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

Abstract

Anti-CTLA-4 binding proteins and methods of use thereof are provided. [Solution] Provided herein are antigen binding proteins (ABPs) that selectively bind to CTLA-4 and its isoforms and homologs, as well as compositions comprising the ABPs. Also provided are methods of using the ABPs, such as therapeutic and diagnostic methods. Provided herein are novel ABPs that have binding specificity for CTLA-4 and methods of using such ABPs. The ABPs specifically bind to human CTLA-4 (SEQ ID NO: 7001) or fragments of human CTLA-4.

Description

[Technical Field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 047,785, filed July 2, 2020, and U.S. Provisional Application No. 63 / 107,376, filed October 29, 2020, which applications are incorporated herein by reference in their entireties.

[0002] 2. Sequence Listing This application contains a Sequence Listing with 12088 sequences submitted via EFS-Web, which is hereby incorporated by reference in its entirety. The ASCII copy, created on July 2, 2021, is named "49228WO Sequence_Listing" and is 1.86 megabytes in size.

[0003] 3.Technical Field Provided herein are antigen binding proteins (ABPs) having binding specificity for CTLA-4, and compositions comprising such ABPs, including pharmaceutical compositions, diagnostic compositions, and kits. 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] 4.Background technology 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. CTLA-4 is constitutively expressed by regulatory T cells (Tregs) and 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 not only prevents autoimmune diseases but also 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 greater affinity and avidity than CD28, allowing it to outcompete 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), contributing to their inhibitory function. T cell activation through the T cell receptor and CD28 results in increased expression of CTLA-4. The mechanism by which CTLA-4 acts in T cells remains somewhat controversial. Biochemical evidence has shown that CTLA-4 recruits phosphatases to the T cell receptor (TCR), thereby attenuating signals. This study remains unconfirmed in the literature since its initial publication. More recent studies have shown that CTLA-4 may function in vivo by capturing and removing B7-1 and B7-2 from the membrane of antigen-presenting cells, thus making them unavailable for CD28 triggering.

[0006] Variants in CTLA-4 have been associated with insulin-dependent diabetes mellitus, Graves' disease, Hashimoto's thyroiditis, celiac disease, systemic lupus erythematosus, thyroid-associated orbitopathy, primary biliary cirrhosis, and other autoimmune diseases. The relatively high binding affinity of CTLA-4 for CD80 and CD86 makes it a potential therapy for autoimmune diseases. A soluble fusion protein of CTLA-4 and an antibody (CTLA-4-Ig) is being used in clinical trials for rheumatoid arthritis.

[0007] Recently, CTLA-4 antibodies have been used with mixed efficacy to treat several types of cancer. 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 ligands 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 and induce 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, 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. PCT application PCT / US2019 / 068820, filed December 27, 2019, and published as publication number WO2020 / 140084A1, describes CTLA-4 ABPs, and that application is incorporated herein by reference in its entirety. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Publication No. 2020 / 140084 Summary of the Invention [Means for solving the problem]

[0010] 5. Summary of the Invention Provided herein are novel ABPs that have binding specificity for CTLA-4 and methods of using such ABPs. The ABPs specifically bind to human CTLA-4 (SEQ ID NO: 7001) or fragments of human CTLA-4.

[0011] In particular, in one aspect, the present disclosure provides novel CTLA-4 monoclonal antibodies that have minimal ability to block CTLA-4 binding to its CD80 / CD86 ligands, yet have excellent anti-tumor activity with reduced toxicity. The anti-CTLA4 antibodies have been shown to induce less peripheral Treg proliferation and more efficient intratumoral Treg depletion in mouse models expressing human CTLA-4.

[0012] The present disclosure also provides that the anti-CTLA-4 monoclonal antibody binds to CTLA-4 at an epitope distinct from that of other known anti-CTLA-4 antibodies (e.g., ipilimumab) and has limited checkpoint inhibitor activity, making it a weak checkpoint inhibitor. Surprisingly, the efficacy of the anti-CTLA-4 antibody presented herein was found to be associated with FcR-mediated Treg depletion in the tumor microenvironment. The anti-CTLA-4 antibody also induces less Treg proliferation and has increased ability to induce in vitro FcR signaling and in vivo depletion of intratumoral Tregs. The experimental results described herein indicate that the enhanced FcR activity of weak checkpoint inhibitors likely contributes to their enhanced anti-tumor activity. They also demonstrate that weak checkpoint inhibition is associated with lower toxicity in mouse models.

[0013] In some embodiments, upon binding to CTLA-4, the ABP makes contact with amino acids K130, Y139, L141, and I143, but not with amino acid R70 of CTLA-4, or R70 is not a major energetic contributor to the interaction between CTLA-4 and the ABP, and / or CTLA-4 upon binding 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.

[0014] 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.

[0015] 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 ) is included.

[0016] In some embodiments, the ABP comprises an scFv or a full-length monoclonal antibody. In some embodiments, the ABP comprises an immunoglobulin constant region.

[0017] 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 or 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.

[0018] In some embodiments, the ABP is an IgG1 ABP. In some embodiments, the ABP comprises an IGHG1*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.

[0019] In some embodiments, the ABP comprises a hypofucosylated Fc region.

[0020] In some embodiments, the ABP is produced from cells containing the bacterial protein RMD (GDP-6-deoxy-D-lyxo-4-hexulose reductase) or a modification thereof. In some embodiments, the cells are cultured in the absence of fucose.

[0021] 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 a fucosylation inhibitor, 2-fluorofucose (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.

[0022] 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 hypofucosylated monoclonal antibody.

[0023] Aspects of the present disclosure also include pharmaceutical compositions comprising an ABP of the present disclosure and a pharmaceutically acceptable excipient.

[0024] 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.

[0025] 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.

[0026] Aspects of the present disclosure provide methods of treating a disease, comprising administering to a subject in need thereof an effective amount of any ABP of the present disclosure or a pharmaceutical composition thereof.

[0027] In some embodiments, the disease is selected from the group consisting of cancer, AIDS, Alzheimer's disease, and a viral or bacterial infection.

[0028] 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.

[0029] 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 the fucosylation inhibitor 2-fluorofucose (2FF). In some embodiments, the host cell overexpresses glycosyltransferase (GnTIII).

[0030] 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.

[0031] 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-fluorofucose (2FF).

[0032] CTLA-4, including administering an effective dose of ABP or a pharmaceutical composition. HI Described in this disclosure are methods for reducing Tregs in subjects with limited expansion of remaining Tregs.

[0033] 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.

[0034] 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.

[0035] 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), wherein 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: 12014, a CDR3-H consisting of SEQ ID NO: 12016, and a CDR3-H consisting of SEQ ID NO: 12018; (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: 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; 015, CDR3-L consisting of SEQ ID NO: 12025, CDR1-H consisting of SEQ ID NO: 12040, CDR2-H consisting of SEQ ID NO: 12050, and CDR3-H consisting of SEQ ID NO: 12060; (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: 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; or (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.

[0036] 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 ) is included.

[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 or 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 comprises an IGHG1*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 hypofucosylated 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 modifications 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 a fucosylation inhibitor, 2-fluorofucose (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.

[0042] 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 hypofucosylated monoclonal antibody. In some embodiments, the hypofucosylated Fc region has less than 30% fucosylation, and less than 30% fucosylation enhances FcgRIII (Fc gamma receptor III) signaling. In some embodiments, the hypofucosylated Fc region has less than 30% fucosylation, and less than 30% fucosylation enhances FcgRIIIa (Fc gamma receptor IIIa) signaling. In some embodiments, the hypofucosylated Fc region has less than 30% fucosylation, and less than 30% fucosylation enhances proteins encoded by proteins encoded by FcgR3a.

[0043] An aspect of the present disclosure includes a pharmaceutical composition comprising an ABP of the present disclosure and a pharmaceutically acceptable excipient.

[0044] 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.

[0045] 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.

[0046] Aspects of the present disclosure provide methods of treating a disease, comprising administering to a subject in need thereof an effective amount of any ABP of the present disclosure or a pharmaceutical composition thereof.

[0047] In some embodiments, the disease is selected from the group consisting of cancer, AIDS, Alzheimer's disease, and a viral or bacterial infection.

[0048] 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.

[0049] 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 the fucosylation inhibitor 2-fluorofucose (2FF). In some embodiments, the host cell overexpresses glycosyltransferase (GnTIII).

[0050] Aspects of the present disclosure provide methods of treating cancer, comprising administering to a subject in need thereof an effective amount of an ABP or pharmaceutical composition of the present disclosure. In some embodiments, the subject has a malignant tumor. In some embodiments, upon administration, the ABP or pharmaceutical composition of the present disclosure induces increased Fc receptor (FcR) signaling compared to ipilimumab, and the administration increases CTLA-4 expression in the subject. HI Reduces the amount of Tregs. In some embodiments, the administering reduces the proliferation of peripheral Tregs in the subject compared to ipilimumab.

[0051] 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.

[0052] In some embodiments, the ABP comprises a hypofucosylated Fc region with less than 30% fucosylation, wherein less than 30% fucosylation enhances FcgRIII signaling. In some embodiments, the ABP comprises a hypofucosylated 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. In some embodiments, the hypofucosylated Fc region has less than 30% fucosylation, wherein less than 30% fucosylation enhances a protein encoded by a protein encoded by FcgR3a.

[0053] 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 the fucosylation inhibitor 2-fluorofucose (2FF). In some embodiments, the host cell overexpresses glycosyltransferase (GnTIII).

[0054] 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 hypofucosylated Fc.

[0055] 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-fluorofucose (2FF).

[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), comprising an IGHG1*01 human heavy chain constant region gene segment.

[0057] 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) CDR2-H consisting of sequence number 5014 and CDR3-H consisting of sequence number 6014, (b) CDR1-L consisting of sequence number 12004, CDR2-L consisting of sequence number 12014, CDR3-L consisting of sequence number 12024, CDR1-H consisting of sequence number 12039, CDR2-H consisting of sequence number 12049 and CDR3-H consisting of sequence number 12059, (c) CDR1-L consisting of sequence number 12005, CDR2-L consisting of sequence number 12015, CDR3-H consisting of sequence number 12025 3-L, CDR1-H consisting of SEQ ID NO: 12040, CDR2-H consisting of SEQ ID NO: 12050, and CDR3-H consisting of SEQ ID NO: 12060; (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-H consisting of SEQ ID NO: 12017 or (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) is included.

[0058] 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 ) is included.

[0059] 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 hypofucosylated 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 modification 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 a fucosylation inhibitor, 2-fluorofucose (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 portion. In some embodiments, the ABP is a hypofucosylated monoclonal antibody.

[0060] An aspect of the present disclosure includes a pharmaceutical composition comprising an ABP of the present disclosure and a pharmaceutically acceptable excipient.

[0061] 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.

[0062] 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.

[0063] Aspects of the present disclosure provide methods of treating a disease, comprising administering to a subject in need thereof an effective amount of an ABP or a pharmaceutical composition.

[0064] In some embodiments, the disease is selected from the group consisting of cancer, AIDS, Alzheimer's disease, and a viral or bacterial infection.

[0065] 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.

[0066] 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 the fucosylation inhibitor 2-fluorofucose (2FF). In some embodiments, the host cell overexpresses glycosyltransferase (GnTIII).

[0067] 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.

[0068] 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-fluorofucose (2FF).

[0069] Aspects of the present disclosure include administering an effective dose of ABP or a pharmaceutical composition to a subject, such as a patient with CTLA-4 HIMethods for reducing Tregs in subjects with limited expansion of remaining Tregs are provided.

[0070] 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 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.

[0071] In another aspect, the disclosure provides an isolated antigen binding protein (ABP) that specifically binds to an antigen, comprising an IGHG1*01 human heavy chain constant region gene segment.

[0072] 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.

[0073] In some embodiments, the antibody comprises a hypofucosylated Fc region. In some embodiments, the antibody is produced from cells comprising the bacterial protein RMD (GDP-6-deoxy-D-lyxo-4-hexulose reductase) or modifications thereof. In some embodiments, the cell is cultured in the absence of fucose. In some embodiments, the antibody is produced from cells lacking or having reduced expression of Fut8. In some embodiments, the antibody is produced from cells cultured in the presence of a fucosylation inhibitor, 2-fluorofucose (2FF).

[0074] 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 hypofucosylated monoclonal antibody.

[0075] 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, and a PI3K antibody or antigen-binding fragment thereof.

[0076] 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) CDR2-H consisting of sequence number 5014 and CDR3-H consisting of sequence number 6014, (b) CDR1-L consisting of sequence number 12004, CDR2-L consisting of sequence number 12014, CDR3-L consisting of sequence number 12024, CDR1-H consisting of sequence number 12039, CDR2-H consisting of sequence number 12049 and CDR3-H consisting of sequence number 12059, (c) CDR1-L consisting of sequence number 12005, CDR2-L consisting of sequence number 12015, CDR3-H consisting of sequence number 12025 3-L, CDR1-H consisting of SEQ ID NO: 12040, CDR2-H consisting of SEQ ID NO: 12050, and CDR3-H consisting of SEQ ID NO: 12060; (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-H 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.

[0077] 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 ) is included.

[0078] In some embodiments, the ABP comprises 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.

[0079] In some embodiments, the ABP comprises a variable light chain (V) comprising a sequence 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 ) is included.

[0080] 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.

[0081] Aspects of the present disclosure include methods of treating a disease comprising administering to a subject in need thereof an effective amount of an ABP or a pharmaceutical composition.

[0082] 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 method further comprises administering one or more additional therapeutic agents to the subject.

[0083] 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.

[0084] 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 the fucosylation inhibitor 2-fluorofucose (2FF). In some embodiments, the host cell overexpresses glycosyltransferase (GnTIII).

[0085] 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.

[0086] 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-fluorofucose (2FF).

[0087] Aspects of the present disclosure include administering an effective dose of ABP or a pharmaceutical composition to a subject, such as a patient with CTLA-4 HI Methods for reducing Tregs in subjects with limited expansion of remaining Tregs are provided.

[0088] 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.

[0089] 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.

[0090] Aspects of the present disclosure include CTLA-4 HI Provided is a method for reducing Tregs in a subject with limited proliferation of remaining Tregs, comprising administering to the subject an effective dose of an antigen binding protein (ABP) that specifically binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).

[0091] 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.

[0092] In some embodiments, the method further comprises administering one or more additional therapeutic agents to the subject.

[0093] 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 ) is included.

[0094] 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.

[0095] 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: 101 to 128. H ) is included. 6. Brief description of the drawings In an embodiment of the present invention, for example, the following items are provided: (Item 1) 1. An isolated antigen binding protein (ABP) that specifically binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), comprising: the ABP contacts amino acids K130, Y139, L141, I143 but does not contact amino acid R70 of the CTLA-4; and / or said CTLA-4, when bound to said ABP, may associate with CD80 / CD86; and / or the interaction between the ABP and amino acid L74A and / or E68 of the CTLA-4 is greater than the interaction between ipilimumab and amino acid L74A of CTLA-4 , ABP. (Item 2) 2. The ABP of item 1, wherein 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. (Item 3) 2. The ABP of item 1, wherein 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. (Item 4) 2. The ABP according to item 1, wherein 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. (Item 5) 2. The ABP according to item 1, wherein 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. (Item 6) 2. The ABP of item 1, wherein 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. (Item 7) 2. The ABP of item 1, wherein 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. (Item 8) The ABP is 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 3. The ABP according to item 1 or 2, comprising: (Item 9) 9. Any of items 1 to 8, wherein the ABP comprises an scFv or a full-length monoclonal antibody. 1. The ABP according to claim 1. (Item 10) 10. The ABP according to any one of items 1 to 9, wherein the ABP comprises an immunoglobulin constant region. (Item 11) The ABP has a K of less than 500 nM as measured by surface plasmon resonance. D binds to human CTLA-4 at The ABP has a K of less than 200 nM as measured by surface plasmon resonance. D binds to human CTLA-4 at The ABP has a K of less than 25 nM as measured by surface plasmon resonance. D Dehi binds to CTLA-4, or The ABP has a K D 11. The ABP according to any one of items 1 to 10, which binds to human CTLA-4 on the cell surface at (Item 12) 12. The ABP according to any one of items 1 to 11, wherein the ABP is an IgG1 ABP. (Item 13) 13. The ABP of any one of items 1 to 12, wherein the ABP comprises an IGHG1*01 human heavy chain constant region gene segment. (Item 14) 14. The ABP according to any one of items 1 to 13, wherein the ABP comprises a lysine at amino acid position 97 (R97) according to the IMGT exon numbering. (Item 15) 14. The ABP according to any one of items 1 to 13, wherein the ABP comprises a lysine at amino acid position 97 (R214) according to EU numbering. (Item 16) 16. The ABP of any one of items 1 to 15, comprising a hypofucosylated Fc region. (Item 17) 17. The ABP according to any one of items 1 to 16, produced from a cell comprising the bacterial protein RMD (GDP-6-deoxy-D-lyxo-4-hexulose reductase) or a modification thereof. (Item 18) 14. The ABP of item 13, wherein the cells are cultured in the absence of fucose. (Item 19) 19. The ABP according to any one of items 1 to 18, which is produced from cells lacking or having reduced expression of Fut8. (Item 20) 20. The ABP of any one of items 1 to 19, produced from cells cultured in the presence of a fucosylation inhibitor, 2-fluorofucose (2FF). (Item 21) 21. The ABP according to any one of items 1 to 20, which is produced from cells overexpressing glycosyltransferase (GnTIII). (Item 22) 22. The ABP according to any one of items 1 to 21, isolated on the basis of its fucosylation state. (Item 23) 23. The ABP according to any one of items 1 to 22, comprising an Fc region that lacks core fucosylation of N-glycans of the Fc portion. (Item 24) 24. The ABP according to any one of items 1 to 23, wherein the ABP is a hypofucosylated monoclonal antibody. (Item 25) 25. A pharmaceutical composition comprising the ABP according to any one of items 1 to 24 and a pharmaceutically acceptable excipient. (Item 26) 26. The pharmaceutical composition of item 25, wherein less than 50% of the ABP is fucosylated. (Item 27) 27. The pharmaceutical composition of item 26, wherein less than 40% of the ABP is fucosylated. (Item 28) 28. The pharmaceutical composition of item 27, wherein less than 30% of the ABP is fucosylated. (Item 29) 29. The pharmaceutical composition of item 28, wherein less than 20% of the ABP is fucosylated. (Item 30) 30. The pharmaceutical composition of item 29, wherein less than 10% of the ABP is fucosylated. (Item 31) 26. The pharmaceutical composition of item 25, wherein more than 30% of the ABP is fucosylated. (Item 32) 32. The pharmaceutical composition of claim 31, wherein more than 40% of the ABP is fucosylated. (Item 33) 33. The pharmaceutical composition of item 32, wherein more than 50% of the ABP is fucosylated. (Item 34) 34. The pharmaceutical composition of item 33, wherein more than 60% of the ABP is fucosylated. (Item 35) 35. The pharmaceutical composition of item 34, wherein more than 70% of the ABP is fucosylated. (Item 36) 36. The pharmaceutical composition of item 35, wherein more than 80% of the ABP is fucosylated. (Item 37) 37. The pharmaceutical composition of item 36, wherein more than 90% of the ABP is fucosylated. (Item 38) 38. The pharmaceutical composition according to any one of items 25 to 37, having a pH of 5.0 to 6.5. (Item 39) 39. The pharmaceutical composition according to any one of items 25 to 38, comprising 20 mM histidine or citrate buffer. (Item 40) 40. The pharmaceutical composition according to any one of items 25 to 39, comprising 50 mM NaCl. (Item 41) 41. The pharmaceutical composition according to any one of items 25 to 40, comprising sucrose at a concentration of 170 mM to 270 mM. (Item 42) 42. The pharmaceutical composition of item 41, comprising 170 mM or 270 mM sucrose. (Item 43) 43. The pharmaceutical composition according to any one of items 25 to 42, comprising 5 mg / mL to 20 mg / mL of the ABP. (Item 44) 44. The pharmaceutical composition according to any one of items 26 to 43, comprising 20 mg / mL of the ABP. (Item 45) 45. The pharmaceutical composition according to any one of items 26 to 44, comprising 5 mg / mL of the ABP. (Item 46) 1. A method of treating a disease, comprising: A method comprising the step of administering to a subject in need thereof an effective amount of the ABP according to any one of items 1 to 24 or the pharmaceutical composition according to any one of items 25 to 45. (Item 47) 47. The method of claim 46, wherein the disease is selected from the group consisting of cancer, AIDS, Alzheimer's disease, and viral or bacterial infections. (Item 48) 48. The method of claim 46 or 47, further comprising administering to the subject one or more additional therapeutic agents. (Item 49) 49. The method of item 48, wherein the additional therapeutic agent is 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. (Item 50) 25. An isolated polynucleotide encoding the ABP according to any one of items 1 to 24. (Item 51) A vector comprising the isolated polynucleotide of item 50. (Item 52) 52. A host cell comprising the isolated polynucleotide of item 50 or the vector of item 51. (Item 53) 53. The host cell of item 52, wherein the host cell further comprises the bacterial protein RMD (GDP-6-deoxy-D-lyxo-4-hexulose reductase). (Item 54) 54. The host cell according to item 52 or 53, wherein the host cell is cultured in the absence of fucose. (Item 55) 55. The host cell of any one of items 52 to 54, wherein expression of Fut8 is absent or reduced. (Item 56) 56. The host cell of any one of items 52 to 55, cultured in the presence of the fucosylation inhibitor 2-fluorofucose (2FF). (Item 57) 57. The host cell of any one of items 52 to 56, which overexpresses glycosyltransferase (GnTIII). (Item 58) 58. 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 the host cell of any one of paragraphs 52 to 57, and isolating the ABP. (Item 59) 59. The method of claim 58, further comprising isolating the ABP based on its fucosylation state. (Item 60) 60. The method of claim 58 or 59, wherein the host cell is cultured in a culture medium containing a fucosylation inhibitor. (Item 61) Item 61. The method of item 60, wherein the fucosylation inhibitor is 2-fluorofucose (2FF). (Item 62) CTLA-4 HI 46. ​​A method for reducing Tregs in a subject with limited proliferation of remaining Tregs, comprising administering to the subject an effective dose of the ABP of any one of items 1 to 24 or the pharmaceutical composition of any one of items 25 to 45. (Item 63) 63. The method of claim 62, wherein 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. (Item 64) 64. The method of claim 62 or 63, further comprising administering to the subject one or more additional therapeutic agents. (Item 65) 65. The method of item 64, wherein the additional therapeutic agent is anti-PD-L1 or anti-PD1, or a combination thereof. (Item 66) 1. An isolated antigen binding protein (ABP) that specifically binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), said ABP comprising: (a) 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; (b) CDR1-L consisting of SEQ ID NO: 1014, CDR2-L consisting of SEQ ID NO: 2014, CDR3-L consisting of SEQ ID NO: 3014, CDR1-H consisting of SEQ ID NO: 4014, CDR2-H consisting of SEQ ID NO: 5014, and CDR3-H consisting of SEQ ID NO: 6014; (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, CDR3-L consisting of SEQ ID NO: 12025, CDR1-H consisting of SEQ ID NO: 12040, CDR2-H consisting of SEQ ID NO: 12050, and CDR3-H consisting of SEQ ID NO: 12060; (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, 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) An ABP comprising 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. (Item 67) The ABP is 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 67. The ABP according to item 66, comprising: (Item 68) 68. The ABP of item 66 or 67, wherein the ABP comprises an scFv or a full-length monoclonal antibody. (Item 69) 69. The ABP according to any one of items 66 to 68, wherein the ABP comprises an immunoglobulin constant region. (Item 70) The ABP has a K of less than 500 nM as measured by surface plasmon resonance. D binds to human CTLA-4 at The ABP has a K of less than 200 nM as measured by surface plasmon resonance. D in binds to human CTLA-4, or The ABP has a K of less than 25 nM as measured by surface plasmon resonance. D binds to human CTLA-4 at The ABP has a K D 70. The ABP according to any one of items 66 to 69, which binds to human CTLA-4 on the cell surface at (Item 71) 71. The ABP according to any one of items 66 to 70, wherein the ABP is an IgG1 ABP. (Item 72) 72. The ABP of any one of items 66 to 71, wherein the ABP comprises an IGHG1*01 human heavy chain constant region gene segment. (Item 73) 73. The ABP according to any one of items 66 to 72, wherein the ABP comprises a lysine at amino acid position 97 (R97) according to the IMGT exon numbering. (Item 74) 74. The ABP according to any one of items 66 to 73, wherein the ABP comprises a lysine at amino acid position 97 (R214) according to EU numbering. (Item 75) 75. The ABP of any one of items 66 to 74, comprising a hypofucosylated Fc region. (Item 76) 76. The ABP according to any one of items 66 to 75, produced from a cell containing the bacterial protein RMD (GDP-6-deoxy-D-lyxo-4-hexulose reductase) or a modification thereof. (Item 77) 77. The ABP of item 76, wherein the cells are cultured in the absence of fucose. (Item 78) 78. The ABP according to any one of items 66 to 77, which is produced from cells lacking or having reduced expression of Fut8. (Item 79) 79. The ABP of any one of items 66 to 78, produced from cells cultured in the presence of a fucosylation inhibitor, 2-fluorofucose (2FF). (Item 80) 80. The ABP according to any one of items 66 to 79, which is produced from cells overexpressing glycosyltransferase (GnTIII). (Item 81) 81. The ABP according to any one of items 66 to 80, isolated on the basis of its fucosylation state. (Item 82) 82. The ABP according to any one of items 58 to 81, comprising an Fc region that lacks core fucosylation of N-glycans of the Fc portion. (Item 83) 83. The ABP according to any one of items 66 to 82, wherein the ABP is a hypofucosylated monoclonal antibody. (Item 84) 84. The ABP of item 83, wherein the hypofucosylated Fc region has less than 30% fucosylation, and less than 30% fucosylation enhances Fc gamma receptor IIIa (FcgRIIIa) signaling or a protein encoded by a protein encoded by FcgR3a. (Item 85) The ABP according to any one of Items 66 to 84, and a pharmaceutically acceptable excipient. , pharmaceutical compositions. (Item 86) 86. The pharmaceutical composition of item 85, wherein less than 50% of the ABP is fucosylated. (Item 87) 87. The pharmaceutical composition of item 86, wherein less than 40% of the ABP is fucosylated. (Item 88) 88. The pharmaceutical composition of item 87, wherein less than 30% of the ABP is fucosylated. (Item 89) 89. The pharmaceutical composition of item 88, wherein less than 20% of the ABP is fucosylated. (Item 90) 90. The pharmaceutical composition of item 89, wherein less than 10% of the ABP is fucosylated. (Item 91) 86. The pharmaceutical composition of item 85, wherein more than 30% of the ABP is fucosylated. (Item 92) 92. The pharmaceutical composition of item 91, wherein more than 40% of the ABP is fucosylated. (Item 93) 93. The pharmaceutical composition of item 92, wherein more than 50% of the ABP is fucosylated. (Item 94) 94. The pharmaceutical composition of item 93, wherein more than 60% of the ABP is fucosylated. (Item 95) 95. The pharmaceutical composition of item 94, wherein more than 70% of the ABP is fucosylated. (Item 96) 96. The pharmaceutical composition of item 95, wherein more than 80% of the ABP is fucosylated. (Item 97) 97. The pharmaceutical composition of item 96, wherein more than 90% of the ABP is fucosylated. (Item 98) 98. The pharmaceutical composition according to any one of items 85 to 97, having a pH of 5.0 to 6.5. (Item 99) 99. The pharmaceutical composition according to any one of items 85 to 98, comprising 20 mM histidine or citrate buffer. (Item 100) 99. The pharmaceutical composition according to any one of items 85 to 99, comprising 50 mM NaCl. (Item 101) 101. The pharmaceutical composition according to any one of items 85 to 100, comprising sucrose in a concentration of 170 mM to 270 mM. (Item 102) 102. The pharmaceutical composition of item 101, comprising 170 mM or 270 mM sucrose. (Item 103) 103. The pharmaceutical composition according to any one of items 85 to 102, comprising 20 mg / mL of said ABP. (Item 104) 104. The pharmaceutical composition according to any one of items 85 to 103, comprising 5 mg / mL of said ABP. (Item 105) 1. A method of treating cancer, comprising: A method comprising the step of administering to a subject in need thereof an effective amount of the ABP according to any one of items 66 to 84 or the pharmaceutical composition according to any one of items 85 to 104. (Item 106) Item 106. The method of item 105, wherein the subject has a malignant tumor. (Item 107) the ABP, when administered, induces increased Fc receptor (FcR) signaling compared to ipilimumab, and the administering increases CTLA-4 in the subject. HI The method of item 105, wherein the amount of Tregs is reduced. (Item 108) 108. The method of claim 107, wherein said administering reduces peripheral Treg proliferation in said subject compared to ipilimumab. (Item 109) 109. The method of any one of items 105 to 108, further comprising administering one or more additional therapeutic agents to the subject. (Item 110) 110. The method of claim 109, wherein the additional therapeutic agent is 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. (Item 111) 111. The method of any one of items 105 to 110, wherein the ABP comprises a hypofucosylated Fc region with less than 30% fucosylation, wherein the less than 30% fucosylation enhances Fc gamma receptor IIIa (FcgRIIIa) signaling or a protein encoded by FcgR3a. (Item 112) 111. The method of any one of items 105 to 110, wherein the ABP comprises a fucosylated Fc region with more than 70% fucosylation. (Item 113) An isolated polynucleotide encoding the ABP according to any one of items 66 to 84. (Item 114) A vector comprising the isolated polynucleotide of Item 113. (Item 115) A host cell comprising the isolated polynucleotide of Item 113 or the vector of Item 114. (Item 116) 116. The host cell of item 115, wherein the host cell has been engineered to have reduced fucosylation compared to a non-engineered host cell. (Item 117) 117. The host cell of item 116, wherein the host cell further comprises the bacterial protein RMD (GDP-6-deoxy-D-lyxo-4-hexulose reductase). (Item 118) 118. The host cell according to any one of items 115 to 117, wherein the host cell is cultured in the absence of fucose. (Item 119) 119. The host cell of any one of items 115 to 118, wherein expression of Fut8 is absent or reduced. (Item 120) 120. The host cell of any one of items 115 to 119, cultured in the presence of the fucosylation inhibitor 2-fluorofucose (2FF). (Item 121) 121. The host cell of any one of items 115 to 120, which overexpresses glycosyltransferase (GnTIII). (Item 122) 122. 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 the host cell of any one of paragraphs 115 to 121 and isolating the ABP, wherein the ABP comprises a hypofucosylated Fc. (Item 123) 123. The method of claim 122, further comprising isolating the ABP based on its fucosylation state. (Item 124) 124. The method of any one of items 115 to 123, wherein the host cell is cultured in a culture medium containing a fucosylation inhibitor. (Item 125) Item 125. The method of item 124, wherein the fucosylation inhibitor is 2-fluorofucose (2FF). (Item 126) 1. An isolated antigen binding protein (ABP) that specifically binds to an antigen, comprising an IGHG1*01 human heavy chain constant region gene segment, optionally wherein the antigen is human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). (Item 127) The ABP is (a) 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; (b) CDR1-L consisting of SEQ ID NO: 1014, CDR2-L consisting of SEQ ID NO: 2014, CDR3-L consisting of SEQ ID NO: 3014, CDR1-H consisting of SEQ ID NO: 4014, CDR2-H consisting of SEQ ID NO: 5014, and CDR3-H consisting of SEQ ID NO: 6014; (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, CDR3-L consisting of SEQ ID NO: 12025, CDR1-H consisting of SEQ ID NO: 12040, CDR2-H consisting of SEQ ID NO: 12050, and CDR3-H consisting of SEQ ID NO: 12060; (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, 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) The ABP according to item 126, comprising 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. (Item 128) The ABP is A variable light chain (V) comprising a sequence at least 97% identical to SEQ ID NO: 14 L ), and SEQ ID NO: 11 A variable heavy chain (V) containing a sequence at least 97% identical to H 128. The ABP according to item 127, comprising (Item 129) The ABP is (a) The ABP according to Item 126, comprising 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: 2001 to 2028, a CDR1-H consisting of any one of SEQ ID NOs: 3001 to 3028. (Item 130) The ABP is A variable light chain (V) comprising a sequence at least 97% identical to any one of SEQ ID NOs: 1 to 28. L ), and a variable heavy chain (V) comprising a sequence at least 97% identical to SEQ ID NOs: 1 to 128. H 128. The ABP according to item 127, comprising (Item 131) 127. The ABP of item 126, wherein the ABP comprises a lysine at amino acid position 97 (R97) according to the IMGT exon numbering. (Item 132) 127. The ABP according to item 126, wherein the ABP comprises a lysine at amino acid position 97 (R214) according to EU numbering. (Item 133) 133. The ABP according to any one of items 126 to 132, comprising a hypofucosylated Fc region. (Item 134) 134. The ABP according to any one of items 126 to 133, produced from a cell containing the bacterial protein RMD (GDP-6-deoxy-D-lyxo-4-hexulose reductase) or a modification thereof. (Item 135) 135. The ABP of item 134, wherein the cells are cultured in the absence of fucose. (Item 136) 136. The ABP of any one of items 126 to 135, which is produced from cells lacking or having reduced expression of Fut8. (Item 137) 137. The ABP of any one of items 126 to 136, produced from cells cultured in the presence of a fucosylation inhibitor, 2-fluorofucose (2FF). (Item 138) 138. The ABP according to any one of items 126 to 137, which is produced from cells overexpressing glycosyltransferase (GnTIII). (Item 139) 139. The ABP according to any one of items 126 to 138, isolated on the basis of its fucosylation state. (Item 140) 139. The ABP according to any one of items 126 to 139, comprising an Fc region that lacks core fucosylation of N-glycans of the Fc portion. (Item 141) 141. The ABP according to any one of items 126 to 140, wherein the ABP is a hypofucosylated monoclonal antibody. (Item 142) The ABP is an antibody selected from the group consisting of an anti-CTLA-4 antibody or an antigen-binding fragment thereof, an anti-PD-L1 antibody or an antigen-binding fragment thereof, an anti-PD1 antibody or an antigen-binding fragment thereof, a TIGIT antibody or an antigen-binding fragment thereof, an LAG-3 antibody or an antigen-binding fragment thereof, a CD47 antibody or an antigen-binding fragment thereof, and a BRAF antibody or an antigen-binding fragment thereof. 142. The ABP according to any one of items 131 to 141, wherein the ABP is selected from a PI3K antibody, a MEK antibody or an antigen-binding fragment thereof, and a PI3K antibody or an antigen-binding fragment thereof. (Item 143) The ABP is (a) 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; (b) CDR1-L consisting of SEQ ID NO: 1014, CDR2-L consisting of SEQ ID NO: 2014, CDR3-L consisting of SEQ ID NO: 3014, CDR1-H consisting of SEQ ID NO: 4014, CDR2-H consisting of SEQ ID NO: 5014, and CDR3-H consisting of SEQ ID NO: 6014; (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, CDR3-L consisting of SEQ ID NO: 12025, CDR1-H consisting of SEQ ID NO: 12040, CDR2-H consisting of SEQ ID NO: 12050, and CDR3-H consisting of SEQ ID NO: 12060; (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, 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) The ABP according to any one of items 131 to 141, comprising 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. (Item 144) The ABP is 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 142. The ABP according to any one of items 131 to 141, comprising: (Item 145) The ABP is (a) The ABP according to any one of items 131 to 141, comprising 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. (Item 146) The ABP is A variable light chain (V) comprising a sequence 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 142. The ABP according to any one of items 131 to 141, comprising: (Item 147) 147. A pharmaceutical composition comprising the ABP according to any one of items 126 to 146 and a pharmaceutically acceptable excipient. (Item 148) 148. The pharmaceutical composition of item 147, wherein less than 50% of the ABP is fucosylated. (Item 149) 149. The pharmaceutical composition of item 148, wherein less than 40% of the ABP is fucosylated. (Item 150) 150. The pharmaceutical composition of claim 149, wherein less than 30% of the ABP is fucosylated. (Item 151) 151. The pharmaceutical composition of item 150, wherein less than 20% of the ABP is fucosylated. (Item 152) 152. The pharmaceutical composition of claim 151, wherein less than 10% of the ABP is fucosylated. (Item 153) 148. The pharmaceutical composition of item 147, wherein more than 30% of the ABP is fucosylated. (Item 154) 154. The pharmaceutical composition of claim 153, wherein more than 40% of the ABP is fucosylated. (Item 155) 155. The pharmaceutical composition of item 154, wherein more than 50% of the ABP is fucosylated. (Item 156) 156. The pharmaceutical composition of item 155, wherein more than 60% of the ABP is fucosylated. (Item 157) 157. The pharmaceutical composition of item 156, wherein more than 70% of the ABP is fucosylated. (Item 158) 158. The pharmaceutical composition of item 157, wherein more than 80% of the ABP is fucosylated. (Item 159) 159. The pharmaceutical composition of item 158, wherein more than 90% of the ABP is fucosylated. (Item 160) 159. The pharmaceutical composition according to any one of items 147 to 159, having a pH of 5.0 to 6.5. (Item 161) 161. The pharmaceutical composition according to any one of items 147 to 160, comprising 20 mM histidine or citrate buffer. (Item 162) 162. The pharmaceutical composition according to any one of items 147 to 161, comprising 50 mM NaCl. (Item 163) 163. The pharmaceutical composition according to any one of items 162 to 162, comprising sucrose in a concentration of 170 mM to 270 mM. (Item 164) 164. The pharmaceutical composition according to item 163, comprising 170 mM or 270 mM sucrose. (Item 165) 165. The pharmaceutical composition according to any one of items 147 to 164, comprising 20 mg / mL of said ABP. (Item 166) 1. A method of treating a disease, comprising: A method comprising administering to a subject in need thereof an effective amount of an ABP according to any one of items 125 to 145 or a pharmaceutical composition according to any one of items 147 to 165. . (Item 167) 167. The method of claim 166, wherein the disease is selected from the group consisting of cancer, AIDS, Alzheimer's disease, and viral or bacterial infections. (Item 168) 168. The method of claim 166 or 167, further comprising administering to the subject one or more additional therapeutic agents. (Item 169) 169. The method of claim 168, wherein the additional therapeutic agent is 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. (Item 170) 147. An isolated polynucleotide encoding the ABP according to any one of items 126 to 146. (Item 171) A vector comprising the isolated polynucleotide of Item 170. (Item 172) 171. A host cell comprising the isolated polynucleotide of item 170 or the vector of item 170. (Item 173) 173. The host cell of item 172, wherein the host cell further comprises the bacterial protein RMD (GDP-6-deoxy-D-lyxo-4-hexulose reductase). (Item 174) 174. The host cell according to item 172 or 173, wherein the host cell is cultured in the absence of fucose. (Item 175) 175. The host cell of any one of items 172 to 174, wherein expression of Fut8 is absent or reduced. (Item 176) 176. The host cell of any one of items 172 to 175, cultured in the presence of the fucosylation inhibitor 2-fluorofucose (2FF). (Item 177) 177. The host cell of any one of items 172 to 176, which overexpresses glycosyltransferase (GnTIII). (Item 178) 178. 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 according to any one of paragraphs 171 to 177, and isolating the ABP. (Item 179) 179. The method of claim 178, further comprising isolating the ABP based on its fucosylation state. (Item 180) 179. The method of claim 178, wherein the host cell is cultured in a culture medium containing a fucosylation inhibitor. (Item 181) 181. The method of claim 180, wherein the fucosylation inhibitor is 2-fluorofucose (2FF). (Item 182) CTLA-4 HI16. A method for reducing Tregs in a subject with limited proliferation of remaining Tregs, comprising administering an effective dose of an ABP according to any one of items 126 to 146 or a pharmaceutical composition according to any one of items 141 to 164. (Item 183) 183. The method of claim 182, wherein 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. (Item 184) 184. The method of claim 182 or 183, further comprising administering to the subject one or more additional therapeutic agents. (Item 185) 185. The method of item 184, wherein the additional therapeutic agent is anti-PD-L1 or anti-PD1, or a combination thereof. (Item 186) 183. The method of claim 182, wherein 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. (Item 187) CTLA-4 HI 16. A method for reducing Tregs in a subject with limited proliferation of remaining Tregs, the method comprising administering an effective dose of an ABP according to any one of items 126 to 146 or a pharmaceutical composition according to any one of items 146 to 164. (Item 188) 188. The method of claim 187, wherein 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. (Item 189) 189. The method of claim 187 or 188, further comprising administering to the subject one or more additional therapeutic agents. (Item 190) 188. The method of claim 187, wherein 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. (Item 191) A method for reducing CTLA-4HI Tregs in a subject with limited proliferation of remaining Tregs, comprising administering to the subject an effective dose of an antigen binding protein (ABP) that specifically binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). (Item 192) 192. The method of claim 191, wherein 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. (Item 193) 193. The method of claim 191 or 192, further comprising administering to the subject one or more additional therapeutic agents. (Item 194) 192. The ABP of item 191, wherein the 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. (Item 195) The ABP is (a) CDR1-L consisting of any one of SEQ ID NOs: 1001 to 1028, CDR2-L consisting of any one of SEQ ID NOs: 3001 to 3028 192. The ABP according to Item 191, comprising a CDR3-L, 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. (Item 196) The ABP is 192. The ABP of Item 191, comprising a variable light chain (VL) comprising a sequence at least 97% identical to any one of SEQ ID NOs: 1 to 28, and a variable heavy chain (VH) comprising a sequence at least 97% identical to any one of SEQ ID NOs: 101 to 128. (Item 197) The ABP is (a) The ABP according to Item 191, comprising 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. (Item 198) The ABP is A variable light chain (V) comprising a sequence 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 192. The ABP according to item 191, comprising [Brief explanation of the drawings]

[0096] [Figure 1] This article summarizes a method for generating an scFv library from B cells isolated from fully human mice 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, respectively, in order of appearance. [Figure 2] The scFv amplification procedure is shown. First, a mixture of primers for the IgK C region, IgG C region, and all V regions is used to amplify IgK and IgH separately. Second, the VH and CK primers contain regions of complementarity that result in the formation of an overlap-extension amplicon, which is the fusion product between IgK and IgH. The region of complementarity 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. [Figure 3] Included is a schematic diagram of the monoclonal antibodies sorted into their epitope bins. [Figure 4A]Figure 4 includes plots from histopathological staining of hCTLA-4 KI mice bearing MC38 tumors treated with PBS or anti-CTLA-1 ABP. Plots show H&E (Figure 4A), immunoglobulin (Ig) (Figures 4B and 4C), and C3 staining (Figures 4D and 4E) from the right kidney. ipi is ipilimumab, and CTLA4.A14.2a is antibody A14 cloned into a murine IgG2a backbone. [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 4D] Same as above. [Figure 4E] Same as above. [Figure 5] 1 includes plots showing alkaline phosphatase levels in treated hCTLA4 KI mice bearing MC38 tumors. IPI is ipilimumab, and CTLA4.A14.2a is antibody A14 cloned into a murine IgG2a backbone. U / L is units per liter. [Figure 6] Plots of the percentage of intratumoral regulatory T cells (Treg) and intratumoral natural killer (NK) cells after the indicated treatments are included. [Figure 7] 1 includes plots showing the change in body weight of hCTLA4 mice receiving the indicated treatments. Ipi is ipilimumab, and CTLA4.A14.2a is antibody A14 cloned into a murine IgG2a backbone. Error bars represent + / - standard error of the mean. [Figure 8A] Figure 8 includes plots showing the effect of control, Ipi, and anti-CTLA4 treatment on the percentages of the 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). Ipi is ipilimumab, and CTLA4.A14.2a is antibody A14 cloned into a murine IgG2a backbone. [Figure 8B] Same as above. [Figure 8C] Same as above. [Figure 8D] Same as above. [Figure 8E] Same as above. [Figure 8F] Same as above. [Figure 9A] Included are plots showing the effect of control, Ipi, and anti-CTLA4 treatment on the percentages of the indicated cell populations, including CD8+ cells (FIG. 9A), CD69+ cells (FIG. 9B), ICOS+ cells (FIG. 9C), and PD1+ cells (FIG. 9D). Ipi is ipilimumab, and CTLA4.A14.2a is antibody A14 cloned into a murine IgG2a backbone. [Figure 9B] Same as above. [Figure 9C] Same as above. [Figure 9D] Same as above. [Figure 10] Included are plots showing the effect of control, Ipi, and anti-CTLA4 treatment on the percentage of dendritic cells (DCs) and activated dendritic cells (CD86+). Ipi is ipilimumab, and CTLA4.A14.2a is antibody A14 cloned into a murine IgG2a backbone. [Figure 11] Included are plots showing mean tumor volumes after treatment with 0.3 mg / kg of the indicated anti-CTLA4. [Figure 12A]Figure 12A shows that FcR effector function is required for ipilimumab antitumor efficacy in mouse models. Figure 12A shows the frequency of CD4+FOXP3- T cells from the LNs of individual control (C57BL / 6) or hCTLA4-KI mice that were CD44LoCD62L+ as determined by flow cytometry (mean + / - SEM). Figure 12B shows hCTLA-4 KI mice bearing MC38 tumors were randomized when tumors were 50–151 mm3 (day 0) and treated biweekly with the indicated antibody at 5 mg / kg for five doses. The plot shows tumor volume (mean ± SEM) of MC38 tumors over time treated with the indicated antibody. Tumor volumes from mice euthanized due to tumor burden exceeding 3000 mm3 were carried forward. 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, for which 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 for isotype, aCTLA-4.28, and Ipi-N297Q; n = 7 for 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 12B] Same as above. [Figure 13A]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 ability of CTLA-4 mAbs to block CD80 or CD86 binding was measured using a plate-based ELISA method. CTLA-4 was used to coat plates, followed by incubation of antibody samples, His-tagged CD80 or CD86, and the amount of ligand available to bind to CTLA-4 was measured. Blocking mAbs that prevent CD80 or CD86 from binding to CTLA-4 reduce the absorbance signal due to the lack of CD80 or CD86 binding to CTLA-4. Weak blocking mAbs 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 as described in (Figure 13A), compared to ipilimumab and CTLA-4.28. Absorbance values ​​were normalized to the anti-PD-1 control (pembrolizumab) and shown 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. The crystal structure (Protein Database [PDB] 1I8L) of the complex between CD80 (blue) and CTLA-4 (gray) is shown, with the CTLA-4 epitope residues shared between ipilimumab and GIGA-564 colored orange and the key differentiating residue R70 colored red (visualized in Pymol). In addition, G142 was identified as a secondary residue in the epitope of ipilimumab but not GIGA-564. Figure 13D. A table showing the key amino acids on CTLA-4 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 of those proteins. [Figure 13B] Same as above. [Figure 13C] Same as above. [Figure 13D] Same as above. [Figure 14A]GIGA-564 inhibits tumor growth in a mouse model. Figure 14A. hCTLA-4 KI mice bearing MC38 tumors were randomized when tumors were 50–150 mm3 (day 0) and treated biweekly with the indicated antibodies at 5 mg / kg for five doses. The plot shows the tumor volume (mean ± SEM) of MC38 tumors over time treated with the indicated antibodies. Tumor volumes from mice euthanized due to tumor burden exceeding 3000 mm3 were carried forward. 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 for GIGA-564, n = 6 for vehicle and commercial ipilimumab. Figure 14B. hCTLA-4 KI mice bearing RM-1 tumors were randomized when tumors were 40-125 mm (day 0) and treated with the indicated antibody at 5 mg / kg on days 0, 3, and 6. The plot shows tumor volume (mean ± SEM) of RM-1 tumors over time treated with the indicated antibody. Tumor volumes from mice euthanized due to tumor burden exceeding 3000 mm were carried forward until no mice from that group survived (thin black vertical line). The thin vertical line indicates censored data. The thin gray vertical line indicates one mouse from the ipilimumab-treated group was euthanized due to tumor ulceration. n = 11 for ipilimumab and GIGA-564 treatment, n = 7 for isotype treatment. Figure 14C (also shown in Figure 11). hCTLA-4 KI mice bearing MC38 tumors were randomized when tumors were 65–125 mm (day 0) and treated with 0.3 mg / kg of the indicated antibody on days 0, 3, and 6. The plot shows the tumor volume (mean ± SEM) of MC38 tumors over time treated with the indicated antibody. Tumor volumes from mice euthanized due to tumor burden exceeding 3000 mm were carried forward until no mice from that group survived (thin black vertical line). n = 13 for ipilimumab and GIGA-564, n = 8 for isotype-treated animals. P values ​​are indicated for statistically significant differences in the change in tumor volume measured longitudinally (linear mixed-effects model). [Figure 14B] Same as above. [Figure 14C] Same as above. [Figure 15A]These results show that GIGA-564 induces less peripheral Treg proliferation but potently mediates intratumoral Treg depletion. Figure 15A. hCTLA-4 KI mice (n = 12) were treated with 5 mg / kg of hIgG1 isotype control, ipilimumab, or GIGA-564 on days 0, 3, and 6 and euthanized on day 7 for flow cytometry analysis. Two samples in the GIGA-564 group were excluded from analysis due to low cell counts. The percentages of 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) expressing Ki67 were determined by flow cytometry (first three panels). The right panel shows the fold change in the percent of cells of each subtype expressing Ki67 in ipilimumab- or GIGA-564-treated mice compared to the average 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 Tregs may further enhance efficacy compared to ipilimumab in patients. Figures 15B-15E. hCTLA-4 KI mice bearing established MC38 tumors were randomized (n=6) and treated once with 5 mg / kg of hIgG1 isotype control, ipilimumab, or GIGA-564. Cells from non-draining LNs and tumors were analyzed by flow cytometry the following day. Figure 15B. Frequency of Tregs (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) in Tregs in LNs (left) and tumors (right). Figure 15D. Representative contour plots of CD4 T cells in each treatment group. The X and Y axes correspond to FOXP3 and CTLA-4, respectively. Figure 15E. Ratio of CD8 T cells to Tregs in LNs (left) and tumors (right). Unless otherwise indicated, p values ​​were calculated using the Wilcoxon rank sum test, and horizontal lines indicate the mean.Flow cytometry showed that GIGA-564 was more efficient than ipilimumab in depleting CTLA-4+ Tregs in the tumor microenvironment. The data from Figure 15D are shown again in Figure 32. [Figure 15B] Same as above. [Figure 15C] Same as above. [Figure 15D] Same as above. [Figure 15E] Same as above. [Figure 16A] Figures 16A-16D show that GIGA-564 induces more FcR signaling than ipilimumab. Target CHO cells expressing human CTLA-4 with the Y201G mutation, which enhances 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, which disrupts 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 then added. Cells were incubated at 37° C. for 6 hours, and then luciferase activity was measured. Data shown are relative light units (RLU) emitted from effector cells and are plotted as the average of two technical replicates. [Figure 16B] Same as above. [Figure 16C] Same as above. [Figure 16D] Same as above. [Figure 17A]Figure 17A shows that GIGA-564 provides protection in a mouse tumor rechallenge model. hCTLA-4 KI mice bearing MC38 tumors were randomized when tumors were 60–120 mm3 (day 8) and then treated with the indicated antibody at 1 mg / kg every 3 days for three doses. The plot shows the tumor volume (mean ± SEM) of MC38 tumors over time treated with the indicated antibody. Tumor volumes from mice euthanized due to tumor burden exceeding 3000 mm3 were carried forward (thin vertical black line). n=8 for isotype and commercial ipilimumab, n=9 for GIGA-564. Figure 17B. MC38 cells were implanted into the contralateral flank of naive C57BL / 6 mice and mice from mice previously treated with ipilimumab or GIGA-564 and with a durable complete response (0 mm3 tumor volume) at day 43 (Figure 17A). The plot shows the tumor volume (mean ± SEM) of MC38 tumors over time in mice previously treated 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 challenge model and against the naive control group in the rechallenge model. Compared to the naive group, previous treatment with ipilimumab (p = 0.0089) or GIGA-564 (p = 0.0088) limited tumor growth. [Figure 17B] Same as above. [Figure 18A]We demonstrate that GIGA-564 and pembrolizumab induce less toxicity than ipilimumab and pembrolizumab in a mouse model. 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 and ipilimumab (Ipi), or pembrolizumab and GIGA-564. One week after the last dose, mice were euthanized, and tissues were collected for pathological analysis. Figure 18A shows the percent change in body weight over time in mice treated with the indicated regimens (mean ± SEM, n = 10). Four mice from the pembrolizumab and ipilimumab group died on day 12, while three mice from the pembrolizumab and GIGA-564 treatment group died on day 12 and one mouse died on day 15, all likely due to ADA-induced hypersensitivity after administration. A mixed-effects model revealed no statistically significant differences in the percent body weight change between groups. Figures 18B-18C. Plots show the skin inflammation (Figure 18B) or colonic epithelial damage (colitis, Figure 18C) scores (mean ± SEM) induced by each treatment regimen. Horizontal lines indicate medians. Adjusted p-values ​​were calculated using the Benjamini-Hochberg step-down procedure to account for multiple comparisons. [Figure 18B] Same as above. [Figure 18C] Same as above. [Figure 19] A model illustrating the mechanism of action of ipilimumab and GIGA-564 is shown. Top panel: Ipilimumab blocks CTLA-4 interaction with CD80 / CD86, allowing antigen-presenting cells (APCs) to costimulate peripheral Tregs and enhance their proliferation. GIGA-564 weakly blocks CTLA-4 interaction with CD80 / CD86, thereby inducing less Treg proliferation. Bottom panel: Ipilimumab and GIGA-564 bind to CTLA-4 on intratumoral Tregs and induce Treg killing through interaction with Fc receptors (FcR) on effector cells. GIGA-564 induces stronger FcR signaling and thus depletes intratumoral Tregs more efficiently than ipilimumab. [Figure 20A]In vitro characterization of scFvs reformatted as full-length antibodies is shown. Figure 20A. Clonal cluster analysis of FACS-enriched anti-CTLA-4 scFv clones. 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. Full-length antibody scFv clones described in this study are labeled with 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 the indicated antibodies at 10 μg / ml. A PE-conjugated anti-human IgG secondary antibody was used to detect cells labeled with the indicated anti-CTLA-4 antibodies, and 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. A cell-based CTLA-4 Blockade 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 mAbs. 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 in each Promega bioassay kit, multiple plates were used to analyze this set of aCTLA-4 mAbs.To control for plate-to-plate variability 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, while plates C and D were run separately at a later time. E. Correlation between antibody affinity and cell-based assay activity. Antibody affinity (KD) for CTLA-4 was compared to the blocking EC50 and RLU maximum signal, and no correlation was found when the data was fit by linear regression. Each data point represents a single antibody, and the following antibodies are distinguished as follows: ipilimumab analog (red square), GIGA-564 (inverted triangle), and aCTLA-4.28 (star). Data are from (Figure 20D) and Table 23. [Figure 20B] Same as above. [Figure 20C] Same as above. [Figure 20D-1] Same as above. [Figure 20D-2] Same as above. [Figure 20E] Same as above. [Figure 21] Figure 1 provides validation of the N297Q mutant for binding to cell surface CTLA-4. CHO cells with and without human CTLA-4 expression were incubated with 10 μg / mL 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. Histograms show staining of CTLA-4+ or CTLA-4- cells by the indicated antibody as determined by flow cytometry. [Figure 22A]Figure 22C shows that costimulation enhances Treg proliferation. Histograms show CellTrace Violet signal (gated on live CD3+CD4+ cells) in Figure 22A. Treg or Figure 22B. Tconv cells cultured in the presence of M-450 tosyl activation beads coated with anti-CD3 antibody with or without CD80, or Figure 22C. Treg or Figure 22D. Tconv cells activated with M-450 tosyl activation beads coated with anti-CD3 antibody and CD80 in the presence of rhCTLA-4 (Abatacept) and / or anti-CTLA-4 mAb (aCTLA-4.28). [Figure 22B] Same as above. [Figure 22C] Same as above. [Figure 22D] Same as above. [Figure 23A] Figures 23A and 23C show that anti-CTLA-4 depletes intratumoral Tregs in hCTLA-4 KI mice. Flow cytometry analysis of cells from MC38 tumor-bearing hCTLA-4 KI mice receiving 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 in the GIGA-564 group were excluded from analysis due to low cell counts. Figures 23A-23B. 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-23D. 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 represent mean + / - SEM. [Figure 23B] Same as above. [Figure 23C] Same as above. [Figure 23D] Same as above. [Figure 24A]Figure 24A shows that GIGA-564 induces more FcR signaling than ipilimumab. Figure 24A shows that purified CTLA-4 antibody was diluted to a starting concentration of 5 μg / mL for an 8-point, 5-fold titration series in different pH buffers and then added to wells coated with rhCTLA-4-Fc. Bound antibody was detected with anti-constant kappa-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-24D show that GIGA-564 from three different productions 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, and PN-4261.01 was generated from a stable expression pool of CHOZN clones (EP-1, enriched pool 1). Figure 24C. Fucosylation levels, as determined by UPLC analysis, are shown for each sample. Figures 24D-24E. Graphs show human FcγRIIIA (V variant) signaling as determined in a cell-based assay 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 24B] Same as above. [Figure 24C] Same as above. [Figure 24D] Same as above. [Figure 24E] Same as above. [Figure 25A]GIGA-564 results in lower toxicity than ipilimumab in a mouse model. Figures 25A-25D show that 4- to 5-week-old hCTLA-4 / hPD-1 double KI mice on a BALB / c background were treated every 3 days for 9 doses with vehicle, pembrolizumab, pembrolizumab and ipilimumab, or pembrolizumab and GIGA-564. One week after the last dose, mice were euthanized, and tissues were collected for pathological analysis. Graphs show colon length (Figure 25A) or spleen weight (Figure 25B) from mice in each group at the time of euthanasia. The number of CD45+ cells / mm2 counted on randomly selected CD45-stained cardiac FFPE sections (Figure 25C) or cardiac pathology scores determined by a pathologist (Figure 25D) are shown. Figure 25E. hCTLA-4 KI mice bearing MC38 tumors were treated twice weekly with vehicle (PBS) or 5 mg / kg ipilimumab or GIGA-564 for five doses (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 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 percent 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-4E. Lines indicate mean + / - SEM. Adjusted p values ​​were calculated using the Benjamini-Hochberg stepdown procedure, which accounts for multiple comparisons. [Figure 25B] Same as above. [Figure 25C] Same as above. [Figure 25D] Same as above. [Figure 25E] Same as above. [Figure 26] Figure 26 shows that the checkpoint inhibitor ipilimumab increases the percentage of expanding Tregs in mice expressing humanized CTLA-4. The data from Figure 26 are from the same experiment as Figure 15A. [Figure 27] These results demonstrate that intratumor Treg depletion, rather than checkpoint inhibition, 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 from Figure 27 includes data as shown in Figures 12B and 14A. [Figure 28] This shows that the GIGA-564 anti-CTLA-4 antibody has weak checkpoint inhibitor activity but strong affinity for CTLA-4. A schematic diagram showing the conventional mechanism versus the current mechanism of action 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 of CTLA-4 and its ligand. [Figure 29] Figure 29 shows that GIGA-564 weakly blocks the CD80 / CD86 binding interaction to CTLA-4 compared to ipilimumab. Figure 29 shows the data shown in Figure 20D (Plate A). [Figure 30] This shows that GIGA-564 has superior antitumor activity compared to ipilimumab. Humanized CTLA-4 knockin mice (n=8-13) bearing MC38 tumors were administered GIGA-564 or ipilimumab at 0.3 mpk on days 0, 3, and 6 of the study. After administration of GIGA-564, CTLA-4 knockin mice bearing MC38 tumors showed reduced tumor volume and increased survival rates compared to ipilimumab and isotype. The superior efficacy of GIGA-564 confirms that its weakness is its inability to inhibit checkpoints. The experiment described in Figure 30 is also described in Figures 11 and 14C. The tumor growth data from Figure 30 includes the data shown in Figures 11 and 14C. [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. [Figure 32] Representative contour plots of CD4 T cells in each treatment group. The X and Y axes correspond to FOXP3 and CTLA-4, respectively. Figure 32 repeats the results from Figure 15D. [Figure 33] These results show 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 with ipilimumab. Further experiments ruled out that the difference was due to Fc-FcR affinity or CTLA-4 cell surface cycling. Figure 33 shows some of the data from Figure 16B again. [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 antigens on the cell surface that are bound by anti-CTLA-4 clones L3D10 or BNI3, validating the expression of cyno or human CTLA-4 on the surface of these cell lines, respectively. [Figure 34-2] Same as above. [Figure 34-3] Same as above. [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 abilities to bind to hCTLA-4, but compared to Ipi, GIGA-564 has a significantly reduced ability to bind to cyno CTLA-4 expressed on the cell surface. Data from atezo binding (negative control) are shown again on multiple plots for reference. [Figure 35-2] Same as above. [Figure 35-3] Same as above. [Figure 35-4] Same as above. [Figure 36A]GIGA-564 has been shown to have reduced binding to several presentations of cytotoxic CTLA-4 compared to ipilimumab when tested using ELISA assays. These ELISAs tested the binding of GIGA-564 to various CTLA-4 proteins supplied by multiple manufacturers, including Fc-chimeric and His-tagged formats. 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 one 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 on the other half of each plate at 1 μg / mL. 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 on one half of separate ELISA plates at 1 μg / mL. RhCTLA-4-Fc (ACROBiosystems, CT4-H5229, Figures 36D-E) was coated on the other half of each of these plates at 1 μg / mL. After coating, the plates were incubated overnight at 4°C. The next day, the plates were blocked with 5% milk in PBST for 1 hour on a plate shaker at room temperature. A titration series of ipilimumab, atezolizumab (negative control), and 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 with an HRP-conjugated anti-kappa light chain antibody (0.5 μg / mL, Southern Biotech 2060-50). After incubation on a plate shaker for 1 hour at room temperature and washing, the plate was developed with TMB substrate.After a sufficient signal was achieved, 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 absorbance versus the logarithm of concentration using Prism (GraphPad). The results indicate that the Ipi and GIGA-564 epitopes are indeed different, as Ipi has similar binding to human and cyno CTLA-4, whereas GIGA-564 has a reduced ability to bind to cyno CTLA-4 compared to human CTLA-4. [Figure 36B] Same as above. [Figure 36C] Same as above. [Figure 36D] Same as above. [Figure 36E] Same as above. [Figure 37] 1 provides response plots generated to determine optimized formulations of GIGA-564. The response plots showed the effects of pH and sucrose concentration when NaCl and buffer concentrations were fixed at 100 mM and 30 mM, respectively. [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 formulations of GIGA-564 antibody as indicated. [Figure 39] 1 shows a Pareto analysis that provided which variables have the most impact on the formulation of GIGA-564. [Figure 40-1] 1 is a conformation 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, and sucrose has some effect, with higher being better. [Figure 40-2] 1 is a conformation 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, and sucrose has some effect, with higher being better. [Figure 41] This shows that GIGA-2328 is designed to have low fucosylation for the purpose of enhancing FcγRIIIa signaling. An ADCC reporter bioassay for human FcγRIIIa, V variant (G7011) from Promega Corporation was performed according to the manufacturer's instructions. Briefly, CHO target cells stably expressing human CTLA-4 with the Y201G mutation, which enhances cell surface expression, were suspended in RPMI 1640 + 4% FBS medium containing 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 included Bio-Glo luciferase assay reagent and a SpectraMax i3x reader. Luciferase activity, measured in relative light units (RLU), was plotted against antibody concentration. [Figure 42A]We demonstrate that GIGA-564 induces antibody-dependent cell-mediated 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 allowed to recover 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, which enhances cell surface expression, were stained with CellTrace Violet (Thermo Fisher Scientific) and then incubated with either GIGA-564 or a human IgG1 isotype control at the indicated concentrations 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 for 4 hours at 37°C to allow ADCC. Samples were then washed with MACS buffer, stained with the viability dye 7-aminoactinomycin D (7-AAD), which labels dead cells, and analyzed by flow cytometry. (Figure 42A) Representative gating strategy for determining ADCC. Samples were first gated on target cells (CellTrace Violet+), then single cells, and then dead cells (7-AAD+). (Figure 42B) Plot of the percent dead cells at each concentration of GIGA-564 and human IgG1 isotype. GIGA-564 results in a higher percent dead CTLA-4+ target cells than the isotype control. [Figure 42B] Same as above. [Figure 43A]These results demonstrate that IgG1 allotypes can affect signaling through the FcγRIIIa receptor. Figure 43A shows the 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 positions are given 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, which use the IGHG1*01 allotype. These results demonstrate that allotypes can affect FcγRIIIa signaling. The 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 the Y201G mutation, which enhances cell surface expression, were suspended in RPMI 1640 + 4% FBS medium containing 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 included Bio-Glo luciferase assay reagent and a SpectraMax i3x reader. Luciferase activity, measured in relative light units (RLU), was plotted against antibody concentration. [Figure 43B] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0097] Table 23. In vitro characterization of scFvs reformatted as full-length antibodies. The k, k, and K of the indicated CTLA-4 mAbs for soluble human CTLA-4 were determined by SPR (Carterra). CTLA-4 detected by secondary human IgG antibody and flow cytometry.+ or CD27 + Geometric MFI of the indicated CTLA-4 mAbs bound to CHO cells. The affinity (KD) of the indicated CTLA-4 mAbs for soluble cynomolgus CTLA-4 was determined by single antigen concentration BLI measurements (Octet). The ability of each aCTLA-4 mAb to block CTLA-4 binding to CD80 / CD86 was determined by a cell-based assay (Promega). Due to sample size constraints in each Promega bioassay kit, this set of aCTLA-4 mAbs was analyzed using multiple plates (plate letters correspond to plates A–D in Figure 20D). To control for plate-to-plate variability in maximum signal, an ipilimumab analog was run on each plate, and a representative sample was used for this table.

[0098] Table 24. GIGA-564 has minimal ability to block CD80 or CD86 from binding to CTLA-4. Summary of blocking ELISA data from Figure 13B. Absorbance values ​​from detected CD80 or CD86 were normalized to the signal from the ELISA plate incubated with pembrolizumab. The data were then analyzed and fitted by nonlinear regression. The normalized maximum and minimum signals, as well as the IC50 of blocking, are provided.

[0099] Table 25. Fc receptor signaling for GIGA-564 and ipilimumab. Summary of FcR bioassay results from Figure 16. Luminescence (RLU) data from the bioassay was fitted by non-linear regression. Maximum signal and EC50 of FcR signaling are provided.

[0100] Table 26. CTLA-4 antibody sequences. The amino acid sequences of the IgK and IgG variable regions of 14 characterized full-length CTLA-4 antibodies are provided.

[0101] Table 27. Reagent antibodies for immunophenotyping. Lists antibody clone details used for immunophenotyping.

[0102] Table 32-33. A14 light chain CDR sequences and A14 heavy chain CDR sequences of GIGA-564 identified by different algorithms (Antibody A14):

[0103] 7. MODE FOR CARRYING OUT THE INVENTION 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, unless otherwise indicated, as described in various general and more specific references cited and discussed throughout the specification. 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, 1999, incorporated herein by reference. 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). Enzymatic reactions and purification techniques are either commonly accomplished in the art or, as described herein, are performed according to manufacturer's specifications. 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, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0104] The following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0105] The terms "CTLA-4," "CTLA-4 protein," and "CTLA-4 antigen" are used interchangeably herein to refer to human CTLA-4, or any variants (e.g., splice variants and allelic variants), isoforms, and species homologs of human CTLA-4 naturally expressed by cells or expressed by cells 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).

[0106] 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 structure of immunoglobulins is well characterized. 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 .

[0107] 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" is an ABP provided herein 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 between or among CTLA-4 proteins from different species.

[0108] 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 Antibody is one type of ABP.

[0109] The terms "hypofucosylation" or "hypofucosylated" 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.

[0110] When a fucosylation ratio is given in the context of a composition comprising an antibody, the ratio refers to 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 hypofucosylated.

[0111] The term "alternative scaffold" refers to a molecule in which one or more regions can be diversified to produce 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 similar specificity and affinity as a naturally occurring antibody. Exemplary alternative scaffolds include fibronectin (e.g., Adnectins™), β-sandwich (e.g., iMabs), lipocalin (e.g., Anticalins®), EETI-II / AGRP, BPTI / LACI-D1 / ITI-D2 (e.g., Kunitz domain), thioredoxin peptide aptamer, Protein A (e.g., Affibody®), ankyrin repeat (e.g., DARPins), gamma-B-crystallin / ubiquitin (e.g., affilin), CTLD3 (e.g., tetranectin), Fynomers, and (LDLR-A module) (e.g., avimers). Additional information about 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.

[0112] The term "antigen-binding domain" refers to a portion of an ABP capable of specifically binding to an antigen or epitope.

[0113] 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 a naturally occurring antibody structure and having a heavy chain that includes an Fc region.

[0114] 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 Fc region that has been modified as described elsewhere in this disclosure.

[0115] V H and V L The regions can be further subdivided into regions of hypervariability (also called "hypervariable regions (HVRs)") interspersed with more conserved regions, which are called framework regions (FRs). H and V L Generally, an antibody comprises three CDRs and four FRs arranged in the following order (N-terminus to C-terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDRs are involved in antigen binding and influence 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, which is incorporated by reference in its entirety. of Health, Bethesda, MD.

[0116] 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.

[0117] 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 differences in sequence and function. Humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0118] The amino acid sequence boundaries of the CDRs can be determined by one of skill in the art using any of a number of 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.

[0119] 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.

[0120] CDRs can be assigned using antibody numbering software such as Abnum, for example, 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]

[0121] 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).

[0122] "Antibody fragments" include portions of an intact antibody, such as the antigen-binding or variable region 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.

[0123] An "Fv" fragment comprises a non-covalently bound dimer of one heavy- and one light-chain variable domain.

[0124] A "Fab" fragment contains the heavy and light chain variable domains 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.

[0125] An "F(ab')2" fragment comprises two Fab' fragments linked near the hinge region by a disulfide bond. F(ab')2 fragments can be produced, for example, by recombinant methods or by pepsin digestion of intact antibodies. F(ab')2 fragments can be dissociated, for example, by treatment with β-mercaptoethanol.

[0126] "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.

[0127] 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 linked to the C-terminus of the scFv. The Fc domain can be linked to the C-terminus of the scFv, depending on the orientation of the variable domains (i.e., V H -V L or V L -V H ) depending on V H or V L Any suitable Fc domain known in the art or described herein may be used. In some cases, the Fc domain comprises an IgG4 Fc domain.

[0128] The term "single domain antibody" refers to a molecule in which one variable domain of an antibody specifically binds to an antigen without the presence 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.

[0129] 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, which is bivalent but recognizes the same epitope in each antigen-binding domain. The binding specificity can be present in any suitable valency.

[0130] The term "monoclonal antibody" refers to an antibody from a population of substantially homogeneous antibodies. A population of substantially homogeneous antibodies contains antibodies that are substantially similar and bind to the same epitope, except for variants that may normally arise during the production of monoclonal antibodies. Such variants are generally present only in small amounts. Monoclonal antibodies are typically obtained by a process that includes selection of a single antibody from a plurality of antibodies. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of 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.

[0131] 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.

[0132] "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 have been 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, having the desired specificity, affinity, or biological effect. In some cases, 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.

[0133] A "human antibody" is one having 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.

[0134] 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 material. 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 sequenator. In some embodiments, an isolated ABP is purified to homogeneity by gel electrophoresis (e.g., SDS-PAGE) under reducing or non-reducing conditions and detected by Coomassie blue or silver staining. An isolated ABP includes an ABP in situ within a recombinant cell, since at least one component of the ABP's natural environment is absent. 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% to 100% ABP or nucleic acid by weight. In some embodiments, the isolated ABP or isolated nucleic acid is provided as a solution comprising at least 85%, 90%, 95%, 98%, 99% to 100% ABP or nucleic acid by volume.

[0135] "Affinity" refers to the strength of the sum 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 dynamic component that contributes to the dissociation equilibrium constant is described in more detail below. Affinity can be measured by common methods known in the art, including those described herein. Affinity can be determined, for example, using surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®).

[0136] With respect to the binding of an ABP to a target molecule, the terms "bind," "specific binding," "specifically binds," "specific," "selectively binds," and "selective" to a particular antigen (e.g., a polypeptide target) or epitope on a particular antigen refer to binding that is measurably different 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 the non-target molecule. Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule. Specific binding is then indicated if 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 a 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 a 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 a 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 a non-target molecule is less than about 0.1% of its affinity for CTLA-4.

[0137] 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 also known as k off is called the value.

[0138] As used herein, "k a " (M -1 ×sec -1 The term k ) refers to the association rate constant of a particular ABP-antigen interaction. This value is also known as k on is called the value.

[0139] 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 .

[0140] As used herein, "K A " (M -1 The term K ) refers to the dissociation equilibrium constant of a particular ABP-antigen interaction. A =k a / k d .

[0141] 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 produced 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. H and V LAffinity maturation by domain shuffling. Random mutagenesis of CDR and / or framework residues is described, for example, by 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.

[0142] An "immunoconjugate" is an ABP conjugated to one or more heterologous molecules.

[0143] "Effector function" refers to a biological activity mediated by the Fc region of an antibody, which activity can 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).

[0144] 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.

[0145] 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 and 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 and 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 with different point mutations or chimeric CTLA-4 variants.

[0146] 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 in a variety of ways 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.

[0147] 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]

[0148] 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."

[0149] The term "treating" (and variations thereof, such as "treat" or "treatment") refers to clinical intervention in an attempt to alter the natural history of a disease or condition in a subject in need thereof. Treatment can be performed both prophylactically and during the course of clinical pathology. Desirable effects of treatment include preventing the occurrence or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.

[0150] 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.

[0151] 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.

[0152] The term "package insert" is used to refer to instructions customarily included in the commercial packaging of a therapeutic or diagnostic product (e.g., a kit) that contain information about the indications, uses, dosage, administration, concomitant therapy, contraindications, and / or warnings regarding the use of such therapeutic or diagnostic product.

[0153] 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.

[0154] "Chemotherapeutic agents" refer to chemical compounds useful in the treatment of cancer. Chemotherapeutic agents include "antihormonal agents" or "endocrine therapeutic agents" that act to regulate, reduce, block, or inhibit the effects of hormones that can promote cancer growth.

[0155] The term "cytostatic agent" refers to a compound or composition that arrests cell growth 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%.

[0156] 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.

[0157] 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.

[0158] The terms "modulate" and "modulation" refer to decreasing or inhibiting or alternatively activating or increasing the recited variable.

[0159] 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.

[0160] 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.

[0161] The term "agonize" refers to the activation of receptor signaling to elicit a biological response associated with receptor activation. An "agonist" is an entity that binds to and agonizes a receptor.

[0162] The term "antagonize" refers to the inhibition of receptor signaling that inhibits the biological response associated with receptor activation. An "antagonist" is an entity that binds to and antagonizes a receptor.

[0163] 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 additional information regarding effector T cells, see Seder and Ahmed, Nature Immunol., 2003, 4:835-842, which is incorporated by reference in its entirety.

[0164] 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 additional information regarding regulatory T cells, see Nocentini et al., Br. J. Pharmacol., 2012, 165:2089-2099, which is incorporated by reference in its entirety.

[0165] The term "dendritic cell" refers to professional antigen-presenting cells that can activate naive T cells and stimulate the proliferation and differentiation of B cells.

[0166] 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 into, deleted from, and / or substituted into the amino acid sequence 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 binding assays as described herein). Variants of the present disclosure include fragments, analogs, recombinant polypeptides, synthetic polypeptides, and / or fusion proteins.

[0167] 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, for example, polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, glycosylation, etc. 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.

[0168] 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 that 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 regulated nucleic acid 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 can be found, for example, in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA, and Baron et al. al., 1995, Nucleic Acids Res. 23:3605-06.

[0169] 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. Exemplary host cells include CS-9 cells, the COS-7 line of monkey kidney cells (ATCC CRL 1651) (see Gluzman et al., 1981, Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cell lines or derivatives thereof, such as Veggie CHO and related cell lines grown 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 derived from the African green monkey kidney cell line CV1 (ATCC CCL 70) (see McMahan et al., 1991, EMBO J. 10:2821), human embryonic kidney cells, such as 293, 293 EBNA, or MSR. Host cells include, but are not limited to, cell lines derived from in vitro culture of HL-60, U937, HaK, or Jurkat cells, including, but not limited to, 293, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, primary tissue, primary explants, or HL-60, U937, HaK, or Jurkat cells. Typically, host cells are cultured cells that can be transformed or transfected with a polypeptide-encoding nucleic acid that can then be expressed in the host cell.

[0170] The phrase "recombinant host cell" can be used to refer to a host cell that has been transformed or transfected with a nucleic acid to be expressed. A host cell can also be a cell that contains a nucleic acid but does not express it at a desired level unless a regulatory sequence is introduced into the host cell so that it 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 successive generations due, for example, to mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell but still be within the scope of the term as used herein.

[0171] In some embodiments, the host cells are used in adoptive cell therapy for delivery of an ABP to a subject.

[0172] 7.2. Other Interpretation Rules Ranges recited herein are understood to be shorthand for all of the values ​​within the range, inclusive of 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.

[0173] Unless otherwise indicated, reference to a compound having one or more stereocenters contemplates each stereoisomer and all combinations of those stereoisomers.

[0174] 7.3. Antigen-binding proteins In one aspect, the present disclosure provides antigen binding proteins (e.g., antibodies, antibody fragments, antibody derivatives, antibody muteins, and antibody variants). In some embodiments, the ABP binds to CTLA-4. In particular, 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 does not include R70. In some embodiments, the ABP contacts amino acids K130, Y139, L141, and I143, but does not contact amino acid R70 of CTLA-4. In some embodiments, the ABP can bind to CTLA-4 while CTLA-4 interacts with CD80 / CD86. In some embodiments, 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.

[0175] 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 can be represented 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 labeled in the sequence below. That is, for example, "A1HC" represents a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 101, "A1LC" represents 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 represented by at least two members of a group include all members of the group between and including the end range members. Thus, the group range A1-A28 includes all members A1-A28, as well as members A1 and A28 themselves. The group range A4-A6 includes members A4, A5, and A6, etc. In certain embodiments, ABP is A14.

[0176] In some embodiments, the antigen binding protein comprises a variable (V(D)J) region 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 a variable (V(D)J) region of the heavy or light chain sequence 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.

[0177] Also shown below are the locations of the CDRs (underlined), which form part of the antigen-binding site, and the framework regions (FRs) that are intervening segments of these variable domain sequences. In both the light chain variable region and the heavy chain variable region, there are three CDRs (CDR1-3) and four FRs (FR1-4). The CDR regions of each light and heavy chain are also grouped by antibody type (A1, A2, A3, etc.). Antigen binding proteins of the disclosure include, for example, L1H1 (antibody A1, used interchangeably herein as "aCTLA-4.9"), L2H2 (antibody A2, used interchangeably herein as "aCTLA-4.4"), L3H3 (antibody A3, used interchangeably herein as "aCTLA-4.2"), L4H4 (antibody A4, used interchangeably herein as "aCTLA-4.29"), L5H5 (antibody A5, used interchangeably herein as "aCTLA-4.28"), L6H6 (antibody A6, used interchangeably herein as "aCTLA-4.26"), L7H7 (antibody A7, used interchangeably herein as "aCTLA-4.3"), L8H8 (antibody A8, used interchangeably herein as "aCTLA-4.1"), L9H9 (antibody A9, used interchangeably herein as "aCTLA-4.29"), L10H10 (antibody A1, used interchangeably herein as "aCTLA-4.9"), L11H11 (antibody A1, used interchangeably herein as "aCTLA-4.9"), L2H2 (antibody A2, used interchangeably herein as "aCTLA-4.4"), L3H3 (antibody A3, used interchangeably herein as "aCTLA-4.2"), L4H4 (antibody A4, used interchangeably herein as "aCTLA-4.29"), L5H5 (antibody A5, used interchangeably herein as "aCTLA-4.28"), L6H6 (antibody A6, used interchangeably herein as "aCTLA-4.26"), L7H7 (antibody A7, used interchangeably herein as "aCTLA-4.3"), L8H8 (antibody A8, used interchangeably herein as "aCTLA-4.1"), L12H12 (antibody A1, used interchangeably herein as "aCTLA-4.1"), L13H13 (antibody A1 L9H9 (antibody A9, used interchangeably herein as "aCTLA-4.24"), L10H10 (antibody A10, used interchangeably herein as "aCTLA-4.22"), L11H11 (antibody A11, used interchangeably herein as "aCTLA-4.31"), L12H12 (antibody A12, used interchangeably herein as "aCTLA-4.12"), L13H13 (antibody A13, used interchangeably herein as "aCTLA-4.14"), L13H13 (antibody A13, used interchangeably herein as "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 as "aCTLA-4.11"), L15H15 (antibody A15, used interchangeably herein as "aCTLA-4.18"), L16H16 (antibody A16, used interchangeably herein as "aCTLA-4.5"), and L17H17 (antibody A17, used interchangeably herein as "aCTLA-4.17"). In some embodiments, an ABP of the disclosure comprises L14H14 (antibody A14, used interchangeably herein as "aCTLA-4.15" or GIGA-564).

[0178] Antigen binding proteins of the disclosure include, for example, L19H19 (antibody A19, used interchangeably herein as "aCTLA-4.7"), L20H20 (antibody A20, used interchangeably herein as "aCTLA-4.25"), L21H21 (antibody A21, used interchangeably herein as "aCTLA-4.10"), L22H22 (antibody A22, used interchangeably herein as "aCTLA-4.21"), L23H23 (antibody A23, used interchangeably herein as "aCTLA-4.23"), L23H24 (antibody A24, used interchangeably herein as "aCTLA-4.24"), L23H25 (antibody A25, used interchangeably herein as "aCTLA-4.25"), L23H26 (antibody A26, used interchangeably herein as "aCTLA-4.26"), L23H27 (antibody A27, used interchangeably herein as "aCTLA-4.27"), L23H28 (antibody A28, used interchangeably herein as "aCTLA-4.28"), L23H29 (antibody A29, used interchangeably herein as "aCTLA-4.29 ... In some embodiments, the ABP of the present disclosure comprises the light and heavy chain variable domains of L14H14 (antibody A14, used interchangeably herein as "aCTLA-4.15" or GIGA-564).

[0179] In some embodiments, the antigen binding protein comprises all six CDR sequences (the three CDRs of the light chain and the 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 (the three CDRs of the light chain or the 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.

[0180] 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 selected from the group consisting of L1-L28, 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 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 of L1 through L28.

[0181] 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 only 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.

[0182] 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 the sequence of a heavy chain variable domain selected from the group consisting of H1 to H28, wherein each such sequence difference is independently either 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 the sequence of a heavy chain variable domain 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.

[0183] In one embodiment, the 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 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 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.

[0184] 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 the light chain CDR1 sequence shown above. In another embodiment, the antigen binding protein comprises the light chain CDR2 sequence shown above. In another embodiment, the antigen binding protein comprises the light chain CDR3 sequence shown above. In another embodiment, the antigen binding protein comprises the heavy chain CDR1 sequence shown above. In another embodiment, the antigen binding protein comprises the heavy chain CDR2 sequence shown above. In another embodiment, the antigen binding protein comprises the heavy chain CDR3 sequence shown above.

[0185] 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 5, 4, 3, 2, or 1 amino acid residue.

[0186] In some embodiments, at least one of the CDR1 sequences of the antigen binding protein is a CDR1 sequence from A1 to A28, CDR1-L1 to 28, or CDR1-H1 to 28 as shown in Table 5. In some embodiments, at least one of the CDR2 sequences of the antigen binding protein is a CDR2 sequence from A1 to A28, CDR2-L1 to 28, or CDR2-H1 to 28 as shown in Table 5. In some embodiments, at least one of the CDR3 sequences of the antigen binding protein is a CDR3 sequence from A1 to A28, CDR3-L1 to 28, or CDR3-H1 to 28 as shown in Table 5.

[0187] In another embodiment, the light chain CDR3 sequence of the antigen-binding protein is a light chain CDR3 sequence from A1 to A28 or CDR3-L1 to 28 as shown in Table 5, and the heavy chain CDR3 sequence of the antigen-binding protein is a heavy chain sequence from A1 to A28 or CDR-H1 to 28 as shown in Table 5.

[0188] 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).

[0189] 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).

[0190] 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.

[0191] In another embodiment, the antigen binding protein comprises 1, 2, 3, 4, or 5 CDR sequences that each independently differ by 6, 5, 4, 3, 2, 1, or 0 single amino acid additions, substitutions, and / or deletions from the CDR sequences of A1-A23, and the antigen binding protein further comprises 1, 2, 3, 4, or 5 CDR sequences that each independently differ by 6, 5, 4, 3, 2, 1, or 0 single amino acid additions, substitutions, and / or deletions from the CDR sequences. In some embodiments, the antigen binding protein comprises 1, 2, 3, 4, or 5 CDR sequences that each 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.

[0192] The nucleotide sequence of A1-A28 or the amino acid sequence of A1-A28 can be modified, for example, by random mutagenesis or by site-directed mutagenesis (e.g., oligonucleotide-directed site-directed mutagenesis) to generate modified polynucleotides containing one or more specific nucleotide substitutions, deletions, or insertions compared to the unmutated polynucleotide. Examples of techniques for making such modifications are described in Walder et al., 1986, Gene 42:133; Bauer et al., 1985, Gene 37:73; Craik, BioTechniques, January 1985, 12-19; ​​Smith et al., 1981, Genetic Engineering: Principles and Methods, Plenum Press, and U.S. Patent Nos. 4,518,584 and 4,737,462. These and other methods can be used to generate derivatives of anti-CTLA-4 antibodies that have desirable properties, such as increased affinity, avidity, or specificity for CTLA-4, increased activity or stability in vivo or in vitro, or reduced in vivo side effects, compared to the underivatized antibody.

[0193] 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 containing heterologous polypeptides fused to the N- or C-terminus of the anti-CTLA-4 antibody polypeptide. For example, the conjugated peptide can be a heterologous signal (or leader) polypeptide, such as the yeast alpha-factor leader, or a peptide such as an epitope tag. Antigen-binding protein-containing fusion proteins can include peptides added to facilitate purification or identification of the antigen-binding protein (e.g., poly-His). The antigen-binding protein can also bind to the FLAG peptide Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DYKDDDDK) (SEQ ID NO: 7002), as described by Hopp et al., Bio / Technology 6:1204, 1988, and U.S. Pat. No. 5,011,912. The FLAG peptide is highly antigenic and provides an epitope that is reversibly bound by a specific monoclonal antibody (mAb), allowing for rapid assay and facile purification of expressed recombinant proteins. Reagents useful for preparing fusion proteins in which the FLAG peptide is fused to a given polypeptide are commercially available (Sigma, St. Louis, MO).

[0194] One suitable Fc polypeptide, described in PCT application WO93 / 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 WO93 / 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 exhibits reduced affinity for Fc receptors.

[0195] In other embodiments, the variable portions of the heavy and / or light chains of an anti-CTLA-4 antibody can be substituted for the variable portions of the antibody heavy and / or light chains.

[0196] Oligomers containing one or more antigen-binding proteins can be used as CTLA-4 antagonists or agonists. The oligomers can be in the form of covalently or non-covalently linked dimers, trimers, or higher-order oligomers. Oligomers containing two or more antigen-binding proteins are contemplated for use, with one example being a homodimer. Other oligomers include heterodimers, homotrimers, heterotrimers, homotetramers, heterotetramers, and the like.

[0197] One embodiment is directed 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 properties that promote oligomerization. Leucine zippers and certain polypeptides derived from antibodies are among the peptides that can promote oligomerization of antigen-binding proteins linked thereto, as described in more detail below.

[0198] In certain embodiments, the oligomer comprises two to four antigen-binding proteins. The antigen-binding proteins of the oligomer can 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.

[0199] In one embodiment, oligomers are prepared using polypeptides derived from immunoglobulins. The preparation of fusion proteins comprising specific heterologous polypeptides fused to various portions of antibody-derived polypeptides (including Fc domains) is described, for example, in Ashkenazi et al. al., 1991, PNAS USA 88:10535, Byrn et al., 1990, Nature 344:677, and Hollenbaugh et al., 1992 Curr. Prots in Immunol., Suppl. 4, pages 10.19.1-10.19.11.

[0200] One embodiment of the present disclosure is directed 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. The dimer can be created, for example, by inserting a gene fusion encoding the fusion protein into an appropriate expression vector, expressing the gene fusion in a host cell transformed with the recombinant expression vector, and allowing the expressed fusion protein to assemble similarly to antibody molecules, after which interchain disulfide bonds form between the Fc portions, resulting in a dimer.

[0201] 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.

[0202] Another method for preparing oligomeric antigen-binding proteins involves the use of leucine zippers. Leucine zipper domains are peptides that promote oligomerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., 1988, Science 240:1759) and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and their derivatives that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble oligomeric proteins are described in PCT application WO 94 / 10308, and the leucine zipper derived from pulmonary surfactant protein D (SPD) is described in Hoppe et al., 1994, FEBS Letters 344:191, incorporated herein by reference. The use of a modified leucine zipper that allows for stable trimerization of a heterologous protein fused thereto is described in Fanslow et al., 1994, Semin. Immunol. 6:267-78. In one approach, a recombinant fusion protein containing an anti-CTLA-4 antibody fragment or derivative fused to a leucine zipper peptide is expressed in suitable host cells, and the soluble oligomeric anti-CTLA-4 antibody fragment or derivative that is formed is recovered from the culture supernatant.

[0203] 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 according to 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 one embodiment, human anti-CTLA-4 monoclonal antibodies generated by a procedure involving immunization of transgenic mice are used to treat such conditions.

[0204] 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.

[0205] Additional embodiments include chimeric antibodies, e.g., humanized versions of non-human (e.g., murine) monoclonal antibodies. Such humanized antibodies can be prepared by known techniques and can offer the advantage of reduced immunogenicity when the antibody is administered to humans. In one embodiment, a humanized monoclonal antibody comprises the variable domain (or all or part of its antigen-binding site) of a murine antibody and the constant domain derived from a human antibody. Alternatively, a humanized antibody fragment can comprise the antigen-binding site of a murine monoclonal antibody and a variable domain fragment (lacking the antigen-binding site) derived from a human antibody. Procedures for the production of chimeric and further engineered monoclonal antibodies include those described in Riechmann et al., 1988, Nature 332:323; Liu et al., 1987, Proc. Nat. Acad. Sci. USA 84:3439; Larrick et al., 1989, Bio / Technology 7:934; and Winter et al., 1993, TIPS 14:139. In one embodiment, the chimeric antibody is a CDR-grafted antibody. Techniques for humanizing antibodies are discussed, for example, in U.S. Pat. Nos. 5,869,619, 5,225,539, 5,821,337, 5,859,205, 6,881,557, Padlan et al., 1995, FASEB J. 9:133-39, and Tamura et al., 2000, J. Immunol. 164:1432-41.

[0206] Procedures have been developed to generate human or partially human antibodies in non-human animals. For example, mice have been prepared in which one or more endogenous immunoglobulin genes have been inactivated by various means. Human immunoglobulin genes have been introduced into the mice to replace the inactivated mouse genes. Antibodies produced in the animals incorporate human immunoglobulin polypeptide chains encoded by the human genetic material introduced into the animal. In 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 generated in the animal.

[0207] One example of a suitable immunogen is soluble human CTLA-4, such as a polypeptide comprising the extracellular domain of the protein having the following sequence: SEQ ID NO: 7001 or other immunogenic fragments of the protein. Examples of techniques for the production and use of transgenic animals for the production of human or partially human antibodies are described in U.S. Pat. Nos. 5,814,318, 5,569,825, and 5,545,806; Davis et al., 2003, Production of human antibodies from transgenic mice in Lo, ed. Antibody Engineering: Methods and Protocols, Humana Press, NJ:191-200; Kellermann et al., 2002, Curr Opin Biotechnol. 13:593-97; Russell et al., 2000, Infect Immun. 68:1820-26; Gallo et al., 2000, Eur J Immun. 30:534-40; Davis et al., 1999, Cancer Metastasis Rev. 18:421-25; Green, 1999, J Immunol. Methods.231:11-23, Jakobovits,1998,Advanced Drug Delivery Reviews 31:33-42, Green et al.,1998,J Exp Med.188:483-95, Jakobovits A,1998,Exp.Opin.Invest.Drugs.7:607-14, Tsuda et al. al.,1997,Genomics.42:413-21, Mendez et al.,1997,Nat Genet.15:146-56, Jakobovits,1994,Curr Biol.4:761-63, Arbones et al.,1994,Immunity.1:247-60, Green et al.,1994,Nat Genet.7:13-21, Jakobovits et. al.,1993,Nature.362:255-58,Jakobovits et al.,1993,Proc Natl Acad Sci US A.90:2551-55.Chen,J.,M.Trounstine,FWAlt,F.Young,C.Kurahara,J.Loring,D.Huszar.Inter'l Immunol.5(1993):647-656; al.,1996,Nature Biotech.14:845-51,Harding et al.,1995,Annals of the New York Academy of Sciences、Lonberg et al.,1994,Nature 368:856-59; 113:49-101;Lonberg et al.,1995,Internal Review of Immunology 13:65-93;Neuberger,1996,Nature Biotechnology 14:826;Taylor et al.,1992,Nucleic Acids Res.20:6287-95; al.,1994,Inter'l Immunol.6:579-91;Tomizuka et al.,1997,Nature Genetics 16:133-43; al.,1993,Pro.Nat'lAcad.Sci.USA 90:3720-24;

[0208] The antigen-binding proteins (e.g., antibodies, antibody fragments, and antibody derivatives) of the present disclosure can comprise any constant region known in the art. The light chain constant region can be, for example, a kappa- or lambda-type light chain constant region, such as a human kappa- or lambda-type light chain constant region. The heavy chain constant region can be, for example, an alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region, such as a human alpha-, delta-, epsilon-, gamma-, or mu-type 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.

[0209] Techniques for deriving antibodies of a different subclass or isotype from an antibody of interest, 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 techniques can 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 Lantto et al., 2002, Methods Mol. Biol. 178:303-16.

[0210] 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 kappa light chain constant region of any of A1 to A28 (L1 to L28), or a fragment of the kappa light chain constant region of any 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 any of A1 to A28 (H1 to H28), or a fragment thereof, and a kappa light chain domain of any of A1 to A28 (L1 to L28), or a fragment thereof.

[0211] In another embodiment, the antigen binding protein of the present disclosure comprises both IgG1 heavy chain domains A1 to A28 (H1 to H28) or fragments thereof. In some embodiments, the IgG1 heavy chain domain comprises a lysine at amino acid position 97 (K97) according to the IMGT exon numbering system. In another embodiment, the IgG1 heavy chain domain comprises a lysine at amino acid position 214 (K214) according to the EU numbering system. In some embodiments, the IgG1 heavy chain domain comprises an arginine at amino acid position 97 (R97) according to the IMGT exon numbering system. In another embodiment, the IgG1 heavy chain domain comprises an arginine at amino acid position 214 (R214) according to the EU numbering system.

[0212] In some embodiments, the antigen binding protein of the disclosure comprises the IgG1 heavy chain domain or a fragment of the IgG1 heavy chain domain of A14(H14) (SEQ ID NO: 114) and the IgG1 light chain domain or a fragment of the IgG1 light chain domain of A14(H14) (SEQ ID NO: 14).

[0213] Thus, antigen binding proteins of the disclosure include, for example, those comprising the variable domain combinations L1H1, L2H2, L3H3, L4H4, L5H5, L6H6, L7H7, L8H8, L9H9, L10H10, L11H11, L12H12, L13H13, ... and L28H28, and those having a desired isotype (e.g., IgA, IgG1, IgG2, IgG3, IgG4, IgM, IgE, and IgD) and Fab or F(ab')2 fragments thereof. Further, if IgG4 is desired, see Bloom et al., 1997, Protein Science, incorporated herein by reference. 6:407, to reduce the tendency for intra-H chain disulfide bonds to form, which can lead to heterogeneity in IgG4 antibodies.

[0214] In one embodiment, the antigen binding protein is present in an amount of 1 x 10 -4 s -1 The following K off 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 substantially the same K 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 K as an antibody comprising one of the amino acid sequences set forth above. off In another embodiment, the antigen binding protein binds to CTLA-4 with substantially the same K as an antibody comprising one or more CDRs from an antibody comprising one of the amino acid sequences set forth above. off binds to CTLA-4.

[0215] 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.

[0216] 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. Such single-chain Fvs (scFvs) consist of two variable domain polypeptides (V L and V H These variable domains were prepared by fusing DNA encoding a peptide linker between DNA encoding the different V domains. The resulting polypeptides can fold back to form antigen-binding monomers or multimers (e.g., dimers, trimers, or tetramers) depending on the length of the flexible linker between the two variable domains (Kortt et al., 1997, Prot. Eng. 10:423; Kortt et al., 2001, Biomol. Eng. 18:95-108; Bird et al., 1988, Science 242:423-26; and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83). L and V H By combining the containing polypeptides, multimeric scFvs that bind to different epitopes can be formed (Kriangkum et al., 2001, Biomol. Eng. 18:31-40). Techniques developed for the production of single-chain antibodies include those described in U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879; Ward et al., 1989, Nature 334:544; de Graaf et al., 2002, Methods Mol. Biol. 178:379-87. ScFvs containing the variable domain combinations L1H1, L2H2, L3H3, L4H4, L5H5, L6H6, ..., and L28H28 are encompassed by the present disclosure.

[0217] 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 supernatant of the host cell culture using a heparin HP column using a salt gradient.

[0218] 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 can administer host cells transfected with a gene encoding an anti-CTLA-4 antibody.

[0219] An antigen-binding protein can have the structure of, for example, a naturally occurring immunoglobulin. An "immunoglobulin" is a tetrameric molecule. In naturally occurring immunoglobulins, each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The amino-terminal portion of each chain contains a variable region of approximately 100-110 amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 or more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)), incorporated by reference in its entirety for all purposes. The variable regions of each light / heavy chain pair form the antibody binding site, such that an intact immunoglobulin has two binding sites.

[0220] In one aspect, the present disclosure provides an ABP comprising a human heavy chain constant region gene segment of IGHG1*01. In some embodiments, the IGHG1*01 Fc antibody comprises an scFv. In some embodiments, the ABP is specific for CTLA-4, PD-1, PD-L1, LAG-3, CD47, TIGIT, or other antigens. In some embodiments, the ABP is specific for CTLA-4. In some embodiments, the ABP comprises an antigen-binding domain of an antibody therapeutic that is approved or under regulatory review. In some embodiments, the ABP comprises the antigen-binding domain of ipilimumab, toripalimab, amivantamab, dostallimab, cemiplimab, durvalumab, atezolizumab, or pembrolizumab.

[0221] In some embodiments, the ABP has enhanced FcR signaling or Fc effector function by including a human heavy chain constant region gene segment of IGHG1*01. Accordingly, 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*01. The present disclosure also provides a method of improving FcR signaling or Fc effector function of an ABP by introducing a human heavy chain constant region gene segment of IGHG1*01 into the ABP.

[0222] In one aspect, antigen binding proteins according to the present disclosure include antigen binding proteins that inhibit the biological activity of CTLA-4.

[0223] In some embodiments, an antigen binding protein according to the present disclosure comprises an IGHG1*01 Fc anti-CTLA-4 antibody or an antigen-binding fragment thereof that enhances FcR signaling or Fc effector function. In some embodiments, the IGHG1*01 Fc anti-CTLA-4 antibody comprises an scFv.

[0224] Different antigen-binding proteins may bind to different domains of CTLA-4 or may act by 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: amino acids at positions 4 and 12, as well as 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 of CTLA-4-binding antigen-binding proteins in treating particular diseases is by way of example only, and the methods presented herein are not constrained thereby.)

[0225] 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).

[0226] The polynucleotide and polypeptide sequences of particular light and heavy chain variable domains are described below. Antibodies comprising light and heavy chains are designated by combining the name of the light chain 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.

[0227] In other embodiments, an antibody may contain a specific 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 (such as CTLA-4) via a specific light or heavy chain, and 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., Rare, high-affinity mouse anti-CTLA-4 antibodies that function in checkpoint blockade,discovered using microfluidics and molecular genomics,MAbs(2017).

[0228] 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 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.

[0229] The term "human antibody," also referred to as "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 immunization with the antigen of interest of mice that have been genetically modified to express antibodies derived from human heavy and / or light chain-encoding genes, examples of which are described below.

[0230] 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 elicit an immune response and / or elicits a less severe immune response when administered to a human subject compared to an antibody derived from the non-human species. 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 immunogenic potential of the non-human antibody when administered to a human subject, either because the altered amino acid residues are not important for immunospecific binding of the antibody to its antigen, or because the changes made to the amino acid sequence are conservative changes such that binding of the humanized antibody to the antigen is not significantly worse than 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.

[0231] 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 in 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).

[0232] Antibody fragments or analogs can be readily prepared by those skilled in the art following the teachings of this specification using techniques well known in the art. Preferred amino and carboxy termini of fragments or analogs occur near boundaries of functional domains. Structural and functional domains can be identified by comparison of nucleotide and / or amino acid sequence data with public or proprietary sequence databases. Computerized comparison methods can be used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and / or function. Methods for identifying protein sequences that fold into known three-dimensional structures are known. See, e.g., Bowie et al., 1991, Science 253:164.

[0233] Antigen-binding fragments derived from antibodies can be obtained, for example, by proteolytic hydrolysis of antibodies, e.g., pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment called F(ab')2. This fragment can be further cleaved using a thiol reducing agent to produce a 3.5S Fab' monovalent fragment. Optionally, the cleavage reaction can be carried out using a blocking group for the sulfhydryl groups resulting from cleavage of disulfide bonds. Alternatively, enzymatic cleavage using papain directly produces two monovalent Fab fragments and one Fc fragment. These methods are described, for example, by Goldenberg, U.S. Pat. No. 4,331,647; Nisonoff et al., Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959; Edelman et al., in Methods in Enzymology 1:422 (Academic Press 1967); and Andrews, S. M. and Titus, J. A. in Current Protocols in Immunology (Coligan J. E., et al., eds), John Wiley & Sons, New York (2003), pages 2.8.1 2.8.10 and 2.10A.1 2.10A.5. Other methods for cleaving antibodies may also be used, such as separating heavy chains to form monovalent light-heavy chain fragments (Fd), further cleavage of fragments, or other enzymatic, chemical, or genetic techniques, so long as the fragment binds to the antigen recognized by the intact antibody.

[0234] An antibody fragment may also be any synthetic or genetically engineered protein, including, for example, an isolated fragment consisting of the light chain variable region, an "Fv" fragment consisting of the heavy and light chain variable regions, or a recombinant single-chain polypeptide molecule in which the light and heavy variable regions are connected by a peptide linker (scFv protein).

[0235] Another form of antibody fragment is a peptide comprising 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, either covalently or non-covalently, to make it an antigen-binding protein. CDRs can be obtained by constructing a polynucleotide encoding the CDR of interest. Such polynucleotides are prepared, for example, by using the polymerase chain reaction to synthesize 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 See Antibodies: Principles and Applications, Birch et al., (eds.), page 137 (Wiley Liss, Inc. 1995).

[0236] 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 have any size or amino acid composition and will generally comprise at least one CDR sequence involved in 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. In general terms, a variable (V) region domain is a CDR sequence that is associated with an immunoglobulin heavy (V) domain. H ) and / or light (V L ) chain variable domains can be any suitable arrangement. Thus, for example, the V region domains are monomers and can be at least 1 x 10 as described below. 7 V, which can independently bind to human CTLA-4 with an affinity of less than or equal to 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 comprise at least one V region that can be non-covalently associated. H and at least one V L chain (hereinafter, F V If desired, the chains can be covalently linked either directly, e.g., 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).

[0237] 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 particular antibody variable region by insertions, deletions, or changes in or to the amino acid sequence of the particular antibody. A particular example includes an engineered variable region domain 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.

[0238] The variable region domain may be covalently linked at the C-terminal amino acid to at least one other antibody domain or fragment thereof. H The V domain may bind to an immunoglobulin CH1 domain, or a fragment thereof. L The domain may be linked to a CK domain or a fragment thereof. Thus, for example, an antibody may comprise an antigen-binding domain covalently linked at their C-termini to the CH1 and CK domains, respectively. 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.

[0239] In some embodiments, the ABP comprises an Fc region that lacks fucose sugar units on the N-glycans.

[0240] In some embodiments, the ABP comprises a glycine at amino acid position 201.

[0241] In some embodiments, the ABP is produced from cells containing the bacterial protein RMD (GDP-6-deoxy-D-lyxo-4-hexulose reductase) or modifications 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 a fucosylation inhibitor, 2-fluorofucose (2FF).

[0242] 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.

[0243] In some embodiments, the ABP is a hypofucosylated monoclonal antibody.

[0244] 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 enhance 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 the form of a monomer, dimer, tetramer, or other form. In one embodiment, one or more water-soluble polymers are attached to one or more specific positions of the binder, e.g., the amino terminus.

[0245] In another example, individual V from an antibody (i.e., a CTLA-4 antibody) L or V H The V chain can be used to form other V antigen-binding fragments (or Fab) with the same specificity. H or V L Therefore, V H Chain 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 Parent V combined with the strand library 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 (such as radiolabeled CTLA-4). See, for example, Portolano et al., J. Immunol. V. 150(3) pp.880-887 (1993).

[0246] 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 create diabodies with two different antigen-binding sites. Similarly, tribodies and tetrabodies are antibodies that contain three and four polypeptide chains, respectively, forming three and four antigen-binding sites, which may be the same or different.

[0247] 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 Publication No. 2005 / 0238646, which are single-chain polypeptides.

[0248] 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 capabilities 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.

[0249] In some embodiments, the ABP of the present disclosure is a monoclonal antibody that binds to CTLA-4. The monoclonal antibodies of the present disclosure can be produced using a variety of known techniques. In general, monoclonal antibodies that bind to a specific antigen can be obtained by methods known to those skilled in the art (see, for example, Kohler et al., Nature 256:495, 1975; Coligan et al. (eds.), Current Protocols in Immunology, 1:2.5.12.6.7 (John Wiley & Sons 1991); U.S. Patent Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993; Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.) (1980); and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press (1988); Picksley et al., "Production of monoclonal antibodies"). (See, for example, "antibody fragments against proteins expressed in E. coli," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), page 93 (Oxford University Press 1995)). Antibody fragments can be derived therefrom using any suitable standard technique, such as proteolytic digestion, or optionally by proteolytic digestion (e.g., using papain or pepsin), followed by mild reduction of disulfide bonds and alkylation. Alternatively, such fragments can also be produced by recombinant genetic engineering techniques as described herein.

[0250] Monoclonal antibodies can be obtained by injecting animals, such as rats, hamsters, rabbits, or preferably mice, including transgenic or knockout animals known in the art, with an immunogen comprising human CTLA-4 [sequence of SEQ ID NO: 7001] or a fragment thereof, according to methods known in the art and described herein. The presence of specific antibody production can be monitored by obtaining serum samples after the initial injection and / or after booster injections and detecting the presence of antibodies that bind to human CTLA-4 or the peptide using any one of several immunodetection methods known in the art and described herein. From animals producing the desired antibodies, lymphoid cells, most commonly cells from the spleen or lymph nodes, are removed to obtain B lymphocytes. The B lymphocytes are then fused with a drug-sensitized myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal and optionally has other desirable properties (e.g., inability to express endogenous Ig gene products, e.g., P3X63-Ag 8.653 (ATCC No. CRL 1580), NSO, SP20), to produce an immortal eukaryotic cell line, a hybridoma.

[0251] Lymphoid (e.g., spleen) cells and myeloma cells are combined for several minutes with a membrane fusion promoter, such as polyethylene glycol or a non-ionic detergent, and then seeded at low density onto a selective medium that supports the growth of hybridoma cells but not unfused myeloma cells. A preferred selective medium is HAT (hypoxanthine, aminopterin, thymidine). After a sufficient period of time, usually about 1-2 weeks, cell colonies are observed. Single colonies are isolated, and antibodies produced by the cells can be tested for binding activity to human CTLA-4 using any one of a variety of immunoassays known in the art and described herein. Hybridomas are cloned (e.g., by limiting dilution cloning or soft agar plaque isolation), and positive clones producing antibodies specific to CTLA-4 are selected and cultured. Monoclonal antibodies from the hybridoma cultures can be isolated from the supernatant of the hybridoma cultures.

[0252] An alternative method for producing murine monoclonal antibodies is to inject hybridoma cells into the peritoneal cavity of syngeneic mice, e.g., mice that have been treated (e.g., pristane-primed) to promote the formation of ascites containing the monoclonal antibodies. Monoclonal antibodies can be isolated and purified by a variety of well-established techniques. Such isolation techniques include affinity chromatography with protein A Sepharose, size-exclusion chromatography, and ion-exchange chromatography (see, e.g., Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al., "Purification of Monoclonal Antibodies"). Immunoglobulin G (IgG),” in Methods in Molecular Biology, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992). Monoclonal antibodies can be purified by affinity chromatography using an appropriate ligand selected based on particular properties of the antibody (e.g., heavy or light chain isotype, binding specificity, etc.). Examples of suitable ligands immobilized on a solid support include protein A, protein G, anti-constant region (light or heavy chain) antibodies, anti-idiotypic antibodies, and TGF-beta binding proteins, or fragments or variants thereof.

[0253] 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 preferably do not produce antibodies, have high fusion efficiency, and possess enzyme deficiencies that render them unable to grow 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.

[0254] The antibodies of the present disclosure can 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.

[0255] 7.4. Nucleic acids 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, such as one or both chains of an antibody of the present disclosure, or fragments, derivatives, muteins, or variants thereof; hybridization probes; PCR or sequencing primers for identifying, analyzing, mutating, or amplifying polynucleotides encoding polypeptides; antisense nucleic acids for inhibiting expression of polynucleotides; and polynucleotides sufficient for use as complementary sequences to the foregoing. Nucleic acids can be of any length. They can be, for example, 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 can include one or more additional sequences, e.g., regulatory sequences, and / or can be part of a larger nucleic acid, e.g., a vector. Nucleic acids can be single- or double-stranded and can comprise RNA and / or DNA nucleotides, as well as artificial variants thereof (e.g., peptide nucleic acids).

[0256] Nucleic acids encoding antibody polypeptides (e.g., heavy or light chains, variable domains only, or full length) can be isolated from B cells of mice immunized with CTLA-4. Nucleic acids can be isolated by conventional procedures such as polymerase chain reaction (PCR).

[0257] 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 numerous 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.

[0258] The present disclosure further provides nucleic acids that hybridize to other nucleic acids (e.g., nucleic acids comprising any nucleotide sequence of the CTLA-4 gene) under specific hybridization conditions. Methods for hybridizing nucleic acids are well known in the art. See, for example, Curr. Prot. in Mol. Biol., John Wiley & Sons, NY (1989), 6.3.1-6.3.6. As defined herein, moderately stringent hybridization conditions use a pre-wash solution containing 5x sodium chloride / sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), a hybridization buffer of about 50% formamide, 6x SSC, and a hybridization temperature of 55°C (or other similar hybridization solutions, such as those containing about 50% formamide, and a hybridization temperature of 42°C), and wash conditions of 60°C in 0.5x SSC, 0.1% SDS. Stringent hybridization conditions are hybridization in 6×SSC at 45° C., followed by one or more washes in 0.1×SSC, 0.2% SDS at 68° C. Moreover, one of skill in the art can manipulate the hybridization and / or wash conditions to increase or decrease the stringency of hybridization, such that nucleic acids containing nucleotide sequences that are at least 65, 70, 75, 80, 85, 90, 95, 98, or 99% identical to each other will typically remain hybridized to each other.Basic parameters influencing the selection of hybridization conditions, and guidance for devising suitable conditions, are described, for example, by Sambrook, Fritsch, and Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, chapters 9 and 11, and Curr. Prot. in Mol. Biol. 1995, Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4), and can be readily determined by one of skill in the art, for example, based on the length and / or base composition of the DNA.

[0259] Changes can be introduced into a nucleic acid by mutation, thereby resulting in a change in the amino acid sequence of the encoded polypeptide (e.g., an antigen-binding protein). Mutations can be introduced using any technique known in the art. In one embodiment, one or more specific amino acid residues are altered, for example, using a site-directed mutagenesis protocol. In another embodiment, one or more randomly selected residues are altered, for example, using a random mutagenesis protocol. However generated, the mutant polypeptides can be expressed and screened for the desired property (e.g., binding to CTLA-4).

[0260] Mutations can be introduced into a nucleic acid without significantly altering the biological activity of the encoded polypeptide. For example, nucleotide substitutions resulting in amino acid substitutions at non-essential amino acid residues can be made. In one embodiment, the 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 amino acid residues indicated herein as residues at which two or more sequences for CTLA-4 differ. 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 encoded polypeptide. For example, the mutations can quantitatively or qualitatively alter the biological activity. Examples of quantitative changes include increasing, reducing, or eliminating activity. Examples of qualitative changes include altering the antigen specificity of an antigen-binding protein.

[0261] 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 only an active portion of a polypeptide of the present disclosure (e.g., a CTLA-4 binding portion).

[0262] Probes based on the sequences of the nucleic acids of the present disclosure can be used to detect nucleic acids or similar nucleic acids, such as transcripts encoding the polypeptides of the present disclosure. Probes can contain a label group, such as a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used to identify cells that express the polypeptides.

[0263] 7.5. Expression Vectors 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, e.g., recombinant expression vectors.

[0264] Another aspect of the present disclosure provides expression vectors containing the nucleic acid molecules and polynucleotides of the present disclosure, as well as 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 (or inducing the expression of) a polypeptide from a polynucleotide sequence. A vector for expressing a polypeptide contains the minimum sequences necessary for vector propagation and expression of a cloned insert. An expression vector contains a transcription unit comprising (1) one or more genetic elements, such as a promoter or enhancer, that have a regulatory role in gene expression; (2) a polypeptide and protein-encoding sequence that is transcribed into mRNA and translated into protein; and (3) an assembly of appropriate transcription start and stop 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.

[0265] 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.

[0266] Recombinant expression vectors contain one or more regulatory sequences operably linked to the nucleic acid sequence to be expressed, selected based on the host cell to be used for expression. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells (e.g., the SV40 early gene enhancer, the Rous sarcoma virus promoter, and the cytomegalovirus promoter), those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences; see Voss et al., 1986, Trends Biochem. Sci. 11:287; Maniatis et al., 1987, Science 236:1237, the entire contents of which are incorporated herein by reference), and those that direct inducible expression of a nucleotide sequence in response to a particular treatment or condition (e.g., the metallothionine promoter in mammalian cells and tet-responsive and / or streptomycin-responsive promoters in both prokaryotic and eukaryotic systems (see ibid.)). It will be understood by one of ordinary skill in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the present disclosure can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by the nucleic acids described herein.

[0267] 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 generated 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 generated by using 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.

[0268] The present disclosure further provides methods for producing polypeptides. A variety of other expression / host systems can be utilized. Vector DNA can be introduced into prokaryotic or eukaryotic systems via conventional transformation or transfection techniques. These systems include, but are not limited to, microorganisms such as bacteria (e.g., E. coli) transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors, yeast transformed with yeast expression vectors, insect cell systems infected with viral expression vectors (e.g., baculovirus), plant cell systems transfected with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems.Mammalian cells useful for recombinant protein production include VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, or derivatives thereof, such as Veggie CHO and related cell lines grown in serum-free medium (see Rasmussen et al., 1998, Cytotechnology 28:31), or the CHO line DX-B11, which is DHFR-deficient (Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216-20), COS cells, such as the monkey kidney cell line COS-7 (ATCC CRL 1651) (see Gluzman et al., 1981, Cell 23:175), W138, BHK, HepG2, 3T3 (ATCC CCL Mammalian expression includes, but is not limited to, 163), RIN, MDCK, A549, PC12, K562, L cells, C127 cells, BHK (ATCC CRL 10) cell line, the CV1 / EBNA cell line derived from the African green monkey kidney cell line CV1 (ATCC CCL 70) (see McMahan et al., 1991, EMBO J. 10:2821), human embryonic kidney cells such as 293, 293 EBNA, or MSR 293, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, primary tissues, primary explants, and cell lines derived from in vitro culture of HL-60, U937, HaK, or Jurkat cells. Mammalian expression allows for the production of secreted or soluble polypeptides that can be recovered from the growth medium.

[0269] In some embodiments, the mammalian cells used for 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 for 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 for recombinant protein production comprise CHO cells. In some embodiments, the mammalian cells are the GlymaxX® cell line. In certain embodiments, the cell line produces hypofucosylated recombinant proteins. In certain embodiments, the mammalian cells have less or reduced fucosylation, e.g., if 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 for recombinant protein production are engineered to overexpress a glycosyltransferase. In certain embodiments, the glycosyltransferase is beta-1,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase.

[0270] In some embodiments, the ABP comprises a hypofucosylated Fc region, hi some embodiments, the antibody is hypofucosylated (e.g., the N-glycans of the Fc region of the antibody do not have a core fucose sugar unit).

[0271] 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).

[0272] 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).

[0273] 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 can 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 grown 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).

[0274] The transformed cells can be cultured under conditions that promote expression of the polypeptide, and the polypeptide can be recovered by conventional protein purification procedures (defined above). One such purification procedure includes, for example, the use of affinity chromatography over a matrix having all or a portion (e.g., the extracellular domain) of CTLA-4 bound thereto. Polypeptides contemplated for use herein include substantially homogeneous recombinant mammalian anti-CTLA-4 antibody polypeptides that are substantially free from contaminating endogenous material.

[0275] In some cases, such as expression using prokaryotic systems, the expressed polypeptides of the present disclosure may need to be "refolded" and oxidized to the proper tertiary structure and generated disulfide bonds to be biologically active. Refolding can be achieved using a number of procedures well known in the art. Such methods include, for example, exposing the solubilized polypeptide to a pH typically above 7 in the presence of a chaotropic agent. The choice of chaotrope is similar to that used for inclusion body solubilization, although the chaotrope is typically used at a lower concentration. Exemplary chaotropic agents are guanidine and urea. In most cases, the refolding / oxidation solution will also contain a reducing agent and its oxidized form in a specific ratio that generates a specific redox potential that allows disulfide shuffling to occur due to the formation of cysteine ​​bridges. Some commonly used redox couples include cysteine / cystamine, glutathione / dithiobis(GSH), cupric chloride, dithiothreitol / dithiane-DTT, and 2-mercaptoethanol (bME) / dithio-bME. Often, a cosolvent can be used to increase the efficiency of refolding. Commonly used cosolvents include glycerol, polyethylene glycols of various molecular weights, and arginine.

[0276] Additionally, polypeptides can be synthesized in solution or on a solid support according to conventional techniques. A variety of automated synthesizers are commercially available and can be used according to known protocols. See, for example, Stewart and Young, Solid Phase Synthesis. See Peptide Synthesis, 2d. Ed., Pierce Chemical Co. (1984); Tam et al., J Am Chem Soc, 105:6442, (1983); Merrifield, Science 232:341-347 (1986); Barany and Merrifield, The Peptides, Gross and Meienhofer, eds., Academic Press, New York, 1-284; Barany et al., Int J Pep Protein Res, 30:705-739 (1987).

[0277] 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, at one level, involve crude fractionation of protein and non-protein fractions. After separating 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). As used herein, the term "purified polypeptide" is intended to refer to a composition in which the polypeptide has been purified to any degree relative to its naturally available state, isolatable from other components. Thus, a purified polypeptide also refers to a polypeptide 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 will refer to a peptide or polypeptide composition in which the polypeptide or peptide forms a major component of the composition, e.g., comprises about 50%, about 60%, about 70%, about 80%, about 85%, or about 90% or more of the protein in the composition.

[0278] 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, hydroxyapatite, hydrophobic interaction chromatography, isoelectric focusing, gel electrophoresis, and combinations of these techniques. As is generally known in the art, it is contemplated that the order in which the various purification steps are performed may be varied, or certain steps may be omitted, and still result in a method suitable for preparing a substantially purified polypeptide. Exemplary purification steps are provided in the Examples below.

[0279] 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 a 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 and compare it to the binding activity of the initial extract, thereby calculating the degree of purification, 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 chosen after purification and whether the polypeptide or peptide exhibits detectable binding activity.

[0280] 7.6. Methods for generating antibodies Fully human monoclonal antibodies can be produced by any number of techniques 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 (including, for example, 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, by 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; Jakobovits et al., 1995 Ann. NY Acad. Sci. 764:525-35. In this technique, elements of 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, human immunoglobulin transgenes can be minigene constructs or translocations on yeast artificial chromosomes, which undergo B cell-specific DNA rearrangements and hypermutation in mouse lymphoid tissues. Fully human monoclonal antibodies can be obtained by immunizing transgenic mice, which can then produce human antibodies specific for CTLA-4. Lymphocyte 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.

[0281] Another method for producing the human antibodies of the present disclosure involves immortalizing human peripheral blood cells by EBV transformation. See, e.g., U.S. Pat. No. 4,464,456. Such immortalized B cell lines (or lymphoblastoid cell lines) that produce monoclonal antibodies that specifically bind to CTLA-4 can be identified by immunodetection methods provided herein, e.g., ELISA, and then isolated by standard cloning techniques. The stability of lymphoblastoid cell lines that produce 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 (e.g., Glasky et al., J. Immunol. 2004, 103:111-114). (See, e.g., Boerner et al., 1991 J. Immunol. 147:86-95.) 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 fusion of the primed B cells with a heterohybrid fusion partner.

[0282] In certain embodiments, B cells producing anti-human CTLA-4 antibodies are selected, and the light and heavy chain variable regions are cloned from the B cells according to molecular biology techniques known in the art (WO92 / 02551, U.S. Patent No. 5,627,052, Babcook et al., Proc. Natl. Acad. Sci. USA 93:7843-48 (1996)) and described herein. B cells from immunized animals can be isolated from the spleen, lymph nodes, or peripheral blood samples by selecting cells producing antibodies that specifically bind to CTLA-4. B cells can also be isolated from humans, for example, from peripheral blood samples.

[0283] Methods for detecting single B cells producing antibodies with a desired specificity, for example, by plaque formation, fluorescence-activated cell sorting, in vitro stimulation followed by detection of specific antibodies, etc., are well known in the art. Methods for selecting specific antibody-producing B cells include, for example, preparing a single-cell suspension of B cells in soft agar containing human CTLA-4. Binding of the specific antibody produced by the B cells to the antigen results in the formation of a complex, which may be visible as an immunoprecipitate.

[0284] In some embodiments, specific antibody-producing B cells are selected using a method that allows for the identification of naturally paired antibodies. For example, 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), incorporated herein by reference in its entirety, can be employed. The method combines microfluidics, molecular genomics, yeast single-chain variable fragment (scFv) display, fluorescence-activated cell sorting (FACS), and deep sequencing, as summarized in Figure 1, adapted from Adler et al. Briefly, B cells can be isolated from immunized animals and then pooled. The B cells are encapsulated in droplets with oligo-dT beads and lysate, and the mRNA-bound beads are purified from the droplets and then injected into a second emulsion containing an OE-RT-PCR amplification mix, which purifies DNA amplicons encoding scFvs with the native pairing of heavy and light Ig chains. The library of naturally paired amplicons is then electroporated into yeast for scFv display. FACS is used to identify high-affinity scFvs. Finally, deep antibody sequencing can be used to identify all clones in the scFv library before and after sorting.

[0285] After B cells producing the desired antibody have been selected, 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.

[0286] Methods for obtaining antibodies of the present disclosure can also employ various phage display techniques 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. For example, a library containing a plurality of polynucleotide sequences encoding Ig variable region fragments can be inserted into the genome of a filamentous bacteriophage, such as M13 or a variant thereof, in frame with a sequence encoding a phage coat protein. The fusion protein can be a fusion of the coat protein with a light chain variable region domain and / or a heavy chain variable region domain. According to certain embodiments, immunoglobulin Fab fragments can also be displayed on phage particles (see, e.g., U.S. Patent No. 5,698,426).

[0287] Antibody fragments fused to another protein, such as a minor coat protein, can also be used to enrich for antigen-bearing phage. Rearranged heavy chain (V) fragments from mice immunized with the antigen (e.g., CTLA-4) can then be isolated. H ) and light chain (V L Using random combinatorial libraries of antibodies, 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).

[0288] 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). Briefly, mRNA is isolated from a B cell population and used to generate heavy and light chain immunoglobulin cDNA expression libraries in λImmunoZap(H) and λImmunoZap(L) vectors. These vectors can be screened individually or coexpressed to form Fab fragments or antibodies (see Huse et al., supra; see also Sastry et al., supra). Positive plaques can then be converted to non-lytic plasmids that allow high-level expression of monoclonal antibody fragments from E. coli.

[0289] 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 skilled in the art or purchased from commercially available sources (e.g., V Ha , V Hb , V Hc , V Hd , C H1, V L , and C L (See Stratagene (La Jolla, California), which sells primers for mouse and human variable regions, including primers for the V region.) These primers can be used to amplify the heavy or light chain variable regions, which can then be inserted into vectors such as ImmunoZAP™ H or ImmunoZAP™ L (Stratagene), respectively. These vectors can then be introduced into E. coli, yeast, or mammalian-based systems for expression. H and V L Large amounts of single-chain proteins containing fusions of the domains can be produced using these methods (see Bird et al., Science 242:423-426, 1988).

[0290] Once cells producing antibodies according to this disclosure are obtained using any of the above immunization and other techniques, the specific antibody gene can be cloned by isolating and amplifying DNA or mRNA therefrom according to standard procedures described herein. The antibodies produced therefrom can be sequenced, the CDRs identified, and the DNA encoding the CDRs can be manipulated as previously described to generate other antibodies according to this disclosure.

[0291] 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 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.

[0292] 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 cross-block the antibodies described herein and / or are cross-blocked from binding to CTLA-4 by one of the antibodies described in this application. The CDR regions from these antibodies are then used to insert into a suitable biocompatible framework to generate CTLA-4 binding agents. The non-CDR portion of the binding agent can be composed of amino acids or can 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.

[0293] Other antibodies according to the present disclosure may be obtained by conventional immunization and cell fusion procedures described herein and known in the art.

[0294] The molecular evolution of the complementarity-determining regions (CDRs) at the center of the antibody binding site has also been studied by Schier et al. As described by [End Page 110] et al., 1996, J. Mol. Biol. 263:551, techniques have been used to isolate antibodies with increased affinity, for example, antibodies with increased affinity for c-erbB-2. Thus, 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 to purify CTLA-4 protein by immunoaffinity chromatography.

[0295] While human, partially human, or humanized antibodies are preferred for many applications, particularly those involving administration of antibodies to human subjects, other types of antigen-binding proteins may be suitable for certain applications. Non-human antibodies of the present disclosure can be derived from any antibody-producing animal, such as, for example, mouse, rat, rabbit, goat, donkey, or non-human primate (such as a monkey (e.g., a cynomolgus or rhesus monkey) or ape (e.g., a chimpanzee)). Antibodies from a particular species can be generated, 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 generate antibodies of that species (e.g., a bacterial or phage display-based system to generate 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 another species, or by replacing one or more amino acid residues of an antibody to more closely resemble the sequence of an antibody from another species. In one embodiment, the antibody is a chimeric antibody, comprising amino acid sequences derived from antibodies from two or more different species.

[0296] Antigen binding proteins can be prepared and screened for desired properties by any of a number of conventional techniques. Particular techniques involve isolating a nucleic acid encoding the polypeptide chain (or portion thereof) of an 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 can be produced in recombinant expression systems 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).

[0297] Any expression system known in the art can be used to produce the recombinant polypeptides of the present disclosure. Expression systems have been comprehensively detailed above. Generally, a host cell is transformed with a recombinant expression vector containing DNA encoding the desired polypeptide. Among the host cells that can be used are prokaryotes, yeast, or higher eukaryotic cells. Prokaryotes include gram-negative or gram-positive organisms, such as E. coli or Bacilli. Higher eukaryotic cells include insect cells and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines 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), and the CVI / EBNA cell line, derived from the African green monkey kidney cell line CVI (ATCC CCL 70) described by McMahan et al., 1991, EMBO J. 10:2821. Suitable cloning and expression vectors for use in bacterial, fungal, yeast, and mammalian cell hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985).

[0298] 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. Accordingly, 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, typically incorporated by chemical peptide synthesis rather than synthesis in a biological system. These include peptidomimetics and other reversed 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.

[0299] Non-conservative substitutions may involve the replacement of a member of one class of amino acids or amino acid mimetics with a member from 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.

[0300] 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 destroyed, undesirably reduced, or inappropriate activity, the variant with such a change 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.

[0301] Those skilled in the art will be able to determine suitable variants of the polypeptides described herein using well-known techniques. In certain embodiments, those skilled in the art can identify suitable regions of the molecule that can be altered without destroying activity by targeting regions that are not believed to be important for activity. In certain embodiments, residues and portions of the molecule that are conserved among similar polypeptides can be identified. In certain embodiments, even regions that may be important for biological activity or structure can be subjected to conservative amino acid substitutions without destroying biological activity or adversely affecting the polypeptide structure.

[0302] In addition, one skilled in the art can review structure-function studies that identify residues in similar polypeptides that are important for activity or structure. In light of such comparisons, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues important for the activity or structure of the similar protein. One skilled in the art can select chemically similar amino acid substitutions for such predicted important amino acid residues.

[0303] One skilled in the art can also analyze the three-dimensional structure and amino acid sequence relative to that structure in similar polypeptides. Given such information, one skilled in the art can predict the alignment of amino acid residues of an antibody with respect to its three-dimensional structure. In certain embodiments, one skilled in the art may choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules.

[0304] Many scientific publications deal with the prediction of secondary structure. Moult J., Curr. Op. in Biotech., 7(4):422-427(1996), Chou et al. al.,Biochem.,13(2):222-245(1974), Chou et al.,Biochem.,113(2):211-222(1974),Chou See, e.g., Chou et al., Adv. Enzymol. Relat. Areas Mol. Biol., 47:45-148 (1978); Chou et al., Ann. Rev. Biochem., 47:251-276; and Chou et al., Biophys. J., 26:367-384 (1979). Furthermore, computer programs are now available to assist in predicting secondary structure. One method for predicting secondary structure is based on homology modeling. For example, two polypeptides or proteins with greater than 30% sequence identity or greater than 40% similarity often have similar structural topologies. The recent growth of the protein structural database (PDB) has provided enhanced predictability of secondary structure, including the number of potential folds within a polypeptide's or protein's structure. See, e.g., Holm et al., Nucl. Acid. Res., 27(1):244-247 (1999). It has been proposed that there are a limited number of folds for a given polypeptide or protein, and that once a critical number of structures are resolved, structure prediction becomes dramatically more accurate (Brenner et al., Curr. Op. Struct. Biol., 7(3):369-376 (1997)).

[0305] Additional methods for predicting secondary structure include "threading" (Jones, D., Curr. Opin. Struct. Biol., 7(3):377-87 (1997); Sippl et al., Structure, 4(1):15-19 (1996)), "profile analysis" (Bowie et al., Science, 253:164-170 (1991); Gribskov et al., Meth. Enzym., 183:146-159 (1990); Gribskov et al., Proc. Nat. Acad. Sci., 84(13):4355-4358 (1987)), and "evolutionary linkage" (see Holm, supra (1999) and Brenner, supra (1997)).

[0306] In certain embodiments, antibody variants include glycosylation variants in which the number and / or type of glycosylation sites are altered compared to the amino acid sequence of the parent polypeptide. In certain embodiments, variants contain more or fewer N-linked glycosylation sites than the native protein. N-linked glycosylation sites are characterized by the sequence Asn-X-Ser or Asn-X-Thr, where the amino acid residue designated as X can be any amino acid residue except proline. Substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions that eliminate this sequence will remove an existing N-linked carbohydrate chain. Rearrangements of N-linked carbohydrate chains are also provided in which one or more N-linked glycosylation sites (typically naturally occurring ones) are removed and one or more new N-linked sites are created. Additional preferred antibody variants include cysteine ​​variants in which one or more cysteine ​​residues are deleted from or substituted with another amino acid (e.g., serine) compared to the parent amino acid sequence. Cysteine ​​variants can be useful when antibodies need to be refolded into a biologically active conformation, such as after isolation of insoluble inclusion bodies. Cysteine ​​variants generally have fewer cysteine ​​residues than the native protein, and typically have an even number to minimize interactions due to unpaired cysteines.

[0307] Desired amino acid substitutions (conservative or non-conservative) can be determined by one of skill in the art at the time such substitutions are desired. In certain embodiments, amino acid substitutions can be used to identify key residues of antibodies to CTLA-4 or to increase or decrease the affinity of the antibodies to CTLA-4 described herein.

[0308] According to certain embodiments, preferred amino acid substitutions are those that (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity, and / or (4) confer or modify other physicochemical or functional properties to such polypeptides. According to certain embodiments, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) can be made in the naturally occurring sequence (in certain embodiments, in portions of the polypeptide outside the domains that form intermolecular contacts). In certain embodiments, conservative amino acid substitutions typically may not substantially alter the structural features of the parent sequence (e.g., the replacement amino acid should tend not to disrupt helices occurring in the parent sequence or other types of secondary structure that characterize the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W.H. Freeman and Company, New York (1984)), Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991)), and Thornton et al. Nature 354:105 (1991), each of which is incorporated herein by reference.

[0309] In certain embodiments, the antibodies of the present disclosure may be chemically conjugated to polymers, lipids, or other moieties.

[0310] The binding agent can comprise at least one of the CDRs described herein incorporated into a biocompatible framework structure. In one example, the biocompatible framework structure comprises a polypeptide or portion thereof sufficient to form a conformationally stable structural support, or framework, or scaffold, capable of displaying one or more sequences of amino acid sequences that bind to an antigen (e.g., CDRs, variable regions, etc.) at localized surface regions. Such structures can be naturally occurring polypeptides or polypeptide "folds" (structural motifs) or can have one or more modifications, such as additions, deletions, or substitutions of amino acids, compared to naturally occurring polypeptides or folds. These scaffolds can be derived from polypeptides of any species (or multiple species), such as humans, other mammals, other vertebrates, invertebrates, plants, bacteria, or viruses.

[0311] Typically, biocompatible framework structures are based on protein scaffolds or skeletons other than immunoglobulin domains, e.g., those based on fibronectin, ankyrin, lipocalin, neocarzinostein, cytochrome b, CP1 zinc finger, PST1, coiled coil, LACI-D1, Z domain, and tendamistat domain can be used (see, e.g., Nygren and Uhlen, 1997, Curr. Opin. in Struct. Biol., 7, 463-469).

[0312] Humanized antibodies can be produced using techniques known to those skilled in the art (Zhang, W., et al., Molecular Immunology. 42(12):1445-1451, 2005; Hwang W. et al., Methods. 36(1):35-42, 2005; Dall'Acqua WF, et al., Methods 36(1):43-60, 2005; and Clark, M., Immunology Today. 21(8):397-402, 2000).

[0313] Additionally, those skilled in the art will recognize that suitable binding agents include portions of these antibodies, such as one or more of CDR1-L1-28 having SEQ ID NOS: 1001-1028, CDR2-L1-28 having SEQ ID NOS: 2001-2028, CDR3-L1-28 having SEQ ID NOS: 3001-3028, CDR1-H1-28 having SEQ ID NOS: 4001-4028, CDR2-H1-28 having SEQ ID NOS: 5001-5028, and CDR3-H1-28 having SEQ ID NOS: 6001-6028, as specifically disclosed herein. At least one of the CDR regions may have at least one amino acid substitution from the sequence provided herein, provided that the antibody retains the binding specificity of the unsubstituted CDR. The non-CDR portion of the antibody may be a non-protein molecule, and the binding agent cross-blocks binding of the antibodies disclosed herein to CTLA-4 and / or neutralizes CTLA-4. The non-CDR portion of the antibody can be a non-protein molecule, and the antibody exhibits a binding pattern to a human CTLA-4 peptide similar to that exhibited by at least one of antibodies A1-A28 in a competitive binding assay and / or neutralizes CTLA-4. The non-CDR portion of the antibody can be composed of amino acids, and the antibody is a recombinant binding protein or a synthetic peptide, and the recombinant binding protein cross-blocks the binding of the antibodies disclosed herein to CTLA-4 and / or neutralizes CTLA-4. The non-CDR portion of the antibody can be composed of amino acids, and the antibody is a recombinant antibody, and the recombinant antibody exhibits a binding pattern to a human CTLA-4 peptide similar to that exhibited by at least one of antibodies A1-A28 in a human CTLA-4 peptide epitope competitive binding assay (described below) and / or neutralizes CTLA-4.

[0314] When an antibody contains one or more of the above-described CDR1-H, CDR2-H, CDR3-H, CDR1-L, CDR2-L, and CDR3-L, it can be obtained by expression from a host cell containing DNA encoding these sequences. DNA encoding each CDR sequence can be determined based on the amino acid sequence of the CDR and, if necessary, can be synthesized together with any desired antibody variable region framework and constant region DNA sequences using oligonucleotide synthesis techniques, site-directed mutagenesis, and polymerase chain reaction (PCR) techniques. DNA encoding variable region frameworks and constant regions is widely available to those skilled in the art from gene sequence databases such as GenBank®.

[0315] Once synthesized, DNA encoding the antibodies or fragments thereof of the present disclosure can be propagated and expressed using any number of known expression vectors according to any of a variety of well-known procedures for nucleic acid excision, ligation, transformation, and transfection. Thus, in certain embodiments, expression of antibody fragments may be preferred in prokaryotic hosts such as Escherichia coli (see, e.g., Pluckthun et al., 1989 Methods Enzymol. 178:497-515). In certain other embodiments, expression of antibodies or fragments thereof may be preferred in eukaryotic host cells, including yeast (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris), animal cells (including mammalian cells), or plant cells. Examples of suitable animal cells include, but are not limited to, myeloma (such as the murine NSO strain), COS, CHO, or hybridoma cells. Examples of plant cells include tobacco, corn, soybean, and rice cells.

[0316] One or more replicable expression vectors containing DNA encoding antibody variable and / or constant regions can be prepared and used to transform a suitable cell line, e.g., a non-producing myeloma cell line such as the mouse NSO strain, or bacteria such as E. coli, in which antibody production will occur. To obtain efficient transcription and translation, the DNA sequence in each vector should contain appropriate regulatory sequences, particularly a promoter and leader sequence operably linked to the variable domain sequence. Specific methods for producing antibodies in this manner are generally well known and routinely used. For example, basic molecular biology procedures are described in Maniatis et al. al. (Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, New York, 1989; Maniatis et al., 3rd ed., Cold Spring Harbor Laboratory, New York, (2001). DNA sequencing can be performed as described in Sanger et al. (PNAS 74:5463, (1977)) and Amersham International plc sequencing handbook, and site-directed mutagenesis can be performed according to methods known in the art (Kramer et al., Nucleic Acids Res. 12:9441, (1984); Kunkel Proc. Natl. Acad. Sci. USA 82:488-92 (1985); Kunkel et al., Methods in Enzymol. 154:367-82 (1987); the Anglian Biotechnology Ltd. handbook). In addition, numerous publications describe techniques suitable for manipulating DNA, constructing expression vectors, and preparing antibodies by transforming and culturing suitable cells (Mountain A and Adair, JR in Biotechnology and Genetic Engineering Reviews (ed. Tombs, MP, 10, Chapter 1, 1992, Intercept, Andover, UK); "Current Protocols in Molecular Biology", 1999, FMA Usubel (ed.), Wiley Interscience, New York).

[0317] If it is desired to improve the affinity of an antibody according to the present disclosure, including one or more of the above CDRs can be achieved by maintaining the CDRs (Yang et al., J. Mol. Biol., 254, 392-403, 1995), chain shuffling (Marks et al., Bio / Technology, 10, 779-783, 1992), using mutant strains of E. coli (Low et al., J. Mol. Biol., 250, 350-368, 1996), DNA shuffling (Patten et al., Curr. Opin. Biotechnol., 8, 724-733, 1997), phage display (Thompson et al., J. Mol. Biol., 256, 7-88, 1996), and sexual PCR (Crameri, et al., J. Mol. Biol., 256, 7-88, 1996). These affinity maturation methods are discussed in detail in Vaughan et al. (Nature Biotech., 16, 535-539, 1998).

[0318] It will be understood by those skilled in the art that some proteins, such as antibodies, can undergo various post-translational modifications. The type and extent of these modifications often depend on the host cell line and culture conditions used to express the protein. Such modifications can include variations in glycosylation, methionine oxidation, diketopiperidine formation, aspartate isomerization, and asparagine deamidation. A frequent modification is the loss of a carboxy-terminal basic residue (such as lysine or arginine) by the action of carboxypeptidases (as described in Harris, RJ Journal of Chromatography 705:129-134, 1995).

[0319] Arrays Antibodies A1 to A28 comprise heavy and light chain V(J)D polynucleotides (also referred to herein as L1 to L28 and H1 to H28, respectively). Antibodies A1 to A28 comprise the sequences listed in Table 5. For example, antibody A1 comprises a light chain L1 (SEQ ID NO: 1) and a heavy chain H1 (SEQ ID NO: 101). The CDR sequences in the light chains (L1 to L28) and heavy chains (H1 to H28) are also provided by the specific SEQ ID NOs. For example, the three CDR sequences of L1 (CDR1, CDR2, and CDR3) are CDR1-L1 (sequence number 1001), CDR2-L1 (sequence number 2001), and CDR3-L1 (sequence number 3001), respectively, and the three CDR sequences of H1 (CDR1, CDR2, and CDR3) are CDR1-H1 (sequence number 4001), CDR2-H1 (sequence number 5001), and CDR3-H1 (sequence number 6001). [Table 5-1] [Table 5-2] [Table 5-3]

[0320] Pharmaceutical Compositions Pharmaceutical compositions containing the proteins and polypeptides of the present disclosure are also provided. Specifically, the present disclosure provides pharmaceutical compositions comprising anti-CTLA ABPs. In some embodiments, the pharmaceutical compositions comprise GIGA-564. In some embodiments, the pharmaceutical compositions comprise GIGA-2328. Such compositions comprise a therapeutically or prophylactically effective amount of the polypeptide or protein in admixture with pharmaceutically acceptable and physiologically acceptable formulation materials.

[0321] Pharmaceutical compositions may include formulation materials to modify, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution rate or release rate, adsorption or penetration of the composition.

[0322] Suitable formulation materials include amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrate, phosphate, other organic acids, etc.); bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colors; flavorings and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (such as glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as Pluronic®, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, Triton, tromethamine, lecithin, cholesterol, tyloxapal, etc.); stability enhancers (sucrose or sorbitol); isotonicity enhancers (such as alkali metal halides (preferably sodium chloride or potassium chloride, mannitol, sorbitol)); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. Neutral buffered saline or saline mixed with conspecific serum albumin are examples of suitable diluents. Preservatives such as benzyl alcohol may also be added in accordance with appropriate industry standards. The composition may be formulated as a lyophilizate using appropriate excipient solutions (e.g., sucrose) as diluents. Suitable ingredients are non-toxic to recipients at the dosages and concentrations used. Further examples of ingredients that may be used in pharmaceutical formulations are found in Remington's Pharmaceutical Sciences, 16th Ed.(1980)and 20 th Ed. (2000), Mack Publishing Company, Easton, PA.

[0323] In some embodiments, less than 50% of the ABPs in the pharmaceutical composition are fucosylated. In some embodiments, less than 40% of the ABPs in the pharmaceutical composition are fucosylated. In some embodiments, less than 30% of the ABPs in the pharmaceutical composition are fucosylated. In some embodiments, less than 20% of the ABPs in the pharmaceutical composition are fucosylated. In some embodiments, less than 10% of the ABPs in the pharmaceutical composition are fucosylated.

[0324] In some embodiments, greater than 99% of the ABP in the pharmaceutical composition are fucosylated. In some embodiments, greater than 95% of the ABP in the pharmaceutical composition are fucosylated. In some embodiments, greater than 90% of the ABP in the pharmaceutical composition are fucosylated. In some embodiments, greater than 85% of the ABP in the pharmaceutical composition are fucosylated. In some embodiments, greater than 80% of the ABP in the pharmaceutical composition are fucosylated. In some embodiments, greater than 75% of the ABP in the pharmaceutical composition are fucosylated. In some embodiments, greater than 70% of the ABP in the pharmaceutical composition are fucosylated. In some embodiments, greater than 65% of the ABP in the pharmaceutical composition are fucosylated. In some embodiments, greater than 60% of the ABP in the pharmaceutical composition are fucosylated. In some embodiments, greater than 50% of the ABP in the pharmaceutical composition are fucosylated. In some embodiments, greater than 40% of the ABP in the pharmaceutical composition are fucosylated. In some embodiments, more than 30% of the ABPs in the pharmaceutical composition are fucosylated. In some embodiments, more than 20% of the ABPs in the pharmaceutical composition are fucosylated. In some embodiments, more than 10% of the ABPs in the pharmaceutical composition are fucosylated.

[0325] In some embodiments, 30-70% of the ABP in the pharmaceutical composition are fucosylated. In some embodiments, 20-50% of the ABP in the pharmaceutical composition are fucosylated. In some embodiments, 10-40% of the ABP in the pharmaceutical composition are fucosylated. In some embodiments, 10-30% of the ABP in the pharmaceutical composition are fucosylated. In some embodiments, 5-20% of the ABP in the pharmaceutical composition are fucosylated. In some embodiments, 1-10% of the ABP in the pharmaceutical composition are fucosylated.

[0326] In some embodiments, the pharmaceutical formulation comprises one or more of histidine buffer, citrate buffer, sucrose, sodium chloride, succinate, polysorbate 20, and polysorbate-80. In certain embodiments, the pharmaceutical formulation comprises 1-20 mM histidine or citrate buffer. In certain embodiments, the pharmaceutical formulation comprises 100-350 mM sucrose. In certain embodiments, the pharmaceutical formulation comprises 0-75 mM sucrose. In certain embodiments, the pharmaceutical formulation comprises 0.002-0.1% by weight polysorbate-20. In certain embodiments, the pharmaceutical formulation comprises 0.002-0.1% by weight polysorbate-80. In certain embodiments, the pharmaceutical formulation comprises 20 mM citrate or histidine, 170-270 mM sucrose, 0-50 mM sodium chloride, and 0.02 wt% polysorbate-20. In certain embodiments, the pharmaceutical formulation comprises 20 mM citrate or histidine, 170-270 mM sucrose, 0-50 mM sodium chloride, and 0.02 wt% polysorbate-80.

[0327] 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 5.5 to 6.5.

[0328] In some embodiments, the pharmaceutical composition comprises 20 mM histidine or citrate buffer.

[0329] In some embodiments, the pharmaceutical composition comprises 50 mM NaCl.

[0330] In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of 170 mM to 270 mM.

[0331] In some embodiments, the pharmaceutical composition comprises 170 mM or 270 mM sucrose.

[0332] In some embodiments, the pharmaceutical composition comprises 20 mg / mL of ABP. In some embodiments, the pharmaceutical composition comprises 5 mg / mL of ABP. In some embodiments, the pharmaceutical composition comprises 5 mg / mL to 20 mg / mL of ABP.

[0333] In some embodiments, the pharmaceutical composition comprises trehalose.

[0334] In some embodiments, the pharmaceutical composition comprises about 0.04-30 mg / ml of ABP, 0.9% Sodium Chloride Injection, USP, or 5% Dextrose Injection, USP.

[0335] In some embodiments, the ABP is formulated at a concentration of 1 mg / ml to 80 mg / ml, hi certain embodiments, the ABP is formulated at a concentration of 5 mg / ml to 20 mg / ml.

[0336] In some embodiments, the pharmaceutical composition comprises a pH of about 5 to 7. In some embodiments, the pharmaceutical formulation comprises a pH of about 5.0 to 6.5. In some embodiments, the pharmaceutical formulation comprises a pH of about 5.0, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.

[0337] Optionally, the composition additionally comprises one or more bioactive agents, e.g., an anti-angiogenic agent, a chemotherapeutic agent (such as capecitabine, 5-fluorouracil, or doxorubicin), an analgesic agent, etc., non-exclusive examples of which are provided herein. In various specific embodiments, the composition comprises 1, 2, 3, 4, 5, or 6 bioactive agents in addition to the CTLA-4 binding protein.

[0338] In another embodiment of the present disclosure, the compositions disclosed herein can be formulated in a neutral or salt form. Exemplary pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) and are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount.

[0339] Carriers can further include any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to humans.

[0340] The optimal pharmaceutical composition will be determined by one of skill in the art depending, for example, on the intended route of administration, delivery format, and desired dosage. See, e.g., Remington's Pharmaceutical Sciences, supra. Such compositions can influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the polypeptide. For example, a suitable composition can be water for injection or physiological saline for parenteral administration.

[0341] 7.8.1. Pharmaceutically Active Ingredient Content In typical embodiments, the active ingredient (i.e., proteins and polypeptides, ABPs of the present disclosure) is present in the pharmaceutical composition at a concentration of at least 0.01 mg / ml, at least 0.005 mg / ml, at least 0.004 mg / ml, at least 0.05 mg / ml, 0.04 mg / ml, 0.1 mg / ml, at least 0.5 mg / ml, or at least 1 mg / ml. In certain embodiments, the active ingredient is present in the pharmaceutical composition at a concentration of at least 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, 25 mg / ml, or 30 mg / ml. In certain embodiments, the active ingredient is present in the pharmaceutical composition at a concentration of at least 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, 50 mg / ml, 55 mg / ml, 60 mg / ml, 65 mg / ml, 70 mg / ml, 75 mg / ml, 80 mg / ml, 85 mg / ml, 90 mg / ml, 95 mg / ml, or 100 mg / ml. In certain embodiments, the active ingredient is present in the pharmaceutical composition at a concentration ranging from 1 mg / ml to 80 mg / ml. In certain embodiments, the active ingredient is present in the pharmaceutical composition at a concentration ranging from 5 mg / ml to 20 mg / ml. In certain embodiments, the active ingredient is present in the pharmaceutical composition at a concentration ranging from 0.04 mg / ml to 30 mg / ml.

[0342] In some embodiments, the pharmaceutical composition comprises, in addition to a protein or polypeptide of the present disclosure, one or more additional active ingredients, which may be drugs that target different checkpoint receptors, such as a PD-1 inhibitor (e.g., an anti-PD-1 antibody), a PD-L1 inhibitor, a LAG-3 inhibitor, a CD47 inhibitor, or a TIGIT inhibitor (e.g., an anti-TIGIT antibody).

[0343] 7.8.2. General Formulation Pharmaceutical compositions may be in any form suitable for human or veterinary medicine, including liquids, oils, emulsions, gels, colloids, aerosols, or solids.

[0344] The pharmaceutical compositions can be formulated for administration by any route of administration suitable for human or veterinary medicine, including enteral and parenteral routes of administration.

[0345] In various embodiments, the pharmaceutical composition is formulated for administration by inhalation. In certain of these embodiments, the pharmaceutical composition is formulated for administration by a vapor inhaler. In certain of these embodiments, the pharmaceutical composition is formulated for administration by a nebulizer. In certain of these embodiments, the pharmaceutical composition is formulated for administration by an aerosol generator.

[0346] In various embodiments, the pharmaceutical compositions are formulated for oral, buccal, or sublingual administration.

[0347] In some embodiments, the pharmaceutical compositions are formulated for intravenous, intramuscular, or subcutaneous administration.

[0348] In some embodiments, the pharmaceutical compositions are formulated for intrathecal or intracerebroventricular administration.

[0349] In some embodiments, the pharmaceutical composition is formulated for injection.

[0350] 7.8.3. Pharmaceutical Compositions Adapted for Injection For intravenous, cutaneous, or subcutaneous injection, or injection at the affected site, the active ingredient will be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity, and stability. Those skilled in the art are well able to prepare suitable solutions using isotonic vehicles such as, for example, sodium chloride injection, Ringer's injection, lactated Ringer's injection, etc. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as necessary.

[0351] In various embodiments, the unit dosage form is a vial, an ampoule, a bottle, or a pre-filled syringe. In some embodiments, the unit dosage form contains 0.005 mg, 0.05 mg, 0.01 mg, 0.1 mg, 0.5 mg, 1 mg, 2.5 mg, 5 mg, 10 mg, 12.5 mg, 25 mg, 50 mg, 75 mg, or 100 mg of the pharmaceutical composition. In some embodiments, the unit dosage form contains 125 mg, 150 mg, 175 mg, or 200 mg of the pharmaceutical composition. In some embodiments, the unit dosage form contains 250 mg of the pharmaceutical composition.

[0352] In typical embodiments, the pharmaceutical composition in the unit dosage form is in liquid form. In various embodiments, the unit dosage form contains 0.1 mL to 50 mL of the pharmaceutical composition. In some embodiments, the unit dosage form contains 1 mL, 2.5 mL, 5 mL, 7.5 mL, 10 mL, 25 mL, or 50 mL of the pharmaceutical composition.

[0353] In certain embodiments, the unit dosage form is a vial containing 1 ml of pharmaceutical composition at a concentration of 0.01 mg / ml, 0.1 mg / ml, 0.5 mg / ml, or 1 mg / ml, hi some embodiments, the unit dosage form is a vial containing 2 ml of pharmaceutical composition at a concentration of 0.01 mg / ml, ...

Claims

[Claim 1] The invention described in the specification.

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