90%, 95%, or 99% by volume. In some embodiments, the isolated ABP or isolated nucleic acid is provided as a solution comprising at least 85%, 90%, 95%, 98%, 99%, or 100% by volume of the ABP or nucleic acid. In some embodiments, the isolated ABP or isolated nucleic acid is provided as a solution comprising at least 85%, 90%, 95%, 98%, 99%, or 100% by volume of the ABP or nucleic acid.

[0210] "Affinity" refers to the overall strength of non-covalent interactions between a single binding site of a molecule (e.g., an ABP) and its binding partner (e.g., an antigen or epitope). Unless otherwise indicated, as used herein, "affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an ABP and an antigen or epitope). The affinity of a molecule X for its partner Y is determined by the dissociation equilibrium constant (K D) The kinetic components that contribute to the dissociation equilibrium constant are described in more detail below. Affinity can be measured by common methods known in the art, including those described herein. Affinity can be measured, for example, using surface plasmon resonance (SPR) (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®).

[0211] With respect to the binding of an ABP to a target molecule, the terms "bind," "specific binding," "specifically binds," "specific for," "selectively binds," and "selective for" a particular antigen (e.g., a polypeptide target) or epitope on a particular antigen refer to binding that is distinct from nonspecific or nonselective interactions (e.g., with a non-target molecule). Specific binding can be measured, for example, by measuring binding to the target molecule and comparing it to binding to a non-target molecule. Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule. In this case, specific binding is indicated when binding of the ABP to the target molecule is competitively inhibited by the control molecule. In some embodiments, the affinity of the CTLA-4 ABP for the non-target molecule is less than about 50% of its affinity for CTLA-4. In some embodiments, the affinity of the CTLA-4 ABP for the non-target molecule is less than about 40% of its affinity for CTLA-4. In some embodiments, the affinity of the CTLA-4 ABP for the non-target molecule is less than about 30% of its affinity for CTLA-4. In some embodiments, the affinity of the CTLA-4 ABP for the non-target molecule is less than about 20% of its affinity for CTLA-4. In some embodiments, the affinity of the CTLA-4 ABP for the non-target molecule is less than about 10% of its affinity for CTLA-4. In some embodiments, the affinity of the CTLA-4 ABP for the non-target molecule is less than about 1% of its affinity for CTLA-4. In some embodiments, the affinity of the CTLA-4 ABP for the non-target molecule is less than about 0.1% of its affinity for CTLA-4.

[0212] As used herein, "k d "" (sec -1 The term k ) refers to the dissociation rate constant of a particular ABP-antigen interaction. This value is off Also called value.

[0213] The term "k" a " (M -1 ×sec -1 ) as used herein refers to the association rate constant of a particular ABP-antigen interaction. This value is known as k on Also called value.

[0214] As used herein, "K D The term "(M)" refers to the dissociation equilibrium constant of a particular ABP-antigen interaction. D =k d / k a .

[0215] As used herein, "K A " (M -1 The term K ) refers to the association equilibrium constant of a particular ABP-antigen interaction. A =k a / k d .

[0216] An "affinity matured" ABP is one that has one or more modifications (e.g., in one or more CDRs or FRs) that result in an improvement in the affinity of the ABP for its antigen compared to a parent ABP that does not have the modifications. In one embodiment, the affinity matured ABP has nanomolar or picomolar affinity for the target antigen. Affinity matured ABPs can be generated using a variety of methods known in the art. For example, Marks et al. (Bio / Technology, 1992, 10:779-783, incorporated by reference in its entirety) describe affinity matured ABPs, such as V H and V LAffinity maturation by domain shuffling and random mutagenesis of CDR and / or framework residues are described, for example, in Barbas et al. (Proc. Nat. Acad. Sci. USA, 1994, 91:3809-3813), Schier et al., Gene, 1995, 169:147-155, Yelton et al., J. Immunol., 1995, 155:1994-2004, Jackson et al., J. Immunol., 1995, 154:3310-33199, and Hawkins et al., J. Mol. Biol., 1992, 226:889-896, each of which is incorporated by reference in its entirety.

[0217] An "immunoconjugate" is an ABP conjugated to one or more heterologous molecules.

[0218] "Effector function" refers to a biological activity mediated by the Fc region of an antibody, which may vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding, which activates complement-dependent cytotoxicity (CDC), Fc receptor binding, which activates antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP).

[0219] As used herein in the context of two or more ABPs, the terms "compete" or "cross-compete" indicate that two or more ABPs compete for binding to an antigen (e.g., CTLA-4). In one exemplary assay, CTLA-4 is coated on a surface and contacted with a first CTLA-4 ABP, followed by the addition of a second CTLA-4 ABP. In another exemplary assay, the first CTLA-4 ABP is coated on a surface and contacted with CTLA-4, followed by the addition of a second CTLA-4 ABP. In either assay, ABPs compete if the presence of the first CTLA-4 ABP reduces the binding of the second CTLA-4 ABP. The term "compete" also includes combinations of ABPs in which one ABP reduces the binding of another ABP, but no competition is observed when the ABPs are added in the reverse order. However, in some embodiments, the first and second ABPs inhibit each other's binding regardless of the order in which they are added. In some embodiments, one ABP reduces the binding of another ABP to its antigen by at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95%. One skilled in the art can select the concentration of antibody used in a competition assay based on the affinity of the ABP for CTLA-4 and the valency of the ABP. The assays described in this definition are exemplary, and one skilled in the art can utilize any suitable assay to determine whether antibodies compete with each other.Suitable assays are described, for example, in Cox et al., "Immunoassay Methods," in Assay Guidance Manual [Internet], Updated December 24, 2014 (www(dot)ncbi(dot)nlm(dot)nih(dot)gov / books / NBK92434 / ; accessed September 29, 2015), Silman et al., Cytometry, 2001, 44:30-37; and Finco et al., J. Pharm. Biomed. Anal., 2011, 54:351-358, each of which is incorporated by reference in its entirety.

[0220] The term "epitope" refers to a portion of an antigen that specifically binds to an ABP. Epitopes often consist of surface-accessible amino acid residues and / or sugar side chains and may have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter may be lost in the presence of denaturing solvents. An epitope may include amino acid residues directly involved in binding as well as other amino acid residues not directly involved in binding. The epitope to which an ABP binds can be determined using known techniques for epitope determination, such as testing ABP binding to CTLA-4 variants or chimeric CTLA-4 variants with different point mutations.

[0221] The percent "identity" between a polypeptide sequence and a reference sequence is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to those in the reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved by a variety of methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0222] A "conservative substitution" or "conservative amino acid substitution" refers to the replacement of an amino acid with a chemically or functionally similar amino acid. Conservative substitution tables providing similar amino acids are well known in the art. By way of example, the groups of amino acids provided in Tables 2-4 are, in some embodiments, considered conservative substitutions for one another. [Table 2] [Table 3] [Table 4]

[0223] Additional conservative substitutions can be found, for example, in Creighton, Proteins: Structures and Molecular Properties 2nd ed. (1993) W.H. Freeman & Co., New York, NY. ABPs generated by making one or more conservative substitutions of amino acid residues in a parent ABP are referred to as "conservatively modified variants."

[0224] The term "treating" (and variations thereof, such as "treat" or "treatment") refers to clinical intervention in an attempt to alter the natural course of a disease or condition in a subject in need thereof. Treatment can be prophylactic or during the clinical pathological course. Desirable effects of treatment include prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, improvement or palliation of the disease state, and remission or improved prognosis.

[0225] As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount of an ABP or pharmaceutical composition provided herein that, when administered to a subject, is effective to treat a disease or disorder.

[0226] As used herein, the term "subject" means a mammalian subject. Exemplary subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats, rabbits, and sheep. In certain embodiments, the subject is a human. In some embodiments, the subject has a disease or condition that can be treated with an ABP provided herein. In some embodiments, the disease or condition is cancer. In some embodiments, the disease or condition is a viral infection.

[0227] The term "package insert" is used to refer to instructions customarily included in commercially available packaging for a therapeutic or diagnostic product (e.g., a kit), which contain information about the indications, usage, dosage, administration, concomitant therapy, contraindications and / or warnings for the use of such therapeutic or diagnostic product.

[0228] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction.

[0229] "Chemotherapeutic agent" refers to a chemical compound useful in the treatment of cancer. Chemotherapeutic agents include "antihormonal agents" or "endocrine therapy agents" that act to regulate, reduce, block, or inhibit the effects of hormones that can promote cancer growth.

[0230] The term "cytostatic agent" refers to a compound or composition that arrests cell proliferation either in vitro or in vivo. In some embodiments, a cytostatic agent is an agent that reduces the percentage of cells in S phase. In some embodiments, a cytostatic agent reduces the percentage of cells in S phase by at least about 20%, at least about 40%, at least about 60%, or at least about 80%.

[0231] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive when referred to herein. The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. In some embodiments, the cell proliferative disorder is cancer.

[0232] The term "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective in treating a subject, and that does not contain additional ingredients that are unacceptably toxic to the subject.

[0233] The terms "modulate" and "modulation" refer to the reduction or inhibition, or activation or increase, of the recited variable.

[0234] The terms "increase" and "activate" refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or greater increase in the recited variable.

[0235] The terms "reduce" and "inhibit" refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater decrease in the recited variable.

[0236] The term "stimulate" refers to the activation of receptor signaling to induce a biological response associated with receptor activation. An "agonist" is a component that binds to and stimulates a receptor.

[0237] The term "antagonize" refers to the inhibition of receptor signaling to inhibit a biological response associated with receptor activation. An "antagonist" is a component that binds to and antagonizes a receptor.

[0238] The term "effector T cells" includes T helper (i.e., CD4+) cells and cytotoxic (i.e., CD8+) T cells. CD4+ effector T cells contribute to the development of several immunological processes, including the maturation of B cells into plasma cells and memory B cells, and the activation of cytotoxic T cells and macrophages. CD8+ effector T cells destroy virus-infected cells and tumor cells. For more information regarding effector T cells, see Seder and Ahmed, Nature Immunol., 2003, 4:835-842, which is incorporated by reference in its entirety.

[0239] The term "regulatory T cells" includes cells that regulate immune tolerance, for example, by suppressing effector T cells. In some embodiments, regulatory T cells have a CD4+CD25+Foxp3+ phenotype. In some embodiments, regulatory T cells have a CD8+CD25+ phenotype. For more information regarding regulatory T cells, see Nocentini et al., Br. J. Pharmacol., 2012, 165:2089-2099, which is incorporated by reference in its entirety.

[0240] The term "dendritic cell" refers to an antigen-presenting cell that can activate naive T cells and stimulate the proliferation and differentiation of B cells.

[0241] A "variant" of a polypeptide (e.g., an antibody) comprises an amino acid sequence in which one or more amino acid residues have been inserted, deleted, and / or substituted compared to the native polypeptide sequence, and retains essentially the same biological activity as the native polypeptide. The biological activity of a polypeptide can be measured using standard techniques in the art (e.g., if the variant is an antibody, its activity can be tested by the binding assays described herein). Variants of the present disclosure include fragments, analogs, recombinant polypeptides, synthetic polypeptides, and / or fusion proteins.

[0242] A "derivative" of a polypeptide is a polypeptide (e.g., an antibody) that has been chemically modified, for example, through conjugation to another chemical moiety, such as polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation. Unless otherwise indicated, the term "antibody" includes antibodies comprising two full-length heavy chains and two full-length light chains, as well as derivatives, variants, fragments, and muteins thereof, examples of which are described below.

[0243] A nucleotide sequence is "operably linked" to a regulatory sequence if the regulatory sequence affects the expression (e.g., level, timing, or location of expression) of the nucleotide sequence. A "regulatory sequence" is a nucleic acid that affects the expression (e.g., level, timing, or location of expression) of a nucleic acid to which it is operably linked. A regulatory sequence can exert its effect, for example, directly on the nucleic acid being regulated or through the action of one or more other molecules (e.g., polypeptides that bind to the regulatory sequence and / or nucleic acid). Examples of regulatory sequences include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Further examples of regulatory sequences are described, for example, in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA, and Baron et al., 1995, Nucleic Acids Res. 23:3605-06.

[0244] A "host cell" is a cell that can be used to express a nucleic acid, e.g., a nucleic acid of the present disclosure. A host cell can be a prokaryote, e.g., E. coli, or a eukaryote, e.g., a unicellular eukaryote (e.g., yeast or other fungi), a plant cell (e.g., a tobacco or tomato plant cell), an animal cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell), or a hybridoma. Examples of host cells include CS-9 cells, the monkey kidney cell line COS-7 (ATCC CRL 1651) (see Gluzman et al., 1981, Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells or their derivatives, such as Veggie CHO and related cell lines that grow in serum-free medium (see Rasmussen et al., 1998, Cytotechnology 28:31), HeLa cells, the BHK (ATCC CRL 10) cell line, the CV1 / EBNA cell line (ATCC CCL 70) derived from the African green monkey kidney cell line CV1 (see McMahan et al., 1991, EMBO J. 10:2821), 293, 293 EBNA, or MSR cells. Examples of host cells include human embryonic kidney cells such as 293, human epithelial A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, primary tissue, primary explants, cell lines derived from in vitro culture of HL-60, U937, HaK, or Jurkat cells. Typically, host cells are cultured cells that can be transformed or transfected with a nucleic acid encoding a polypeptide that can then be expressed in the host cell.

[0245] The phrase "recombinant host cell" may be used to refer to a host cell that has been transformed or transfected with a nucleic acid to be expressed. A host cell may also be a cell that contains a nucleic acid but does not express the nucleic acid at a desired level unless a regulatory sequence is introduced into the host cell so that the nucleic acid is operably linked to the nucleic acid. It is understood that the term host cell refers not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in subsequent generations due to, for example, mutations or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0246] In some embodiments, the host cells are used in adoptive cell therapy for delivery of an ABP to a subject. 7.2. Other Interpretation Rules

[0247] Ranges recited herein are understood to be shorthand for all values ​​within the range, including the recited endpoints. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[0248] Unless otherwise indicated, a reference to a compound having one or more stereocenters contemplates each and every stereoisomer thereof and all combinations of stereoisomers. 7.3. Antigen-binding proteins

[0249] In one aspect, the present disclosure provides antigen binding proteins (“ABPs”) (e.g., antibodies, antibody fragments, antibody derivatives, antibody muteins, and antibody variants). In some embodiments, the ABPs bind to CTLA-4.

[0250] In some embodiments, the present disclosure provides an ABP that binds to a different epitope of CTLA-4 than ipilimumab. In some embodiments, the epitope includes K130, Y139, L141, and I143, but not R70. In some embodiments, the ABP contacts amino acids K130, Y139, L141, and I143 of CTLA-4, but not amino acid R70. In some embodiments, the ABP can bind to CTLA-4 even while CTLA-4 is interacting with CD80 / CD86. In some embodiments, the interaction between the ABP and amino acids L74A and / or E68 of CTLA-4 is greater than the interaction between ipilimumab and amino acid L74A of CTLA-4.

[0251] In some embodiments, the present disclosure provides antigen binding proteins comprising a light chain variable region selected from the group consisting of A1LC-A28LC or a heavy chain variable region selected from the group consisting of A1HC-A28HC, as well as fragments, derivatives, muteins, and variants thereof. Such antigen binding proteins may be designated using the nomenclature "LxHy," where "x" corresponds to the number of the light chain variable region and "y" corresponds to the number of the heavy chain variable region, as represented in the following sequence: For example, "A1HC" denotes a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 101, "A1LC" denotes a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1, etc. More generally, "L2H1" refers to an antigen binding protein having a light chain variable region comprising the amino acid sequence of L2 (SEQ ID NO: 2) and a heavy chain variable region comprising the amino acid sequence of H1 (SEQ ID NO: 101). For clarity, all ranges recited by at least two members of a group include all members of the group between and including the members of the extreme ranges. Thus, the group range A1-A28 includes all members between A1 and A28, as well as members A1 and A28 themselves. The group range A4-A6 includes members A4, A5, and A6, etc. In certain embodiments, the ABP is A14. In some embodiments, the ABP comprises the six CDR sequences of A14 (GIGA-564). In some embodiments, the ABP comprises the heavy and light chain sequences of A14 (GIGA-564). In some embodiments, the ABP comprises the heavy chain variable domain and light chain variable domain of A14 (GIGA-564).

[0252] In some embodiments, the ABP comprises a heavy chain variable domain having a sequence at least 95% identical to that of A14 (GIGA-564). In some embodiments, the ABP comprises a heavy chain variable domain having a sequence at least 96% identical to that of A14 (GIGA-564). In some embodiments, the ABP each comprise a heavy chain variable domain having a sequence at least 97% identical to that of A14 (GIGA-564). In some embodiments, the ABP each comprise a heavy chain variable domain having a sequence at least 98% identical to that of A14 (GIGA-564). In some embodiments, the ABP each comprise a heavy chain variable domain having a sequence at least 99% identical to that of A14 (GIGA-564).

[0253] In some embodiments, the ABPs each comprise a light chain variable domain having a sequence at least 95% identical to that of A14 (GIGA-564). In some embodiments, the ABPs each comprise a light chain variable domain having a sequence at least 96% identical to that of A14 (GIGA-564). In some embodiments, the ABPs each comprise a light chain variable domain having a sequence at least 957% identical to that of A14 (GIGA-564). In some embodiments, the ABPs each comprise a light chain variable domain having a sequence at least 98% identical to that of A14 (GIGA-564). In some embodiments, the ABPs each comprise a light chain variable domain having a sequence at least 99% identical to that of A14 (GIGA-564).

[0254] In some embodiments, the antigen binding protein comprises the variable (V(D)J) regions of both the heavy and light chain sequences identical to one of the clones in the library of CTLA-4 binding clones deposited under ATCC Accession No. PTA-125512. In some embodiments, the antigen binding protein comprises the variable (V(D)J) regions of either the heavy or light chain sequences identical to one of the clones in the library of CTLA-4 binding clones deposited under ATCC Accession No. PTA-125512. In some embodiments, the antigen binding protein is expressed from an expression vector in one of the clones in the library of CTLA-4 binding clones deposited under ATCC Accession No. PTA-125512.

[0255] The positions of the CDRs (underlined), which form part of the antigen-binding site, are also shown below, with the framework regions (FRs) being the intervening segments of these variable domain sequences. In both the light chain variable region and the heavy chain variable region, there are three CDRs (CDRs 1-3) and four FRs (FRs 1-4). The CDR regions of each light and heavy chain are also classified according to antibody type (A1, A2, A3, etc.). Antigen binding proteins of the disclosure include, for example, L1H1 (antibody A1, used interchangeably herein with "aCTLA-4.9"), L2H2 (antibody A2, used interchangeably herein with "aCTLA-4.4"), L3H3 (antibody A3, used interchangeably herein with "aCTLA-4.2"), L4H4 (antibody A4, used interchangeably herein with "aCTLA-4.29"), L5H5 (antibody A5, used interchangeably herein with "aCTLA-4.28"), L6H6 (antibody A6, used interchangeably herein with "aCTLA-4.26"), L7H7 (antibody A7, used interchangeably herein with "aCTLA-4.3"), L8H8 (antibody A8, used interchangeably herein with "aCTLA-4.1"), L9H9 (antibody A9, used interchangeably herein with "aCTLA-4.2"), L10H10 (antibody A1, used interchangeably herein with "aCTLA-4.9"), L11H10 (antibody A1, used interchangeably herein with "aCTLA-4.9"), L2H20 (antibody A2, used interchangeably herein with "aCTLA-4.4"), L3H3 (antibody A3, used interchangeably herein with "aCTLA-4.2"), L4H4 (antibody A4, used interchangeably herein with "aCTLA-4.29"), L5H5 (antibody A5, used interchangeably herein with "aCTLA-4.28"), L6H6 (antibody A6, used interchangeably herein with "aCTLA-4.26"), L7H7 (antibody A7, used interchangeably herein with "aCTLA-4.3"), L8H8 (antibody A8, used interchangeably herein with "aCTLA-4.1"), L11H10 (antibody A1, used interchangeably herein with "aCTLA-4.1"), L12H10 (antibody A1 L9H9 (antibody A9, used interchangeably herein with "aCTLA-4.24"), L10H10 (antibody A10, used interchangeably herein with "aCTLA-4.22"), L11H11 (antibody A11, used interchangeably herein with "aCTLA-4.31"), L12H12 (antibody A12, used interchangeably herein with "aCTLA-4.12"), L13H13 (antibody A13, used interchangeably herein with "aCTLA-4.14"), L13H13 (antibody A13, used interchangeably herein with "aCTLA-4.14"), ...and L28H28 (antibody A28).Antigen binding proteins of the disclosure include, for example, antigen binding proteins having light and heavy chain variable domains selected from the group consisting of L18H18 (antibody A18, used interchangeably herein with "aCTLA-4.11"), L15H15 (antibody A15, used interchangeably herein with "aCTLA-4.18"), L16H16 (antibody A16, used interchangeably herein with "aCTLA-4.5"), and L17H17 (antibody A17, used interchangeably herein with "aCTLA-4.17"). In some embodiments, an ABP of the disclosure comprises L14H14 (antibody A14, used interchangeably herein with "aCTLA-4.15" or GIGA-564).

[0256] Antigen binding proteins of the disclosure include, for example, L19H19 (antibody A19, used interchangeably herein with "aCTLA-4.7"), L20H20 (antibody A20, used interchangeably herein with "aCTLA-4.25"), L21H21 (antibody A21, used interchangeably herein with "aCTLA-4.10"), L22H22 (antibody A22, used interchangeably herein with "aCTLA-4.21"), L23H23 (antibody A23, used interchangeably herein with "aCTLA-4.23"), L23H24 (antibody A24, used interchangeably herein with "aCTLA-4.24"), L23H25 (antibody A25, used interchangeably herein with "aCTLA-4.25"), L23H26 (antibody A26, used interchangeably herein with "aCTLA-4.26"), L23H27 (antibody A27, used interchangeably herein with "aCTLA-4.27"), L23H28 (antibody A28, used interchangeably herein with "aCTLA-4.28"), L23H29 (antibody A29, used interchangeably herein with "aCTLA-4.29 ... In some embodiments, the ABP of the present disclosure comprises an antigen binding protein having light and heavy chain variable domains selected from the group consisting of L14H14 (antibody A14, used interchangeably herein with "aCTLA-4.15" or GIGA-564).

[0257] In some embodiments, the antigen binding protein comprises all six CDR sequences (three CDRs of the light chain and three CDRs of the heavy chain) identical to one of the clones in the library of CTLA-4 binding clones deposited under ATCC Accession No. PTA-125512. In some embodiments, the antigen binding protein comprises three of the six CDR sequences (three CDRs of the light chain or three CDRs of the heavy chain) identical to one of the clones in the library of CTLA-4 binding clones deposited under ATCC Accession No. PTA-125512. In some embodiments, the antigen binding protein comprises one, two, three, four, or five of the six CDR sequences identical to one of the clones in the library of CTLA-4 binding clones deposited under ATCC Accession No. PTA-125512.

[0258] In one embodiment, the present disclosure provides an antigen binding protein comprising a light chain variable domain comprising a sequence of amino acids that differs by 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residue from a light chain variable domain sequence selected from the group consisting of L1-L28, wherein each such sequence difference is independently a deletion, insertion, or substitution of one amino acid residue. In another embodiment, the light chain variable domain comprises a sequence of amino acids that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a light chain variable domain sequence selected from the group consisting of L1-L28. In another embodiment, the light chain variable domain comprises a sequence of amino acids encoded by a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence encoding a light chain variable domain selected from the group consisting of L1-L28 (including L1, L2, L3, L4, L5, L6, L7, L8, L9, L10, L11, L12, L13, L14, ... and L28). In another embodiment, the light chain variable domain comprises a sequence of amino acids encoded by a polynucleotide that hybridizes under moderately stringent conditions to the complement of a polynucleotide encoding a light chain variable domain selected from the group consisting of L1-L28. In another embodiment, the light chain variable domain comprises a sequence of amino acids encoded by a polynucleotide that hybridizes under moderately stringent conditions to the complement of a polynucleotide encoding a light chain variable domain selected from the group consisting of L1-L28. In another embodiment, the light chain variable domain comprises a sequence of amino acids encoded by a polynucleotide that hybridizes under moderately stringent conditions to the complement of a light chain polynucleotide L1 through L28.

[0259] In one embodiment, the present disclosure provides an antigen binding protein comprising a light chain variable domain comprising a sequence of amino acids that differs by 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residue from the sequence of a light chain variable domain encoded by one of the clones in the library of CTLA-4 binding clones deposited under ATCC Accession No. PTA-125512, wherein each such sequence difference is independently either a deletion, insertion, or substitution of a single amino acid residue. In another embodiment, the light chain variable domain comprises a sequence of amino acids that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of a light chain variable domain encoded by one of the clones in the library of CTLA-4 binding clones deposited under ATCC Accession No. PTA-125512. In another embodiment, the light chain variable domain comprises a sequence of amino acids encoded by a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of one of the clones in the library of CTLA-4 binding clones deposited under ATCC Accession No. PTA-125512.

[0260] In another embodiment, the present disclosure provides an antigen binding protein comprising a heavy chain variable domain comprising a sequence of amino acids that differs by 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residue from a heavy chain variable domain sequence selected from the group consisting of H1 to H28, wherein each such sequence difference is independently a deletion, insertion, or substitution of a single amino acid residue. In another embodiment, the heavy chain variable domain comprises a sequence of amino acids that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a heavy chain variable domain sequence selected from the group consisting of H1 to H28. In another embodiment, the heavy chain variable domain comprises a sequence of amino acids encoded by a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence encoding a heavy chain variable domain selected from the group consisting of H1-H28. In another embodiment, the heavy chain variable domain comprises a sequence of amino acids encoded by a polynucleotide that hybridizes under moderately stringent conditions to the complement of a polynucleotide encoding a heavy chain variable domain selected from the group consisting of H1-H28. In another embodiment, the heavy chain variable domain comprises a sequence of amino acids encoded by a polynucleotide that hybridizes under moderately stringent conditions to the complement of a polynucleotide encoding a heavy chain variable domain selected from the group consisting of H1-H28. In another embodiment, the heavy chain variable domain comprises a sequence of amino acids encoded by a polynucleotide that hybridizes under moderately stringent conditions to the complement of a heavy chain polynucleotide disclosed herein.

[0261] In one embodiment, the present disclosure provides an antigen binding protein comprising a heavy chain variable domain comprising a sequence of amino acids that differs by only 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residue from the sequence of a heavy chain variable domain encoded by one of the clones in the library of CTLA-4 binding clones deposited under ATCC Accession No. PTA-125512, wherein each such sequence difference is independently either a deletion, insertion, or substitution of one amino acid residue. In another embodiment, the heavy chain variable domain comprises a sequence of amino acids that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of a heavy chain variable domain encoded by one of the clones in the library of CTLA-4 binding clones deposited under ATCC Accession No. PTA-125512. In another embodiment, the heavy chain variable domain comprises a sequence of amino acids encoded by a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of one of the clones in the library of CTLA-4 binding clones deposited under ATCC Accession No. PTA-125512.

[0262] Certain embodiments of the antigen binding proteins of the present disclosure comprise one or more amino acid sequences that are identical to the amino acid sequences of one or more of the CDRs and / or FRs referenced herein. In one embodiment, the antigen binding protein comprises a light chain CDR1 sequence exemplified above. In another embodiment, the antigen binding protein comprises a light chain CDR2 sequence exemplified above. In another embodiment, the antigen binding protein comprises a light chain CDR3 sequence exemplified above. In another embodiment, the antigen binding protein comprises a heavy chain CDR1 sequence exemplified above. In another embodiment, the antigen binding protein comprises a heavy chain CDR2 sequence exemplified above. In another embodiment, the antigen binding protein comprises a heavy chain CDR3 sequence exemplified above.

[0263] In one embodiment, the disclosure provides an antigen binding protein comprising one or more CDR sequences that differ from the CDR sequences set forth above by no more than 5, 4, 3, 2, or 1 amino acid residue.

[0264] In some embodiments, at least one of the CDR1 sequences of the antigen binding protein is the CDR1 sequence of A1 to A28, CDR1-L1 to 28, or CDR1-H1 to 28 shown in Table 5. In some embodiments, at least one of the CDR2 sequences of the antigen binding protein is the CDR2 sequence of A1 to A28, CDR2-L1 to 28, or CDR2-H1 to 28 shown in Table 5. In some embodiments, at least one of the CDR3 sequences of the antigen binding protein is the CDR3 sequence of A1 to A28, CDR3-L1 to 28, or CDR3-H1 to 28 shown in Table 5.

[0265] In another embodiment, the light chain CDR3 sequence of the antigen-binding protein is a light chain CDR3 sequence of A1 to A28 or CDR3-L1 to 28 shown in Table 5, and the heavy chain CDR3 sequence of the antigen-binding protein is a heavy chain sequence of A1 to A28 or CDR-H1 to 28 shown in Table 5.

[0266] In some embodiments, at least one of the CDR1 sequences of the antigen binding protein is the light chain CDR1 sequence of QSVSSSYLA (SEQ ID NO: 12078 or 1014). In some embodiments, at least one of the CDR2 sequences of the antigen binding protein is the light chain CDR2 sequence of GASSRAT (SEQ ID NO: 12079 or 2014). In some embodiments, at least one of the CDR3 sequences of the antigen binding protein is the light chain CDR3 sequence of QQYGSSPWT (SEQ ID NO: 12080 or 3014).

[0267] In some embodiments, at least one of the CDR1 sequences of the antigen binding protein is the heavy chain CDR1 sequence of GFTFSSY (SEQ ID NO: 12075 or 4014). In some embodiments, at least one of the CDR2 sequences of the antigen binding protein is the heavy chain CDR2 sequence of WYEGRN (SEQ ID NO: 12076 or 5014). In some embodiments, at least one of the CDR3 sequences of the antigen binding protein is the heavy chain CDR3 sequence of AGDLGAFDI (SEQ ID NO: 12077 or 6014).

[0268] In some embodiments, the ABP comprises a CDR1-L consisting of SEQ ID NO: 12004, a CDR2-L consisting of SEQ ID NO: 12014, a CDR3-L consisting of SEQ ID NO: 12024, a CDR1-H consisting of SEQ ID NO: 12039, a CDR2-H consisting of SEQ ID NO: 12049, and a CDR3-H consisting of SEQ ID NO: 12059. In some embodiments, the ABP comprises a CDR1-L consisting of SEQ ID NO: 12005, a CDR2-L consisting of SEQ ID NO: 12015, a CDR3-L consisting of SEQ ID NO: 12025, a CDR1-H consisting of SEQ ID NO: 12040, a CDR2-H consisting of SEQ ID NO: 12050, and a CDR3-H consisting of SEQ ID NO: 12060. In some embodiments, the ABP comprises a CDR1-L consisting of SEQ ID NO: 12006, a CDR2-L consisting of SEQ ID NO: 12016, a CDR3-L consisting of SEQ ID NO: 12026, a CDR1-H consisting of SEQ ID NO: 12041, a CDR2-H consisting of SEQ ID NO: 12051, and a CDR3-H consisting of SEQ ID NO: 12061. In some embodiments, the ABP comprises a CDR1-L consisting of SEQ ID NO: 12007, a CDR2-L consisting of SEQ ID NO: 12017, a CDR3-L consisting of SEQ ID NO: 12027, a CDR1-H consisting of SEQ ID NO: 12042, a CDR2-H consisting of SEQ ID NO: 12052, and a CDR3-H consisting of SEQ ID NO: 12062. In some embodiments, the ABP comprises a CDR1-L consisting of SEQ ID NO: 12008, a CDR2-L consisting of SEQ ID NO: 12018, a CDR3-L consisting of SEQ ID NO: 12028, a CDR1-H consisting of SEQ ID NO: 12043, a CDR2-H consisting of SEQ ID NO: 12053, and a CDR3-H consisting of SEQ ID NO: 12063.

[0269] In another embodiment, the antigen binding protein comprises 1, 2, 3, 4, or 5 CDR sequences that each independently differ from the CDR sequences of A1-A23 by 6, 5, 4, 3, 2, 1, or 0 single amino acid additions, substitutions, and / or deletions, and the antigen binding protein further comprises 1, 2, 3, 4, or 5 CDR sequences that each independently differ from the CDR sequences by 6, 5, 4, 3, 2, 1, or 0 single amino acid additions, substitutions, and / or deletions. In some embodiments, the antigen binding protein comprises 1, 2, 3, 4, or 5 CDR sequences that have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the CDR sequences of A1-A28, respectively.

[0270] The nucleotide sequence of A1-A28, or the amino acid sequence of A1-A28, can be modified, for example, by random mutagenesis or site-directed mutagenesis (e.g., oligonucleotide site-directed mutagenesis) to generate modified polynucleotides containing one or more specific nucleotide substitutions, deletions, or insertions compared to the unmutated polynucleotide.

[0271] Other derivatives of anti-CTLA-4 antibodies within the scope of the present disclosure include covalent or aggregative conjugates of anti-CTLA-4 antibodies or fragments thereof with other proteins or polypeptides, such as by expression of recombinant fusion proteins comprising a heterologous polypeptide fused to the N-terminus or C-terminus of the anti-CTLA-4 antibody polypeptide.

[0272] One suitable Fc polypeptide, described in WO 93 / 10151 (incorporated herein by reference), is a single-chain polypeptide extending from the N-terminal hinge region to the native C-terminus of the Fc region of a human IgG1 antibody. Another useful Fc polypeptide is the Fc mutein described in U.S. Pat. No. 5,457,035 and Baum et al., 1994, EMBO J. 13:3992-4001. The amino acid sequence of this mutein is identical to that of the native Fc sequence presented in WO 93 / 10151, except that amino acid 19 is changed from Leu to Ala, amino acid 20 is changed from Leu to Glu, and amino acid 22 is changed from Gly to Ala. The mutein has reduced affinity for Fc receptors.

[0273] In other embodiments, the variable portions of the heavy and / or light chains of an anti-CTLA-4 antibody may be substituted for the variable portions of an antibody heavy and / or light chain.

[0274] Oligomers containing one or more antigen-binding proteins can be used as CTLA-4 antagonists or agonists. The oligomers may be in the form of covalently or non-covalently linked dimers, trimers, or higher-order oligomers. The use of oligomers containing two or more antigen-binding proteins is contemplated, and one example is a homodimer. Other oligomers include heterodimers, homotrimers, heterotrimers, homotetramers, heterotetramers, etc.

[0275] One embodiment relates to oligomers comprising multiple antigen-binding proteins linked via covalent or non-covalent interactions between peptide moieties fused to the antigen-binding proteins. Such peptides can be peptide linkers (spacers) or peptides with oligomerization-promoting properties. Leucine zippers and certain polypeptides derived from antibodies are peptides that can promote oligomerization of antigen-binding proteins attached to them, as described in more detail below.

[0276] In certain embodiments, the oligomer comprises two to four antigen-binding proteins. The antigen-binding proteins of the oligomer may be in any form, such as any of the forms described above, e.g., variants or fragments. Preferably, the oligomer comprises an antigen-binding protein that has CTLA-4 binding activity.

[0277] One embodiment of the present disclosure relates to a dimer comprising two fusion proteins created by fusing a CTLA-4 binding fragment of an anti-CTLA-4 antibody to the Fc region of an antibody.

[0278] Alternatively, the oligomer is a fusion protein comprising multiple antigen-binding proteins, with or without peptide linkers (spacer peptides). Among suitable peptide linkers are those described in U.S. Patent Nos. 4,751,180 and 4,935,233.

[0279] Another method for preparing oligomeric antigen binding proteins involves the use of leucine zippers.

[0280] In one aspect, the present disclosure provides antigen-binding proteins that interfere with the binding of CTLA-4 to its ligand. Such antigen-binding proteins can be directed against CTLA-4, or fragments, variants, or derivatives thereof, and screened in conventional assays for their ability to interfere with the binding of CTLA-4 to its ligand. Examples of suitable assays are assays that test antigen-binding proteins for their ability to inhibit the binding of a CTLA-4 ligand to cells expressing CTLA-4, or assays that test antigen-binding proteins for their ability to reduce a biological or cellular response resulting from the binding of a CTLA-4 ligand to cell-surface CTLA-4. For example, antibodies can be screened for their ability to bind to an immobilized antibody surface (CTLA-4). Antigen-binding proteins that block the binding of CTLA-4 to its ligand can be used to treat any CTLA-4-associated condition, including, but not limited to, cancer. In certain embodiments, human anti-CTLA-4 monoclonal antibodies generated by a procedure involving immunization of transgenic mice are used in treating such conditions.

[0281] Antigen-binding fragments of the antigen-binding proteins of the present disclosure can be produced by conventional techniques. Examples of such fragments include, but are not limited to, Fab and F(ab')2 fragments. Antibody fragments and derivatives produced by genetic engineering techniques are also contemplated.

[0282] Further embodiments include chimeric antibodies, eg, humanized versions of non-human (eg, murine) monoclonal antibodies.

[0283] Procedures have been developed for producing human or partially human antibodies in non-human animals, hi one embodiment, a non-human animal, such as a transgenic mouse, is immunized with a CTLA-4 polypeptide such that antibodies against the CTLA-4 polypeptide are produced in the animal.

[0284] One example of a suitable immunogen is a polypeptide comprising soluble human CTLA-4, eg, the extracellular domain of the protein having SEQ ID NO: 7001, or other immunogenic fragments of this protein.

[0285] Antigen binding proteins (e.g., antibodies, antibody fragments, and antibody derivatives) of the present disclosure may comprise any constant region known in the art. The light chain constant region can be, for example, a kappa or lambda light chain constant region, e.g., a human kappa or lambda light chain constant region. The heavy chain constant region can be, for example, an alpha, delta, epsilon, gamma, or mu heavy chain constant region, e.g., a human alpha, delta, epsilon, gamma, or mu heavy chain constant region. In one embodiment, the light or heavy chain constant region is a fragment, derivative, variant, or mutein of a naturally occurring constant region.

[0286] Techniques for inducing antibodies of a different subclass or isotype from that of a desired antibody, i.e., subclass switching, are known. Thus, an IgG antibody can be derived from, for example, an IgM antibody, and vice versa. Such techniques allow for the preparation of new antibodies that have the antigen-binding properties of a given antibody (parent antibody), but also exhibit biological properties associated with an antibody isotype or subclass different from that of the parent antibody. Recombinant DNA technology may also be used. Cloned DNA encoding a specific antibody polypeptide, for example, DNA encoding the constant domain of an antibody of a desired isotype, can be used in such procedures. See also Lantto et al., 2002, Methods Mol. Biol. 178:303-16.

[0287] In one embodiment, the antigen-binding protein of the present disclosure comprises an IgG1 heavy chain domain of any of A1 to A28 (H1 to H28) or a fragment of an IgG1 heavy chain domain of any of A1 to A28 (H1 to H28). In another embodiment, the antigen-binding protein of the present disclosure comprises a κ light chain constant chain region of A1 to A28 (L1 to L28) or a fragment of the κ light chain constant region of A1 to A28 (L1 to L28). In another embodiment, the antigen-binding protein of the present disclosure comprises both an IgG1 heavy chain domain of A1 to A28 (H1-H28) or a fragment thereof, and a κ light chain domain of A1 to A28 (L1 to L28) or a fragment thereof.

[0288] In another embodiment, the antigen binding protein of the present disclosure comprises both IgG1 heavy chain domains A1 to A28 (H1-H28) or fragments thereof. In some embodiments, the IgG1 heavy chain domain comprises a lysine (K97) at amino acid position 97 according to the IMGT exon numbering system. In another embodiment, the IgG1 heavy chain domain comprises a lysine (K214) at amino acid position 214 according to the EU numbering system. In some embodiments, the IgG1 heavy chain domain comprises an arginine (R97) at amino acid position 97 according to the IMGT exon numbering system. In another embodiment, the IgG1 heavy chain domain comprises an arginine (R214) at amino acid position 214 according to the EU numbering system.

[0289] In some embodiments, the antigen binding protein of the disclosure comprises the IgG1 heavy chain domain of A14(H14) (SEQ ID NO: 114) or a fragment of the IgG1 heavy chain domain, and the IgG1 light chain domain of A14(H14) (SEQ ID NO: 14) or a fragment of the IgG1 light chain domain.

[0290] Thus, antigen binding proteins of the disclosure include those that comprise a combination of variable domains of, for example, L1H1, L2H2, L3H3, L4H4, L5H5, L6H6, L7H7, L8H8, L9H9, L10H10, L11H11, L12H12, L13H13, ... and L28H28, and have a desired isotype (e.g., IgA, IgG1, IgG2, IgG3, IgG4, IgM, IgE, and IgD), as well as Fab or F(ab')2 fragments thereof. Furthermore, if an IgG4 is desired, it may also be desirable to introduce a point mutation (CPSCP (SEQ ID NO: 11969) -> CPPCP (SEQ ID NO: 11970) in the hinge region as described in Bloom et al., 1997, Protein Science 6:407, incorporated herein by reference) to reduce the tendency to form intra-H chain disulfide bonds that can lead to heterogeneity in IgG4 antibodies.

[0291] In one embodiment, the Kd off is 1 x 10 -4 s -1 In another embodiment, K off is 5 x 10 -5 s -1 In another embodiment, K off In another embodiment, the antigen binding protein has a Kd that is substantially the same as an antibody comprising one or more CDRs from an antibody having a combination of light chain and heavy chain variable domain sequences selected from the group of combinations consisting of L1H1, L2H2, L3H3, L4H4, L5H5, L6H6, ... and L23H28. off In another embodiment, the antigen binding protein binds to CTLA-4 with substantially the same Kd as an antibody comprising one of the amino acid sequences exemplified above. off In another embodiment, the antigen binding protein binds to CTLA-4 with substantially the same Kd as an antibody comprising one or more CDRs from an antibody comprising one of the amino acid sequences exemplified above.off binds to CTLA-4.

[0292] In one aspect, the present disclosure provides antigen-binding fragments of the anti-CTLA-4 antibodies of the present disclosure. Such fragments may consist entirely of sequences derived from the antibody or may contain additional sequences. Examples of antigen-binding fragments include Fab, F(ab'), single-chain antibodies, diabodies, triabodies, tetrabodies, and domain antibodies. Other examples are provided in Lunde et al., 2002, Biochem. Soc. Trans. 30:500-06.

[0293] Single-chain antibodies (scFv) can be formed by linking heavy and light chain variable domain (Fv region) fragments via an amino acid bridge (a short peptide linker, e.g., a synthetic sequence of amino acid residues), resulting in a single polypeptide chain. ScFvs containing the variable domain combinations L1H1, L2H2, L3H3, L4H4, L5H5, L6H6, ..., and L28H28 are encompassed by the present disclosure.

[0294] The ABP provided herein can be an anti-CTLA-4 antibody purified from host cells transfected with a gene encoding the antibody by eluting the filtered host cell culture supernatant using a heparin HP column with a salt gradient.

[0295] In some embodiments, the host cells are used in adoptive cell therapy for delivery of an ABP to a subject. In some embodiments, the methods described herein may involve administering host cells transfected with a gene encoding an anti-CTLA-4 antibody.

[0296] The antigen-binding protein may, for example, have the structure of a naturally occurring immunoglobulin.

[0297] In one aspect, the present disclosure provides IGHG1 * In some embodiments, the ABP comprises a human heavy chain constant region gene segment of IGHG1. *The O1 Fc antibody comprises an scFv. In some embodiments, the ABP is specific for CTLA-4. In some embodiments, the ABP comprises the antigen-binding domain of an antibody therapeutic that is approved or under review for approval. In some embodiments, the ABP comprises the antigen-binding domain of ipilimumab, toripalimab, amivantamab, dostallimab, cemiplimab, durvalumab, atezolizumab, or pembrolizumab.

[0298] In some embodiments, the ABP is a human heavy chain constant region gene segment, IGHG1 * 01, thereby enhancing FcR signaling or Fc effector function. * The present disclosure further provides a method of inducing FcR-mediated Treg depletion in a tumor microenvironment, comprising administering an ABP comprising a human heavy chain constant region gene segment of IGHG1. * Also provided is a method for improving FcR signaling or Fc effector function of an ABP by introducing a human heavy chain constant region gene segment of O1 into the ABP.

[0299] In one aspect, antigen binding proteins according to the present disclosure include antigen binding proteins that inhibit the biological activity of CTLA-4.

[0300] In some embodiments, the antigen binding protein according to the present disclosure is an IGHG1 that enhances FcR signaling or Fc effector function. * In some embodiments, the antibody comprises an Fc anti-CTLA-4 antibody or an antigen-binding fragment thereof. * The 01 Fc anti-CTLA-4 antibody comprises an scFv.

[0301] Different antigen-binding proteins may bind to different domains of CTLA-4 or may act via different mechanisms of action. As specifically noted herein, domain regions are designated to include groups unless otherwise indicated. For example, amino acids 4-12 refers to nine amino acids, the amino acids at positions 4 and 12, plus the seven intervening amino acids in the sequence. Other examples include antigen-binding proteins that inhibit the binding of CTLA-4 to its ligand. An antigen-binding protein need not completely inhibit CTLA-4-induced activity to find use in the present disclosure; rather, antigen-binding proteins that reduce a particular activity of CTLA-4 are also contemplated for use. (Discussion herein of specific mechanisms of action for CTLA-4-binding antigen-binding proteins in the treatment of particular diseases is for illustrative purposes only, and the methods presented herein are not constrained thereby.)

[0302] Fab fragments are V L , V H , C L and C H1 A F(ab')2 fragment is a monovalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region, and an Fd fragment is a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region. H and C H1 The Fv fragment has a V domain, and the Fv fragment has a V domain of a single arm of the antibody. L and V H domain, and the dAb fragment has a V H Domain, V L Domain or V H or V L and antigen-binding fragments of the domain (U.S. Patent Nos. 6,846,634, 6,696,245, U.S. Patent Application Publication Nos. 05 / 0202512, 04 / 0202995, 04 / 0038291, 04 / 0009507, 03 / 0039958, Ward et al., Nature 341:544-546, 1989).

[0303] The polynucleotide and polypeptide sequences of specific light and heavy chain variable domains are set forth below. Antibodies comprising light and heavy chains are named by combining the name of the light chain variable domain and the name of the heavy chain variable domain. For example, "L4H7" refers to an antibody comprising the light chain variable domain of L4 (comprising the sequence of SEQ ID NO:4) and the heavy chain variable domain of H7 (comprising the sequence of SEQ ID NO:107). The light chain variable sequences are provided in SEQ ID NOs:1-28, and the heavy chain variable sequences are provided in SEQ ID NOs:101-128.

[0304] In other embodiments, an antibody may comprise a particular heavy or light chain, but the complementary light or heavy chain variable domain remains unspecified. In particular, certain embodiments herein include antibodies that bind to a specific antigen (e.g., CTLA-4) by a specific light or heavy chain, such that the complementary heavy or light chain may be promiscuous or even unrelated, but may be determined, for example, by screening a combinatorial library. Portolano et al.,J.Immunol.V.150 (3),pp.880-887 (1993);Clackson et al.,Nature v.352 pp.624-628 (1991);Adler et al.,A natively paired antibody library yields drug leads with higher sensitivity and specificity than a randomly paired antibody library,MAbs (2018)), Adler et al. al.,Rare,high-affinity mouse anti-CTLA-4 antibodies that function in checkpoint blockade,discovered using microfluidics and molecular genomics,MAbs (2017).

[0305] Naturally occurring immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FR) connected by three hypervariable regions, also called complementarity-determining regions or CDRs. From the N-terminus to the C-terminus, both light and heavy chains contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain follows the definition in Kabat et al. in Sequences of Proteins of Immunological Interest, 5th Ed., US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242, 1991.

[0306] The term "human antibody," also called "fully human antibody," includes all antibodies having one or more variable and constant regions derived from human immunoglobulin sequences. In one embodiment, all of the variable and constant domains are derived from human immunoglobulin sequences (fully human antibody). These antibodies can be prepared in a variety of ways, including through immunization with an antigen of interest of mice that have been genetically modified to express antibodies derived from genes encoding human heavy and / or light chains, examples of which are described below.

[0307] A humanized antibody has a sequence that differs from that of an antibody derived from a non-human species by one or more amino acid substitutions, deletions, and / or additions, such that the humanized antibody is less likely to induce an immune response and / or induces a less severe immune response when administered to a human subject compared to the non-human species antibody. In one embodiment, specific amino acids in the framework and constant domains of the heavy and / or light chains of a non-human species antibody are mutated to produce a humanized antibody. In another embodiment, a constant domain from a human antibody is fused to a variable domain of a non-human species. In another embodiment, one or more amino acid residues in one or more CDR sequences of a non-human antibody are altered to reduce the likelihood that the non-human antibody will be immunogenic when administered to a human subject, where the altered amino acid residues are not important for immunospecific binding of the antibody to its antigen, or the changes made to the amino acid sequence are conservative changes such that binding of the humanized antibody to the antigen is not significantly impaired compared to binding of the non-human antibody to the antigen. Examples of methods for making humanized antibodies can be found in US Pat. Nos. 6,054,297, 5,886,152, and 5,877,293.

[0308] The term "chimeric antibody" refers to an antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies. In one embodiment, one or more of the CDRs are derived from a human anti-CTLA-4 antibody. In another embodiment, all of the CDRs are derived from a human anti-CTLA-4 antibody. In another embodiment, CDRs from two or more human anti-CTLA-4 antibodies are mixed and matched to form a chimeric antibody. For example, a chimeric antibody may contain CDR1 from the light chain of a first human anti-CTLA-4 antibody, CDR2 and CDR3 from the light chain of a second human anti-CTLA-4 antibody, and CDRs from the heavy chain of a third anti-CTLA-4 antibody. Furthermore, the framework regions may be derived from one of the same anti-CTLA-4 antibodies, one or more different antibodies, such as a human antibody, or a humanized antibody. In one example of a chimeric antibody, a portion of the heavy and / or light chain is identical to, homologous to, or derived from an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to, homologous to, or derived from an antibody from another species or belonging to another antibody class or subclass. Also included are fragments of such antibodies that exhibit the desired biological activity (i.e., the ability to specifically bind to CTLA-4).

[0309] Antibody fragments or analogs can be readily prepared by one of ordinary skill in the art following the teachings herein and using techniques well known in the art.

[0310] Another form of antibody fragment is a peptide containing one or more complementarity-determining regions (CDRs) of an antibody. CDRs (also called "minimal recognition units" or "hypervariable regions") can be incorporated into a molecule by either covalent or non-covalent bonding to form an antigen-binding protein. CDRs can be obtained by constructing a polynucleotide encoding the desired CDR. Such polynucleotides are prepared, for example, by polymerase chain reaction, which synthesizes the variable region using mRNA from antibody-producing cells as a template (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology 2:106, 1991; Courtenay Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166 (Cambridge University Press 1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), page 137 (Wiley-Liss, Inc. 1995)).

[0311] Thus, in one embodiment, the binding agent comprises at least one CDR described herein. The binding agent may comprise at least two, three, four, five, or six CDRs described herein. The binding agent may further comprise at least one variable region domain of an antibody described herein. The variable region domain may be of any size or amino acid composition and generally comprises at least one CDR sequence responsible for binding to human CTLA-4, e.g., CDR1-H, CDR2-H, CDR3-H, CDR1-L, CDR2-L, and CDR3-L, specifically described herein, adjacent to or in frame with one or more framework sequences. Generally speaking, a variable (V) region domain is an immunoglobulin heavy chain variable domain (V H ) and / or a light chain variable domain (V L ) in any suitable arrangement. Thus, for example, the V region domain may be a monomer, and as described below, may be at least 1 x 10 7 V, which can independently bind human CTLA-4 with an affinity equal to or less than M H or V L Alternatively, the V region domain may be a dimer, H V H , V H V L or V L V L A V region dimer may contain at least one V domain that can non-covalently associate. H chain and at least one V L chain (hereinafter, F V If desired, the chains can be covalently linked, for example, directly via a disulfide bond between the two variable domains, or via a linker, e.g., a peptide linker, to form a single-chain Fv (scFv).

[0312] The variable region domain can be any naturally occurring variable domain or an engineered version thereof. By engineered version is meant a variable region domain created using recombinant DNA engineering techniques. Such engineered versions include, for example, those created from a specific antibody variable region by insertions, deletions, or changes in or to the amino acid sequence of the specific antibody. Particular examples include engineered variable region domains comprising at least one CDR and optionally one or more framework amino acids from a first antibody and the remainder of the variable region domain from a second antibody.

[0313] The variable region domain may be covalently linked at the C-terminal amino acid to at least one other antibody domain or fragment thereof. Thus, for example, a V present in the variable region domain may be covalently linked to at least one other antibody domain or fragment thereof. H The V domain may be linked to an immunoglobulin CH1 domain or a fragment thereof. L The domains can be linked to a CK domain or fragment thereof. Thus, for example, antibodies can be prepared by combining the antigen-binding domains of interest with the CH1 and CK domains covalently linked at their C-termini to the relevant V domains. H and V L The CH1 domain may be an Fab fragment containing the CH1 domain, which may be extended with additional amino acids, for example to provide a hinge region or part of a hinge region domain as found in an Fab' fragment, or to provide additional domains such as antibody CH2 and CH3 domains.

[0314] In some embodiments, the ABP comprises an Fc region that lacks fucose sugar units on the N-glycans.

[0315] In some embodiments, the ABP comprises a glycine at amino acid position 201.

[0316] In some embodiments, the ABP is produced from cells containing the bacterial protein RMD (GDP-6-deoxy-D-lyxo-4-hexulose reductase) or a variant thereof. In certain embodiments, the cells are cultured in the absence of fucose. In some embodiments, the ABP is produced from cells lacking or having reduced expression of Fut8. In some embodiments, the ABP is produced from cells cultured in the presence of 2-Fluorfucose (2FF), a fucosylation inhibitor.

[0317] In some embodiments, the ABP is produced from cells that overexpress glycosyltransferase (GnTIII). In some embodiments, the ABP is isolated based on its fucosylation state.

[0318] In some embodiments, the ABP is a defucosylated monoclonal antibody.

[0319] As described herein, an antibody comprises at least one of these CDRs. For example, one or more CDRs can be incorporated into a known antibody framework region (e.g., IgG1, IgG2, etc.) or conjugated to a suitable vehicle to extend its half-life. Suitable vehicles include, but are not limited to, Fc, polyethylene glycol (PEG), albumin, transferrin, etc. These and other suitable vehicles are known in the art. Such conjugated CDR peptides can be in a monomeric, dimeric, tetrameric, or other form. In one embodiment, one or more water-soluble polymers are attached to one or more specific positions of the binder, for example, at the amino terminus.

[0320] In another example, individual V from an antibody (i.e., a CTLA-4 antibody) L or V H The chains can be linked to other V fragments capable of forming antigen-binding fragments (or Fab) with the same specificity. H or V L can be used to search for the V H and VL Random combinations of chain Ig genes can be expressed as antigen-binding fragments in bacteriophage libraries (such as fd or lambda phage). For example, combinatorial libraries can be used to generate antigen-binding specific V L or V H The parent V L or V H The combinatorial library can be generated by utilizing a strand library. The combinatorial library can then be screened by conventional techniques, for example, by using a radiolabeled probe (e.g., radiolabeled CTLA-4). See, for example, Portolano et al., J. Immunol. V. 150 (3) pp. 880-887 (1993).

[0321] Diabodies are bivalent antibodies containing two polypeptide chains, each of which has a V domain connected by a linker that is too short to allow pairing between the two domains on the same chain. H and V L A diabody comprises three or four polypeptide chains, each of which can pair with a complementary domain on another polypeptide chain (see, e.g., Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-48, and Poljak et al., 1994, Structure 2:1121-23). ​​If the two polypeptide chains of a diabody are identical, the resulting diabody will have two identical antigen-binding sites. Polypeptide chains with different sequences can be used to generate diabodies with two different antigen-binding sites. Similarly, tribodies and tetrabodies are antibodies that contain three or four polypeptide chains, respectively, forming three or four antigen-binding sites, which may be the same or different.

[0322] Antibody polypeptides, including fibronectin polypeptide monobodies, are also disclosed in U.S. Patent No. 6,703, 199. Other antibody polypeptides are disclosed in U.S. Patent Application Publication No. 2005 / 0238646, which are single-chain polypeptides.

[0323] In certain embodiments, the antibody comprises one or more water-soluble polymer conjugates, including, but not limited to, polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol. See, e.g., U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, and 4,179,337. In certain embodiments, the derivative binder comprises one or more of monomethoxy-polyethylene glycol, dextran, cellulose, or other carbohydrate-based polymers, poly-(N-vinylpyrrolidone)-polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyol (e.g., glycerol), and polyvinyl alcohol, and mixtures of such polymers. In certain embodiments, one or more water-soluble polymers are randomly attached to one or more side chains. In certain embodiments, PEG can act to improve the therapeutic capacity of a binder, such as an antibody. Certain such methods are discussed, for example, in US Pat. No. 6,133,426, which is incorporated herein by reference for any purpose.

[0324] In some embodiments, the ABP of the present disclosure is a monoclonal antibody that binds to CTLA-4.

[0325] Monoclonal antibodies can be produced using any technique known in the art, for example, by immortalizing spleen cells harvested from the transgenic animals after the immunization schedule is complete. The spleen cells can be immortalized using any technique known in the art, for example, by fusing them with myeloma cells to produce hybridomas. Hybridoma cell lines that produce antibodies that bind to CTLA-4 polypeptides are identified. Such hybridoma cell lines and the anti-CTLA-4 monoclonal antibodies produced by them are encompassed by the present disclosure. Myeloma cells for use in the hybridoma production fusion procedure are preferably non-antibody-producing, have high fusion efficiency, and possess enzyme deficiencies that prevent them from growing in specific selective media that support the growth of only the desired fused cells (hybridomas). Examples of cell lines suitable for use in mouse fusions include Sp-20, P3-X63 / Ag8, P3-X63-Ag8.653, NS1 / 1.Ag 4 1, Sp210-Ag14, FO, NSO / U, MPC-11, MPC11-X45-GTG 1.7, and S194 / 5XX0 Bul; examples of cell lines used in rat fusions include R210.RCY3, Y3-Ag 1.2.3, IR983F, and 4B210. Other cell lines useful for cell fusion are U-266, GM1500-GRG2, LICR-LON-HMy2, and UC729-6. Hybridomas or mAbs can be further screened to identify mAbs with specific properties, such as the ability to block CTLA-4-induced activity.

[0326] The antibodies of the present disclosure may also be fully human monoclonal antibodies. Provided are isolated, fully human antibodies that specifically bind to CTLA-4, wherein the antigen-binding protein has at least one in vivo biological activity of a human anti-CTLA-4 antibody. 7.4. Nucleic acids

[0327] In one aspect, the present disclosure provides isolated nucleic acid molecules. Nucleic acids include, for example, polynucleotides encoding all or a portion of an antigen-binding protein, e.g., one or both chains of an antibody of the present disclosure, or a fragment, derivative, mutein, or variant thereof; polynucleotides sufficient for use as hybridization probes, PCR primers, or sequencing primers to identify, analyze, mutate, or amplify polynucleotides encoding the polypeptide; antisense nucleic acids for inhibiting expression of a polynucleotide; and complementary sequences of the foregoing. Nucleic acids can be of any length. For example, it may be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1,000, 1,500, 3,000, 5,000 or more nucleotides in length, and / or may include one or more additional sequences, e.g., regulatory sequences, and / or may be part of a larger nucleic acid, e.g., a vector. Nucleic acids may be single- or double-stranded and may comprise RNA and / or DNA nucleotides, as well as artificial variants thereof (e.g., peptide nucleic acids).

[0328] Nucleic acids encoding antibody polypeptides (eg, heavy or light chains, variable domains only, or full length) can be isolated from B cells of mice immunized with CTLA-4.

[0329] Nucleic acid sequences encoding the variable regions of the heavy and light chain variable regions are provided herein. Those skilled in the art will understand that due to the degeneracy of the genetic code, each of the polypeptide sequences disclosed herein is encoded by many other nucleic acid sequences. The present disclosure provides degenerate nucleotide sequences encoding each antigen-binding protein of the present disclosure. In some embodiments, the nucleic acid sequences are codon-optimized. In some embodiments, the nucleic acid sequences are codon-optimized for expression in mammalian cells.

[0330] The disclosure further provides nucleic acids that hybridize to other nucleic acids (eg, nucleic acids comprising any nucleotide sequence of the CTLA-4 gene) under specific hybridization conditions.

[0331] Changes can be introduced into a nucleic acid by mutation, thereby resulting in a change in the amino acid sequence of the polypeptide (e.g., antigen binding protein) that it encodes. Mutations can be introduced using any technique known in the art.

[0332] Mutations can be introduced into a nucleic acid without significantly altering the biological activity of the polypeptide it encodes. For example, nucleotide substitutions resulting in amino acid substitutions at non-essential amino acid residues can be made. In one embodiment, a nucleotide sequence provided herein for CTLA-4, or a desired fragment, variant, or derivative thereof, is mutated to encode an amino acid sequence containing one or more deletions or substitutions of the amino acid residues set forth herein for CTLA-4, such that two or more sequences result in different residues. Alternatively, one or more mutations can be introduced into a nucleic acid that selectively alter the biological activity (e.g., CTLA-4 binding) of the polypeptide it encodes. For example, a mutation can quantitatively or qualitatively alter a biological activity. Examples of quantitative changes include increasing, decreasing, or eliminating an activity. Examples of qualitative changes include changing the antigen specificity of an antigen-binding protein.

[0333] In another aspect, the present disclosure provides nucleic acid molecules suitable for use as primers or hybridization probes for the detection of nucleic acid sequences of the present disclosure. The nucleic acid molecules of the present disclosure can include only a portion of a nucleic acid sequence encoding a full-length polypeptide of the present disclosure, e.g., a fragment that can be used as a probe or primer, or a fragment that encodes an active portion of a polypeptide of the present disclosure (e.g., a CTLA-4 binding portion). 7.5. Expression Vectors

[0334] The present disclosure provides a vector comprising a nucleic acid encoding a polypeptide of the present disclosure or a portion thereof. Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors, and expression vectors, such as recombinant expression vectors.

[0335] In another aspect of the present disclosure, expression vectors containing the nucleic acid molecules and polynucleotides of the present disclosure are also provided, as are host cells transformed with such vectors and methods for producing the polypeptides. The term "expression vector" refers to a plasmid, phage, virus, or vector for expressing (e.g., or inducing) a polypeptide derived from a polynucleotide sequence. A vector for polypeptide expression contains the minimum sequences necessary for vector propagation and expression of a cloned insert. An expression vector contains a transcription unit comprising an assembly of (1) genetic elements that play a regulatory role in gene expression, such as a promoter or enhancer, (2) polypeptide and protein-encoding sequences that are transcribed into mRNA and translated into protein, and (3) appropriate transcription start and termination sequences. These sequences may further include a selectable marker. Suitable vectors for expression in host cells are readily available, and nucleic acid molecules are inserted into the vector using standard recombinant DNA techniques. Such vectors may include promoters that function in specific tissues and viral vectors for expression of polypeptides in targeted human or animal cells.

[0336] The recombinant expression vector of the present disclosure can include a nucleic acid of the present disclosure in a form suitable for expression of the nucleic acid in a host cell. Thus, in one aspect, the present disclosure provides a host cell comprising a polynucleotide or vector encoding an ABP of the present disclosure. The host cell can be used to produce the ABP ex vivo. In some embodiments, the host cell is administered to a subject to induce expression of the ABP in vivo. In some embodiments, the host cell is used as a therapeutic agent for the treatment of a disease.

[0337] In some embodiments, the expression vector is an expression vector purified from one of the clones in the library of CTLA-4 binding clones deposited under ATCC Accession No. PTA-125512. In some embodiments, the expression vector is made by genetic modification of one of the expression vectors in one of the clones purified from the library of CTLA-4 binding clones deposited under ATCC Accession No. PTA-125512. In some embodiments, the expression vector is made by using the heavy and light chain variable region sequences of one of the clones in the library of CTLA-4 binding clones deposited under ATCC Accession No. PTA-125512.

[0338] The disclosure further provides methods for producing the polypeptides. A variety of other expression / host systems can be utilized.

[0339] In some embodiments, the mammalian cells used in recombinant protein production comprise engineered cells that produce a reduced amount of core fucosylation (e.g., compared to the amount of core fucosylation in unengineered cells). In some embodiments, the mammalian cells used in recombinant protein production comprise engineered cells that produce a reduced amount of fucose (e.g., compared to the amount of fucose in unengineered cells). In some embodiments, the mammalian cells used in recombinant protein production comprise CHO cells. In some embodiments, the mammalian cells are the GlymaxX® cell line. In certain embodiments, the cell line produces defucosylated recombinant protein. In certain embodiments, the mammalian cells have less or reduced fucosylation, e.g., the mammalian cells produce a reduced amount of fucose. In certain embodiments, during cell culture, the method comprises adding a fucosylation inhibitor to the medium in which the cells are grown. Non-limiting examples of fucosylation inhibitors include fucosyltransferase (FUT) inhibitors, 2-fluoroperacetylated fucose (2FF), 2-fluorofucose (SGN-2FF), Fucotrim I (PD-Rha6F2-1P), Fucotrim II (PD-Rha6F3-1P), A2FF1P, and B2FF1. In some embodiments, mammalian cells used in recombinant protein production are engineered to overexpress a glycosyltransferase. In certain embodiments, the glycosyltransferase is β-1,4-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase.

[0340] In some embodiments, the ABP comprises a defucosylated Fc region, hi some embodiments, the antibody is defucosylated (e.g., the N-glycans of the Fc region of the antibody do not have a core fucose sugar unit).

[0341] In certain embodiments, the glycosyltransferase competes with FuT8. In some embodiments, the mammalian cell line used for recombinant protein production produces ABPs with reduced fucosylation (e.g., less than 70% fucosylation, less than 65% fucosylation, less than 60% fucosylation, less than 55% fucosylation, less than 50% fucosylation, less than 45% fucosylation, less than 40% fucosylation, less than 35% fucosylation, less than 30% fucosylation, less than 25% fucosylation, less than 20% fucosylation, less than 15% fucosylation, less than 10% fucosylation, less than 5% fucosylation, or less than 2.5% fucosylation).

[0342] In some embodiments, the mammalian cell line used for recombinant protein production produces ABPs with increased fucosylation (e.g., greater than 99% fucosylation, greater than 95% fucosylation, greater than 90% fucosylation, greater than 85% fucosylation, greater than 80% fucosylation, greater than 75% fucosylation, greater than 70% fucosylation, greater than 65% fucosylation, greater than 60% fucosylation, greater than 55% fucosylation, greater than 50% fucosylation, greater than 45% fucosylation, greater than 40% fucosylation, greater than 35% fucosylation, greater than 30% fucosylation, greater than 25% fucosylation, greater than 20% fucosylation, greater than 15% fucosylation, greater than 10% fucosylation, greater than 5% fucosylation, or greater than 2.5% fucosylation).

[0343] For stable transfection of mammalian cells, it is known that, depending on the expression vector and transfection technique used, only a small fraction of cells may integrate foreign DNA into their genome. To identify and select these integrants, a gene encoding a selectable marker (e.g., for antibiotic resistance) is generally introduced into the host cells along with the gene of interest. Once such cells are transformed with a vector containing the selectable marker and the desired expression cassette, the cells may be grown, for example, in an enriched medium before being switched to a selective medium. The selectable marker is designed to allow the growth and recovery of cells that successfully express the introduced sequence. Resistant clumps of stably transformed cells can be propagated using tissue culture techniques appropriate for the cell line used. A review of recombinant protein expression can be found in *Methods of Enzymology*, v. 185, Goeddell, DV, ed., Academic Press (1990). Preferred selectable markers include those that confer resistance to drugs such as G418, hygromycin, and methotrexate. Cells stably transfected with the introduced nucleic acid can be identified by, among other methods, drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).

[0344] The transformed cells can be cultured under conditions that promote expression of the polypeptide, which can be recovered by conventional protein purification procedures (defined above). One such purification procedure includes, for example, the use of affinity chromatography on a matrix to which all or a portion of CTLA-4 (e.g., the extracellular domain) is bound. Polypeptides contemplated for use herein include substantially homogeneous recombinant mammalian anti-CTLA-4 antibody polypeptides that are substantially free from contaminating endogenous material.

[0345]

[0346] The polypeptides and proteins of the present disclosure can be purified according to protein purification techniques well known to those of skill in the art. These techniques include, at one level, crude fractionation of proteinaceous and non-proteinaceous fractions. After separation of the peptide polypeptide from other proteins, the peptide or polypeptide of interest can be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). The term "purified polypeptide," as used herein, is intended to refer to a composition that is isolatable from other components, where the polypeptide is purified to any degree relative to its naturally obtainable state. Thus, a purified polypeptide also refers to a polypeptide that is free from the environment in which it may naturally occur. Generally, "purified" refers to a polypeptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. When the term "substantially purified" is used, this designation refers to a peptide or polypeptide composition in which the polypeptide or peptide forms the majority of the composition, such as comprising more than about 50%, about 60%, about 70%, about 80%, about 85%, or about 90% of the protein in the composition.

[0347] Various techniques suitable for use in purification are well known to those skilled in the art. These include, for example, precipitation with ammonium sulfate, PEG, antibodies (immunoprecipitation), or heat denaturation followed by centrifugation, chromatography, for example, affinity chromatography (protein A column), ion exchange, gel filtration, reverse phase, hydroxylapatite, hydrophobic interaction chromatography, isoelectric focusing, gel electrophoresis, and combinations of these techniques. As is generally known in the art, the order in which the various purification steps are performed may be varied or certain steps may be omitted, and still result in a suitable method for preparing a substantially purified polypeptide. Exemplary purification steps are provided in the Examples below.

[0348] Various methods for quantifying the degree of purification of a polypeptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific binding activity of an active fraction or assessing the amount of peptide or polypeptide within the fraction by SDS / PAGE analysis. A preferred method for assessing the purity of a polypeptide fraction is to calculate the binding activity of the fraction, compare it to the binding activity of the initial extract, and then calculate the degree of purification, which is assessed herein by "fold purification." The actual units used to express the amount of binding activity will, of course, depend on the particular assay technique selected after purification and whether the polypeptide or peptide exhibits detectable binding activity. 7.6. Antibody Production Method

[0349] Fully human monoclonal antibodies can be produced by any technique familiar to those skilled in the art. Such methods include, but are not limited to, Epstein-Barr virus (EBV) transformation of human peripheral blood cells (e.g., including B lymphocytes), in vitro immunization of human B cells, fusion of spleen cells from immunized transgenic mice carrying inserted human immunoglobulin genes, isolation from a human immunoglobulin V-region phage library, or other procedures known in the art and based on the disclosure herein. For example, fully human monoclonal antibodies can be obtained from transgenic mice engineered to produce specific human antibodies in response to antigen challenge. Methods for obtaining fully human antibodies from transgenic mice are described, for example, in Green et al., Nature Genet. 7:13, 1994; Lonberg et al., Nature 368:856, 1994; Taylor et al., Int. Immun. 6:579, 1994; U.S. Patent No. 5,877,397; Bruggemann et al., 1997 Curr. Opin. Biotechnol. 8:455-58; and Jakobovits et al., 1995 Ann. NY Acad. Sci. 764:525-35. In this technique, elements of the human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines containing targeted disruptions of the endogenous heavy and light chain loci (see also Bruggemann et al., Curr. Opin. Biotechnol. 8:455-58 (1997)). For example, the human immunoglobulin transgene can be a minigene construct or a transgene locus on a yeast artificial chromosome that undergoes B cell-specific DNA rearrangement and hypermutation in mouse lymphoid tissues. Fully human monoclonal antibodies can be obtained by immunizing transgenic mice, which can then produce human antibodies specific to CTLA-4. Lymphoid cells of the immunized transgenic mice can be used to produce human antibody-secreting hybridomas according to the methods described herein. Polyclonal serum containing fully human antibodies can also be obtained from the blood of immunized animals.

[0350] Another method for generating the human antibodies of the present disclosure involves immortalizing human peripheral blood cells by EBV transformation. See, for example, U.S. Patent No. 4,464,456. Such immortalized B cell lines (or lymphoblastoid cell lines) producing monoclonal antibodies that specifically bind to CTLA-4 can be identified by immunodetection methods such as ELISA, as provided herein, and then isolated by standard cloning techniques. The stability of lymphoblastoid cell lines producing anti-CTLA-4 antibodies can be improved by fusing the transformed cell line with a mouse myeloma to generate a mouse-human hybrid cell line, according to methods known in the art (see, for example, Glasky et al., Hybridoma 8:377-89 (1989)). Yet another method for generating human monoclonal antibodies is in vitro immunization, which involves priming human splenic B cells with human CTLA-4, followed by fusing the primed B cells with a heterohybrid fusion partner. See, e.g., Boerner et al., 1991 J. Immunol. 147:86-95.

[0351] In certain embodiments, B cells producing anti-human CTLA-4 antibodies are selected, and the light chain variable region and heavy chain variable region are cloned from the B cells according to molecular biology techniques known in the art and described herein (WO 92 / 02551; U.S. Pat. No. 5,627,052; Babcook et al., Proc. Natl. Acad. Sci. USA 93:7843-48 (1996)).

[0352] In some embodiments, B cells producing specific antibodies are selected by using a method that allows for the identification of naturally paired antibodies, such as the method described in Adler et al., A natively paired antibody library yields drug leads with higher sensitivity and specificity than a randomly paired antibody library, MAbs (2018), which is incorporated herein by reference in its entirety.

[0353] After selection of B cells producing the desired antibody, the specific antibody gene can be cloned by isolating and amplifying the DNA or mRNA according to methods known in the art and described herein.

[0354] Methods for obtaining antibodies of the present disclosure can also employ various phage display methods known in the art. See, e.g., Winter et al., 1994 Annu. Rev. Immunol. 12:433-55; Burton et al., 1994 Adv. Immunol. 57:191-280. Combinatorial libraries of human or mouse immunoglobulin variable region genes can be generated in phage vectors that can be screened to select Ig fragments (Fab, Fv, sFv, or multimers thereof) that specifically bind to a CTLA-4 binding protein or a variant or fragment thereof. See, e.g., U.S. Pat. No. 5,223,409; Huse et al., 1989 Science 246:1275-81; Sastry et al., Proc. Natl. Acad. Sci. USA 86:5728-32 (1989); Alting-Mees et al., Strategies in Molecular Biology 3:1-9 (1990); Kang et al., 1991 Proc. Natl. Acad. Sci. USA 88:4363-66; Hoogenboom et al., 1992 J. Molec. Biol. 227:381-388; Schlebusch et al., 1997 Hybridoma 16:47-52, and references cited therein.

[0355] An antibody fragment fused to another protein, e.g., a minor coat protein, can also be used to enrich phage with antigen. Next, rearranged heavy chains (V) from mice immunized against the antigen (e.g., CTLA-4) are isolated. H ) and light chain (V L Using random combinatorial libraries such as those described above, diverse libraries of antibody fragments are displayed on the surface of phage. These libraries can be screened for complementary variable domains, and the domains can be purified, for example, by affinity columns. See Clackson et al., Nature, Vol. 352, pp. 624-628 (1991).

[0356] Heavy and light chain immunoglobulin cDNA expression libraries can also be prepared in lambda phage using, for example, λImmunoZap™(H) and λImmunoZap™(L) vectors (Stratagene, La Jolla, California).

[0357] In one embodiment, in a hybridoma, the variable regions of the genes expressing the monoclonal antibody of interest are amplified using nucleotide primers. These primers can be synthesized by one of skill in the art or purchased from commercial sources. (For example, Stratagene (La Jolla, California) offers, among others, the V Ha , V Hb , V Hc , V Hd , C H1 , V L and C L (Primers for mouse and human variable regions, such as the heavy and light chain variable regions, are commercially available.) These primers can be used to amplify the heavy or light chain variable region, which can then be inserted into a vector such as ImmunoZAP™ H or ImmunoZAP™ L (Stratagene), respectively.

[0358] Once cells producing an antibody according to the present disclosure have been obtained using any of the above immunization and other techniques, the specific antibody gene may be cloned by isolating and amplifying the DNA or mRNA therefrom according to standard procedures described herein. The antibody produced may then be sequenced, the CDRs identified, and the DNA encoding the CDRs manipulated as described above to generate other antibodies according to the present disclosure.

[0359] CTLA-4 binding agents of the present disclosure preferably modulate CTLA-4 function in the cell-based assays described herein and / or in the in vivo assays described herein, and / or bind to one or more of the domains described herein, and / or cross-block the binding of one of the antibodies described herein, and / or are cross-blocked from binding to CTLA-4 by one of the antibodies described herein. Accordingly, such binding agents can be identified using the assays described herein.

[0360] In certain embodiments, antibodies are generated by first identifying antibodies that bind to one or more of the domains provided herein and / or that neutralize in the cell-based and / or in vivo assays described herein and / or that cross-block the antibodies described herein and / or that are cross-blocked from binding to CTLA-4 by one of the antibodies described herein. The CDR regions from these antibodies are then used to insert into a suitable biocompatible framework to generate a CTLA-4 binding agent. The non-CDR portion of the binding agent may be composed of amino acids or may be a non-protein molecule. The assays described herein allow for characterization of the binding agent. Preferably, the binding agents of the present disclosure are antibodies as defined herein.

[0361] Other antibodies according to the present disclosure can be obtained by conventional immunization and cell fusion procedures described herein and known in the art.

[0362] Molecular evolution of the complementarity-determining region (CDR) at the center of the antibody binding site has also been used to isolate antibodies with increased affinity, for example, antibodies with increased affinity for c-erbB-2 as described by Schier et al., 1996, J. Mol. Biol. 263:551. Therefore, such techniques are useful in preparing antibodies against CTLA-4. Antigen-binding proteins against CTLA-4 can be used, for example, in assays to detect the presence of CTLA-4 polypeptide either in vitro or in vivo. Antigen-binding proteins can also be used in purifying CTLA-4 protein by immunoaffinity chromatography.

[0363] While human, partially human, or humanized antibodies are suitable for many applications, particularly those involving administration of antibodies to human subjects, other types of antigen-binding proteins may also be suitable for certain applications. Non-human antibodies of the present disclosure may be derived, for example, from any antibody-producing animal, such as a mouse, rat, rabbit, goat, donkey, or non-human primate, such as a monkey (e.g., a cynomolgus or rhesus monkey) or an ape (e.g., a chimpanzee). Antibodies from a particular species can be produced, for example, by immunizing an animal of that species with a desired immunogen (e.g., a CTLA-4 polypeptide), or by using an artificial system to produce antibodies of that species (e.g., a bacterial or phage display-based system to produce antibodies of a particular species), or by converting an antibody from one species to an antibody from another species, for example, by replacing the antibody's constant region with a constant region from the other species, or by substituting one or more amino acid residues of the antibody to more closely resemble the sequence of an antibody from the other species. In one embodiment, the antibody is a chimeric antibody, comprising amino acid sequences derived from antibodies of two or more different species.

[0364] Antigen binding proteins can be prepared and screened for desired properties by any of a number of conventional techniques. Certain techniques involve isolating a nucleic acid encoding the polypeptide chain (or portion thereof) of the antigen binding protein of interest (e.g., an anti-CTLA-4 antibody) and manipulating the nucleic acid by recombinant DNA technology. The nucleic acid can be fused to another nucleic acid of interest or can be modified (e.g., by mutagenesis or other conventional techniques) to, for example, add, delete, or substitute one or more amino acid residues. Furthermore, antigen binding proteins can be purified from cells that naturally express them (e.g., antibodies can be purified from the hybridoma that produces them) or produced in a recombinant expression system using any technique known in the art. See, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980), and Antibodies: A Laboratory Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988).

[0365] Any expression system known in the art can be used to produce the recombinant polypeptides of the present disclosure. Expression systems have been comprehensively described in detail above. Generally, host cells are transformed with a recombinant expression vector containing DNA encoding the desired polypeptide. Host cells that can be used include prokaryotes, yeast, or higher eukaryotic cells. Prokaryotes include gram-negative or gram-positive bacteria, such as Escherichia coli or Bacillus. Higher eukaryotic cells include insect cells and established cell lines of mammalian origin. Examples of suitable mammalian host strains include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., 1981, Cell 23:175), L cells, 293 cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, BHK (ATCC CRL 10) cell lines, and the CVI / EBNA cell line derived from the African green monkey kidney cell line CVI (ATCC CCL 70) as described by McMahan et al., 1991, EMBO J. 10:2821. Suitable cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985).

[0366] It will be understood that the antibodies of the present disclosure may have at least one amino acid substitution, provided that the antibody retains its binding specificity. Therefore, modifications to the antibody structure are encompassed within the scope of the present disclosure. These may include amino acid substitutions, which may be conservative or non-conservative, that do not destroy the antibody's ability to bind to CTLA-4. Conservative amino acid substitutions may include non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than synthesis in biological systems. These include peptidomimetics and other reverse or inverted forms of amino acid moieties. Conservative amino acid substitutions may also include substitutions of natural amino acid residues with standard residues, such that there is little or no effect on the polarity or charge of the amino acid residue at that position.

[0367] Non-conservative substitutions may involve exchanging a member of one class of amino acids or amino acid mimetics for a member of another class having different physical properties (e.g., size, polarity, hydrophobicity, charge). Such substituted residues may be introduced into regions of the human antibody that are homologous with the non-human antibody, or into the non-homologous regions of the molecule.

[0368] Furthermore, one skilled in the art can generate test variants containing single amino acid substitutions at each desired amino acid residue. The variants can then be screened using activity assays known to those skilled in the art. Such variants can be used to gather information about suitable variants. For example, if it is discovered that a change to a particular amino acid residue results in impaired activity, undesirably reduced activity, or inappropriate activity, variants with such changes can be avoided. In other words, based on the information gathered from such routine experiments, one skilled in the art can easily determine amino acids for which further substitutions should be avoided, either alone or in combination with other mutations.

[0369] Those skilled in the art can use well-known techniques to determine suitable variants of polypeptides as set forth herein. In certain embodiments, those skilled in the art can identify suitable regions of the molecule that can be changed without impairing activity by targetin...

Claims

1. A composition for use in the treatment of cancer, comprising an antigen-binding protein (anti-CTLA-4 ABP) that specifically binds to human cytotoxic T lymphocyte-associated protein 4, wherein the anti-CTLA-4 ABP comprises CDR1-L consisting of SEQ ID NO: 12078, CDR2-L consisting of SEQ ID NO: 12079, CDR3-L consisting of SEQ ID NO: 12080, CDR1-H consisting of SEQ ID NO: 12075, CDR2-H consisting of SEQ ID NO: 12076, and CDR3-H consisting of SEQ ID NO: 12077, the composition.

2. wherein the cancer is resistant to anti-PD-1 or anti-PD-L1 therapy, and / or therapy with an anti-PD-1 antibody or an anti-PD-L1 antibody and, optionally, the cancer patient has progressed or relapsed after anti-PD-1 or anti-PD-L1 therapy, and / or has melanoma, RCC (renal cell carcinoma), NSCLC (non-small cell lung cancer), Merkel cell carcinoma, cSCC, mesothelioma, hepatocellular carcinoma, esophageal cancer, breast cancer, sarcoma, MSI-Hi / dMMR colorectal cancer, ovarian cancer, or cervical cancer, bladder cancer, prostate cancer, TMB-HI tumors of any origin, tumors that are MSI, tumors that are dMMR, T cell leukemia / lymphoma, NHL, or tumors that express CTLA-4 by cancer cells, The composition according to claim 1.

3. wherein the cancer is resistant to anti-PD-1 therapy or anti-PD-L1 therapy, and / or the composition is administered in combination with an antigen-binding protein (anti-PD-1 ABP or anti-PD-L1 ABP) that specifically binds to human PD-1 or PD-L1, and optionally, the PD-1 ABP is pembrolizumab, The composition according to any one of claims 1 to 2.

4. The anti-CTLA-4 ABP and the anti-PD-1 ABP are used in a weight ratio selected from 3:1, 3:10, 1:3, 1:10, 10:1, 10:3, 9:1, and 1:1, or are used in a weight ratio selected from 2:1 to 10:1 or 1:1 to 1:10, and optionally, the anti-CTLA-4 ABP and the anti-PD-1 ABP are used on the same day or on different days, The composition according to claim 3.

5. The effective amount of the anti-CTLA-4 ABP is 0.01 mg / kg to 30 mg / kg; At least 0.01 mg / kg, 0.03 mg / kg, 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, 9 mg / kg or 27 mg / kg; 0.5 mg / kg to 30 mg / kg; 1 mg / kg to 18 mg / kg; 1 mg / kg to 10 mg / kg; 1 mg / kg, 3 mg / kg, or 30 mg / kg; 50 mg to 2500 mg; 70 mg to 150 mg, 150 mg to 500 mg, 500 mg to 800 mg, 700 mg to 900 mg, 800 mg to 1200 mg, 1200 mg to 1500 mg, or 1500 mg to 2500 mg; or 80 mg, 240 mg, 720 mg, 800 mg, 1440 mg, 2160 mg, 50 mg, 100 mg, 150 mg, 250 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1500 mg, 2000 mg, or 2500 mg The composition according to claim 1, which is.

6. The anti-CTLA-4 ABP is A variable light chain (V Ｌ ) comprising a sequence that is at least 97% identical to SEQ ID NO: 14, and a variable heavy chain (V Ｈ ) comprising a sequence that is at least 97% identical to SEQ ID NO: 114, the composition according to claim 1.

7. The anti-CTLA-4 ABP contains a scFv or a full-length monoclonal antibody, and the ABP is An immunoglobulin constant region; IGHG1 * 01 human heavy chain constant region gene segment; Lysine at amino acid position 97 (R97) according to IMGT exon numbering; Lysine at amino acid position 97 (R214) according to EU numbering; A defucosylated Fc region; and An Fc region lacking core fucosylation of the N-glycan in the Fc portion The composition according to claim 1, which contains one or more of.

8. The anti-CTLA-4 ABP is Cells containing the bacterial protein RMD (GDP-6-deoxy-D-lyxo-4-hexulose reductase) or a variant thereof; Cells lacking the expression of Fut8 or having reduced expression thereof; Cells overexpressing glycosyltransferase (GnTIII); or Cells cultured in the presence of the fucosylation inhibitor 2-Fluorofucose (2FF) Produced from, and the cells are cultured in the absence of fucose, the composition according to claim 7.

9. The anti-CTLA-4 ABP is isolated based on its fucosylation state, and optionally the anti-CTLA-4 ABP is a defucosylated monoclonal antibody, the composition according to claim 1.

10. The CTLA-4 ABP is 20 mM histidine or citrate buffer; 50 mM NaCl; Sucrose at a concentration of 170 mM to 270 mM; 0.1 to 1 mg / mL of polysorbate 20; and / or 5 mg / mL to 20 mg / mL of said anti-CTLA-4 ABP The composition according to claim 1, which is contained in a pharmaceutical composition.

11. The composition according to claim 10, wherein the pharmaceutical composition has a pH of 5.0 to 6.

5.

12. The composition according to claim 10, wherein the pharmaceutical composition contains 20 mM of histidine, 270 mM of sucrose, and 0.2 mg / mL of polysorbate 20 and has a pH of 6.

2.

13. The use of said anti-CTLA-4 ABP is at least 2 times, 3 times, 4 times, or more; weekly, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, or every 7 weeks; every 1 to 2 weeks, every 2 to 3 weeks, every 3 to 4 weeks, every 4 to 5 weeks, every 5 to 6 weeks, every 6 to 7 weeks, every 7 to 8 weeks, every 8 to 9 weeks, every 9 to 10 weeks, every 10 to 11 weeks, every 11 to 12 weeks, every 12 to 13 weeks, every 13 to 14 weeks, or every 14 to 15 weeks; or every 1 to 2 months, every 2 to 3 months, every 3 to 4 months, every 4 to 5 months, or every 5 to 6 months The composition according to claim 12, characterized in that it is repeated.

14. The composition according to claim 10, characterized in that the anti-PD-1 antibody or anti-PD-L1 antibody is used in combination with the anti-CTLA-4 ABP in each of the repeated administrations.

15. A pharmaceutical composition comprising an anti-CTLA-4 ABP and a pharmaceutically acceptable excipient, wherein the anti-CTLA-4 ABP is an isolated antigen-binding protein (ABP) that specifically binds to human cytotoxic T lymphocyte-associated protein 4 (CTLA-4), and comprises CDR1-L consisting of SEQ ID NO: 12078, CDR2-L consisting of SEQ ID NO: 12079, CDR3-L consisting of SEQ ID NO: 12080, CDR1-H consisting of SEQ ID NO: 12075, CDR2-H consisting of SEQ ID NO: 12076, and CDR3-H consisting of SEQ ID NO: 12077.

16. The anti-CTLA-4 ABP comprises a variable light chain (V Ｌ ) comprising the sequence of SEQ ID NO: 14 and a variable heavy chain (V Ｈ ) comprising the sequence of SEQ ID NO: 114, and the pharmaceutical composition according to claim 15.

17. 0.1 to 1 mg / mL of polysorbate 20, 20 mM of histidine, 270 mM of sucrose, and 0.02% of PS-20 5 mg / mL to 20 mg / mL of said anti-CTLA-4 ABP comprising, and having a pH in the range of 5.0 to 6.5, The pharmaceutical composition according to claim 15.

18. Less than 50% of the anti-CTLA-4 ABP is fucosylated; Is less than 40% of the anti-CTLA-4 ABP fucosylated? Is less than 30% of the anti-CTLA-4 ABP fucosylated? Is less than 20% of the anti-CTLA-4 ABP fucosylated? Is less than 10% of the anti-CTLA-4 ABP fucosylated? Or 3% to 30% of the anti-CTLA-4 ABP is fucosylated, The pharmaceutical composition according to claim 15.

19. The pharmaceutical composition according to claim 15, formulated for injection or iv infusion.

20. A unit dosage form of the pharmaceutical composition according to claim 15, optionally 50 mg to 5000 mg of the anti-CTLA-4 ABP; 50 mg to 2500 mg of the anti-CTLA-4 ABP; 70 mg to 150 mg, 150 mg to 500 mg, 500 mg to 800 mg, 700 mg to 900 mg, 800 mg to 1200 mg, 1200 mg to 1500 mg, or 1500 mg to 2500 mg of the anti-CTLA-4 ABP; Or 80 mg, 240 mg, 720 mg, 800 mg, 1440 mg or 2160 mg of the anti-CTLA-4 ABP comprising a unit dosage form.

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