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

Abstract

Antigen binding proteins (ABPs) that selectively bind to CTLA-4 and its subtypes and homologs, and compositions comprising the ABPs are provided herein. Methods of using the ABPs, such as therapeutic and diagnostic methods, are also provided.

Description

[0001] 1. Cross-reference to related applications

[0002] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 785,659, filed December 27, 2018, the entire contents of which are incorporated herein by reference.

[0003] 2. Sequence Listing

[0004] This application contains a sequence listing of 11,998 sequences, which has been submitted via EFS-Web, the entire contents of which are incorporated herein by reference. The ASCII copy was created on December 20, 2019, is named GGN-010WO_SL.txt, and is 1,927,908 bytes in size. 3. Technical field

[0006] 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 preparing CTLA-4 ABPs and methods of using CTLA-4 ABPs, e.g., for therapeutic, diagnostic, and research purposes. 4. Background Technology

[0008] CTLA-4, also known as cytotoxic T lymphocyte-associated protein 4 and CD152 (cluster of differentiation 152), is a cell surface receptor that inhibits the inflammatory activity of T cells. CTLA-4 is constitutively expressed by regulatory T cells (Tregs) and is upregulated in stimulated T cells. CD80 and CD86, 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 the immune response and promoting self-tolerance by inhibiting the inflammatory activity of T cells. This activity can prevent autoimmune diseases and block the immune system from killing cancer cells.

[0009] CTLA-4, a member of the immunoglobulin superfamily, is expressed by activated T cells and transmits inhibitory signals to them. CTLA-4 binds to CD80 and CD86 with higher affinity and avidity than CD28, enabling it to outcompete CD28 for its ligands. CTLA-4 transmits inhibitory signals to T cells, whereas CD28 transmits stimulatory signals. CTLA-4 is also present on regulatory T cells (Tregs) and contributes to their suppressive function. Activation of T cells via the T cell receptor and CD28 leads to increased CTLA-4 expression. The mechanism of action of CTLA-4 in T cells remains controversial. Biochemical evidence suggests that CTLA-4 recruits phosphatases to the T cell receptor (TCR), thereby attenuating signaling. Since its initial publication, this work has remained unconfirmed in the literature. Recent work suggests that CTLA-4 may function in vivo by capturing and removing B7-1 and B7-2 from the membranes of antigen-presenting cells, rendering them unavailable for CD28 triggering.

[0010] Variants of CTLA-4 are 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. CTLA-4's high binding affinity for CD80 and CD86 makes it a potential therapy for autoimmune diseases. Soluble fusion proteins and antibodies to CTLA-4 (CTLA-4-Ig) have been used in clinical trials for rheumatoid arthritis.

[0011] Tumor cells suppress anti-tumor immune responses through various mechanisms, including upregulation of Tregs. Recently, CTLA-4 inhibitors have been shown to antagonize the binding of CTLA-4 to its ligands, thereby activating the immune system to attack tumors. CTLA-4 antibodies have also been used to induce antibody-dependent cell-mediated cytotoxicity (ADCC) of Tregs specific to the tumor microenvironment, thereby reducing immune tolerance to tumors. As a result, CTLA-4 antibodies have achieved varying degrees of success in treating certain types of cancer.

[0012] Therefore, there is a need to develop CTLA-4 ABPs that can be used to treat, diagnose, and study various diseases, including cancer and autoimmune diseases. 5. Summary of the Invention

[0014] Provided herein are novel ABPs with binding specificity for CTLA-4 and methods of using such ABPs. The CTLA-4 is human CTLA-4 (SEQ ID: 7001) or a fragment of human CTLA-4.

[0015] The ABP may comprise an antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the ABP comprises an antibody fragment. In some embodiments, the ABP comprises an alternative scaffold. In some embodiments, the ABP comprises a single-chain variable fragment (scFv).

[0016] The ABPs provided herein can induce various biological effects associated with the inhibition of CTLA-4. In some embodiments, the ABPs provided herein prevent the binding between CTLA-4 and its ligand. In some embodiments, the ABPs provided herein prevent the inhibition of effector T cells by Treg. In some embodiments, the ABPs directly kill or induce the killing of Treg or other CTLA-4 expressing cells in the tumor microenvironment through ADCC and / or ADCP, for example, by the binding mediation of CD16 expressed by NK cells and the ABP Fc domain. In some embodiments, the ABPs inhibit the inhibition of effector T cells by regulatory T cells by directly killing Treg. In some embodiments, the tissue is a tumor. In some embodiments, the ABPs activate CTLA-4, resulting in Treg expression and activation.

[0017] Also provided are kits comprising one or more pharmaceutical compositions comprising an ABP and instructions for use of the pharmaceutical compositions.

[0018] Also provided are isolated polynucleotides encoding the ABPs provided herein, and portions thereof.

[0019] Vectors comprising such polynucleotides are also provided.

[0020] Recombinant host cells comprising such polynucleotides and recombinant host cells comprising such vectors are also provided.

[0021] Also provided are methods of producing ABP using the polynucleotides, vectors, or host cells provided herein.

[0022] Also provided are pharmaceutical compositions comprising the ABP and a pharmaceutically acceptable excipient.

[0023] Also provided are methods for treating or preventing a disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of an ABP as provided herein, or a pharmaceutical composition comprising such an ABP. In some aspects, the disease or condition is cancer or an autoimmune disease. In some aspects, the disease or condition is a viral or bacterial infection. In some aspects, the method further comprises administering one or more additional therapeutic agents. In some aspects, the additional therapeutic agent is an immunostimulant.

[0024] More specifically, the present disclosure provides an isolated antigen binding protein (ABP) that specifically binds to human cytotoxic T lymphocyte-associated protein 4 (CTLA-4), the ABP comprising: (a) a CDR3-L having a sequence selected from the group consisting of SEQ ID NOs: 3001-3028, and a CDR3-H having a sequence selected from the group consisting of SEQ ID NOs: 6001-6028; or (b) a CDR3-L having a sequence selected from the group consisting of SEQ ID NOs: 9984-10479, and a CDR3-H having a sequence selected from the group consisting of SEQ ID NOs: 11472-11967; or (c) a CDR3-L having the sequence of CD3-L of any clone in the library deposited with ATCC Accession No. PTA-125512, and a CDR3-L having the sequence of CD3-L of any clone in the library deposited with ATCC Accession No. PTA-125512. In some embodiments, the CDR3-L and the CDR3-H are a cognate pair.

[0025] In some embodiments, the ABP comprises (a) a CDR1-L having a sequence selected from the group consisting of SEQ ID NOs: 1001-1028, and a CDR2-L having a sequence selected from the group consisting of SEQ ID NOs: 2001-2028; and a CDR1-H having a sequence selected from the group consisting of SEQ ID NOs: 4001-4028; and a CDR2-H having a sequence selected from the group consisting of SEQ ID NOs: 5001-5028; or (b) a CDR1-L having a sequence selected from the group consisting of SEQ ID NOs: 8992-9487; and a CDR2-L having a sequence selected from the group consisting of SEQ ID NOs: 9488-9983; and a CDR1-H having a sequence selected from the group consisting of SEQ ID NOs: 10480-10975; and a CDR2-H having a sequence selected from the group consisting of SEQ ID NOs: 10481-10976. NO:10976-11471; or (c) CDR1-L having the sequence of CDR1-L of any clone in the library deposited with ATCC Accession No. PTA-125512; and CDR2-L having the sequence of CDR2-L of any clone in the library deposited with ATCC Accession No. PTA-125512; and CDR1-H having the sequence of CDR1-H of any clone in the library deposited with ATCC Accession No. PTA-125512; and CDR2-H having the sequence of CDR2-H of any clone in the library deposited with ATCC Accession No. PTA-125512.

[0026] In some embodiments, the ABP comprises CDR1-L, CDR2-L, CDR3-L, CDR1-H, CDR2-H, and CDR3-H, wherein the CDR1-L consists of SEQ ID NO: 1001, the CDR2-L consists of SEQ ID NO: 2001, the CDR3-L consists of SEQ ID NO: 3001, the CDR1-H consists of SEQ ID NO: 4001, the CDR2-H consists of SEQ ID NO: 5001, and the CDR3-H consists of SEQ ID NO: 6001; or the CDR1-L consists of SEQ ID NO: 1002, the CDR2-L consists of SEQ ID NO: 2002, the CDR3-L consists of SEQ ID NO: 3002, the CDR1-H consists of SEQ ID NO: 4002, the CDR2-H consists of SEQ ID NO: 5002, and the CDR3-H consists of SEQ ID NO: NO: 6002; or the CDR1-L consists of SEQ ID NO: 1003, the CDR2-L consists of SEQ ID NO: 2003, the CDR3-L consists of SEQ ID NO: 3003, the CDR1-H consists of SEQ ID NO: 4003, the CDR2-H consists of SEQ ID NO: 5003, and the CDR3-H consists of SEQ ID NO: 6003; or the CDR1-L consists of SEQ ID NO: 1004, the CDR2-L consists of SEQ ID NO: 2004, the CDR3-L consists of SEQ ID NO: 3004, the CDR1-H consists of SEQ ID NO: 4004, the CDR2-H consists of SEQ ID NO: 5004, and the CDR3-H consists of SEQ ID NO: 6004; or the CDR1-L consists of SEQ ID NO: 1005, the CDR2-L consists of SEQ ID NO: NO: 2005, the CDR3-L consists of SEQ ID NO: 3005, the CDR1-H consists of SEQ ID NO: 4005, the CDR2-H consists of SEQ ID NO: 5005, and the CDR3-H consists of SEQ ID NO: 6005; or the CDR1-L consists of SEQ ID NO: 1006, the CDR2-L consists of SEQ ID NO: 2006, the CDR3-L consists of SEQ ID NO: 3006, the CDR1-H consists of SEQ ID NO: 4006, the CDR2-H consists of SEQ ID NO: 5006, and the CDR3-H consists of SEQ ID NO: 6006;or the CDR1-L consists of SEQ ID NO: 1007, the CDR2-L consists of SEQ ID NO: 2007, the CDR3-L consists of SEQ ID NO: 3007, the CDR1-H consists of SEQ ID NO: 4007, the CDR2-H consists of SEQ ID NO: 5007, and the CDR3-H consists of SEQ ID NO: 6007; or the CDR1-L consists of SEQ ID NO: 1008, the CDR2-L consists of SEQ ID NO: 2008, the CDR3-L consists of SEQ ID NO: 3008, the CDR1-H consists of SEQ ID NO: 4008, the CDR2-H consists of SEQ ID NO: 5008, and the CDR3-H consists of SEQ ID NO: 6008; or the CDR1-L consists of SEQ ID NO: 1009, the CDR2-L consists of SEQ ID NO: 5010, and the CDR3-H consists of SEQ ID NO: 6011; NO: 2009, the CDR3-L consists of SEQ ID NO: 3009, the CDR1-H consists of SEQ ID NO: 4009, the CDR2-H consists of SEQ ID NO: 5009, and the CDR3-H consists of SEQ ID NO: 6009; or the CDR1-L consists of SEQ ID NO: 1010, the CDR2-L consists of SEQ ID NO: 2010, the CDR3-L consists of SEQ ID NO: 3010, the CDR1-H consists of SEQ ID NO: 4010, the CDR2-H consists of SEQ ID NO: 5010, and the CDR3-H consists of SEQ ID NO: 6010; or the CDR1-L consists of SEQ ID NO: 1011, the CDR2-L consists of SEQ ID NO: 2011, the CDR3-L consists of SEQ ID NO: 3011, the CDR1-H consists of SEQ ID NO: NO:4011, the CDR2-H consists of SEQ ID NO:5011, and the CDR3-H consists of SEQ ID NO:6011; or the CDR1-L consists of SEQ ID NO:1012, the CDR2-L consists of SEQ ID NO:2012, the CDR3-L consists of SEQ ID NO:3012, the CDR1-H consists of SEQ ID NO:4012, the CDR2-H consists of SEQ ID NO:5012, and the CDR3-H consists of SEQ ID NO:6012;or the CDR1-L consists of SEQ ID NO: 1013, the CDR2-L consists of SEQ ID NO: 2013, the CDR3-L consists of SEQ ID NO: 3013, the CDR1-H consists of SEQ ID NO: 4013, the CDR2-H consists of SEQ ID NO: 5013, and the CDR3-H consists of SEQ ID NO: 6013; or the CDR1-L consists of SEQ ID NO: 1014, the CDR2-L consists of SEQ ID NO: 2014, the CDR3-L consists of SEQ ID NO: 3014, the CDR1-H consists of SEQ ID NO: 4014, the CDR2-H consists of SEQ ID NO: 5014, and the CDR3-H consists of SEQ ID NO: 6014; or the CDR1-L consists of SEQ ID NO: 1015, the CDR2-L consists of SEQ ID NO: 1016, the CDR2-H consists of SEQ ID NO: 1017, the CDR3-L consists of SEQ ID NO: 1018, the CDR2- NO: 2015, the CDR3-L consists of SEQ ID NO: 3015, the CDR1-H consists of SEQ ID NO: 4015, the CDR2-H consists of SEQ ID NO: 5015, and the CDR3-H consists of SEQ ID NO: 6015; or the CDR1-L consists of SEQ ID NO: 1016, the CDR2-L consists of SEQ ID NO: 2016, the CDR3-L consists of SEQ ID NO: 3016, the CDR1-H consists of SEQ ID NO: 4016, the CDR2-H consists of SEQ ID NO: 5016, and the CDR3-H consists of SEQ ID NO: 6016; or the CDR1-L consists of SEQ ID NO: 1017, the CDR2-L consists of SEQ ID NO: 2017, the CDR3-L consists of SEQ ID NO: 3017, the CDR1-H consists of SEQ ID NO: NO:4017, the CDR2-H consists of SEQ ID NO:5017, and the CDR3-H consists of SEQ ID NO:6017; or the CDR1-L consists of SEQ ID NO:1018, the CDR2-L consists of SEQ ID NO:2018, the CDR3-L consists of SEQ ID NO:3018, the CDR1-H consists of SEQ ID NO:4018, the CDR2-H consists of SEQ ID NO:5018, and the CDR3-H consists of SEQ ID NO:6018;Or the CDR1-L consists of SEQ ID NO: 1019, the CDR2-L consists of SEQ ID NO: 2019, the CDR3-L consists of SEQ ID NO: 3019, the CDR1-H consists of SEQ ID NO: 4019, the CDR2-H consists of SEQ ID NO: 5019, and the CDR3-H consists of SEQ ID NO: 6019; or the CDR1-L consists of SEQ ID NO: 1020, the CDR2-L consists of SEQ ID NO: 2020, the CDR3-L consists of SEQ ID NO: 3020, the CDR1-H consists of SEQ ID NO: 4020, the CDR2-H consists of SEQ ID NO: 5020, and the CDR3-H consists of SEQ ID NO: 6020; or the CDR1-L consists of SEQ ID NO: 1021, the CDR2-L consists of SEQ ID NO: 5021 NO: 2021, the CDR3-L consists of SEQ ID NO: 3021, the CDR1-H consists of SEQ ID NO: 4021, the CDR2-H consists of SEQ ID NO: 5021, and the CDR3-H consists of SEQ ID NO: 6021; or the CDR1-L consists of SEQ ID NO: 1022, the CDR2-L consists of SEQ ID NO: 2022, the CDR3-L consists of SEQ ID NO: 3022, the CDR1-H consists of SEQ ID NO: 4022, the CDR2-H consists of SEQ ID NO: 5022, and the CDR3-H consists of SEQ ID NO: 6022; or the CDR1-L consists of SEQ ID NO: 1023, the CDR2-L consists of SEQ ID NO: 2023, the CDR3-L consists of SEQ ID NO: 3023, the CDR1-H consists of SEQ ID NO: NO: 4023, the CDR2-H consists of SEQ ID NO: 5023, and the CDR3-H consists of SEQ ID NO: 6023; or the CDR1-L consists of SEQ ID NO: 1024, the CDR2-L consists of SEQ ID NO: 2024, the CDR3-L consists of SEQ ID NO: 3024, the CDR1-H consists of SEQ ID NO: 4024, the CDR2-H consists of SEQ ID NO: 5024, and the CDR3-H consists of SEQ ID NO: 6024;or the CDR1-L consists of SEQ ID NO: 1025, the CDR2-L consists of SEQ ID NO: 2025, the CDR3-L consists of SEQ ID NO: 3025, the CDR1-H consists of SEQ ID NO: 4025, the CDR2-H consists of SEQ ID NO: 5025, and the CDR3-H consists of SEQ ID NO: 6025; or the CDR1-L consists of SEQ ID NO: 1026, the CDR2-L consists of SEQ ID NO: 2026, the CDR3-L consists of SEQ ID NO: 3026, the CDR1-H consists of SEQ ID NO: 4026, the CDR2-H consists of SEQ ID NO: 5026, and the CDR3-H consists of SEQ ID NO: 6026; or the CDR1-L consists of SEQ ID NO: 1027, the CDR2-L consists of SEQ ID NO: 5027 NO: 2027, the CDR3-L consists of SEQ ID NO: 3027, the CDR1-H consists of SEQ ID NO: 4027, the CDR2-H consists of SEQ ID NO: 5027, and the CDR3-H consists of SEQ ID NO: 6027; or the CDR1-L consists of SEQ ID NO: 1028, the CDR2-L consists of SEQ ID NO: 2028, the CDR3-L consists of SEQ ID NO: 3028, the CDR1-H consists of SEQ ID NO: 4028, the CDR2-H consists of SEQ ID NO: 5028, and the CDR3-H consists of SEQ ID NO: 6028.

[0027] In some embodiments, the ABP comprises a variable light chain (V L ), which comprises a sequence having at least 97% identity to a sequence selected from SEQ ID NOs: 1-28, and a variable heavy chain (V H ), which comprises a sequence having at least 97% identity to a sequence selected from SEQ ID NOs: 101-128; or a variable light chain (V L ), comprising a sequence having at least 97% identity to a sequence selected from the group consisting of SEQ ID NOs: 8000-8495, and a variable heavy chain (V H ), which comprises a sequence having at least 97% identity to a sequence selected from SEQ ID NOs: 8496-8991; or a variable light chain (V L ), comprising a V clone identical to any clone in the library deposited with ATCC Accession No. PTA-125512 LThe sequences have at least 97% identity, and the variable heavy chain (V H ), comprising a V clone identical to any clone in the library deposited with ATCC Accession No. PTA-125512 H In some embodiments, the V L and the V H are cognate pairs.

[0028] In some embodiments, the ABP comprises a variable light chain (V L ), which comprises a sequence selected from SEQ ID NO: 1-28, and a variable heavy chain (V H ), which comprises a sequence selected from SEQ ID NO: 101-128; or a variable light chain (V L ), which comprises a sequence selected from SEQ ID NO: 8000-8495, and a variable heavy chain (V H ), which comprises a sequence selected from SEQ ID NOs: 8496-8991; or a variable light chain (V L ), comprising a V clone of any one of the clones in the library deposited under ATCC Accession No. PTA-125512 L sequence, and variable heavy chain (V H ), comprising a V clone of any one of the clones in the library deposited under ATCC Accession No. PTA-125512 H In some embodiments, the V L and the V H are cognate pairs.

[0029] In some embodiments, the ABP comprises a scFv or a full-length monoclonal antibody. In some embodiments, the ABP comprises an immunoglobulin constant region.

[0030] In some embodiments, the ABP is expressed as a cytokine with a K of less than 500 nM as measured by surface plasmon resonance. D In some embodiments, the ABP binds to human CTLA-4 with a K of less than 200 nM as measured by surface plasmon resonance. D In some embodiments, the ABP binds to human CTLA-4 with a K of less than 25 nM as measured by surface plasmon resonance. D In some embodiments, the ABP binds to human CTLA-4 with a K of less than 25 nM. D Binds to human CTLA-4 on the cell surface.

[0031] Another aspect of the present disclosure provides a method for treating a disease, comprising the steps of administering to a subject in need thereof an effective amount of an ABP disclosed herein or a pharmaceutical composition disclosed herein. In some embodiments, the disease is selected from the group consisting of cancer, AIDS, Alzheimer's disease, and viral or bacterial infection. In some embodiments, the method further comprises administering to the subject one or more additional therapeutic agents. In some embodiments, the additional therapeutic agent is selected from the group consisting of a CTLA-4 inhibitor, a TIGIT inhibitor, a chemotherapeutic agent, an immunostimulant, radiation, a cytokine, a polynucleotide encoding a cytokine, and a combination thereof. 6. Description of the Figures

[0033] Figure 1 Methods for generating scFv libraries from B cells isolated from fully human mice and selecting B cells expressing antibodies with high affinity for antigens are summarized. Figure 1 SEQ ID NOs 11971-11998 are disclosed respectively in order of appearance.

[0034] Figure 2 The scFv amplification procedure is described. First, a mixture of primers targeting the IgK C region, the IgG C region, and all V regions is used to amplify IgK and IgH, respectively. Second, the VH and CK primers contain complementary regions that result in the formation of overlap extension amplicons, which are fusion products between IgK and IgH. This complementary region contains a DNA sequence encoding a Gly-Ser-rich scFv linker sequence. Finally, a semi-nested PCR is performed to add adapters for Illumina sequencing or yeast display.

[0035] Figure 3 Included is a schematic for sorting monoclonal antibodies into their epitope bins.

[0036] Figure 4 Included are images of histopathological staining from hCTLA-4 KI mice bearing MC38 tumors. The figure shows the scores of H&E, immunoglobulin (Ig), and C3 staining from the right kidney. ipi is ipilimumab, and CTLA4.A14.2a is antibody A14 cloned on a mouse IgG2a backbone.

[0037] Figure 5 Included is a graph showing alkaline phosphatase levels in treated hCTLA4KI mice bearing MC38 tumors. IPI is ipilimumab, and CTLA4.A14.2a is antibody A14 cloned on a mouse IgG2a backbone. U / L is units per liter.

[0038] Figure 6Included are plots of the percentage of intratumoral regulatory T cells (Treg) and intratumoral natural killer (NK) cells after the indicated treatments.

[0039] Figure 7 Included are graphs showing changes in body weight of hCTLA4 mice receiving the indicated treatments. Ipi is ipilimumab, and CTLA4.A14.2a is antibody A14 cloned on a mouse IgG2a backbone. Error bars represent + / - standard error of the mean.

[0040] Figure 8 Included are graphs showing the effects of control, Ipi, and anti-CTLA4 treatment on the percentages of the indicated cell populations. Ipi is ipilimumab, and CTLA4.A14.2a is antibody A14 cloned on a mouse IgG2a backbone.

[0041] Figure 9 Included are graphs showing the effects of control, Ipi, and anti-CTLA4 treatment on the percentage of the indicated cell populations. Ipi is ipilimumab, and CTLA4.A14.2a is antibody A14 cloned on a mouse IgG2a backbone.

[0042] Figure 10 Included are graphs showing the effects of control, Ipi, and anti-CTLA4 treatment on dendritic cells (DC) and the percentage of activated DCs (CD86+). Ipi is ipilimumab, and CTLA4.A14.2a is antibody A14 cloned on a mouse IgG2a backbone.

[0043] Figure 11 Included are graphs showing mean tumor volumes following treatment with 0.3 mg / kg of the indicated anti-CTLA4. 7. Specific Implementation Methods

[0045] 7.1. Definitions

[0046] Unless otherwise defined herein, scientific and technical terms relevant to the present disclosure should have the meaning commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, singular terms should include plural terms, and plural terms should include singular terms. Generally, the nomenclature and techniques associated with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization as described herein are those well known in the art and commonly used. Unless otherwise indicated, the methods and techniques of the present disclosure are generally carried out according to conventional methods well known in the art, and as described in the various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990), which are incorporated herein by reference. Enzymatic reactions and purification techniques are carried out according to the instructions of the manufacturer, as generally accomplished in the art or as described herein. Terms used in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry as described herein, as well as laboratory procedures and techniques thereof are those well known in the art and commonly used. Standard techniques for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery and patient treatment can be used.

[0047] Unless otherwise indicated, the following terms shall be understood to have the following meanings:

[0048] The terms "CTLA-4," "CTLA-4 protein," and "CTLA-4 antigen" are used interchangeably herein to refer to human CTLA-4 or any variant (e.g., splice variants and allelic variants), isoforms, and species homologs of human CTLA-4 that are naturally expressed by cells or expressed by cells transfected with the CTLA4 gene. In some aspects, the CTLA-4 protein is a CTLA-4 protein naturally expressed by primates (e.g., monkeys or humans), rodents (e.g., mice or rats), dogs, camels, cats, cows, goats, horses, or sheep. In some aspects, the CTLA-4 protein is human CTLA-4 (hCTLA-4; SEQ ID NO: 7001).

[0049] The term "immunoglobulin" refers to a class of structurally related proteins that typically comprise two pairs of polypeptide chains: a pair of light (L) chains and a pair of heavy (H) chains. In an "intact immunoglobulin," all four chains are interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, for example, Paul, Fundamental Immunology 7th ed., Ch. 5 (2013) Lippincott Williams & Wilkins, Philadelphia, PA. Briefly, each heavy chain typically comprises a heavy chain variable region (V H ) and heavy chain constant region (C H The heavy chain constant region usually contains three domains, abbreviated as C H1 、C H2 and C H3 Each light chain typically comprises a light chain variable region (V L ) and the light chain constant region. The light chain constant region usually contains a domain, abbreviated as C L .

[0050] 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 the antigen or epitope with a specificity and affinity similar to that of 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 alternative scaffold. In some embodiments, the ABP consists of an alternative scaffold. In some embodiments, the ABP consists essentially of an alternative 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 as 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 ABP provided herein binds to a CTLA-4 epitope that is conserved between or among CTLA-4 proteins from different species.

[0051] The term "antibody" is used herein in its broadest sense to include certain types of immunoglobulin molecules that contain one or more antigen binding domains that specifically bind an antigen or epitope. Antibodies particularly include intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multispecific antibodies. An example of an antigen binding domain is a V-binding domain. H -V L The antigen-binding domain formed by dimers. Antibodies are a type of ABP.

[0052] The term "alternative scaffold" refers to a molecule in which one or more regions can be diversified to generate one or more antigen binding domains that specifically bind to an antigen or epitope. In some embodiments, the antigen binding domain binds to the antigen or epitope with a specificity and affinity similar to that of naturally occurring antibodies. Exemplary alternative scaffolds include those derived from fibronectin (e.g., Adnectins TM ), β-sandwich (e.g., iMab), lipocalin (e.g., ), EETI-II / AGRP, BPTI / LACI-D1 / ITI-D2 (e.g., Kunitz domain), thioredoxin peptide aptamer, protein A (e.g., ), ankyrin repeats (e.g., DARPins), γ-B-crystallin / ubiquitin (e.g., Affilins), CTLD3 (e.g., tetranectin), Fynomers, and (LDLR-A modules) (e.g., Avimers). Other novel alternative scaffolds are 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; the entire contents of each of which are incorporated by reference. Alternative scaffolds are a type of ABP.

[0053] The term "antigen binding domain" refers to the portion of an ABP that is capable of specifically binding an antigen or epitope.

[0054] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody that has a structure substantially similar to a naturally occurring antibody structure and has heavy chains that include an Fc region.

[0055] 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 certain proteins of the complement system. The structures of various immunoglobulin Fc regions and the glycosylation sites contained therein are well known in the art. See Schroeder and Cavacini, J. Allergy Clin. Immunol., 2010, 125: S41-52, the entire contents of which are incorporated by reference. The Fc region may be a naturally occurring Fc region or a modified Fc region as described elsewhere in this disclosure.

[0056] V H and V L The V domains can be further subdivided into regions of hypervariability ("hypervariable regions (HVRs); also called "complementarity determining regions" (CDRs)) interspersed with regions that are more conserved. The more conserved regions are called framework regions (FRs). Each V domain has a H and V LTypically, the antibody comprises three CDRs and four FRs arranged in the following order (from N-terminus to C-terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDRs participate 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, Bethesda, MD, the entire contents of which are incorporated by reference.

[0057] The light chains from any vertebrate species can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the sequences of their constant domains.

[0058] Heavy chains from any vertebrate species can be classified into one of five different types (or isotypes): IgA, IgD, IgE, IgG, and IgM. These classes are also referred to as 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.

[0059] The amino acid sequence boundaries of a CDR can be determined by one skilled in the art using any of a variety of known numbering schemes, including those described by Kabat et al., supra ("Kabat" numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 ("Chothia" numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 ("Contact" numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 ("IMGT" numbering scheme); and Honegge and Plückthun, J. Mol. Biol., 2001, 309:657-70 ("AHo" numbering scheme); the entire contents of each of which are incorporated by reference.

[0060] Table 1 provides 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) and CDR3-H (V HFor CDR1-H, residue numbers are provided using both the Kabat and Chothia numbering schemes.

[0061] For example, CDRs can be assigned using antibody numbering software, such as Abnum available from www.bioinf.org.uk / abs / abnum / and described in Abhinandan and Martin, Immunology, 2008, 45:3832-3839, incorporated by reference in its entirety.

[0062]

[0063] *When numbering using the Kabat numbering convention, the C-terminus of CDR1-H varies between 32 and 34, depending on the length of the CDR.

[0064] When referring to residues in the antibody heavy chain constant region, the "EU numbering scheme" (eg, as reported in Kabat et al., supra) is generally used.

[0065] "Antibody fragments" comprise a portion of an intact antibody, such as the antibody binding or variable region of an intact antibody. Antibody fragments include, for example, Fv fragments, Fab fragments, F(ab')2 fragments, Fab' fragments, scFv (sFv) fragments, and scFv-Fc fragments.

[0066] An "Fv" fragment comprises a dimer of one heavy-chain and one light-chain variable domain non-covalently linked.

[0067] In addition to the heavy and light chain variable domains, a "Fab" fragment contains 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 intact antibodies.

[0068] "F(ab')2" fragments comprise two Fab' fragments linked by a disulfide bond near the hinge region. F(ab')2 fragments can be produced, for example, by recombinant methods or by papain digestion of intact antibodies. F(ab') fragments can be dissociated, for example, by treatment with β-mercaptoethanol.

[0069] "Single-chain Fv," "sFv," or "scFv" antibody fragments comprise the V domain of a single polypeptide chain. H domain and V L Domain. V H and V LTypically linked via a peptide linker. See Plückthun 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, the entire contents of which are incorporated by reference.

[0070] "scFv-Fc" fragments comprise an scFv attached to an Fc domain. For example, the Fc domain can be attached to the C-terminus of the scFv. The Fc domain can be attached to the V H or V L This then depends on the orientation of the variable domains in the scFv (i.e., V H -V L or V L -V H Any suitable Fc domain known in the art or described herein can be used. In some cases, the Fc domain comprises an IgG4 Fc domain.

[0071] 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, the entire contents of each of which are incorporated by reference.

[0072] 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 the naturally occurring IgG molecule, which, although bivalent, recognizes the same epitope in each antigen-binding domain. Binding specificities can exist in any suitable valency.

[0073] The term "monoclonal antibody" refers to an antibody from a group of substantially homogeneous antibodies. Except for the variants that may usually occur during the monoclonal antibody production process, a group of substantially homogeneous antibodies comprises antibodies that are substantially similar and in conjunction with one or more identical epitopes. Such variants are usually present only in small quantities. Monoclonal antibodies are usually obtained by a method comprising selecting a single antibody from a variety of antibodies. For example, a selection method can be to select a unique clone from a plurality of clones, such as hybridoma clones, phage clones, yeast clones, bacterial clones or other recombinant DNA clones. The antibody selected can be further changed, for example, to improve affinity to the target ("affinity mutation"), to humanize the antibody, to improve its output in cell culture and / or to reduce its immunogenicity in a subject.

[0074] 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, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0075] The non-human antibody of " humanization " form is chimeric antibody, and it comprises the minimum sequence that derives from non-human antibody.Humanized antibody is normally human antibody (recipient antibody), and wherein the residue from one or more CDR is replaced by one or more CDR residues from non-human antibody (donor antibody).Donor antibody can be any suitable non-human antibody, as mouse, rat, rabbit, chicken or non-human primate antibody with required specificity, affinity or biological effect.In some cases, the selected framework region residue of receptor antibody is replaced by the corresponding concave region residue from donor antibody.Humanized antibody also includes residue that is not found in receptor antibody or donor antibody.This type of modification can be carried out to further improve 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.

[0076] A "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source using a human antibody library or human antibody coding sequence (e.g., obtained from a human source or designed de novo). Human antibodies specifically exclude humanized antibodies. In some embodiments, rodents are genetically modified to replace their rodent antibody sequences with human antibodies.

[0077] An "isolated ABP" or "isolated nucleic acid" is an ABP or nucleic acid that has been separated and / or recovered from components of its natural environment. Components of the natural environment may include enzymes, hormones, and other proteinaceous or non-proteinaceous materials. In some embodiments, the isolated ABP is purified to a degree sufficient to obtain at least 15 N-terminal or internal amino acid sequence residues, for example, by using a spinning cup sequencer. In some embodiments, the 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. Isolated ABP includes the ABP in situ within recombinant cells, as at least one component of the ABP's natural environment is absent. In some aspects, the isolated ABP or isolated nucleic acid is prepared by at least one purification step. In some embodiments, the 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 in the form of a solution comprising at least 85%, 90%, 95%, 98%, 99% to 100% by weight of the ABP or nucleic acid. In some embodiments, the isolated ABP or isolated nucleic acid is provided in the form of a solution comprising at least 85%, 90%, 95%, 98%, 99% to 100% by volume of the ABP or nucleic acid.

[0078] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., ABP) and its binding partner (e.g., antigen or epitope). Unless otherwise indicated, as used herein, "affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., ABP and antigen or epitope). The affinity of a molecule X for its partner Y can be expressed in terms of the dissociation equilibrium constant (K D ) are represented. The kinetic components that contribute to the dissociation equilibrium constant are described in more detail below. Affinity can be measured by conventional methods known in the art, including those described herein. Surface plasmon resonance (SPR) techniques (e.g., ) or biolayer interferometry (e.g. )Determine affinity.

[0079] With respect to the binding of an ABP to a target molecule, the terms "bind," "specifically binds," "specifically binds to," "specifically for," "selectively binds to," and "selectively for" a specific antigen (e.g., a polypeptide target) or an epitope on a specific antigen refer to binding that is significantly different from non-specific or non-selective interactions (e.g., with non-target molecules). For example, specific binding can be measured by measuring binding to a target molecule and comparing it to binding to a non-target molecule. Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule. In this case, specific binding is indicated if the binding of the ABP to the target molecule is competitively inhibited by the control molecule. In some aspects, the affinity of the CTLA-4 ABP for a non-target molecule is less than about 50% of the affinity for CTLA-4. In some aspects, the affinity of the CTLA-4 ABP for a non-target molecule is less than about 40% of the affinity for CTLA-4. In some aspects, the affinity of the CTLA-4 ABP for a non-target molecule is less than about 30% of the affinity for CTLA-4. In some aspects, the affinity of the CTLA-4 ABP for the non-target molecule CTLA-4 ABP is less than about 20% of its affinity for CTLA-4. In some aspects, the affinity of the CTLA-4 ABP for the non-target molecule CTLA-4 ABP is less than about 10% of its affinity for CTLA-4. In some aspects, the affinity of the CTLA-4 ABP for the non-target molecule CTLA-4 ABP is less than about 1% of its affinity for CTLA-4. In some aspects, the affinity of the CTLA-4 ABP for the non-target molecule CTLA-4 ABP is less than about 0.1% of its affinity for CTLA-4.

[0080] As used herein, the term "k d "(Second -1 ) refers to the dissociation rate constant for a specific ABP-antigen interaction. This value is also called K off value.

[0081] As used herein, the term "k a ”(M -1 × seconds -1 ) refers to the association rate constant for a specific ABP-antigen interaction. This value is also called K on value.

[0082] As used herein, the term "K D "(M)" refers to the dissociation equilibrium constant of a specific ABP-antigen interaction. K D =k d / k a .

[0083] As used herein, the term "K A ”(M -1) refers to the binding equilibrium constant for a specific ABP-antigen interaction. A =k a / k d .

[0084] An "affinity matured" ABP is an ABP that has one or more alterations (e.g., in one or more CDRs or FRs) that result in an increase in the affinity of the ABP for its antigen compared to a parent ABP that does not have the one or more alterations. In one embodiment, the affinity matured ABP has nanomolar or picomolar affinity for the target antigen. Affinity matured ABPs can be produced using various methods known in the art. For example, Marks et al. (Bio / Technology, 1992, 10:779-783, the entire contents of which are incorporated by reference) describe affinity matured ABPs produced by V H and V L Affinity maturation by domain rearrangement. 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; the entire contents of each of which are incorporated by reference.

[0085] An "immunoconjugate" is an ABP conjugated to one or more heterologous molecules.

[0086] "Effector function" refers to the biological activity mediated by the Fc region of an antibody, which activity may vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding to activate complement-dependent cytotoxicity (CDC), Fc receptor binding to activate antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP).

[0087] When used herein in the context of two or more ABPs, the term "competes with" or "cross-competes with" means that the 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, and then a second CTLA-4 ABP is added. In another exemplary assay, a first CTLA-4 ABP is coated on a surface and contacted with CTLA-4, and then a second CTLA-4 ABP is added. If the presence of the first CTLA-4 ABP reduces the binding of the second CTLA-4 ABP, the ABPs compete in either assay. The term "competes with" 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 opposite order. However, in some embodiments, the first and second ABPs inhibit the binding of each other, 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 of skill can select the antibody concentration for use 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 illustrative, and one of skill 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,” Assay Guidance Manual [Internet], updated December 24, 2014 (www.ncbi.nlm.nih.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; the entire contents of each of which are incorporated by reference.

[0088] The term "epitope" refers to the portion of an antigen to which an ABP specifically binds. An epitope is typically composed of surface-accessible amino acid residues and / or sugar side chains and may have specific three-dimensional structural features as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that binding to the former but not the latter may be lost in the presence of denaturing solvents. An epitope may contain amino acid residues that are directly involved in binding, as well as other amino acid residues that are not directly involved in binding. The epitope to which an ABP binds can be determined using known techniques for epitope determination, for example, by testing the binding of the ABP to CTLA-4 variants or chimeric CTLA-4 variants having different point mutations.

[0089] 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 the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percent sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways within the skill in 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 algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0090] "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. For example, the amino acid groups provided in Tables 2-4 are considered to be conservative substitutions of each other in some embodiments.

[0091]

[0092]

[0093]

[0094]

[0095] Other conservative substitutions can be found in, for example, Creighton, Proteins: Structures and Molecular Properties 2nd ed. (1993) WH Freeman & Co., New York, NY. ABPs generated by making one or more conservative amino acid residue substitutions in a parent ABP are termed "conservatively modified variants."

[0096] The term "treating" (and variants thereof, such as "treat" or "treatment") refers to clinical intervention intended to alter the natural course of a disease or condition in a subject in need thereof. Treatment can be performed either prophylactically or during the course of clinical pathology. Desired effects of treatment include preventing the occurrence or recurrence of the disease, alleviating symptoms, alleviating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or relieving the disease state, and alleviating or improving prognosis.

[0097] 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 in treating a disease or condition.

[0098] As used herein, the term "subject" refers to 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 human. In some embodiments, the subject has a disease or condition that can be treated using the ABPs provided herein. In some aspects, the disease or condition is cancer. In some aspects, the disease or condition is a viral infection.

[0099] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic or diagnostic products (e.g., kits), that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic or diagnostic products.

[0100] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes the death or destruction of cells.

[0101] "Chemotherapeutic agents" refer to chemical compounds used to treat cancer. Chemotherapeutic agents include "antihormonal agents" or "endocrine therapeutic agents," which work by regulating, reducing, blocking, or inhibiting the effects of hormones that can promote cancer growth.

[0102] The term "cytostatic" refers to a compound or composition that inhibits cell growth in vitro or in vivo. In some embodiments, the cytostatic is an agent that reduces the percentage of cells in the S phase. In some embodiments, the cytostatic reduces the percentage of cells in the S phase by at least about 20%, at least about 40%, at least about 60%, or at least about 80%.

[0103] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, as well as all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive 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.

[0104] The term "pharmaceutical composition" refers to a preparation that is in such form that the biological activity of the active ingredients contained therein is effective to treat the subject, and that does not contain additional ingredients that are unacceptably toxic to the subject.

[0105] The terms "modulate" and "modulation" refer to the decrease or inhibition, or, the activation or increase, of the recited variable.

[0106] The terms "increase" and "activate" refer to an increase of 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 more of the recited variable.

[0107] 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 more reduction in the recited variable.

[0108] The term "agonism" refers to activation of receptor signaling to induce a biological response associated with receptor activation. An "agonist" is an entity that binds to a receptor and agonizes the receptor.

[0109] The term "antagonize" refers to the inhibition of receptor signaling to suppress the biological response associated with receptor activation. An "antagonist" is an entity that binds to a receptor and antagonizes the receptor.

[0110] 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 a variety of immune 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. See Seder and Ahmed, Nature Immunol., 2003, 4:835-842, the entire contents of which are incorporated by reference for more information on effector T cells.

[0111] The term "regulatory T cells" includes cells that regulate immune tolerance, for example, by suppressing effector T cells. In some aspects, regulatory T cells have a CD4+CD25+Foxp3+ phenotype. In some aspects, regulatory T cells have a CD8+CD25+ phenotype. See Nocentini et al., Br. J. Pharmacol., 2012, 165:2089-2099, the entire contents of which are incorporated by reference for more information on regulatory T cells.

[0112] The term "dendritic cell" refers to professional antigen-presenting cells that are capable of activating naive T cells and stimulating B cell growth and differentiation.

[0113] A "variant" of a polypeptide (e.g., an antibody) comprises an amino acid sequence in which one or more amino acid residues are inserted, deleted, and / or substituted into the amino acid sequence relative to the native polypeptide sequence and substantially retains the same biological activity as the native polypeptide. The biological activity of the polypeptide can be measured using standard techniques in the art (e.g., if the variant is an antibody, its activity can be detected by binding assays, as described herein). Variants of the present disclosure include fragments, analogs, recombinant polypeptides, synthetic polypeptides, and / or fusion proteins.

[0114] A "derivative" of a polypeptide is a polypeptide (e.g., an antibody) that has been chemically modified, for example, by conjugation to another chemical moiety, such as, for example, polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation. Unless otherwise indicated, the term "antibody" includes, in addition to antibodies comprising two full-length heavy chains and two full-length light chains, derivatives, variants, fragments, and muteins thereof, examples of which are described below.

[0115] A nucleotide sequence is "operably linked" to a regulatory sequence if the regulatory sequence affects the expression of the nucleotide sequence (e.g., the level, time, or location of expression). A "regulatory sequence" is a nucleic acid that affects the expression (e.g., the level, time, or location of expression) of a nucleic acid to which it is operably linked. For example, a regulatory sequence can act directly on the regulated nucleic acid, or through one or more other molecules (e.g., a polypeptide that binds to the regulatory sequence and / or nucleic acid). Examples of regulatory sequences include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). For example, other examples of regulatory sequences are described in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA, and Baron et al., 1995, Nucleic Acids Res. 23: 3605–06

[0078] .

[0116] 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 it can be a eukaryotic cell, e.g., a unicellular eukaryote (e.g., a yeast or other fungus), a plant cell (e.g., a tobacco or tomato plant cell), an animal cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell), or a hybridoma. Examples of host cells include CS-9 cells, monkey kidney cell COS-7 cell line (ATCC CRL 1651) (see Gluzman et al., 1981, Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells or 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, BHK (ATCC CRL 10) cell line, CV1 / EBNA cell line derived from 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, 293EBNA or MSR 293, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell lines derived from in vitro cultures of primary tissues, primary explants, HL-60, U937, HaK or Jurkat cells. Generally, the host cell is a cultured cell that can be transformed or transfected with a nucleic acid encoding a polypeptide, which can then be expressed in the host cell.

[0117] The phrase "recombinant host cell" may be used to refer to a host cell that has been transformed or transfected with a nucleic acid to be expressed. A host cell may also be a cell that contains a nucleic acid but does not express it at the desired level unless a regulatory sequence is introduced into the host cell so that it is operably linked to the nucleic acid. It should be understood that the term host cell refers not only to the specific subject cell, but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in subsequent generations due to, for example, mutations or environmental influences, such progeny may not actually be the same as the parent cell, but are still included within the scope of the term as used herein.

[0118] 7.2. Other Understanding Conventions

[0119] 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[0120] Unless otherwise indicated, reference to a compound having one or more stereocenters refers to each stereoisomer thereof and all combinations of stereoisomers thereof.

[0121] Nucleic Acids

[0122] In one aspect, the present disclosure provides isolated nucleic acid molecules. Nucleic acid includes, for example, a polynucleotide encoding all or part of an antigen-binding protein, for example, one or two chains of an antibody of the present disclosure, or a fragment, derivative, mutant or variant thereof, a polynucleotide sufficient to be used as a hybridization probe, PCR primer or sequencing primer to identify, analyze, mutate or amplify a polynucleotide encoding a polypeptide, an antisense nucleic acid for inhibiting polynucleotide expression, and the above-mentioned complementary sequence. The nucleic acid can be of any length. For example, its length can be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1,000, 1,500, 3,000, 5,000 or more nucleotides, and / or can include one or more additional sequences, for example, regulatory sequences, and / or a part of a larger nucleic acid (e.g., a vector). Nucleic acids can be single-stranded or double-stranded, and can comprise RNA and / or DNA nucleotides, as well as artificial variants thereof (eg, peptide nucleic acids).

[0123] 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).

[0124] Nucleic acid sequences encoding heavy chain variable regions and light chain variable regions are shown herein. It will be appreciated by those skilled in the art that, due to the degeneracy of the genetic code, each polypeptide sequence disclosed herein is encoded by a large number of other nucleic acid sequences. The present disclosure provides each degenerate nucleotide sequence encoding each antigen-binding protein of the present disclosure.

[0125] The present disclosure also provides nucleic acids that hybridize to other nucleic acids (e.g., nucleic acids comprising the nucleotide sequence of any 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 comprising 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 a solution comprising about 50% formamide, and a hybridization temperature of 42°C) and 60°C, wash conditions in 0.5X SSC, 0.1% SDS. Stringent hybridization conditions hybridize in 6X SSC at 45°C, followed by one or more washes in 0.1X SSC, 0.2% SDS at 68°C. In addition, those skilled in the art can manipulate hybridization and / or wash conditions to increase or reduce the stringency of hybridization so that nucleic acids comprising nucleotide sequences at least 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical to each other generally remain hybridized to each other. The basic parameters affecting the selection of hybridization conditions and the guiding principles for designing suitable conditions are, for example, 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 easily determined by those of ordinary skill in the art based on, for example, the length and / or base composition of DNA.

[0126] Changes can be introduced into a nucleic acid by mutation, thereby causing changes in the amino acid sequence of the polypeptide (e.g., antigen binding protein) encoded thereby. Mutations can be introduced using any technique known in the art. In one embodiment, one or more specific amino acid residues are altered using, for example, a site-directed mutagenesis protocol. In another embodiment, one or more randomly selected residues are altered using, for example, a random mutagenesis protocol. Regardless of how it is prepared, the mutant polypeptide can be expressed and screened for desired properties (e.g., binding to CTLA-4).

[0127] Mutations can be introduced into nucleic acids without significantly changing the biological activity of the polypeptides encoded therein. For example, nucleotide substitutions can be performed, resulting in amino acid substitutions at non-essential amino acid residues. In one embodiment, the nucleotide sequence for CTLA-4 or a desired fragment, variant, or derivative thereof provided herein is mutated so that it encodes an amino acid sequence comprising a deletion or substitution of one or more amino acid residues, which is shown herein for CTLA-4 as residues in which two or more sequences are different. Alternatively, one or more mutations can be introduced into nucleic acids that selectively change the biological activity of the polypeptides encoded therein (e.g., binding to CTLA-4). For example, mutations can quantitatively or qualitatively change biological activity. Examples of quantitative changes include increasing, decreasing, or eliminating activity. Examples of qualitative changes include changing the antigen specificity of the antigen binding protein.

[0128] In another aspect, the present disclosure provides nucleic acid molecules suitable for use as primers or hybridization probes for detecting nucleic acid sequences of the present disclosure. The nucleic acid molecules of the present disclosure may comprise only a portion of a nucleic acid sequence encoding a full-length polypeptide of the present disclosure, e.g., a fragment useful as a probe or primer, or a fragment encoding an active portion (e.g., a CTLA-4 binding portion) of a polypeptide of the present disclosure.

[0129] Probes based on the nucleic acid sequences of the present disclosure can be used to detect nucleic acids or similar nucleic acids, for example, transcripts encoding the polypeptides of the present disclosure. The probes can contain a labeling group, for example, a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor. Such probes can be used to identify cells expressing the polypeptide.

[0130] 7.4. Expression Vectors

[0131] The present disclosure provides vectors comprising nucleic acids encoding the polypeptides of the present disclosure or portions 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.

[0132] In another aspect of the present disclosure, expression vectors comprising the nucleic acid molecules and polynucleotides of the present disclosure and host cells transformed with such vectors are also provided, as are methods for producing polypeptides. The term "expression vector" refers to a plasmid, phage, virus or vector used to express a polypeptide from a polynucleotide sequence. A vector for expressing a polypeptide contains the minimum sequences required for vector propagation and expression of cloned inserts. An expression vector contains a transcription unit comprising the following components: (1) one or more genetic elements that have a regulatory role in gene expression, such as a promoter or enhancer, (2) sequences encoding polypeptides and proteins that can be transcribed into mRNA and translated into protein, and (3) suitable transcription start and stop sequences. These sequences may further contain a selective marker. Vectors suitable for expression in host cells are readily available, and nucleic acid molecules are inserted into the vector using standard recombinant DNA techniques. Such vectors can include promoters that function in specific tissues, as well as viral vectors for expressing polypeptides in target human or animal cells.

[0133] The recombinant expression vector of the present disclosure can include the nucleic acid of the present disclosure in the form suitable for expressing nucleic acid in host cells.The recombinant expression vector includes one or more regulatory sequences selected based on the host cell to be used for expression, which are operably connected to the nucleic acid sequence to be expressed.Regulatory sequences are included in those sequences (for example, SV40 early gene enhancer, Rous sarcoma virus promoter and cytomegalovirus promoter) that instruct the nucleotide sequence constitutive expression in many types of host cells, those sequences (for example, 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 sequences (for example, metallothionein promoters in mammalian cells and tet responsiveness and / or streptomycin responsiveness promoters (see the same)) that instruct the nucleotide sequence to respond to specific treatments or the patient's condition. Those skilled in the art will recognize that the design of the expression vector can depend on factors such as the choice of the host cell to be transformed, the level of expression of the desired protein, etc. The expression vectors of the present disclosure can be introduced into host cells to produce proteins or peptides, including fusion proteins or peptides, encoded by the nucleic acids described herein.

[0134] In some embodiments, the expression vector is an expression vector purified from one of the clones of the CTLA-4 binding clone library deposited with ATCC Accession No. PTA- 125512. In some embodiments, the expression vector is generated by genetically modifying one of the expression vectors from one of the clones purified from the CTLA-4 binding clone library deposited with ATCC Accession No. PTA- 125512. In some embodiments, the expression vector is generated by using the heavy and light chain variable region sequences of one of the clones of the CTLA-4 binding clone library deposited with ATCC Accession No. PTA-125512.

[0135] The present disclosure also provides methods for preparing polypeptides. A variety of other expression / host systems can be utilized. Vector DNA can be introduced into prokaryotic or eukaryotic systems by conventional transformation or transfection techniques. These systems include, but are not limited to, microorganisms, such as bacteria (e.g., E. coli) transformed using recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed using yeast expression vectors; insect cell systems infected using viral expression vectors (e.g., baculovirus); plant cell systems transfected using viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed using bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Mammalian cells used in the production of recombinant proteins include, but are not limited to, VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines or derivatives thereof, such as VeggieCHO ​​and related cell lines grown in serum-free medium (see Rasmussen et al., 1998, Cytotechnology 28:31) or the CHO cell line DX-B11, which is deficient in DHFR (see Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216-20), COS cells, such as monkey kidney cell COS cell line (ATCC CRL 1651) (see Gluzman et al., 1981, Cell 23:175), W138, BHK, HepG2, 3T3 (ATCC CCL 163), RIN, MDCK, A549, PC12, K562, L cells, C127 cells, BHK (ATCC CRL 10) Cell lines, CV1 / EBNA cell lines 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, 293EBNA or MSR 293, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell lines derived from primary tissue in vitro culture, primary explants, HL-60, U937, HaK or Jurkat cells. Mammalian expression allows the production of secreted or soluble polypeptides that can be recovered from the growth medium.

[0136] For the stable transfection of mammalian cells, it is well known that, depending on the expression vector and transfection technique used, only a small portion of cells can integrate exogenous DNA into their genome. In order to identify and select these integrants, genes encoding selective markers (e.g., for antibiotic resistance) are usually introduced into the host cell together with the target gene. For example, once such cells are transformed with a vector comprising a selective marker and a desired expression cassette, the cells can be grown in an enriched culture medium and then converted to a selective culture medium. The selective marker is designed to allow cells that successfully express the introduced sequence to grow and be recovered. Tissue culture techniques suitable for the cell line used can be used to proliferate resistant clumps of stably transformed cells. For a review of recombinant protein expression, see Methods of Enzymology, v.185, Goeddell, DV, ed., Academic Press (1990). Preferred selective markers include those that confer drug resistance, such as G418, hygromycin, and methotrexate. Cells stably transfected with the introduced nucleic acid can be identified by methods such as drug selection (e.g., cells incorporating the selective marker gene will survive, while other cells die).

[0137] The transformed cells can be cultured under conditions that promote expression of the polypeptide, and the polypeptide (as defined above) can be recovered by conventional protein purification procedures. One such purification procedure includes the use of affinity chromatography, for example, on 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 of contaminating endogenous materials.

[0138] In some cases, such as in expression using prokaryotic systems, the expressed polypeptides of the present disclosure may need to be "refolded" and oxidized to the appropriate tertiary structure and generated disulfide bonds in order to be biologically active. Refolding can be achieved using a variety of procedures well known in the art. Such methods include, for example, exposing the dissolved polypeptide to a pH typically above 7 in the presence of a chaotropic agent. The choice of chaotropic agent is similar to that used for inclusion body solubilization; however, chaotropic agents are typically used at lower concentrations. Exemplary chaotropic agents are guanidine and urea. In most cases, the refolding / oxidation solution will also contain a specific ratio of a reducing agent and its oxidized form to generate a specific redox potential, allowing disulfide rearrangement to form cysteine ​​bridges. Some commonly used redox pairs include cysteine / cystamine, glutathione / dithiobis(GSH), copper chloride, dithiothreitol (DTT) / dithiane (DTT), and 2-mercaptoethanol (bME) / dithio-bME. In many cases, a cosolvent can be used to improve the efficiency of refolding. Common cosolvents include glycerol, polyethylene glycols of various molecular weights, and arginine.

[0139] In addition, polypeptides can be synthesized in solution or on a solid support according to conventional techniques. Various automatic synthesizers are commercially available and can be used according to known protocols. See, for example, Stewart and Young, Solid Phase 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).

[0140] The polypeptides and proteins disclosed herein can be purified according to protein purification techniques well known to those skilled in the art. These techniques, at one level, involve crude fractionation of protein and non-protein fractions. After the peptide polypeptide is separated from other proteins, the target peptide or polypeptide can be further purified using chromatography and electrophoresis 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 that is separated from other components and in which the polypeptide is purified to any degree relative to its naturally available state. Thus, a purified polypeptide also refers to a polypeptide that is separated from the environment in which it may naturally occur. Generally, "purified" will refer to a polypeptide composition that has been subjected to fractionation to remove various other components, and in which the composition substantially retains its expressed biological activity. When the term "substantially purified" is used, the designation will refer to a peptide or polypeptide composition in which the polypeptide or peptide constitutes the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 85%, or about 90% or more of the protein in the composition.

[0141] Various techniques suitable for purification are well known to those skilled in the art. These include, for example, precipitation with ammonium sulfate, PEG, antibodies (immunoprecipitation), or by thermal denaturation followed by centrifugation; chromatography, such as 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 believed that the order in which the various purification steps are performed can be changed, or certain steps can be omitted, and still produce a suitable method for preparing a substantially purified polypeptide. Exemplary purification steps are provided in the following examples.

[0142] In light of the present disclosure, those skilled in the art will recognize various methods for quantifying the degree of purification of a polypeptide. 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 to calculate the degree of purification, herein assessed as "-fold purification." Of course, the actual units used to express the amount of binding activity will depend on the specific assay technique selected for purification and whether the polypeptide or peptide exhibits detectable binding activity.

[0143] 7.5. Antibodies

[0144] CTLA-4 antibodies can be purified from host cells transfected with a gene encoding the antibody by using a heparin HP column and eluting the filtered supernatant of the host cell culture fluid using a salt gradient.

[0145] Fab fragments have V L 、V H 、C L and C H1 The F(ab')2 fragment is a bivalent fragment with two Fab fragments connected by a disulfide bridge at the hinge region; the Fd fragment has a V H and C H1 Domain; Fv fragment has the V domain of a single arm of an antibody L and V H domain; and dAb fragments having V H domain, V L domain or V H or V L Antigen-binding fragments of the IL-63 domain (U.S. Patent Nos. 6,846,634, 6,696,245, U.S. Application Publication Nos. 05 / 0202512, 04 / 0202995, 04 / 0038291, 04 / 0009507, 03 / 0039958, Ward et al., Nature 341:544-546, 1989).

[0146] The polynucleotide and polypeptide sequences of specific light chain and heavy chain variable domains are described below. Antibodies comprising light and heavy chains are named by combining the light chain name and the heavy chain variable domain name. For example, "L4H7" indicates an antibody comprising light chain variable domain L4 (comprising the sequence of SEQ ID NO: 4) and heavy chain variable domain H7 (comprising the sequence of SEQ ID NO: 107). Light chain variable sequences are provided in SEQ ID Nos: 1-28 and heavy chain variable sequences are provided in SEQ ID Nos: 101-128.

[0147] In other embodiments, the antibody may comprise a specific heavy chain or light chain, while the complementary light chain or heavy chain variable domain remains unspecified. In particular, certain embodiments herein include antibodies that bind to a specific antigen (such as CTLA-4) by a specific light chain or heavy chain, such that the complementary heavy chain or light chain may be promiscuous, or even unrelated, but which can be determined by, for example, 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).

[0148] Naturally occurring immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FRs) connected by three hypervariable regions (also called complementarity determining regions or CDRs). From N-terminus to C-terminus, both light and heavy chains contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acid distribution of each domain conforms to the definition of Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., US Dept. of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242, 1991.

[0149] 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 variable and constant domains are derived from human immunoglobulin sequences (fully human antibodies). These antibodies can be prepared in a variety of ways, examples of which are described below, including by immunization with the target antigen of mice that have been genetically modified to express antibodies derived from human heavy and / or light chain encoding genes.

[0150] The sequence of humanized antibodies is different from the sequence of the antibody derived from non-human species, and it is by one or more amino acid replacements, disappearances and / or additions, so that when it is applied to a human subject, compared with non-human species antibodies, humanized antibodies are unlikely to induce an immune response, and / or induce an immune response that is not serious. In one embodiment, certain amino acids in the framework and constant domains of the heavy chain and / or light chain of non-human species antibodies are mutated to produce humanized antibodies. In another embodiment, one or more constant domains of people's antibodies are fused with one or more variable domains of non-human species. In another embodiment, when non-human antibodies are applied to a human subject, one or more amino acid residues in one or more CDR sequences of non-human antibodies are changed to reduce the possible immunogenicity of non-human antibodies, the amino acid residues wherein changed are not critical for the immunospecific binding of antibody and its antigen, or the change made to amino acid sequence is a conservative change, so that the combination of humanized antibodies and antigen is not obviously worse than the combination of non-human antibodies and antigen. Examples of how to prepare humanized antibodies can be found in US Patent Nos. 6,054,297, 5,886,152, and 5,877,293.

[0151] The term "chimeric antibody" refers to an antibody that comprises one or more regions from one antibody and one or more regions from one or more other antibodies. In one embodiment, one or more CDRs are derived from a human anti-CTLA-4 antibody. In another embodiment, all CDRs are derived from a human anti-CTLA-4 antibody. In another embodiment, CDRs from more than one human anti-CTLA-4 antibody are mixed and matched in a chimeric antibody. For example, a chimeric antibody can comprise CDR1 from a light chain of a first human anti-CTLA-4 antibody, CDR2 and CDR3 from a light chain of a second human CTLA-4 antibody, and CDRs from a heavy chain of a third anti-CTLA-4 antibody. In addition, the framework regions can be derived from one of the same anti-CTLA-4 antibodies, from one or more different antibodies (e.g., human antibodies), or from a humanized antibody. In one example of a chimeric antibody, a portion of the heavy and / or light chain is identical, homologous, 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, homologous, 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 (ie, the ability to specifically bind to CTLA-4).

[0152] According to the teachings of this specification and using techniques well known in the art, one of ordinary skill in the art can easily prepare fragments or analogs of antibodies. Preferred fragments or analogs have amino and carboxyl termini located near the boundaries of functional domains. Structural and functional domains can be identified by comparing nucleotide and / or amino acid sequence data with public or proprietary sequence databases. Computerized comparative methods can be used to identify sequence motifs or predicted protein conformational domains present in other proteins of known structure and / or function. Methods for identifying protein sequences that fold into known three-dimensional structures are known. See, for example, Bowie et al., 1991, Science 253:164.

[0153] Antigen-binding fragments derived from antibodies can be obtained, for example, by proteolysis of the antibody, for example, by pepsin or papain digestion of intact antibodies according to conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of the antibody with pepsin to provide a 5S fragment called F(ab')2. This fragment can also be further cleaved using a sulfhydryl reducing agent to produce a 3.5S Fab' monovalent fragment. Optionally, a cleavage reaction can be performed using a protecting group for the sulfhydryl groups generated by the cleavage of disulfide bonds. As an alternative, enzymatic cleavage using papain can directly produce two monovalent Fab fragments and one Fc fragment. These methods are described, for example, in 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., Methods in Enzymology 1:422 (Academic Press 1967); and Andrews, SM and Titus, JA Current Protocols in Immunology (Coligan JE, et al., eds.), John Wiley & Sons, New York (2003), pp. 2.8.1 to 2.8.10 and 2.10A.1 to 2.10A.5. Other methods of cleaving antibodies, such as separating the heavy chains to form monovalent light and heavy chain fragments (Fd), further cleaving the fragments or other enzymatic, chemical or genetic techniques, can also be used, as long as the fragments bind to the antigen recognized by the intact antibody.

[0154] Antibody fragments can also be any synthetic or genetically engineered proteins. For example, antibody fragments include isolated fragments consisting of the variable region of the light chain, "Fv" fragments consisting of the variable regions of the heavy and light chains, and recombinant single-chain polypeptide molecules (scFv proteins) in which the light and heavy chain variable regions are connected by a peptide linker.

[0155] Another form of antibody fragment is a peptide containing one or more complementarity determining regions (CDRs) of an antibody. CDRs (also known as "minimal recognition units" or "hypervariable regions") can be covalently or non-covalently incorporated into molecules to make them antigen-binding proteins. CDRs can be obtained by constructing polynucleotides encoding the CDRs of interest. Such polynucleotides are prepared, for example, by synthesizing the variable region using the polymerase chain reaction with mRNA from antibody-producing cells as a template (see, e.g., Larrick et al., Methods: A Companion to Methods in Enzymology 2:106, 1991; Courtenay Luck, "Genetic Manipulation of Monoclonal Antibodies," Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al., eds., p. 166 (Cambridge University Press 1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies," Monoclonal Antibodies: Principles and Applications, Birch et al., eds., p. 137 (Wiley Liss, Inc. 1995)).

[0156] Thus, in one embodiment, the binding agent comprises at least one CDR as described herein. The binding agent may comprise at least 2, 3, 4, 5, or 6 CDRs as described herein. The binding agent may also comprise at least one variable region domain of an antibody as described herein. The variable region domain may be of any size or amino acid composition and will typically comprise at least one CDR sequence responsible for binding to human CTLA-4 (e.g., CDR1-H, CDR2-H, CDR3-H, CDR1-L, CDR2-L, and CDR3-L, as specifically described herein), and adjacent to or in frame with one or more framework sequences. In general, the variable (V) region domain may be an immunoglobulin heavy (V) region domain. H ) and / or light (V L ) chain variable domains. Thus, for example, the V region domains can be monomeric and can be V H or V L domain, which can be at least equal to 1x10 7 Alternatively, the V region domain may be dimeric and comprise a VH V H 、V H V L or V L V L Dimer. A V region dimer comprises at least one V region that may be non-covalently associated. H and at least one V L Chains (hereinafter referred to as FV). If desired, the chains can be covalently coupled directly, for example, via a disulfide bond between the two variable domains, or via a linker, such as a peptide linker, to form a single-chain Fv (scFV).

[0157] The variable region domain can be any naturally occurring variable domain or an engineered version thereof. An engineered version refers to a variable region domain created using recombinant DNA engineering techniques. Such engineered versions include, for example, those generated from a specific antibody variable region by insertion, deletion, or alteration in the amino acid sequence of a specific antibody. Specific examples include engineered variable region domains comprising at least one CDR and optionally one or more framework amino acids from a first antibody and the remainder of the variable region domain from a second antibody.

[0158] The variable region domain may be covalently linked to at least one other antibody domain or fragment thereof at the C-terminal amino acid. Thus, for example, the V H The V domain can be linked to an immunoglobulin CH1 domain or a fragment thereof. L The V domain may be linked to a CK domain or a fragment thereof. In this manner, for example, the antibody may be a Fab fragment in which the antigen binding domain comprises the associated V domain. H and V L The CH1 domain can be extended with more amino acids, for example to provide a hinge region or a portion of a hinge region domain found in a Fab' fragment, or to provide further domains, such as antibody CH2 and CH3 domains.

[0159] As described herein, antibody comprises at least one of these CDRs.For example, one or more CDRs can be introduced into known antibody framework regions (IgG1, IgG2 etc.), or be conjugated to suitable carriers to enhance their half-life.Suitable carriers include but are not limited to Fc, polyethylene glycol (PEG), albumin, transferrin etc. These and other suitable carriers are well known in the art.The CDR peptide of this type of conjugation can be monomer, dimer, tetramer or other forms.In one embodiment, one or more water-soluble polymers are bonded to one or more specific positions of adhesive, for example, amino terminal.

[0160] In another example, a single V from an antibody (ie, a CTLA-4 antibody) L or V H The V chains can be used to search for other V fragments that can form antigen-binding fragments (or Fabs) with the same specificity. H or V L chain. Therefore, V H and V L Random combinations of chain Ig genes can be expressed as antigen-binding fragments in phage libraries (such as fd or lambda phage). For example, combinatorial libraries can be constructed by using the V L or V H Parental V of the chain library combination L or V H A combinatorial library of these chains is generated. 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).

[0161] Diabodies are bivalent antibodies comprising two polypeptide chains, each comprising a VH and VL domain connected by a linker that is too short to allow pairing between the two domains on the same chain, thereby allowing each domain to pair with a complementary domain on the other 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 diabody resulting from their pairing will have two identical antigen-binding sites. Polypeptide chains with different sequences can be used to prepare diabodies with two different antigen-binding sites. Similarly, tribodies and tetrabodies are antibodies comprising three and four polypeptide chains, respectively, and forming three and four antigen-binding sites, respectively, which may be identical or different.

[0162] Antibody polypeptides, including fibronectin polypeptide monomers, are also disclosed in US Patent No. 6,703, 199. Other antibody polypeptides, which are single-chain polypeptides, are disclosed in US Patent Publication No. 2005 / 0238646.

[0163] In certain embodiments, the antibody comprises one or more water-soluble polymer links, including but not limited to polyethylene glycol, polyoxyethylene glycol or polypropylene glycol. See, for example, 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 derived binding agent includes one or more of monomethoxy-polyethylene glycol, dextran, cellulose or other carbohydrate-based polymers, poly-(N-vinyl pyrrolidone)-polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (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 play a role in improving the therapeutic ability of binding agents (such as antibodies). Certain such methods are discussed, for example, in US Pat. No. 6,133,426, which is incorporated herein by reference for any purpose.

[0164] 7.6. Antigen Binding Proteins

[0165] In one aspect, the disclosure provides antigen binding proteins (e.g., antibodies, antibody fragments, antibody derivatives, antibody muteins, and antibody variants) that bind to CTLA-4.

[0166] Antigen binding proteins can have, for example, naturally occurring immunoglobulin structures. "Immunoglobulins" are tetrameric molecules. In naturally occurring immunoglobulins, each tetramer is composed of two pairs of identical polypeptide chains, each pair having a "light" chain (about 25 kDa) and a "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain contains a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The carboxyl-terminal portion of each chain defines a constant region that is primarily responsible for effector function. Human light chains are divided into kappa light chains and lambda light chains. Heavy chains are divided into μ, δ, γ, α, or ε, and define the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains 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 combining site, such that an intact immunoglobulin has two binding sites.

[0167] Antigen binding proteins according to the present disclosure include antigen binding proteins that inhibit the biological activity of CTLA-4.

[0168] Different antigen binding proteins may bind to different domains of CTLA-4 or act through different mechanisms of action. As specifically noted herein, unless otherwise indicated, a domain region is designated to include that group. For example, amino acids 4-12 refer to 9 amino acids: the amino acids at positions 4 and 12, and the seven intermediate amino acids in the sequence. Other examples include antigen binding proteins that inhibit the binding of CTLA-4 to its ligand. Antigen binding proteins do not need to completely inhibit CTLA-4-induced activity to be used in the present disclosure; on the contrary, antigen binding proteins that reduce the specific activity of CTLA-4 are also contemplated. (The discussion herein of the specific mechanisms of action of CTLA-4 binding antigen binding proteins in treating specific diseases is illustrative only, and the methods proposed herein are not limited thereto.)

[0169] In another aspect, 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, and fragments, derivatives, muteins, and variants thereof. Such antigen-binding proteins can be represented using the nomenclature "LxHy," where "x" corresponds to the number of light chain variable regions and "y" corresponds to the number of heavy chain variable regions, as labeled in the following sequences. 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, and so on. 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, including the end range members. Thus, the group range A1-A28 includes all members between A1 and A28, as well as members A1 and A28 themselves. The group range A4-A6 includes members A4, A5, and A6, and so on.

[0170] In some embodiments, the antigen binding protein comprises a variable (V(D)J) region of a heavy chain and light chain sequence identical to a clone in the CTLA-4 binding clone library deposited under ATCC Accession No. PTA-125512. In some embodiments, the antigen binding protein comprises a variable (V(D)J) region of a heavy chain or light chain sequence identical to a clone in the CTLA-4 binding clone library deposited under ATCC Accession No. PTA-125512. In some embodiments, the antigen binding protein is expressed from an expression vector in a clone in the CTLA-4 binding clone library deposited under ATCC Accession No. PTA-125512.

[0171] The positions of the CDRs (underlined) that form part of the antigen binding site are also shown below, while the framework region (FR) is the middle segment of these variable domain sequences. In the light chain variable region and the heavy chain variable region, there are three CDRs (CDR1-3) and four FRs (FR 1-4). The CDR regions of each light chain and heavy chain are also grouped according to the antibody type (A1, A2, A3, etc.). The antigen binding proteins of the present disclosure include, for example, antigen binding proteins having a combination of light chain and heavy chain variable domains selected from the group consisting of: L1H1 (Antibody A1), L2H2 (Antibody A2), L3H3 (Antibody A3), L4H4 (Antibody A4), L5H5 (Antibody A5), L6H6 (Antibody A6), L7H7 (Antibody A7), L8H8 (Antibody A8), L9H9 (Antibody A9), L10H10 (Antibody A10), L11H11 (Antibody A11), L12H12 (Antibody A12), L13H13 (Antibody A14), L14H14 (Antibody A15), L15H15 (Antibody A16), L16H17 (Antibody A17), L17H18 (Antibody A19), L18H19 (Antibody A20), L19H20 (Antibody A21), L20H21 (Antibody A22), L21H22 (Antibody A23), L22H23 (Antibody A24), L23H24 (Antibody A25), L24H25 (Antibody A26), L25H26 (Antibody A27), L26H27 (Antibody A28), L27H28 (Antibody A29), L28H29 (Antibody A30), L29H30 (Antibody A31), L30H31 (Antibody A32), L31H32 (Antibody A33), L32H33 (Antibody A34), L33H34 (Antibody A35), L34H35 Antibody A13), L14H14 (antibody 14), L15H15 (antibody 15), L16H16 (antibody 16), L17H17 (antibody 17), L18H18 (antibody 18), L19H19 (antibody 19), L20H20 (antibody 20), L21H21 (antibody 21), L22H22 (antibody 22), L23H23 (antibody 23), L24H24 (antibody 24), L25H25 (antibody 25), L26H26 (antibody 26), L27H27 (antibody 27) and L28H28 (antibody 28).

[0172] In some embodiments, the antigen binding protein comprises all six CDR sequences (three CDRs of the light chain and three CDRs of the heavy chain) identical to one of the clones in the CTLA-4 binding clone library deposited with ATCC Accession No. PTA-125512. In some embodiments, the antigen binding protein comprises three of the six CDR sequences (three CDRs of the light chain and three CDRs of the heavy chain) identical to one of the clones in the CTLA-4 binding clone library deposited with 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 CTLA-4 binding clone library deposited with ATCC Accession No. PTA-125512.

[0173] In one embodiment, the disclosure provides antigen-binding proteins comprising a light chain variable domain, the amino acid sequence included in the light chain variable domain is different from the sequence of the light chain variable domain selected from L1 to L28 at only 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues, wherein the difference of each such sequence is independently a deletion, insertion or replacement of an amino acid residue. In another embodiment, the light chain variable domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the sequence of the light chain variable domain selected from the group consisting of L1-L28. In another embodiment, the light chain variable domain comprises an amino acid sequence 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 (which includes L1, L2, L3, L4, L5, L6, L7, L8, L9, L10, L11, L12, L13, L14, L15, L16, L17, L18, L19, L20, L21, L22, L23, L24, L25, L26, L27 and L28). In another embodiment, the light chain variable domain comprises an amino acid sequence encoded by a polynucleotide that hybridizes under moderate stringency 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 an amino acid sequence encoded by a polynucleotide that hybridizes under moderate stringency 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 an amino acid sequence encoded by a polynucleotide that hybridizes under moderate stringency conditions to the complement of a light chain polynucleotide consisting of L1-L28.

[0174] In one embodiment, the disclosure provides an antigen binding protein comprising a light chain variable domain comprising an amino acid sequence that differs from the sequence of a light chain variable domain encoded by one of the clones in the CTLA-4 binding clone library deposited under ATCC Accession No. PTA-125512 at only 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residue, wherein each such sequence difference is independently a deletion, insertion, or substitution of one amino acid residue. In another embodiment, the light chain variable domain comprises an amino acid sequence 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 CTLA-4 binding clone library deposited under ATCC Accession No. PTA-125512. In another embodiment, the light chain variable domain comprises an amino acid sequence 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 CTLA-4 binding clone library deposited under ATCC Accession No. PTA-125512.

[0175] In another embodiment, the disclosure provides antigen-binding proteins comprising a heavy chain variable domain, the amino acid sequence comprising the heavy chain variable domain differs from the sequence of the heavy chain variable domain selected from H1 to H28 at only 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residues, wherein the difference of each such sequence is independently a deletion, insertion or substitution of an amino acid residue. In another embodiment, the heavy chain variable domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequence of the heavy chain variable domain selected from the group consisting of H1-H28. In another embodiment, the heavy chain variable domain comprises an amino acid sequence encoded by a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity 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 an amino acid sequence encoded by a polynucleotide that hybridizes under moderate stringency conditions with the complementary sequence 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 an amino acid sequence encoded by a polynucleotide that hybridizes under moderate stringency conditions with the complementary sequence 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 an amino acid sequence encoded by a polynucleotide that hybridizes under moderate stringency conditions with the complementary sequence of a polynucleotide encoding a heavy chain variable domain selected from the group consisting of H1-H28.

[0176] In one embodiment, the disclosure provides an antigen binding protein comprising a heavy chain variable domain comprising an amino acid sequence that differs from the sequence of a heavy chain variable domain encoded by one of the clones in the CTLA-4 binding clone library deposited under ATCC Accession No. PTA-125512 at only 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residue, wherein each such sequence difference is independently a deletion, insertion, or substitution of one amino acid residue. In another embodiment, the heavy chain variable domain comprises an amino acid sequence 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 CTLA-4 binding clone library deposited under ATCC Accession No. PTA-125512. In another embodiment, the heavy chain variable domain comprises an amino acid sequence 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 CTLA-4 binding clone library deposited under ATCC Accession No. PTA-125512.

[0177] Specific embodiments of the antigen-binding proteins of the present disclosure comprise one or more amino acid sequences identical to the amino acid sequences of one or more CDRs and / or FRs mentioned herein. In one embodiment, the antigen-binding protein comprises a light chain CDR1 sequence as shown above. In another embodiment, the antigen-binding protein comprises a light chain CDR2 sequence as shown above. In another embodiment, the antigen-binding protein comprises a light chain CDR3 sequence as shown above. In another embodiment, the antigen-binding protein comprises a heavy chain CDR1 sequence as shown above. In another embodiment, the antigen-binding protein comprises a heavy chain CDR2 sequence as shown above. In another embodiment, the antigen-binding protein comprises a heavy chain CDR3 sequence as shown above.

[0178] In one embodiment, the present disclosure provides antigen binding proteins comprising one or more CDR sequences that differ from the above-described CDR sequences by no more than 5, 4, 3, 2, or 1 amino acid residues.

[0179] In some embodiments, the CDR1 sequence of at least one antigen binding protein is a CDR1 sequence, CDR1-L1 to 28, or CDR1-H1 to 28 from A1-A28 as shown in Table 5. In some embodiments, the CDR2 sequence of at least one antigen binding protein is a CDR2 sequence, CDR2-L1 to 28, or CDR2-H1 to 28 from A1-A28 as shown in Table 5. In some embodiments, the CDR3 sequence of at least one antigen binding protein is a CDR3 sequence, CDR3-L1 to 28, or CDR3-H1 to 28 from A1-A28 as shown in Table 5.

[0180] In another embodiment, the light chain CDR3 sequence of the antigen binding protein is a CDR3 sequence or CDR3-L1 to 28 from A1-A28 as shown in Table 5, and the heavy chain CDR3 sequence of the antigen binding protein is a heavy chain sequence or CDR3-H1 to 28 from A1-A28 as shown in Table 5.

[0181] In another embodiment, the antigen binding protein comprises 1, 2, 3, 4 or 5 CDR sequences, each of which independently differs from the CDR sequence of A1-A23 by 6, 5, 4, 3, 2, 1 or 0 single amino acid additions, substitutions and / or deletions, and the antigen binding protein further comprises 1, 2, 3, 4 or 5 CDR sequences, each of which independently differs from the CDR sequence by 6, 5, 4, 3, 2, 1 or 0 single amino acid additions, substitutions and / or deletions. In some embodiments, the antigen binding protein comprises 1, 2, 3, 4 or 5 CDR sequences, each of which has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the CDR sequence of A1-A28.

[0182] The nucleotide sequence of A1-A28 or the amino acid sequence of A1-A28 can be altered, for example, by random mutagenesis or by site-directed mutagenesis (e.g., oligonucleotide-directed site-specific mutagenesis) to produce an altered polynucleotide comprising one or more specific nucleotide substitutions, deletions, or insertions compared to the unmutated polynucleotide. Examples of techniques for making such alterations 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 prepare, for example, derivatives of anti-CTLA-4 antibodies that have desired properties, such as increased affinity, avidity, or specificity for CTLA-4, increased in vivo or in vitro activity or stability, or reduced in vivo side effects compared to the underivatized antibody.

[0183] Other derivatives of anti-CTLA-4 antibodies within the scope of the present disclosure include covalent or aggregated conjugates of anti-CTLA-4 antibodies or fragments thereof with other proteins or polypeptides, such as by expressing a recombinant fusion protein comprising a heterologous polypeptide fused to the N-terminus or C-terminus of an anti-CTLA-4 antibody polypeptide. For example, the conjugated peptide can be a heterologous signal (or leader) polypeptide, for example, a yeast alpha factor leader or a peptide such as an epitope tag. Fusion proteins comprising antigen binding proteins can contain added peptides to facilitate purification or identification of the antigen binding protein (e.g., poly-His). Antigen binding proteins can also be linked to the FLAG peptide Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Asp-Lys (DYKDDDDK) (SEQ ID NO: 12191), as described in Hopp et al., Bio / Technology 6: 1204, 1988 and US Patent 5,011,912. The FLAG peptide is highly antigenic and provides an epitope that reversibly binds to a specific monoclonal antibody (mAb), enabling rapid detection and easy purification of the expressed recombinant protein. Reagents that can be used to prepare fusion proteins in which the FLAG peptide is fused to a given polypeptide are commercially available (Sigma, St. Louis, MO).

[0184] A suitable Fc polypeptide described in PCT application WO 93 / 10151 (incorporated herein by reference) is a single-chain polypeptide extending from the N-terminal hinge region of the human IgG1 antibody Fc region to the native C-terminal end. Another useful Fc polypeptide is the Fc mutein described in U.S. Patent No. 5,457,035 and in Baum et al., 1994, EMBO J. 13: 3992-4001. The amino acid sequence of this mutein is identical to the amino acid sequence of the native Fc sequence provided in WO 93 / 10151, except that amino acid 19 has been changed from Leu to Ala, amino acid 20 has been changed from Leu to Glu, and amino acid 22 has been changed from Gly to Ala. The mutein shows reduced affinity for Fc receptors.

[0185] 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.

[0186] Oligomers comprising one or more antigen binding proteins can be used as CTLA-4 antagonists. Oligomers can be covalently or non-covalently linked dimers, trimers, or higher oligomers. Oligomers comprising two or more antigen binding proteins are contemplated, one example of which is a homodimer. Other oligomers include heterodimers, homotrimers, heterotrimers, homotetramers, heterotetramers, and the like.

[0187] One embodiment relates to oligomers comprising multiple antigen-binding proteins linked by 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 antibody-derived polypeptides are examples of peptides that can promote oligomerization of the antigen-binding proteins to which they are attached, as described in more detail below.

[0188] 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, for example, variants or fragments. Preferably, the oligomer comprises an antigen-binding protein that has CTLA-4 binding activity.

[0189] In one embodiment, oligomers are prepared using polypeptides derived from immunoglobulins. For example, Ashkenazi et al., 1991, PNAS USA 88:10535; Byrn et al., 1990, Nature 344:677; and Hollenbaugh et al., 1992 Curr. Prots in Immunol., Suppl. 4, pp. 10.19.1-10.19.11 have described the preparation of fusion proteins comprising certain heterologous polypeptides fused to various portions of antibody-derived polypeptides, including the Fc domain.

[0190] One embodiment of the present disclosure relates to a dimer comprising two fusion proteins produced by fusing a CTLA-4 binding fragment of an anti-CTLA-4 antibody to the Fc region of an antibody. The dimer can be prepared by, for example, inserting a gene fusion encoding the fusion protein into a suitable expression vector, expressing the gene fusion in a host cell transformed with the recombinant expression vector, and allowing the expressed fusion protein to assemble more like an antibody molecule, thereby forming an interchain disulfide bond between the Fc portions to produce a dimer.

[0191] Alternatively, the oligomer is a fusion protein comprising multiple antigen binding proteins, with or without a peptide linker (spacer peptide). Suitable peptide linkers are those described in US Patents 4,751,180 and 4,935,233.

[0192] Another method for preparing oligomeric antigen binding proteins involves the use of leucine zippers. Leucine zipper domains are peptides that promote the oligomerization of proteins discovered. Leucine zippers were initially 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, there are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble oligomeric proteins are described in PCT application WO 94 / 10308, and Hoppe et al., 1994, FEBS Letters 344:191 describe a leucine zipper derived from pulmonary surfactant protein D (SPD), which is incorporated herein by reference. The use of modified leucine zippers that allow the trimerization of heterologous proteins fused thereto is described in Fanslow et al., 1994, Semin. Immunol. 6:267-78. In one approach, a recombinant fusion protein comprising an anti-CTLA-4 antibody fragment or derivative fused to a leucine zipper peptide is expressed in a suitable host cell, and the resulting soluble oligomeric anti-CTLA-4 antibody fragment or derivative is recovered from the culture supernatant.

[0193] 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 produced against CTLA-4 or its fragments, variants or derivatives and screened for the ability to interfere with the binding of CTLA-4 to its ligand in conventional assays. Examples of suitable assays are assays that test the ability of antigen binding proteins to inhibit the binding of ligands to cells expressing CTLA-4, or the ability of antigen binding proteins to reduce the biological or cellular response caused by the binding of CTLA-4 ligands to cell surface CTLA-4. For example, antibodies can be screened based on their ability to bind to fixed antibody surfaces (CTLA-4). Antigen binding proteins that block the binding of CTLA-4 to ligands can be used to treat any CTLA-4-related disorders, including but not limited to cancer. In one embodiment, human anti-CTLA-4 monoclonal antibodies produced by a procedure involving immune transgenic mice are used to treat such disorders.

[0194] 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.

[0195] Other embodiments include chimeric antibodies, for example, humanized forms of non-human (for example, mouse) polyclonal antibodies. Such humanized antibodies can be prepared by known techniques, and provide the advantage of reducing immunogenicity when the antibody is administered to people. In one embodiment, humanized monoclonal antibodies include the variable domains (or all or part of its antigen binding site) of mouse antibodies and the constant domains derived from human antibodies. Alternatively, humanized antibody fragments can include the antigen binding site of mouse monoclonal antibodies and the variable domain fragments (lacking antigen binding site) derived from human antibodies. The program for producing chimeric and further engineered monoclonal antibodies is included 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: those described in 139. In one embodiment, chimeric antibodies are CDR-grafted antibodies. Techniques for humanizing antibodies are discussed in, e.g., U.S. Patent 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.

[0196] Procedures have been developed for producing human or partially human antibodies in non-human animals. For example, mice are 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. The antibodies produced in the animals incorporate human immunoglobulin polypeptide chains encoded by the human genetic material introduced into the animals. In one embodiment, a non-human animal (e.g., a transgenic mouse) is immunized with a CTLA-4 polypeptide to produce antibodies against the CTLA-4 polypeptide in the animal.

[0197] An 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: 7001 or other immunogenic fragments of the protein. Examples of techniques for producing and using transgenic animals for producing human or partially human antibodies are described in U.S. Patents 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, Russel 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., 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, Choi et al., 1993, Nature Genetics 4:117-23, Fishwild et 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, Lonberg, 1994, Transgenic Approaches to Human Monoclonal Antibodies in Handbook of Experimental Pharmacology 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, Taylor et al., 1994, Inter'l Immunol. 6:579-91, Tomizuka et al., 1997, Nature Genetics 16:133-43, Tomizuka et al., 2000, Pro.Nat'lAcad.Sci.USA 97:722-27, Tuaillon et al., 1993, Pro. Nat'l Acad. Sci. USA 90:3720-24, and Tuaillon et al., 1994, J. Immunol. 152:2912-20. .

[0198] Antigen-binding proteins (e.g., antibodies, antibody fragments, and antibody derivatives) of the present disclosure may comprise any constant region known in the art. The light chain constant region may be, for example, a κ or λ type light chain constant region, for example, a human κ or λ type light chain constant region. The heavy chain constant region may be, for example, an α, δ, ε, γ, or μ type heavy chain constant region, for example, a human α, δ, ε, γ, or μ type heavy chain constant region. In one embodiment, the light chain or heavy chain constant region is a fragment, derivative, variant, or mutant protein of a naturally occurring constant region.

[0199] The technology for deriving different subclasses or isotypes of antibodies from a target antibody is well known, i.e., subclass conversion. Thus, an IgG antibody can be derived from an IgM antibody, for example, and vice versa. Such technology can prepare new antibodies with the antigen-binding properties of a given antibody (parent antibody), but also exhibit biological properties related to antibody isotypes or subclasses that are different from the parent antibody. Cloned DNA encoding specific antibody polypeptides can be used in such procedures, for example, DNA encoding the constant domains of antibodies of the desired isotype. Also see Lantto et al., 2002, Methods Mol.Biol.178:303-16.

[0200] In one embodiment, the antigen-binding protein of the present disclosure comprises the IgG1 heavy chain domain of any of A1-A28 (H1-H28) or a fragment of the IgG1 heavy chain domain of any of A1-A28 (H1-H28). In another embodiment, the antigen-binding protein of the present disclosure comprises the κ light chain constant region of A1-A28 (L1-L28) or a fragment of the κ light chain constant region of A1-A28 (L1-L28). In another embodiment, the antigen-binding protein of the present disclosure comprises both the IgG1 heavy chain domain of A1-A28 (L1-L28) or a fragment thereof and the κ light chain domain of A1-A28 (L1-L28) or a fragment thereof.

[0201] Thus, antigen binding proteins of the present disclosure include those comprising, for example, the variable domain combinations L1H1, L2H2, L3H3, L4H4, L5H5, L6H6, L7H7, L8H8, L9H9, L10H10, L11H11, L12H12, L13H13, ... and L28H28, of the desired isotype (e.g., IgA, IgG1, IgG2, IgG3, IgG4, IgM, IgE, and IgD), as well as Fab or F(ab')2 fragments thereof. Furthermore, if IgG4 is desired, it may also be desirable to introduce point mutations in the hinge region (CPSCP (SEQ ID NO: 11969) -> CPPCP (SEQ ID NO: 11970)), as described in Bloom et al., 1997, Protein Science 6: 407 (incorporated herein by reference), to reduce the tendency to form intra-H chain disulfide bonds, which may contribute to the heterogeneity of IgG4 antibodies.

[0202] In one embodiment, the antigen binding protein has 1x10 -4 s -1 or lower K off In another embodiment, K off It's 5x10 -5 s -1 Or lower. In another embodiment, Koff The antigen binding protein is substantially identical to an antibody having a combination of light and heavy chain variable domain sequences selected from the group consisting of: L1H1, L2H2, L3H3, L4H4, L5H5, L6H6, ..., and L28H28. In another embodiment, the antigen binding protein is substantially identical to an antibody comprising one or more CDRs from an antibody having a combination of light and heavy chain variable domain sequences selected from the group consisting of: off Binds to CTLA-4: L1H1, L2H2, L3H3, L4H4, L5H5, L6H6, ... and L23H28. In another embodiment, the antigen binding protein binds to CTLA-4 with a Kp of substantially the same as that of an antibody comprising one of the above amino acid sequences. off In another embodiment, the antigen binding protein binds to CTLA-4 with a KD substantially the same as that of an antibody comprising one or more CDRs from an antibody comprising one of the above amino acid sequences. off Binds to CTLA-4.

[0203] In one aspect, the present disclosure provides antigen-binding fragments of the anti-CTLA-4 antibodies of the present disclosure. Such fragments can be composed entirely of antibody-derived sequences or can contain additional sequences. Examples of antigen-binding fragments include Fab, F(ab')2, single-chain antibodies, diabodies, triabodies, tetrabodies, and domain antibodies. Further examples are provided in Lunde et al., 2002, Biochem. Soc. Trans. 30:500-06.

[0204] 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), thereby generating a single polypeptide chain. Such single-chain Fvs (scFv) have been developed by encoding two variable domain polypeptides (V L and V H ) are fused with a DNA encoding a peptide linker. The resulting polypeptide can fold itself to form an antigen-binding monomer, or it can form a multimer (e.g., a dimer, trimer, or tetramer), 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). By combining different V L and V HCombinations of polypeptides can form multimeric scFvs that bind to different epitopes (Kriangkum et al., 2001, Biomol. Eng. 18:31-40). Technologies developed for the production of single-chain antibodies include those described in U.S. Patent 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. The present disclosure encompasses scFvs comprising the variable domain combinations L1H1, L2H2, L3H3, L4H4, L5H5, L6H6 ... and L28H28.

[0205] 7.7. Monoclonal Antibodies

[0206] In another aspect, the present disclosure provides monoclonal antibodies that bind to CTLA-4. The monoclonal antibodies of the present disclosure can be produced using various well-known techniques. In general, monoclonal antibodies that bind to a specific antigen can be obtained by methods well known to those skilled in the art (see, e.g., 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 against proteins expressed in E. coli," DNA Cloning 2: Expression Systems, 2nd ed., Glover et al. (eds.), p. 93 (Oxford University Press 1995). Antibody fragments can be derived using any suitable standard technique, such as proteolytic digestion, or optionally by proteolytic digestion (e.g., using papain or pepsin) followed by mild reduction and alkylation of disulfide bonds. Alternatively, such fragments can also be produced by recombinant genetic engineering techniques as described herein.

[0207] Monoclonal antibodies can be obtained by injecting an animal, such as a rat, hamster, rabbit, or preferably a mouse, including, for example, a transgenic or knockout version, with an immunogen comprising human CTLA-4 [SEQ ID 7001] or a fragment thereof, as is known in the art and described herein. The presence of specific antibody production can be monitored after the initial injection and / or after booster injections by obtaining serum samples and detecting the presence of antibodies that bind to human CTLA-4 or the peptide using any of several immunoassays known in the art and described herein. Lymphoid cells, most commonly spleen cells or lymph node cells, are removed from the animal producing the desired antibody 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 desired properties (e.g., inability to express endogenous Ig gene products, e.g., P3X63-Ag 8.653 (ATCC No. CRL1580); NSO, SP20), to produce hybridomas, which are immortalized eukaryotic cell lines.

[0208] Lymphoid (e.g., spleen) cells and myeloma cells can be combined with a membrane fusion promoter (such as polyethylene glycol or a nonionic detergent) for a few minutes and then seeded at low density on a selective medium that supports the growth of hybridoma cells rather than unfused myeloma cells. A preferred selective medium is HAT (hypoxanthine, aminopterin, thymidine). After a sufficient period of time, usually about one to two weeks, cell colonies are observed. Single colonies are isolated, and any of the various immunoassays known in the art and described herein can be used to test the binding activity of the antibodies produced by the cells to human CTLA-4. The hybridoma is cloned (e.g., by limiting dilution cloning or by soft agar plaque isolation), and positive clones that produce antibodies specific for CTLA-4 are selected and cultured. Monoclonal antibodies from hybridoma cultures can be isolated from hybridoma culture supernatants.

[0209] Another method for producing mouse monoclonal antibodies is to inject hybridoma cells into the peritoneal cavity of isogenic mice, for example, mice processed (for example, pristane stimulates) to promote ascites formation comprising monoclonal antibodies. Monoclonal antibodies can be separated and purified by various well-established technical separations. Such separation techniques include affinity chromatography, size exclusion chromatography and ion exchange chromatography using protein A agarose (see, for example, Coligan, at pages 2.7.1-2.7.12 and 2.9.1-2.9.3; Baines et al., " Purification of Immunoglobulin G (IgG), " Methods in Molecular Biology, Vol. 10, pp. 79-104 (The Humana Press, Inc. 1992)). Monoclonal antibodies can be purified by affinity chromatography using the appropriate ligand selected based on the specific properties of the antibody (for example, heavy chain or light chain isotype, binding specificity, etc.). Examples of suitable ligands immobilized on a solid support include protein A, protein G, antibody constant region (light or heavy chain) antibodies, anti-idiotypic antibodies, and TGFβ binding protein or fragments or variants thereof.

[0210] Monoclonal antibodies can be produced using any technique known in the art, for example, by immortalizing spleen cells harvested from transgenic animals after an immunization procedure. Splenocytes 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. The present disclosure includes such hybridoma cell lines and the anti-CTLA-4 monoclonal antibodies produced therefrom. Myeloma cells used in the fusion procedure for producing hybridomas are preferably non-antibody-producing, have a high fusion efficiency, and are enzyme-deficient, which prevents them from growing in certain selective media that only support the growth of the desired fused cells (hybridomas). Examples of suitable cell lines for mouse fusion include Sp-20, P3-X63 / Ag8, P3-X63-Ag8.653, NS1 / 1.Ag41, Sp210-Ag14, FO, NSO / U, MPC-11, MPC11-X45-GTG 1.7, and S194 / 5XX0 Bull; Examples of cell lines for rat fusion include R210.RCY3, Y3-Ag 1.2.3, IR983F, and 4B210. Other cell lines that can be used 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.

[0211] The antibodies of the present disclosure may also be fully human monoclonal antibodies. An isolated fully human antibody that specifically binds to CTLA-4 is provided, wherein the antigen binding protein has at least one in vivo biological activity of a human anti-CTLA-4 antibody.

[0212] 7.8. Methods for Producing Antibodies

[0213] Fully human monoclonal antibodies can be produced by various techniques well known to those of ordinary skill in the art. Such methods include but are not limited to Epstein-Barr virus (EBV) transformation of human peripheral blood cells (e.g., comprising B lymphocytes), in vitro immunity of human B cells, fusion of splenocytes from immune transgenic mice carrying inserted human immunoglobulin genes, separation from human immunoglobulin V district phage libraries or as well known in the art and based on other programs of the present disclosure. For example, fully human monoclonal antibodies can be obtained from transgenic mice, which are engineered to produce specific human antibodies in response to antigenic stimulation. Methods for obtaining fully human antibodies from transgenic mice are described in, for example, 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 the human heavy and light chain loci are introduced into a mouse strain derived from an embryonic stem cell line, which contains targeted disruptions of the endogenous heavy and light chain loci (see also Bruggemann et al., Curr. Opin. Biotechnol. 8:455 58 (1997)). For example, a human immunoglobulin transgene can be a minigene construct or a transposition site on a yeast artificial chromosome that undergoes B cell-specific DNA rearrangement and hypermutation in mouse lymphoid tissue. Fully human monoclonal antibodies can be obtained by immunizing transgenic mice, which in turn produce human antibodies specific for CTLA-4. According to the methods described herein, lymphoid cells from immunized transgenic mice can be used to produce hybridomas that secrete human antibodies. Polyclonal serum containing fully human antibodies can also be obtained from the blood of immunized animals.

[0214] Another method for producing human antibodies of the present disclosure includes immortalizing human peripheral blood cells by EBV transformation. See, for example, U.S. Patent No. 4,464,456. This immortalized B cell line (or lymphoblastoid cell line) that produces monoclonal antibodies that specifically bind to CTLA-4 can be identified by immunoassay methods such as ELISA as provided herein and then isolated by standard cloning techniques. According to methods well known in the art, the stability of the lymphoblastoid cell line that produces anti-CTLA-4 antibodies can be improved by fusing the transformed cell line with a mouse myeloma to produce a mouse-human hybrid cell line (see, for example, Glasky et al., Hybridoma 8:377-89 (1989)). Another method for producing human monoclonal antibodies is in vitro immunization, which includes activating human spleen B cells using human CTLA-4. The activated B cells are then fused with a heterologous hybrid fusion partner. See, for example, Boerner et al., 1991 J. Immunol. 147:86-95.

[0215] In certain embodiments, B cells that produce 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 (WO 92 / 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 that produce antibodies that specifically bind to CTLA-4. B cells can also be isolated from humans (e.g., from peripheral blood samples).

[0216] Methods for detecting single B cells that produce antibodies with the desired specificity are well known in the art, for example, by plaque formation, fluorescence-activated cell sorting, in vitro stimulation followed by detection of specific antibodies, etc. Methods for selecting B cells that produce specific antibodies include, for example, preparing a single cell suspension of B cells in soft agar containing human CTLA-4. The binding of specific antibodies produced by B cells to antigens results in the formation of complexes that can be visualized as immunoprecipitates.

[0217] In some embodiments, B cells that produce specific antibodies are selected by using a method that allows 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), the entire contents of which are incorporated herein by reference, can be used. Figure 1 As summarized in, the method combines microfluidics, molecular genomics, yeast single chain variable fragment (scFv) display, fluorescence activated cell sorting (FACS) and deep sequencing.In brief, B cell can be separated from immune animal, then merged.B cell oligo-dT beads and lysate are encapsulated in droplet, from the beads of droplet purification mRNA bonded, then injected into the second emulsion comprising OE-RT-PCR amplification mixture, its generation coding has the DNA amplicon of the scFv right of natural heavy chain and light chain Ig.Then the library of the amplicon of natural pairing is electroporated into yeast for scFv display.FACS is used to identify high-affinity scFv.Finally, deep antibody sequencing can be used to identify all clones in the scFv library before and after the sorting.

[0218] After selection of B cells producing the antibody, the specific antibody gene can be cloned by isolating and amplifying DNA or mRNA according to methods known in the art and described herein.

[0219] Methods for obtaining the disclosed antibodies can also employ various phage display techniques known in the art. See, for example, 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 constructed in phage vectors, and the phage vectors can be screened to select Ig fragments (Fab, Fv, sFv, or multimers thereof) that specifically bind to CTLA-4 binding protein, or variants or fragments thereof. See, e.g., U.S. Patent 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 comprising a plurality of polynucleotide sequences encoding Ig variable region fragments can be inserted into the genome of a filamentous phage such as M13 or a variant thereof in frame with a sequence encoding a phage capsid protein. The fusion protein can be a fusion of a capsid protein with a light chain variable region domain and / or with 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).

[0220] Antibody fragments fused to another protein (such as a minor capsid protein) can also be used to enrich phage with antigen. Rearranged recombinant antibodies (V) from mice immunized against the antigen (e.g., CTLA-4) are then used to enrich phage. H ) and light (V L ) chains, and display diverse antibody fragment libraries on the surface of phage. These libraries can be screened for complementary variable domains and the domains purified, for example, using affinity columns. See Clackson et al., Nature, Vol. 352, pp. 624-628 (1991).

[0221] Heavy and light chain immunoglobulin cDNA expression libraries can also be prepared in lambda phage, for example, using lambdalmmunoZap TM (H) and λImmunoZap TM(L) vectors (Stratagene, La Jolla, California). Briefly, mRNA is re-isolated from B cell populations and used to regenerate heavy and light chain immunoglobulin cDNA expression libraries in the λImmunoZap(H) and λImmunoZap(L) vectors. These vectors can be screened individually or co-expressed to form Fab fragments or antibodies (see Huse et al., supra; see also Sastry et al., supra). Positive plaques can then be converted to a non-lytic plasmid that allows high-level expression of monoclonal antibody fragments from E. coli.

[0222] In one embodiment, nucleotide primers are used to amplify the variable region of the monoclonal antibody gene of interest expressed in a hybridoma. These primers can be synthesized by one of ordinary skill in the art or purchased from commercial sources. (See, for example, Stratagene (La Jolla, California), which sells mouse and human variable region primers, including V Ha 、V Hb 、V Hc 、V Hd 、C H1 、V L and C L These primers can be used to amplify the heavy or light chain variable regions, which can then be inserted into vectors such as ImmunoZAP. TM H or ImmunoZAP TM L (Stratagene). These vectors can then be introduced into E. coli, yeast, or mammalian expression systems. These methods can be used to produce large quantities of V H and V L A single-chain protein that is a fusion protein of the 1,2-diaminobenzyltransferase (1,2-dGly) domain (see Bird et al., Science 242: 423-426, 1988).

[0223] Once cells producing antibodies according to the present disclosure are obtained using any of the above-mentioned immunization and other techniques, specific antibody genes can be cloned by isolating and amplifying DNA or mRNA therefrom according to standard procedures described herein. The antibodies thus produced can be sequenced and the CDRs identified and the DNA encoding the CDRs can be manipulated as previously described to produce other antibodies according to the present disclosure.

[0224] The CTLA-4 binding agents disclosed herein preferably modulate CTLA-4 function and / or bind to one or more of the domains described herein and / or cross-block binding of and / or are cross-blocked from binding to CTLA-4 by one of the antibodies described herein in the cell-based assays described herein and / or in the in vivo assays described herein. Thus, such binding agents can be identified using the assays described herein.

[0225] In certain embodiments, antibodies are generated by first identifying antibodies that bind to one or more of the domains provided herein and / or neutralize and / or cross-block the binding of an antibody described herein in a cell-based assay and / or in an in vivo assay as described herein and / or are cross-blocked by one of the antibodies described herein to CTLA-4. The CDR regions from these antibodies are then used to insert into an appropriate biocompatible framework to generate a CTLA-4 binding agent. 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 the identification of binding agents. Preferably, the binding agents of the present disclosure are antibodies as defined herein.

[0226] Other antibodies according to the present disclosure can be obtained by conventional immunization and cell fusion procedures as described herein and well known in the art.

[0227] Molecular evolution of the complementarity determining regions (CDRs) at the center of the antibody binding site has also been used to isolate antibodies with increased affinity, for example, antibodies with increased affinity for c-erbB-2, as described in Schier et al., 1996, J. Mol. Biol. 263:551. Thus, such techniques can be used to prepare antibodies against CTLA-4. For example, antigen binding proteins against CTLA-4 can be used in assays for the presence of CTLA-4 polypeptides in vitro or in vivo. Antigen binding proteins can also be used to purify CTLA-4 protein by immunoaffinity chromatography.

[0228] Although human antibodies, partially human antibodies, or humanized antibodies are suitable for many applications, particularly those involving the administration of antibodies to human subjects, other types of antigen-binding proteins are also suitable for certain applications. The non-human antibodies of the present disclosure can be derived, for example, from any animal that produces antibodies, such as mice, rats, rabbits, goats, donkeys, or non-human primates (e.g., monkeys (e.g., cynomolgus monkeys or rhesus monkeys) or apes (e.g., chimpanzees)). Antibodies from a particular species can be prepared, for example, by immunizing an animal of that species with a desired immunogen (e.g., a CTLA-4 polypeptide) or using an artificial system for producing antibodies of that species (e.g., a bacterial or phage display-based system for producing antibodies of a particular species), or by converting an antibody from one species into an antibody from another species, for example, by replacing the constant region of the antibody with a constant region from another species, or by replacing one or more amino acid residues of the antibody so that it more closely resembles the antibody sequence from the other species. In one embodiment, the antibody is a chimeric antibody, which comprises an amino acid sequence derived from antibodies from two or more different species.

[0229] Antigen binding proteins can be prepared by any of a variety of conventional techniques and screened for desired properties. Certain techniques involve separating nucleic acids encoding target antigen binding proteins (e.g., anti-CTLA-4 antibodies) polypeptide chains (or portions thereof) and manipulating the nucleic acids by recombinant DNA techniques. For example, nucleic acids can be fused to another target nucleic acid, or altered (e.g., by mutagenesis or other conventional techniques) to add, delete, or replace one or more amino acid residues. In addition, antigen binding proteins can be purified from cells naturally expressing antigen binding proteins (e.g., antibodies can be purified from hybridomas producing them) or 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).

[0230] Any expression system known in the art can be used to prepare the recombinant polypeptides of the present disclosure. Expression vectors are described in full detail above. Typically, a recombinant expression vector containing DNA encoding the desired polypeptide is used to transform a host cell. Useful host cells include prokaryotes, yeast, or higher eukaryotic cells. Prokaryotes include Gram-negative or Gram-positive organisms, such as Escherichia coli or Bacillus. 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) cell lines, and the CVI / EBNA line (ATCC CCL 70) derived from the African green monkey kidney cell line CVI, as described by McMahan et al., 1991, EMBO J. 10: 2821. Suitable cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985).

[0231] It will be appreciated that the antibodies of the present disclosure may have at least one amino acid replacement, provided that the antibody retains binding specificity. Thus, modifications to the antibody structure are included within the scope of the present disclosure. These may include amino acid replacements, which may be conservative or non-conservative (which do not destroy the CTLA-4 binding ability of the antibody). Conservative amino acid replacements may include non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptide mimetics and other reverse or inverted forms of amino acid moieties. Conservative amino acid replacements may also involve replacements 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.

[0232] Non-conservative substitutions may involve exchanging a member of one class of amino acids or amino acid mimetics for a member of another class having different physical properties (e.g., size, polarity, hydrophobicity, charge). Such substituted residues may be introduced into regions of homology between human and non-human antibodies, or into non-homologous regions of the molecule.

[0233] In addition, those skilled in the art can produce test variants comprising a single amino acid substitution at each desired amino acid residue. Then, variants can be screened using activity assays well known to those skilled in the art. Such variants can be used to collect information about suitable variants. For example, if it is found that changes to specific amino acid residues result in activity destruction, undesirable reduction in activity, or inappropriate activity, variants with such changes can be avoided. In other words, based on the information collected from such routine experiments, those skilled in the art can easily determine further substituted amino acids that should be avoided, either alone or in combination with other mutations.

[0234] Those skilled in the art will be able to use well-known techniques to determine suitable variants of the polypeptides described herein. In certain embodiments, those skilled in the art can identify suitable regions of the molecule that can be altered without destroying the activity by targeting regions that are thought to be unimportant 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 the biological activity or having an adverse effect on the polypeptide structure.

[0235] In addition, one skilled in the art can review structure-function studies to identify residues in similar polypeptides that are important for activity or structure. Based on this comparison, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues that are important for activity or structure in similar proteins. One skilled in the art can select chemically similar amino acid substitutions to replace such predicted important amino acid residues.

[0236] Those skilled in the art can also analyze the three-dimensional structure and amino acid sequence related to the structure in similar polypeptides. In view of this information, those skilled in the art can predict the comparison of the amino acid residues of the antibody based on its three-dimensional structure. In certain embodiments, those skilled in the art can choose not to completely change the amino acid residues predicted on the protein surface because these residues may be involved in important interactions with other molecules.

[0237] Many scientific publications have been devoted to the prediction of secondary structure. See Moult J., Curr. Op. in Biotech., 7(4):422-427 (1996), Chou et al., Biochem., 13(2):222-245 (1974); Chou et al., Biochem., 113(2):211-222 (1974); 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). In addition, computer programs are currently available to help predict secondary structure. One method of predicting secondary structure is based on homology modeling. For example, two polypeptides or proteins with a sequence identity greater than 30%, or a similarity greater than 40%, tend to have similar structural topologies. Recent developments in the Protein Structure Database (PDB) have enhanced the predictability of secondary structure, including the number of potential folds within a polypeptide or protein structure. See Holm et al., Nucl. Acid. Res., 27(1):244-247 (1999). It has been suggested (Brenner et al., Curr. Op. Struct. Biol., 7(3):369-376 (1997)) that a given polypeptide or protein has a finite number of folds, and that once a critical number of structures are determined, structure prediction will become more accurate.

[0238] Other 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)), "property 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)).

[0239] 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, the variant contains a greater or lesser number of N-linked glycosylation sites compared to the native protein. N-linked glycosylation sites can be characterized by the following 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 potential new sites for adding N-linked carbohydrate chains. Alternatively, substitutions that eliminate this sequence will remove existing N-linked carbohydrate chains. Also provided is a rearrangement of N-linked carbohydrate chains, in which one or more N-linked glycosylation sites (typically naturally occurring) are eliminated and one or more new N-linked sites are created. Other preferred antibody variants include cysteine ​​variants, in which one or more cysteine ​​residues are deleted or substituted for another amino acid (e.g., serine) compared to the parent amino acid sequence. Cysteine ​​variants may be useful when the antibody must be refolded into a biologically active conformation, for example, after the separation of insoluble inclusion bodies. Cysteine ​​variants typically have fewer cysteine ​​residues than the native protein, and typically have an even number to minimize interactions due to unpaired cysteines.

[0240] Desired amino acid substitutions (whether conservative or non-conservative) can be determined by one skilled in the art when such substitutions are desired. In certain embodiments, amino acid substitutions can be used to identify important residues for antibodies to CTLA-4, or to increase or decrease the affinity of the antibodies to CTLA-4 described herein.

[0241] 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 protein complexes, (4) alter binding affinity, and / or (5) confer or alter other physiochemical or functional properties of such polypeptides. According to certain embodiments, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) can be in the naturally occurring sequence (in certain embodiments, in the portion of the polypeptide outside of one or more domains that form intermolecular contacts). In certain embodiments, conservative amino acid substitutions generally do not significantly alter the structural characteristics of the parent sequence (e.g., the replacing amino acid should not tend to disrupt helices present in the parent sequence, or disrupt other types of secondary structure that characterize the parent sequence). Examples of art-recognized secondary and tertiary structures of polypeptides are described in Proteins, Structure and Molecular Principles (Creighton, Ed., WH 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.

[0242] In certain embodiments, the antibodies of the present disclosure may be chemically bonded to a polypeptide, lipid, or other moiety.

[0243] The binding agent may comprise at least one CDR 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 that is capable of displaying one or more amino acid sequences (e.g., CDRs, variable regions, etc.) that bind to an antigen in a localized surface area. Such a structure may be a naturally occurring polypeptide or polypeptide "fold" (structural motif), or may have one or more modifications relative to a naturally occurring polypeptide or fold, such as the addition, deletion, or substitution of amino acids. These scaffolds may be derived from polypeptides of any species (or more than one species), such as humans, other mammals, other vertebrates, invertebrates, plants, bacteria, or viruses.

[0244] Typically, biocompatible framework structures are based on protein scaffolds or backbones other than immunoglobulin domains. For example, those based on fibronectin, ankyrin, lipocalin, neocarcinomatin, cytochrome b, CP1 zinc finger, PST1, coiled-coil, LACI-D1, Z domains, and tendamist domains can be used (see, e.g., Nygren and Uhlen, 1997, Curr. Opin. in Struct. Biol., 7, 463-469).

[0245] Humanized antibodies can be produced using techniques well known in the art, such as those described herein (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).

[0246] In addition, one skilled in the art will recognize that suitable binding agents include portions of these antibodies, such as CDR1-L1 to 28 having SEQ ID NOs 1001-1028; CDR2-L1 to 28 having SEQ ID NOs 2001-2028; CDR3-L1 to 28 having SEQ ID NOs 3001-3028; CDR1-H1 to 28 having SEQ ID NOs 4001-4028; CDR2-H1 to 28 having SEQ ID NOs 5001-5028; and CDR3-H1 to 28 having SEQ ID NOs 6001-6028, as specifically disclosed herein. At least one region of a CDR region may have at least one amino acid substitution with a 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, wherein the binding agent 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 a non-protein molecule, wherein the antibody exhibits a similar binding pattern to a human CTLA-4 peptide as at least one of antibodies A1-A28 in a competition binding assay, and / or neutralizes CTLA-4. The non-CDR portion of the antibody can be composed of amino acids, wherein the antibody is a recombinant binding protein or a synthetic peptide, and the recombinant binding protein cross-blocks the binding of an antibody disclosed herein to CTLA-4 and / or neutralizes CTLA-4. The non-CDR portion of the antibody can be composed of amino acids, wherein the antibody is a recombinant binding protein or a synthetic peptide, and the recombinant binding protein cross-blocks the binding of an antibody disclosed herein to CTLA-4 and / or neutralizes CTLA-4. The non-CDR portion of the antibody can be composed of amino acids, wherein the antibody is a recombinant binding protein or a synthetic peptide, and the recombinant binding protein cross-blocks the binding of an antibody disclosed herein to CTLA-4 and / or neutralizes CTLA-4. The non-CDR portion of the antibody can be composed of amino acids, wherein the antibody is a recombinant antibody, and the recombinant antibody exhibits a similar binding pattern to a human CTLA-4 peptide in a human CTLA-4 peptide epitope competition binding assay (described below), such as exhibited by at least one of antibodies A1-A28, and / or neutralizes CTLA-4.

[0247] When the antibody comprises one or more of CDR1-H, CDR2-H, CDR3-H, CDR1-L, CDR2-L and CDR3-L as described above, it can be obtained by expressing from a host cell comprising DNA encoding these sequences. The DNA encoding each CDR sequence can be determined based on the amino acid sequence of the CDR and synthesized together with any desired antibody variable region framework and constant region DNA sequence using oligonucleotide synthesis technology, site-directed mutagenesis and polymerase chain reaction (PCR) technology as appropriate. Those skilled in the art can obtain the antibody from gene sequence databases such as DNA encoding the variable region framework and constant regions is widely available.

[0248] Once synthesized, the DNA encoding the antibodies of the present disclosure or their fragments can be propagated and expressed using any number of known expression vectors according to any of the various well-known procedures for nucleic acid excision, ligation, transformation, and transfection. Therefore, in certain embodiments, the expression of the antibody fragment can preferably be in a prokaryotic host, such as Escherichia coli (see, e.g., Pluckthun et al., 1989 Methods Enzymol. 178: 497-515). In certain other embodiments, the expression of the antibody or its fragment can preferably be in a eukaryotic host cell, 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 (e.g., mouse NSO cell line), COS, CHO, or hybridoma cells. Examples of plant cells include tobacco, corn, soybean, and rice cells.

[0249] One or more replicable expression vectors comprising DNA encoding the variable and / or constant regions of an antibody can be prepared and used to transform a suitable cell line, e.g., a non-productive myeloma cell line, such as a mouse NSO cell line, or bacteria, such as E. coli, in which the antibody is to be produced. To achieve 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. (Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, New York, 1989; see also 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 the Amersham International plc sequencing manual, and site-directed mutagenesis can be performed according to methods well 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); Anglian Biotechnology Ltd. manual). In addition, numerous publications describe techniques suitable for preparing antibodies by manipulating DNA, creating expression vectors, and transforming and culturing appropriate cells (Mountain A and Adair, JR, Biotechnology and Genetic Engineering Reviews (Tombs, MP ed., 10, Chapter 1, 1992, Intercept, Andover, UK); "Current Protocols in Molecular Biology", 1999, FM Ausubel (ed.), Wiley Interscience, New York).

[0250] When it is necessary to improve the affinity of the antibodies according to the present disclosure, antibodies comprising one or more of the above-mentioned CDRs can be obtained by a variety of affinity maturation protocols, including retention of CDRs (Yang et al., J. Mol. Biol., 254, 392-403, 1995), chain shuffling (Marks et al., Bio / Technology, 10, 779-783, 1992), use of E. coli mutant strains (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, 788, 1996) and sexual PCR (Crameri et al., Nature, 391, 288-291, 1998). All of these methods of affinity maturation are discussed in Vaughan et al. (Nature Biotech., 16, 535 539, 1998).

[0251] Those skilled in the art will appreciate that some proteins (such as antibodies) can undergo a variety of post-translational modifications. The type and extent of these modifications generally depend on the host cell line and culture conditions used to express the protein. Such modifications may include changes in glycosylation, methionine oxidation, diketopiperazine formation, aspartic acid isomerization, and asparagine deamidation. A common modification is the loss of a basic residue (such as lysine or arginine) at the carboxyl terminus due to the action of a carboxypeptidase (as described in Harris, RJ Journal of Chromatography 705: 129-134, 1995).

[0252] 7.9. Sequence

[0253] Antibodies A1-A28 comprise heavy and light chain V(J)D polynucleotides (referred to herein as L1-L28 and H1-H28, respectively). Antibodies A1-A28 comprise the sequences listed in Table 5. For example, antibody A1 comprises light chain L1 (SEQ ID NO: 1) and heavy chain H1 (SEQ ID NO: 101). The CDR sequences of the light chain (L1-L28) and heavy chain (H1-H28) are also provided with specific SEQ ID NOs. For example, the three CDR sequences (CDR1, CDR2 and CDR3) for L1 are CDR1-L1 (SEQ ID NO: 1001), CDR2-L1 (SEQ ID NO: 2001) and CDR3-L1 (SEQ ID NO: 3001), and the three CDR sequences (CDR1, CDR2 and CDR3) for H1 are CDR1-H1 (SEQ ID NO: 4001), CDR2-H1 (SEQ ID NO: 5001) and CDR3-H1 (SEQ ID NO: 6001).

[0254]

[0255]

[0256]

[0257] 7.10. Pharmaceutical Compositions

[0258] Also provided are pharmaceutical compositions comprising the proteins and polypeptides of the present disclosure. Such compositions comprise a therapeutically or prophylactically effective amount of the polypeptide or protein admixed with pharmaceutically acceptable materials and physiologically acceptable formulation materials.

[0259] The pharmaceutical compositions may contain formulation materials used to alter, maintain or preserve, for example, the pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption or penetration of the composition.

[0260] Suitable formulation materials include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., borates, bicarbonates, Tris-HCl, citrates, phosphates, other organic acids); bulking agents (e.g., mannitol or glycine), chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, or hydroxypropyl-β-D-pyrrolidone); -cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); coloring agents; 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, phenylethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); ); solvents (such as glycerol, 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); stability enhancers (sucrose or sorbitol); tonicity 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 the same serum albumin are examples of suitable diluents. Preservatives such as benzyl alcohol may also be added according to appropriate industry standards. The composition may be formulated as a lyophilizate using a suitable excipient solution (such as sucrose) as a diluent. Suitable components are non-toxic to the recipient at the dosages and concentrations employed. Other examples of components that can be used in pharmaceutical formulations can be found in Remington's Pharmaceutical Sciences, 16th Ed. (1980) and 20th Ed. (2000), Mack Publishing Company, Easton, PA.

[0261] Optionally, the composition further comprises one or more physiologically active agents, e.g., anti-angiogenic substances, chemotherapeutic substances (e.g., capecitabine, 5-fluorouracil, or doxorubicin), analgesic substances, etc., non-exhaustive examples of which are provided herein. In various specific embodiments, the composition comprises 1, 2, 3, 4, 5, or 6 physiologically active agents in addition to the CTLA-4 binding protein.

[0262] In another embodiment of the present disclosure, the compositions disclosed herein can be prepared as addition salts in neutral or salt form. Exemplary pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein), and they are formed with inorganic acids, for example, hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. The salts formed with the free carboxyl groups can also be derived from inorganic bases, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide, and organic bases, such as isopropylamine, trimethylamine, histidine, procaine, etc. When formulated, the solution will be used in a manner compatible with the formulation and in a therapeutically effective amount.

[0263] Carriers may also include any and all solvents, dispersion media, carriers, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active ingredient, their use in therapeutic compositions is contemplated. Supplementary active ingredients may also be added to the composition. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to humans.

[0264] The optimal pharmaceutical composition will be determined by one skilled in the art based on, for example, the intended route of administration, delivery form, and desired dosage. See, for example, Remington's Pharmaceutical Sciences, supra. Such compositions can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the polypeptide. For example, a suitable composition can be water for injection or a physiological saline solution for parenteral administration.

[0265] 7.10.1. Content of active pharmaceutical ingredients

[0266] In typical embodiments, the active ingredient (i.e., proteins and polypeptides of the present disclosure) is present in the pharmaceutical composition at a concentration of at least 0.01 mg / ml, at least 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, or 25 mg / ml. In certain embodiments, the active ingredient is present in the pharmaceutical composition at a concentration of at least 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, or 50 mg / ml.

[0267] In some embodiments, in addition to the protein or polypeptide of the present disclosure, the pharmaceutical composition comprises one or more additional active ingredients. The one or more additional active ingredients can be drugs that target different checkpoint receptors, such as CTLA-4 inhibitors (e.g., anti-CTLA-4 antibodies) or TIGIT inhibitors (e.g., anti-TIGIT antibodies).

[0268] 7.10.2. General preparations

[0269] The pharmaceutical composition may be in any form suitable for human or veterinary use, including liquid, oil, emulsion, gel, colloid, aerosol or solid.

[0270] Pharmaceutical compositions may be formulated for administration by any route of administration suitable for human or veterinary medicine, including enteral and parenteral routes of administration.

[0271] In various embodiments, the pharmaceutical composition is formulated for administration by inhalation. In certain of these embodiments, the pharmaceutical composition is formulated for administration via a vaporizer. In certain of these embodiments, the pharmaceutical composition is formulated for administration via a nebulizer. In certain of these embodiments, the pharmaceutical composition is formulated for administration via an aerosol.

[0272] In various embodiments, the pharmaceutical composition is formulated for oral administration, for buccal administration, or for sublingual administration.

[0273] In some embodiments, the pharmaceutical composition is formulated for intravenous, intramuscular, or subcutaneous administration.

[0274] In some embodiments, the pharmaceutical composition is formulated for intrathecal or intraventricular administration.

[0275] In some embodiments, the pharmaceutical composition is formulated for topical administration.

[0276] 7.10.3. Pharmaceutical compositions suitable for injection

[0277] For intravenous, cutaneous or subcutaneous injection, or injection at the site of disease, the active ingredient will be pyrogen-free and in the form of a parenterally acceptable aqueous solution having suitable pH, isotonicity and stability. One skilled in the art will be well able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included as needed.

[0278] In various embodiments, the unit dosage form is a vial, ampoule, bottle, or prefilled syringe. In some embodiments, the unit dosage form comprises 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 comprises 125 mg, 150 mg, 175 mg, or 200 mg of the pharmaceutical composition. In some embodiments, the unit dosage form comprises 250 mg of the pharmaceutical composition.

[0279] In typical embodiments, the pharmaceutical composition in the unit dosage form is in liquid form. In various embodiments, the unit dosage form contains between 0.1 mL and 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.

[0280] In certain embodiments, the unit dosage form is a vial comprising 1 ml of a pharmaceutical composition having a concentration of 0.01 mg / ml, 0.1 mg / ml, 0.5 mg / ml, or 1 mg / ml. In some embodiments, the unit dosage form is a vial comprising 2 ml of a pharmaceutical composition having a concentration of 0.01 mg / ml, 0.1 mg / ml, 0.5 mg / ml, or 1 mg / ml.

[0281] In some embodiments, the pharmaceutical composition in unit dosage form is in a solid form, such as a lyophilisate suitable for dissolution.

[0282] Unit dosage form embodiments suitable for subcutaneous, intradermal, or intramuscular administration include prefilled syringes, autoinjectors, and autoinjector pens, each containing a predetermined amount of a pharmaceutical composition as described above.

[0283] In various embodiments, the unit dosage form is a preloaded syringe comprising a syringe and a predetermined amount of the pharmaceutical composition. In certain embodiments of the preloaded syringe, the syringe is suitable for subcutaneous administration. In certain embodiments, the syringe is suitable for self-administration. In specific embodiments, the preloaded syringe is a disposable syringe.

[0284] In various embodiments, the preloaded syringe contains about 0.1 mL to about 0.5 mL of the pharmaceutical composition. In certain embodiments, the syringe contains about 0.5 mL of the pharmaceutical composition. In specific embodiments, the syringe contains about 1.0 mL of the pharmaceutical composition. In specific embodiments, the syringe contains about 2.0 mL of the pharmaceutical composition.

[0285] In certain embodiments, the unit dosage form is an autoinjector pen. The autoinjector pen comprises an autoinjector pen comprising a pharmaceutical composition as described herein. In some embodiments, the autoinjector pen delivers a predetermined volume of the pharmaceutical composition. In other embodiments, the autoinjector pen is configured to deliver a volume of the pharmaceutical composition set by the user.

[0286] In various embodiments, the autoinjector pen contains about 0.1 mL to about 5.0 mL of the pharmaceutical composition. In certain embodiments, the autoinjector pen contains about 0.5 mL of the pharmaceutical composition. In certain embodiments, the autoinjector pen contains about 1.0 mL of the pharmaceutical composition. In other embodiments, the autoinjector pen contains about 5.0 mL of the pharmaceutical composition.

[0287] 7.11. Unit Dosage Form

[0288] The pharmaceutical compositions may conveniently be presented in unit dosage form.

[0289] Unit dosage forms are generally adapted for one or more specific routes of administration of the pharmaceutical composition.

[0290] In various embodiments, the unit dosage form is suitable for administration by inhalation. In certain of these embodiments, the unit dosage form is suitable for administration by a vaporizer. In certain of these embodiments, the unit dosage form is suitable for administration by a nebulizer. In certain of these embodiments, the unit dosage form is suitable for administration by an aerosol.

[0291] In various embodiments, the unit dosage form is suitable for oral administration, buccal administration, or sublingual administration.

[0292] In some embodiments, the unit dosage form is suitable for intravenous, intramuscular, or subcutaneous administration.

[0293] In some embodiments, the unit dosage form is suitable for intrathecal or intracerebroventricular administration.

[0294] In some embodiments, the pharmaceutical composition is formulated for topical administration.

[0295] The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.

[0296] 7.12. Usage

[0297] Therapeutic antibodies that specifically bind to intact CTLA-4 can be used.

[0298] In vivo and / or in vitro assays may optionally be employed to help determine the optimal dosage range. The precise dosage to be used in the formulation also depends on the route of administration and the severity of the condition and should be determined based on the judgment of the practitioner and the circumstances of each subject. Effective doses may be extrapolated from dose-response curves in in vitro or animal model test systems.

[0299] An oligopeptide or polypeptide is within the scope of the present disclosure if its amino acid sequence is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to at least one CDR provided herein; and / or to a CDR of at least one CTLA-4 binding agent that cross-blocks binding to CTLA-4 of antibodies A1-A28, and / or to a CDR of a CTLA-4 binding agent where the binding agent can block binding of CTLA-4 to its ligand.

[0300] CTLA-4 binding agent polypeptides and antibodies are within the scope of the disclosure if they have an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the variable region of at least one of antibodies A1-A28, and cross-blocks binding of at least one of antibodies A1-A28 to CTLA-4 and / or is cross-blocked from binding to CTLA-4 by at least one of antibodies A1-A28; and / or can block the inhibitory effects of CTLA-4 on its ligands.

[0301] The antibodies according to the present disclosure may have a CTLA-4 affinity of less than or equal to 5x10 -7 M, less than or equal to 1x10 - 7 M, less than or equal to 0.5x10 -7 M, less than or equal to 1x10 -8 M, less than or equal to 1x10 -10 M, less than or equal to 1x10 -11 M or less than or equal to 1x10 -12 The binding affinity of M.

[0302] The affinity of the antibody or binding partner, and the extent to which the antibody inhibits binding, can be determined by one of ordinary skill in the art using conventional techniques, such as those described by Scatchard et al. (Ann. NY Acad. Sci. 51: 660 672 (1949)), or by surface plasmon resonance (SPR; BIAcore, Biosensor, Piscataway, NJ). For surface plasmon resonance, the target molecule is immobilized on a solid phase and contacted with a ligand in a mobile phase in a flow cell. If the ligand binds to the immobilized target, the local refractive index changes, resulting in a change in the SPR angle, which can be monitored in real time by detecting changes in the intensity of the reflected light. The rate of change of the SPR signal can be analyzed to generate apparent rate constants for the association and dissociation phases of the binding reaction. The ratio of these values ​​gives the apparent equilibrium constant (affinity) (see, for example, Wolff et al., Cancer Res. 53: 2560 65 (1993)).

[0303] Antibodies according to the present disclosure can belong to any immunoglobulin class, such as IgG, IgE, IgM, IgD or IgA. They can be obtained from animals or derived from animals, such as poultry (e.g., chicken) and mammals, including but not limited to mice, rats, hamsters, rabbits or other rodents, cattle, horses, sheep, goats, camels, humans or other primates. The antibodies can be internalizing antibodies. The generation of antibodies is generally disclosed in U.S. Patent Publication No. 2004 / 0146888 A1.

[0304] In the methods described above for generating antibodies according to the present disclosure, including manipulating specific A1-A28 CDRs into new frameworks and / or constant regions, appropriate assays for selecting desired antibodies are available (i.e., assays for determining binding affinity to CTLA-4; cross-blocking assays; Biacore-based competition binding assays; in vivo assays).

[0305] 7.12.1. Methods for Treating Diseases Responsive to CTLA-4 Inhibitors

[0306] In another aspect, a method for treating a subject having a disease responsive to a CTLA-4 inhibitor or activator is provided. The disease can be cancer, AIDS, Alzheimer's disease, or a viral or bacterial infection.

[0307] The terms "treat", "treatment" and the like are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of preventing a disease, condition or its symptoms in whole or in part, and / or therapeutic in terms of partially or completely curing a disease or condition and / or side effects (such as those due to the symptoms of the disease or condition). As used herein, "treatment" encompasses any treatment of a mammal, particularly a human disease or condition, and includes: (a) preventing the occurrence of the disease or condition in a subject who may be susceptible to the disease or condition but has not yet been diagnosed with the disease or condition; (b) suppressing the disease or condition (e.g., preventing its progression); or (c) alleviating the disease or condition (e.g., causing the disease or condition to subside, improving one or more symptoms). The improvement of any condition can be easily assessed according to standard methods and techniques known in the art. The subject population treated by the method of the disease includes a subject suffering from an undesirable condition or disease, and a subject at risk of developing a condition or disease.

[0308] The term "therapeutically effective dose" or "effective amount" refers to a dose or amount that produces the desired effect for which it is administered. The exact dose or amount will depend on the purpose of the treatment and can be determined by one skilled in the art using known techniques (see, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0309] The term "sufficient amount" refers to an amount sufficient to produce the desired effect.

[0310] The term "therapeutically effective amount" is an amount effective to ameliorate symptoms of a disease. A therapeutically effective amount can be a "prophylactically effective amount" since prophylaxis can be considered treatment.

[0311] The term "amelioration" refers to any therapeutically beneficial result in treating a disease state (eg, a neurodegenerative disease state), including prevention, lessening of its severity or progression, remission, or cure.

[0312] The actual dosage, administration rate and time course will depend on the nature and severity of the protein aggregation disease being treated. Treatment prescription (e.g., determination of dosage, etc.) is the responsibility of general practitioners and other physicians, and will generally take into account the condition to be treated, the condition of the individual patient, the delivery site, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

[0313] In some embodiments, the pharmaceutical composition is administered by inhalation, orally, buccally, sublingually, by injection, or by topical application.

[0314] In some embodiments, the pharmaceutical composition is administered in an amount sufficient to modulate neuronal survival or dopamine release. In some embodiments, the principal cannabinoid is administered in an amount of less than 1 g, less than 500 mg, less than 100 mg, or less than 10 mg per dose.

[0315] In some embodiments, the pharmaceutical composition is administered once a day, 2-4 times a day, 2-4 times a week, once a week, or once every two weeks.

[0316] The composition can be administered alone or in combination with other treatments, administered simultaneously or sequentially, depending on the condition to be treated. For example, the pharmaceutical composition can be co-administered with one or more drugs that target different checkpoint receptors, such as CTLA-4 inhibitors (e.g., anti-CTLA-4 antibodies) or TIGIT inhibitors (e.g., anti-TIGIT antibodies).

[0317] 8. Examples

[0318] The following are examples of specific embodiments for implementing the present disclosure. These examples are provided for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but of course some experimental errors and deviations should be allowed for.

[0319] Unless otherwise indicated, the practice of the present disclosure will employ conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology within the skill of the art. These techniques are fully explained in the literature. See, for example, T. Creighton, Proteins: Structures and Molecular Properties (W. H. Freeman and Company, 1993); A. Lehninger, Biochemistry (Worth Publishers, Inc., current edition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry, 3rd edition (Plenum Press), Volumes A and B (1992). In addition, the methods for generating and selecting antibodies explained 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), and Adler et al., Rare, high-affinity mouse anti-CTLA-4 antibodies that function in checkpoint blockade, discovered using microfluidics and molecular genomics, MAbs (2017), the entire contents of which are incorporated herein by reference, can be used.

[0320] 8.1.1. Example 1: Production of Antigen Binding Proteins

[0321] Mouse immunization and sample preparation:

[0322] First, transgenic mice carrying an inserted human immunoglobulin gene were immunized with a soluble CTLA-4 immunogen of SEQ ID NO: 7001 (i.e., His-tagged CTLA-4 protein (R&D Systems)) using TiterMax as an adjuvant. 1 μg of immunogen was injected into each paw, and 3 μg of immunogen was administered intraperitoneally every 3 days for 15 days. Titers were assessed by enzyme-linked immunosorbent assay (ELISA) on 1:2 serial dilutions of each animal's serum starting at a 1:200 dilution. A final intravenous boost of 2.5 μg / paw (without adjuvant) was given to each animal before harvest. After sacrifice, lymph nodes (popliteal, inguinal, axillary, and mesenteric) were surgically removed. Single-cell suspensions were prepared from each animal by manual disruption and then passage through a 70 μm filter. Next, EasySep TM The Mouse Pan B Cell Isolation Kit (Stemcell Technologies) negative selection kit was used to isolate B cells from each sample. Lymph node B cell populations were quantified by counting on a C-Chip blood cell counter (Incyto), and viability was assessed using trypan blue. TM Cells were diluted to 5,000–6,000 cells / mL in phosphate-buffered saline (PBS) with density gradient medium (Sigma). This cell mixture was used for microfluidic encapsulation. Approximately one million B cells from each of six animals were run through an emulsion droplet microfluidics platform.

[0323] Generation of paired heavy and light chain libraries:

[0324] DNA libraries of scFV encoding single-cell RNA with intact natural heavy chain Ig pairings were generated using an emulsion droplet microfluidics platform or vortex emulsions. The methods used to generate DNA libraries were divided into 1) poly (A) + mRNA capture, 2) multiplexed overlap extension reverse transcriptase polymerase chain reaction (OE-RT-PCR), and 3) nested PCR to remove artifacts and add adapters for deep sequencing or yeast display libraries. The scFV library was generated from approximately one million B cells from each animal that reached a positive ELISA titer.

[0325] For poly(A)+ mRNA capture, a custom-designed fused emulsion droplet microfluidic chip fabricated from glass (Dolomite) was used. The microfluidic chip had two input channels for fluorocarbon oil (Dolomite), one input channel for the cell suspension mixture described above, and one input channel for 1.25 mg / ml of oligo-DT beads (NEB) in cell lysis buffer (20 mM Tris pH 7.5, 0.5 M NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.5% Tween-20, and 20 mM dithiothreitol). For most of the chip length, the input channel was etched to 50 μm by 150 μm, narrowed to 55 μm at the droplet junction, and coated with hydrophobic Pico-Glide (Dolomite). Three Mitos P pumps (Dolomite) were used to pump liquid through the chip. Droplet size depended on pressure, but droplets of ~45 mm in diameter were generally the most stable. The emulsion was collected into a cooled 2 mL microfiber tube and incubated at 40°C for 15 minutes for mRNA capture. The beads were extracted from the droplets using a Pico-Glide (Dolomite). In some embodiments, similar single cell partitioning emulsions were prepared using vortexing.

[0326] For multiplexed OE-RT-PCR, a glass Telos droplet emulsion microfluidic chip (Dolomite) was used. The mRNA-bound beads were resuspended in the OE-RT-PCR mixture and injected into the microfluidic chip with a mineral oil-based surfactant mixture (commercially available from GigaGen) under pressure to produce 27 μm droplets. The OE-RT-PCR mixture contained 2× one-step RT-PCR buffer, 2.0 mM MgSO4, SuperScript III reverse transcriptase, and Platinum Taq (Thermo Fisher Scientific), as well as a mixture of primers for the IgK C region, IgG C region, and all V regions ( Figure 2 The overlapping region is a DNA sequence encoding a Gly-Ser-rich scFv linker sequence. DNA fragments are recovered from the droplets using a droplet disruption solution (commercially available from GigaGen) and then purified using a QIAquick PCR purification kit (Qiagen). In some embodiments, a similar OE-RT-PCR emulsion is prepared using vortexing.

[0327] For nested PCR ( Figure 2), first run the purified OE-RT-PCR product on a 1.7% agarose gel at 150V for 80 minutes. The 1200-1500 base pair (bp) band corresponding to the ligation product was cut out and purified using a NucleoSpin gel and PCR Clean-up kit (Macherey Nagel). PCR was then performed to add adapters for Illumina sequencing or yeast display; for sequencing, 7 nucleotides of random material were added to increase base calling accuracy in the subsequent next-generation sequencing step. Nested PCR was performed with 2x NeBNext High-Fidelity Amplification Mix (NEB), which had Illumina adapters containing primers or primers for cloning into yeast expression vectors. The nested PCR products were run on a 1.2% agarose gel at 150V for 50 minutes. The 800-1100 bp band was cut out and purified using a NucleoSpin gel and PCR Clean-up kit (Macherey Nagel).

[0328] In some embodiments, the scFv library is not naturally paired, for example, scFvs are randomly paired by amplifying RNA isolated directly from B cells.

[0329] 8.1.2. Example 2: Isolation of CTLA-4 Binders by Yeast Display

[0330] Library screening:

[0331] Human IgG1-Fc (Thermo Fisher Scientific) and CTLA-4 (R&D Systems) proteins were biotinylated using the EZ-Link Micro Sulfo-NHS-LC-Biotinylation Kit (Thermo Fisher Scientific). The biotinylation reagent was resuspended to 9 mM and added to the protein at a 50-fold molar excess. The reaction was incubated on ice for 2 hours, and then the biotinylation reagent was removed using a Zeba desalting column (Thermo Fisher Scientific). The final protein concentration was calculated using the Bradford assay.

[0332] Next, the six DNA libraries were expressed as surface scFvs in yeast. A yeast surface display vector (pYD) containing the GAL1 / 10 promoter, the Aga2 cell wall tether, and a C-terminal c-Myc tag was constructed. The GAL1 / 10 promoter induces expression of the scFv protein in a medium containing galactose. The Aga2 cell wall tether is required to shuttle the scFv to the yeast cell surface and tether the scFv to the extracellular space. The c-Myc tag is used to stain yeast cells expressing in-frame scFv proteins during flow cytometry. Saccharomyces cerevisiae cells (ATCC) were electroporated (Bio-Rad Gene Pulser II; 0.54 kV, 25 uF, resistance set to infinity) with gel-purified nested PCR products and the linearized pYD vector for in vivo homologous recombination. The transformed cells were amplified and induced with galactose to generate the yeast scFv display library.

[0333] Two million yeast cells from the amplified scFv library were stained with anti-c-Myc (Thermo Fisher Scientific A21281) and AF488-conjugated secondary antibodies (Thermo Fisher Scientific A11039). To select for scFv-expressing cells that bind to CTLA-4, biotinylated CTLA-4 antigen was added to the yeast culture (final 7 nM) during the primary antibody incubation period and then stained with PE-streptavidin (Thermo Fisher Scientific). Yeast cells were flow-sorted for double-positive cells (AF488C / PEC) on a BD Influx (Stanford Shared FACS Facility), and the recovered clones were then plated on SD-CAA plates containing kanamycin, streptomycin, and penicillin (Teknova) for expansion. The amplified first-round FACS clones were then subjected to a second round of FACS using the same antigen at the same molar concentration (final 7 nM). Plasmid minipreps (Zymo Research) were performed using yeast recovered from the final FACS sort. Tail-end PCR was used to add Illumina adapters to the plasmid library for deep sequencing.

[0334] In a typical FACS dot plot, the upper right quadrant contains yeast stained for antigen binding and scFv expression (identified by a C-terminal c-Myc tag). The lower left quadrant contains yeast not stained for antigen or scFv expression. The lower right quadrant contains yeast expressing scFv but not binding antigen. The frequency of binders in each library was estimated by dividing the counts of yeast dually stained for antigen and scFv expression by the counts of yeast expressing scFv. When sorted at a final antigen concentration of 7 nM, libraries generated from immunized mice produced a low percentage of scFv binders (range 0.08%–1.28%). There was no clear correlation between serum titer and the frequency of binders in the library. After expansion of these sorted cells, a second round of FACS using a final antigen concentration of 7 nM was used to increase the specificity of the screen. The frequency of binders in the second FACS was always significantly higher than in the first FACS, ranging from 8.39%–84.4%. In general, a lower frequency of binders in the first sort resulted in a lower frequency of binders in the second sort. Presumably, this is due to lower gating specificity for samples that truly had fewer binders in the original pool.

[0335] Deep library sequencing:

[0336] CTLA-4 binding clones were recovered as a library ("CTLA-4 binding clone library") and subjected to deep library sequencing. The CTLA-4 binding clone library was deposited under the Budapest Treaty on November 20, 2018 with ATCC accession number PTA-125512, ATCC account number 197361 (American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110USA). Each clone in the library contains scFv, which contains paired variable (V (D) J) regions of heavy and light chain sequences derived from a single cell. Deep library sequencing determined the sequences of all paired variable (V (D) J) regions of heavy and light chain sequences. Some heavy and light chain sequences obtained from yeast scFv library sequencing are provided in SEQ ID NO: 1-28 and SEQ ID NO: 101-128. Additional sequences obtained from yeast scFv library sequencing are provided in SEQ ID NOs 8001-8991. Specifically, the variable light chains (V L ) sequences include SEQ ID NO: 8001-8495. Its variable heavy chain (V H ) sequences include SEQ ID NOs:8496-8991.

[0337] The depth antibody sequencing library is quantified and diluted to 17.5 pM using quantitative PCR Illumina library quantitative test kit (KAPA). According to the instructions for producing the manufacturer, the library is sequenced on MiSeq (Illumina) using 500 cycles of MiSeq test kit v2. In order to obtain high-quality sequence readings with maintenance heavy and light chain connections, sequencing is performed in two separate runs. In the first run (" connected run"), the scFv library is directly sequenced to obtain 340 cycles of forward readings for light chain V-genes and CDR3, and 162 cycles of reverse readings covering a part for heavy chain CDR3 and heavy chain V-genes. In the second run (" unconnected run"), the scFv library is first used as a template for PCR, to amplify heavy chain and light chain V-genes respectively. Then, 340 cycles of forward readings and 162 cycles of reverse readings are obtained for heavy chain and light chain Ig, respectively. This generates forward and reverse reads that overlap at the CDR3 and part of the V-gene, which increases confidence in the nucleotide call.

[0338] To eliminate base calling errors, the expected number of errors (E) of the read is calculated based on its Phred score. By default, reads with E>1 are discarded, leaving the reads with the most likely number of base calling errors of zero. As an additional quality filter, singleton nucleotide reads are discarded because sequences found twice or more are likely to be correct. Finally, high-quality connected antibody sequences are generated by merging filtered sequences from connected and unconnected runs. Briefly, a series of scripts that first merge the forward and reverse reads from the unconnected runs are written in Python. Any forward and reverse sequence pairs containing mismatches are discarded. Next, the nucleotide sequences from the connected runs are used to query the merged sequences in the unconnected runs. The final output of the script is a series of full-length, high-quality variable (V(D)J) sequences with natural heavy and light chain Ig pairings.

[0339] To identify reading frames and FR / CDR connections, a carefully curated immunoglobulin sequence database is first processed to generate a position-specific sequence matrix (PSSM) for each FR / CDR connection. These PSSMs are used to identify the FR / CDR connections of each merged nucleotide sequence generated using the above process. This determines the protein reading frame of each nucleotide sequence. CDR sequences with low identification scores for the PSSM are indicated with an exclamation mark. Then, a Python script is used to translate the sequence. Reads need to have valid predicted CDR3 sequences, so, for example, reads with frameshifts between the V and J segments are discarded. Next, UBLAST is run using the scFv nucleotide sequence as a query and the V and J gene sequences from the IMGT database as reference sequences. The UBLAST alignment with the lowest E-value is used to assign V and J gene families and calculate the %ID of the germline.

[0340] After the second FACS selection, each animal generated 38-50 unique scFv sequences present at a frequency of 0.1% or higher, including a total of 28 unique scFv candidate binders (light chain: SEQ ID No: 1-28; heavy chain: SEQ ID No: 101-128). The light chain with the sequence of SEQ ID NO: [n] and the heavy chain with the sequence of SEQ ID NO: [100+n] are homologous pairs from a single cell and form a single scFv. For example, the light chain of SEQ ID NO: 1 and the heavy chain of SEQ ID NO: 101 are homologous pairs, the light chain of SEQ ID NO: 28 and the heavy chain of SEQ ID NO: 128 are homologous pairs, and so on.

[0341] In this approach, two rounds of FACS resulted in the enrichment of CTLA-4-binding scFvs. Furthermore, not many scFvs were detected in sequencing data from naive B cell populations from immunized mice, and most scFvs present in the pre-sorted mouse repertoire were eliminated after FACS. Thus, this work demonstrates that most antibodies present in the repertoire of immunized mice are not strong binders to the immunogen and that this approach can enrich for rare, nanomolar affinity binders from naive B cell populations from immunized mice.

[0342] 8.1.3. Example 3: Biological Characteristics of Antibody Binding Proteins

[0343] scFv sequences that appeared at low frequencies in the pre-sort library and became high frequencies in the post-sort library were then synthesized as full-length mAbs in Chinese hamster ovary (CHO) cells. These mAbs included the 2-3 most abundant sequences in the second round of FACS for each animal. CTLA-4 target binding properties:

[0344] The binding specificity and affinity of each full-length antibody for CTLA-4 were determined using biolayer interferometry (BLI) and / or surface plasmon resonance (SPR). Affinity for anti-cyno CTLA-4 and anti-mouse CTLA-4 was determined using ForteBio (BLI). Affinity for anti-human CTLA-4 was determined using Carterra (SPR).

[0345] For BLI, antibodies were loaded onto anti-human IgG Fc (AHC) biosensors using the Octet Red96 system (ForteBio). The loaded biosensors were immersed in antigen dilutions starting at 300 nM and serially diluted 6 times at a 1:3 ratio. Kinetic analysis was performed using a 1:1 binding model and global fitting.

[0346] For SPR, medium-density (>>1,000 response units) anti-human IgG-Fc reagent (Southern Biotech 2047-01) was amine-conjugated to a Xantec CMD-50M chip (50 nm carboxymethyl dextran medium-density functional group) and activated using 100 mM MES pH 5.5 containing 133 mM EDC (Sigma) and 33.3 mM S-NHS (Thermo Fisher). Then, goat anti-human IgG Fc (Southern Biotech 2047-01) was conjugated at 25 mg / mL in 10 mM sodium acetate pH 4.5 (Carterra Inc.) for 10 minutes. The surface was then deactivated with 1 M ethanolamine pH 8.5 (Carterra Inc.). The running buffer used for plate immobilization was HBS-EPC (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH 7.4; Teknova).

[0347] The sensor chip was then transferred to a continuous flow microspot instrument (CFM; Carterra Inc.) for array capture. The mAb supernatant was diluted 50 times (final concentration was 3-10 mg / mL) into HBS-EPC containing 1 mg / mL BSA. The sample was captured twice with 15-minute and 4-minute capture steps on the first and second prints, respectively, to establish multiple densities using a 65 mL / min flow rate. Running buffer in CFM was also HBS-EPC.

[0348] Next, the sensor chip was loaded onto an SPR reader (MX-96 system; Ibis Technologies) for kinetic analysis. CTLA-4 was injected in a running buffer (HBS-EPC containing 1.0 mg / mL BSA) at five increasing concentrations of a four-fold dilution series of 1.95, 7.8, 31.25, 125, and 500 nM. The CTLA-4 injection time was 5 minutes, and the dissociation was 15 minutes at 8 mL / second in a non-regenerative kinetic series. At the end of the series, 75 mg / mL of goat anti-human IgG Fc capture antibody was injected to verify the capture level of each mAb. Binding data were double-referenced by deducting the interspot surface and blank injections, and ka (association rate), kd (dissociation rate), and KD (affinity) were analyzed using Kinetic Interaction Tool software (Carterra Inc.).

[0349] For cell surface binding studies, Flp-In CHO (Thermo Fisher Scientific) cells stably expressing CTLA-4 were generated and mixed at a 50:50 ratio. One million cells were stained for 30 minutes at 4°C using 200 μl MACS buffer (DPBS containing 0.5% bovine serum albumin and 2 mM EDTA) containing 1 μg of anti-CTLA-4 recombinant antibody. The cells were then co-stained with anti-human irrelevant target APC and anti-human IgG Fc-PE [M1310G05] (BioLegend 41070) antibodies for 30 minutes at 4°C. Anti-human CTLA-4-FITC antibody was used as a control for these mixing experiments, and cell viability was assessed using DAPI. Flow cytometry analysis was performed on a BD Influx at the Stanford Shared FACS Faccility, and data were analyzed using FlowJo.

[0350] Antibodies that specifically bind to CTLA-4 were identified. The activity of each antibody against CTLA-4 (K D The affinity of each antibody for human CTLA-4 is shown in Table 7.

[0351]

[0352]

[0353] CTLA-4 ligand blocking assay:

[0354] To analyze the ability of antibodies to block CTLA-4 / ligand interactions, a CTLA-4 blocking bioassay (Promega) was used according to the manufacturer's instructions. The day before the assay, aAPC / Raji cells expressing CTLA-4 ligands CD80 and CD86 were thawed into 90% Ham F-12 / 10% fetal bovine serum (FBS) and seeded into the inner 60 wells of two 96-well plates. The cells were incubated overnight at 37°C, 5% CO2. On the day of the assay, the antibodies were diluted in 99% RPMI / 1% FBS. The antibody dilution was added to the wells containing aAPC / Raji cells expressing CTLA-4 ligands, followed by the addition of CTLA-4 effector cells (thawed into 99% RPMI / 1% FBS). The cell / antibody mixture was incubated at 37°C, 5% CO2 for 6 hours, followed by the addition of Bio-Glo reagent and read using a Spectramax i3x microplate reader (Molecular Devices). Fold induction was plotted by calculating the ratio [signal with antibody] / [signal without antibody] and these plots were used to calculate EC50 using SoftMax Pro (Molecular Devices). In-house produced Ipilimumab was used as a positive control and an antibody binding to an irrelevant antigen was used as a negative control.

[0355] The combination of CTLA-4 and its ligand leads to the inhibition of T cell signaling. Therefore, antibodies that bind to CTLA-4 and antagonize CTLA-4 / ligand interactions can eliminate this inhibitory effect, thereby allowing activation of T cells. CTLA-4 / ligand checkpoint blocking is tested by in vitro cell activated T cell nuclear factor (NFAT) luciferase reporter analysis. In this assay, the antibody antagonism CTLA-4 / ligand interaction that the anti-CTLA-4 epitope falls within the ligand binding domain leads to an increase in NFAT-luciferase reporter. Full-length mAb candidates that can bind to CTLA-4 expressed in CHO cells were determined. To generate EC50 values ​​for each mAb, multiple concentrations were measured. It was found that some full-length mAbs work in a dose-dependent manner in checkpoint blocking, as summarized in Table 6.

[0356] The ability of the CTLA4 antibodies (shown in Table 8) to block the binding of CD80 or CD86 to plate-bound CTLA4 was assessed using ELISA. The EC50 and % inhibition of each interaction are shown in Table 8. Plates were coated with rhCTLA4-Fc and then blocked with 1xPBST containing 5% w / v nonfat dry milk. After blocking, a dilution series of the indicated antibodies was added to the plates. Then, to determine how much CD80 or CD86 was still able to bind to plate-bound CTLA4, rhCD80-His or rhCD86-His, respectively, was added to the plates after washing the plates. Unbound CD80-His / CD86-His was washed away and mouse anti-His-HRP was added. In the presence of each antibody, TMB was used to determine how much CD80-His / CD86-His bound to the plate bound to CTLA4.

[0357] In some embodiments of the present disclosure, anti-CTLA-4 antibodies exert their pharmacological effects through antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments of the present disclosure, immune-related toxicities associated with anti-CTLA-4 antibody therapy are eliminated by antibodies that function in ADCC but not in checkpoint blockade.

[0358]

[0359] Epitope binning:

[0360] Epitope binning was performed using a modified classical sandwich approach using high-throughput array SPR. The sensor chip was functionalized using Carterra CFM using a similar approach to the SPR affinity studies, except that a CMD-200M chip type (200 nm carboxymethyl dextran, Xantec) was used and mAb was coupled at 50 mg / mL to create a surface with a higher binding capacity (~3,000 response units immobilized). The mAb supernatant was diluted in running buffer at a ratio of 1:1 or 1:10, depending on the mAb concentration in the supernatant.

[0361] The sensor chip was placed in an MX-96 instrument and the captured mAb ("ligand") was cross-linked to the surface using the divalent amine-reactive linker bis(sulfosuccinimidyl) suberate (BS3, ThermoFisher) and injected into water at 0.87 nM over 10 minutes. Excess activated BS3 was neutralized with 1 M ethanolamine, pH 8.5. For each binning cycle, a 7-minute injection of 250 mg / mL human IgG (Jackson ImmunoResearch 009-000-003) was used to block any remaining capacity of the reference surface and target.

[0362] Next, 250 nM CTLA4 protein was injected onto the sensor chip, followed by injection of diluted mAb supernatant ("analyte") or a buffer blank as a negative control. Therefore, if the analyte mAb does not compete with the ligand mAb, it will only bind to the antigen. At the end of each cycle, a one-minute regeneration injection was performed using 4 parts Pierce IgG Elution Buffer (ThermoFisher #21004), one part 5M NaCl (final 0.83M), and 1.25 parts 0.85% H3PO4 (final 0.17%). Through multiple regenerations, only 18 mAbs remained active as ligands, so the bin analysis contained an 18 x 46 competition matrix.

[0363] Then, a network community graph algorithm is used in the SPR epitope data analysis software package (Carterra Inc.) to determine the epitope binning. It is noteworthy that the clustering algorithm separates the clustering of mAbs with only the available analyte data from mAbs with both ligand and analyte data. This phenomenon is an illusion of an incomplete competition matrix. mAbs with both ligand and analyte data have more mAb-mAb measured values, which result in more mAb-mAb connections, thereby resulting in a closer relationship in the community graph.

[0364] Epitope binning showed that all mAbs were in a different bin than ipilimumab ( Figure 3 ).

[0365] 8.4. Example 4: Effect of CTLA-4 ABP on Tumor Growth

[0366] Transgenic mice expressing human CTLA-4 (hCTLA-4 KI mice) were implanted subcutaneously with MC38 tumor cells in the right flank. hCTLA-4 KI mice were treated with 1 mg / kg of the specified CTLA-4 antibody on days 8, 11, and 14 after implantation. Specifically, mice were treated with a control antibody (n=8), ipilimumab (n=8), CTLA4.A2 antibody (n=8), CTLA4.A14 antibody (n=9), CTLA4.A14.2a antibody (n=8), CTLA4.A7 antibody (n=9), CTLA4.A7 antibody (n=9), and CTLA4.A12 antibody (n=8). The CTLA-4.A14.2a antibody is an A14 antibody cloned on a mouse IgG2a backbone that enhances antibody-dependent cellular cytotoxicity (ADCC) activity. Tumor volume was measured and tumor growth inhibition was calculated using the following formula:

[0367] Average inhibition % = (average value (C) - average value (T)) / average value (C) * 100%

[0368] T - the value of the current group

[0369] C-value of the control group

[0370] MC38 tumor cells (1x10 6 Tumors were implanted subcutaneously in the right flank region for tumor development. Cells were harvested in the exponential growth phase before tumor implantation and quantified using a cell counter. Tumor volume was measured twice weekly in two dimensions using a caliper and was expressed in mm using the formula 3 V = (L x W x W) / 2, where V is the tumor volume, L is the tumor length (the longest tumor dimension), and W is the tumor width (the longest tumor dimension perpendicular to L). Dosing and tumor and body weight measurements were performed in a laminar flow cabinet. TM Body weight and tumor volume were measured using software (version 3.1.399.19). Animals were administered intraperitoneally (ip) with a sterile saline solution containing 0.1 mg / ml of the specified protein. Each mouse received 10 microliters of the specified solution per gram of body weight, resulting in a 1 mg / kg dose. Animals were administered at day 0, day 3, and day 6 after randomization.

[0371] Table 9 shows the percentage of mice whose tumors had a complete response (CR) to treatment.

[0372] At least 2 consecutive 0mm 3 Tumor measurements were consistent with CR.

[0373]

[0374] Table 10 shows the percentage of mice that had a CR but subsequently had tumor relapse on day 56. The CTLA4.A14.2a-treated group, which had previously shown a CR, had a 0% relapse rate on day 56, suggesting that ADCC can prolong anti-tumor immunity.

[0375]

[0376] Table 11 shows the mean inhibition of tumor volume over time when hCTLA-4 KI mice implanted with MC38 tumor cells were treated with 1 mg / kg control or 1 mg / kg of the indicated CTLA-4 antibodies.

[0377]

[0378] Example 5: Effect of CTLA4 ABP on systemic anti-tumor immunity

[0379] hCTLA4 KI mice bearing MC38 tumors were treated with the indicated anti-CTLA4 on days 8, 11, and 14 after tumor cell implantation, as described above. Mice that showed CR to their tumors were re-challenged by implanting MC38 cells into the contralateral flank. Table 12 shows the tumor volumes (mm) of individual mice with original or re-challenge tumors on the last day of the study (73 days after original tumor cell implantation and 30 days after re-challenge implantation). 3 In mice whose original tumors remained CR, there was no growth in the rechallenge tumors. The three instances of growth observed in the rechallenge tumors occurred in mice where the original tumors had begun to regrow (see Table 12). The results also demonstrate that CTLA4.A2 can induce protective systemic anti-tumor immunity even if the primary tumor (the original tumor) recurs (see Table 13).

[0380]

[0381]

[0382]

[0383] 8.5. Example 6: Effect of increasing doses of CLTA-4 ABP

[0384] Treatment of MC38 tumors with anti-CTLA-4

[0385] Two to eight transgenic mice expressing human CTLA-4 (hCTLA-4 KI mice) were implanted with MC38 tumor cells in the right flank. When the average tumor size reached 98.5 mm 2 Randomization began at day 0 after randomization. Starting on day 0 after randomization, hCTLA-4 KI mice were treated with 5 mg / kg of the indicated anti-CTLA4 every two weeks for five doses. The antibodies administered are shown in Table 14. CTLA4.A14.2a is an A14 antibody cloned on a mouse IgG2a backbone, which enhances ADCC activity. The 297 suffix indicates that the hIgG1 Fc is mutated at amino acid N297 to eliminate glycosylation, thereby abolishing Fc effector functions, including ADCC.

[0386] A) Tumor Growth Inhibition

[0387] During the study, tumor growth inhibition was determined using the following formula:

[0388] Average inhibition % = (average value (C) - average value (T)) / average value (C) * 100%

[0389] T - the value of the current group

[0390] C-value of the control group

[0391] The results showed that antibodies lacking Fc activity had reduced efficacy overall. These antibodies were still able to induce tumor regression in some animals, indicating that anti-CTLA4 acts through both Fc-dependent and Fc-independent mechanisms of action and that anti-CTLA4 lacking Fc activity, including ADCC and ADCP, can induce anti-tumor responses (Tables 14 and 15).

[0392]

[0393]

[0394] B) Histological analysis:

[0395] hCTLA-4 mice were euthanized and their right kidneys were collected for histological analysis. The tissues were formalin-fixed and paraffin-embedded, cut into 5 μm sections, and placed on slides for standard hematoxylin and eosin (H&E) staining and anti-IgG and anti-C3 immunohistochemistry (IHC) staining. The stained slides were prepared as digital images. A board-certified veterinary pathologist with experience in laboratory animal and toxicological pathology evaluated any findings on the H&E images and assessed the location, intensity, and percentage of positive staining on the anti-IgG and C3 slides. The findings in the H&E images were scored on a scale of 0 to 5 (0 = within the normal range, 1 = minimal findings or discernible minimal changes, 2 = mild findings, 3 = moderate, 4 = significant, 5 = severe or to the maximum possible). The findings in the IHC images were scored on an intensity scale of 1 to 4 (0 = negative, 1 = minimal or slightly positive, 4 = very dark) and as the percentage of positive cells in the glomeruli (at least 5 glomeruli after review).

[0396] H&E, immunoglobulin, or C3-stained images were scored by a blinded pathologist and the results are shown in Figure 4 The main finding of H&E is that leukocytes in the renal interstitium usually do not accumulate in the glomeruli. Ig and C3 precipitation in the glomerular score are also shown. Figure 4 middle.

[0397] C) Alkaline phosphatase:

[0398] hCTLA-4 mice were also analyzed for changes in alkaline phosphatase levels. Serum alkaline phosphatase levels were measured using the integrated diagnostic rotor on the ABAXIS VetScan VS2.

[0399] Studies have found that ipilimumab (IPI) increases alkaline phosphatase levels, which may be an indication of immune-mediated hepatitis. CTLA4 antibodies (e.g., CTLA4.A14.2A) show a decrease in elevated alkaline phosphatase levels ( Figure 5This reduction in alkaline phosphatase elevation induced by the currently disclosed CTLA4 antibodies may indicate that they are less likely to induce immune-mediated hepatitis than treatments such as pembrolizumab.

[0400] 8.6. Example 7: Effect of CTLA-4 ABP on a Second Tumor Model

[0401] Treatment of RM1 tumors with anti-CTLA-4

[0402] RM1 tumor cells were implanted in the right flank of transgenic mice expressing human CTLA-4 (hCTLA-4 KI mice). (Human IgG1 isotype negative control n=7, atezolizumab n=8, all other groups n=11). hCTLA4 KI mice were treated with the antibodies shown in Table 16. CTLA4 antibodies were administered at 5 mg / kg on days 0, 3, and 6 after randomization, and atezolizumab was administered at 5 mg / kg every two weeks starting on day 0 after randomization for 3 weeks. Human IgG1 isotype negative controls were administered at 5 mg / kg on days 0, 3, and 6 after randomization. The average inhibition of tumor growth was determined on days 0, 4, 7, 11, 14, and 18 using the following formula:

[0403] Average Δ inhibition % = (average value (C) - average value (T)) / average value (C) * 100%

[0404] T - the value of the current group

[0405] C-value of the control group

[0406] Table 16 shows the mean inhibition values ​​for control, CTLA4 antibody, and atezolizumab treatment over the course of the study.

[0407]

[0408] 8.7. Example 8: Combination Therapy (Pembrolizumab and Anti-CTLA-4)

[0409] Transgenic mice expressing human CTLA-4 and PD-1 (hCTLA4-hPD1 KI mice, n = 8 per treatment group) were implanted with 1x10 6MC38 tumor cells. hCTLA4-hPD1 KI mice were treated with control (1x phosphate-buffered saline, or PBS); 2 mg / kg pembrolizumab (pembro) or 2 mg / kg pembro + 5 mg / kg anti-CTLA4, administered intraperitoneally. Starting from day 1 after randomization, the dosing volume for each animal was 10 mL / kg, as shown in Table 17, twice a week for three weeks. The mean (%) Δ inhibition of tumor growth induced by each treatment compared to the control treatment was calculated using the following formula, and the results are shown in Table 17.

[0410] Average Δ inhibition %=((average value (C)-average value (C0))-(average value (T)-average value (T0))) / (average value (C)-average value (C0))*100%

[0411] T - the value of the current group

[0412] T0 – initial value of the current group

[0413] C-value of the control group

[0414] C0 – initial value of the control group

[0415]

[0416] The study showed that mice treated with pembro alone showed no tumor growth inhibition at day 24, but adding the designated CTLA4 antibodies increased tumor growth inhibition over the course of the study.

[0417] At the end of the experiment, selected tumors were harvested and flow cytometry was performed to investigate the immune cell population within the tumor. The data showed that anti-CTLA4 reduced the intratumoral Treg cell population while increasing the intratumoral NK cell population ( Figure 6 ).

[0418] 8.8. Example 9: Immune-Related Adverse Events

[0419] Transgenic mice expressing human CTLA-4 (hCTLA-4 KI mice) were implanted with MC38 tumor cells in the right flank. hCTLA-4 KI mice were treated with 1 mg / kg of the indicated CTLA-4 antibodies on days 8, 11, and 14 post-implantation. Mice were weighed on days 8, 11, 14, and 17 post-implantation. The number of animals was: n=8 for ipilimumab, n=9 for A7, n=9 for A2, n=9 for A14, and n=8 for A14.2. The percent weight change of mice receiving the indicated anti-CTLA4 treatment is shown in Figure 2. Figure 7 As shown in .

[0420] Mice treated with CTLA4.A7, CTLA4.A14, and CTLA4.A14.2a did not appear to exhibit weight loss after the final dose of anti-CTLA4 ( Figure 7 This finding was unexpected, as immune-related adverse events (irAEs) have been reported to be greater when anti-CTLA4 with enhanced ADCC (e.g., CTLA.A14.2a) is used. This data suggests that blocking anti-CTLA4 with reduced activity may limit the induction of irAEs even when ADCC is enhanced.

[0421] 8.9. Example 10: Peripheral Flow Cytometry

[0422] Transgenic mice expressing human CTLA-4 (hCTLA-4 KI mice) were implanted with MC38 tumor cells in the right flank. hCTLA-4 KI mice were treated with 1 mg / kg of the indicated CTLA-4 antibodies on days 8, 11, and 14 post-implantation. Peripheral flow cytometry was performed on day 27. 100 μL of blood was used for staining. The results of peripheral flow cytometry are shown in Figure 2. Figure 8-10 As shown in .

[0423] The results showed that CTLA4.A2 and CTLA4.A14 reduced the increase in peripheral T cells (CD3+). Enhancing ADCC with CTLA4.A14.2a increased newly activated T cells (CD69+). CTLA4.A2 and CTLA4.A14 produced fewer unconventional regulatory cells (CD4+PD1+, CD4+ICOS+). (See Figure 8 ).

[0424] The results also showed that CTLA4.A2 and CTLA4.A14 better enhanced CD8+ T cells. Compared with ipilimumab, CTLA4.A2 better enhanced newly activated T cells (CD8+CD69+) and caused less T cell exhaustion (CD8+PD1+). ICOS has been described as a pharmacodynamic marker for anti-CTLA4. Enhancing ADCC with CTLA4.A14.2a appeared to further increase CD8+ICOS+ cells ( Figure 9 The results also showed that CTLA4.A2 and CTLA4.A14.2a resulted in reduced peripheral immune activation relative to ipilimumab, as judged by the frequencies of dendritic cells (DC) and activated DC (CD86+). (See Figure 10 ).

[0425] 8.10. Example 11: Studies using low-dose CTLA-4 therapy

[0426] MC38 tumor cells (1E6) in 0.1 ml PBS were subcutaneously implanted into the right flank region of transgenic mice expressing human CTLA-4 (hCTLA-4 KI mice) for tumor development. Cells in the exponential growth phase were harvested before tumor implantation and quantified using a cell counter. When the average tumor volume was 96.15 mm 3 hCTLA-4 KI mice were randomized and treated with 0.3 mg / kg of the indicated anti-CTLA4, ipilimumab, or human IgG1 isotype control (isotype) on days 0, 3, and 6 after randomization. Tumor volumes and mean % inhibition were determined as described in Example 4. CTLA4.A2 and CTLA4.A14 resulted in significantly higher tumor inhibition over the 18 days of the study. The results are shown in Tables 18 and Figure 11 shown.

[0427]

[0428] 9. Incorporation by Reference

[0429] All publications, patents, patent applications, and other documents cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document were individually indicated to be incorporated by reference for all purposes.

[0430] 10. Equivalents

[0431] Although various specific embodiments have been illustrated and described, the above description is not restrictive. It should be understood that various changes can be made without departing from the spirit and scope of the present disclosure. After reading this description, those skilled in the art will recognize many changes.

[0432] Table 19 provides the sequences of the antibody light chain, antibody heavy chain, CDRs, and human CTLA4

[0433]

[0434]

[0435]

[0436]

[0437]

[0438]

[0439]

[0440]

[0441]

[0442]

[0443] Table 20 provides the sequence identifiers for the light chain, heavy chain, and CDRs of the indicated clones.

[0444]

[0445]

[0446]

[0447]

[0448]

[0449]

[0450]

[0451]

[0452]

[0453]

[0454]

[0455]

[0456]

[0457]

[0458]

[0459]

[0460]

[0461]

Claims

1. An isolated antigen binding protein (ABP) that specifically binds to human cytotoxic T lymphocyte-associated protein 4 (CTLA-4), the ABP comprising CDR1-L, CDR2-L, CDR3-L, CDR1-H, CDR2-H and CDR3-H, wherein The CDR1-L consists of SEQ ID NO: 1014, the CDR2-L consists of SEQ ID NO: 2014, the CDR3-L consists of SEQ ID NO: 3014, the CDR1-H consists of SEQ ID NO: 4014, the CDR2-H consists of SEQ ID NO: 5014 and the CDR3-H consists of SEQ ID NO: 6014.

2. The ABP of claim 1, wherein the CDR3-L and the CDR3-H are a cognate pair.

3. The ABP of claim 1, comprising Variable light chain (V L ), which comprises the sequence of SEQ ID NO: 14, and a variable heavy chain (V H ), which comprises the sequence of SEQ ID NO:

114.

4. The ABP of claim 3, wherein the V L and the V H are cognate pairs.

5. The ABP of claim 1, wherein the ABP comprises a scFv or a full-length monoclonal antibody.

6. The ABP of claim 1, wherein the ABP comprises an immunoglobulin constant region.

7. The ABP of claim 1, wherein the ABP has a K of less than 500 nM as measured by surface plasmon resonance. D Binds human CTLA-4.

8. The ABP of claim 7, wherein the ABP has a K of less than 200 nM as measured by surface plasmon resonance. D Binds human CTLA-4.

9. The ABP of claim 8, wherein the ABP has a K of less than 25 nM as measured by surface plasmon resonance. D Binds human CTLA-4.

10. The ABP of any one of claims 1-9, wherein the ABP has a K of less than 25 nM. D Binds to human CTLA-4 on the surface of cells.

11. A pharmaceutical composition comprising the ABP of any one of claims 1-10 and an excipient.

12. Use of the ABP of any one of claims 1 to 10 or the pharmaceutical composition of claim 11 in the preparation of a medicament for treating cancer in a subject in need thereof.

13. The use of claim 12, wherein one or more additional therapeutic agents are administered to the subject.

14. The method of claim 13, wherein the additional therapeutic agent is selected from the group consisting of a CTLA-4 inhibitor, a TIGIT inhibitor, a chemotherapeutic agent, an immunostimulatory agent, radiation, a cytokine, a polynucleotide encoding a cytokine, and combinations thereof.

15. An isolated polynucleotide encoding the ABP of any one of claims 1-6.

16. A vector comprising the isolated polynucleotide of claim 15.

17. A host cell comprising the isolated polynucleotide of claim 15 or the vector of claim 16.

18. A method for producing an isolated antigen binding protein (ABP) that specifically binds to human CTLA-4, comprising: Expressing the ABP in the host cell of claim 17, and isolating the ABP.

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