Antibodies specific for glycosylated CTLA-4 and methods of use thereof

Abstract

The present application provides antibodies that selectively bind to glycosylated CTLA-4 relative to unglycosylated CTLA-4. In certain aspects, the present application also provides CTLA-4 polypeptides comprising glycosylated amino acid positions. The present application also provides methods for preparing and using such antibodies and polypeptides (e.g., for treating cancer).

Description

[0001] Sequence Listing

[0002] This application contains a sequence listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on September 25, 2020, is named 24258_0013P1_Sequence_Listing.txt, and is 9,405 bytes in size. Technical Field

[0003] The present invention relates generally to the fields of medicine, molecular biology and oncology. More specifically, it relates to antibodies for treating cancer. Background Art

[0004] The persistence of T cell activation has greatly reshaped the treatment of a wide range of malignant cancers. For example, the development of ipilimumab (a CTLA4-specific antibody and the first FDA-approved checkpoint blocker targeting T-cell responses) has made it possible to treat metastatic melanoma (Hodi et al., The New England Journal of Medicine 363, 711-723 (2010)).

[0005] Post-translational modification (PTM) of immune checkpoints (such as CTLA-4) has become an important regulatory mechanism for regulating immunosuppression in cancer patients. Recent studies suggest that, in PTM, glycosylation plays an important role in regulating immune checkpoint protein stability and translocation and protein-protein interactions. Co-inhibitory (inducing immunosuppressive signaling) ligand / receptor pairs, including CTLA4, show significant binding loss after deglycosylation, while costimulatory (inducing immune activation signaling) pairs do not have this performance (Li et al., 2018, Cancer Cell 33, 187-201).

[0006] On this basis, glycosylated CTLA4-specific antibodies may be valuable in cancer therapy. Summary of the Invention

[0007] Provided herein are isolated monoclonal antibodies that selectively bind to glycosylated CTLA-4 (anti-glycCTLA-4 antibodies herein) and inhibit CD80 and / or CD86. In certain aspects, the antibody selectively binds to CTLA-4 glycosylated at positions N113 and / or N145 relative to unglycosylated CTLA-4.

[0008] In certain embodiments, the isolated antibody selectively binds to human CTLA-4 glycosylated at N113. In certain embodiments, the isolated antibody selectively binds to human CTLA-4 glycosylated at N145. In certain embodiments, the isolated antibody selectively binds to human CTLA-4 glycosylated at both N113 and N145.

[0009] In certain aspects, the anti-glycCTLA-4 antibody binds to CTLA-4 and masks or screens one or more glycosylation motifs to block binding or other interactions of the molecule with the motif, and can block glycosylation of CTLA-4 at the glycosylation site. In specific embodiments, the anti-glycCTLA-4 antibody masks glycosylation sites at one or more of N113 and N145.

[0010] In one embodiment, the antibody inhibits the interaction of CTLA-4 with CD80, and specifically inhibits the interaction of glycosylated CTLA-4 expressed by effector T cells with CD80 expressed by antigen-presenting cells. In one embodiment, the antibody inhibits the interaction of CTLA-4 with CD86, and specifically inhibits the interaction of glycosylated CTLA-4 expressed by effector T cells with CD86 expressed by antigen-presenting cells. In one embodiment, the antibody inhibits the interaction of CTLA-4 with CD86 and CD80, and specifically inhibits the interaction of glycosylated CTLA-4 expressed by effector T cells with CD86 and CD80 expressed by antigen-presenting cells.

[0011] In some aspects, the antibody binds (such as selectively) one or more glycosylation motifs. In some aspects, the antibody binds to a glycopeptide comprising a glycosylation motif and a neighboring peptide. In some aspects, the antibody binds to a peptide sequence that is three-dimensionally located near one or more glycosylation motifs.

[0012] In certain aspects, the anti-glycCTLA-4 antibody binds to glycosylated CTLA-4 with a K of 0.1 to 13 nM, 0.1 to 10 nM, or 0.1 nM to 5 nM, inclusive. d Less than the K value of unglycosylated CLTA-4. d In other aspects, the antibody binds to the K of glycosylated CTLA-4. d is the K relative to unglycosylated CTLA-4 d at most one tenth.

[0013] In a specific aspect, an anti-glycCLTA-4 monoclonal antibody STC1807 is provided, which has the amino acid sequences of SEQ ID NOs: 3 and 5, respectively (mature V H and V L The present invention also provides a heavy chain variable domain and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 3, and antigen-binding portions thereof, as well as humanized and chimeric forms thereof. Provided herein are anti-glycCTLA-4 antibodies that compete with STC1807 MAb for binding to glycosylated CTLA-4 and / or bind to the same epitope as STC1807. In other aspects, anti-glycCTLA-4 heavy chain antibodies have a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 3.

[0014] The nucleic acid (DNA) and corresponding amino acid sequences of the heavy and light chain variable (V) domains of STC1807 MAb are provided in Table 3 below. SEQ ID NOs: 2 and 3 are STC1807 V H The nucleotide and amino acid sequences of the STC1807 heavy and light chain V domains are shown in Table 4, and SEQ ID NOs: 4 and 5 are the nucleotide and amino acid sequences of the mature form of the STC1807 kappa light chain variable domain. Table 4 provides the Chothia, AbM, Kabat, and Contact heavy and light chain V domain CDRs of STC1807.

[0015] In one embodiment, an anti-glycCTLA-4 antibody that specifically and preferentially binds to glycosylated CTLA-4 comprises a V having the amino acid sequence of SEQ ID NO: 3. H domain and / or a V domain having an amino acid sequence of SEQ ID NO: 5 L In one embodiment, the anti-glycCTLA-4 antibody competes for specific binding to glycosylated CTLA-4 with an antibody comprising V domain of SEQ ID NO: 3. H Domain and V of SEQ ID NO:5 L In other embodiments, the anti-glycCTLA-4 antibody comprises a V domain that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 3. H domain and / or a V domain having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 5 LThese anti-glycCTLA-4 antibodies can be chimeric antibodies and comprise human constant domains, e.g., human constant domains from human IgG1, IgG2, IgG3, or IgG4.

[0016] In one embodiment, an anti-glycCTLA-4 antibody that specifically and preferentially binds to glycosylated CTLA-4 comprises V H domain, the V H The domain comprises Chothia CDRs 1-3 having the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively; AbM CDRs 1-3 having the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 8, respectively; Kabat CDRs 1-3 having the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 8, respectively; or Contact CDRs 1-3 having the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively, or a combination thereof. In one embodiment, the anti-glycCTLA-4 antibody competes for specific binding to glycosylated CTLA-4 with a specific antibody comprising V H domain, the V H The domain comprises Chothia CDR1-3 having the amino acid sequences of SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively; AbM CDR 1-3 having the amino acid sequences of SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:8, respectively; Kabat CDR 1-3 having the amino acid sequences of SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:8, respectively; or Contact CDR 1-3 having the amino acid sequences of SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15, respectively, or a combination thereof. Preferably, the V H and V L The domains have CDRs of the same class, ie, both have Chothia, AbM, Kabat, or Contact CDRs.

[0017] In other embodiments, the anti-glycCTLA-4-1 antibody has a V region comprising CDR H1, CDR H2, and CDRH3. HThe anti-glycCTLA-4 antibody may have a V domain, wherein the CDR H1, CDR H2, and CDR H3 have an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions in one, two, or three of the following CDRs: a Chothia CDR having an amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively, or an AbM CDR having an amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 8, respectively, or a Cabat CDR having an amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 8, respectively, or a Contact CDR having an amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively. H and V L In certain embodiments, the amino acid substitutions are conservative substitutions.

[0018] Preferably, the aforementioned antibody has human framework regions, ie, is a humanized form of STC1807, and optionally comprises a human constant domain, for example, a human constant domain from human IgG1, IgG2, IgG3 or IgG4.

[0019] One skilled in the art will appreciate that one or more amino acid substitutions can be made in the CDRs and / or framework regions of a humanized antibody to improve binding affinity or other parameters. In one embodiment, the anti-glycCTLA-4-1 antibody competes for specific binding to glycosylated CTLA-4 with an antibody comprising the V H and V L In one embodiment, the anti-glycCTLA-4 antibody binds to the K domain of glycosylated CTLA-4. d In one embodiment, the anti-glycCTLA-4 antibody binds to glycosylated CTLA-4 with a K of 0.1-10 nM or 1-20 nM, inclusive. d Less than the K exhibited by the antibody binding to unglycosylated CTLA-4 d In one embodiment, the anti-glycCTLA-4 antibody binds to the K of glycosylated CTLA-4 protein. d is relative to the K of unglycosylated CTLA-4. d In one embodiment, the anti-glycCTLA-4 antibody binds to a glycosylated CTLA-4 protein with a K of at most one-fifth.d is the K expressed by the binding of the antibody to the unglycosylated CTLA-4 protein d at most one tenth.

[0020] In one embodiment, the antibody inhibits the interaction between CTLA-4 and recombinant human CD86-Fc protein in an antibody neutralization assay, expressed as bound green counts / mm2 to cells expressing wild-type CTLA-4. 2 Green counts of cells expressing unglycosylated CTLA-4 / mm 2 3 times, 5 times, 10 times, 20 times, 50 times or 100 times.

[0021] In one embodiment, the antibody is detectable directly or indirectly by a fluorescent marker or label. In one embodiment, the antibody is directly labeled with a fluorescent marker or label (such as FITC), or is detected by a fluorescently labeled secondary antibody. In one embodiment, the binding affinity of the STC1807 MAb, or a chimeric or humanized form thereof, to glycosylated CTLA-4 is 0.1-13 nM or 0.1-5 nM, including lower and upper limits. In one embodiment, the antibody inhibits the interaction of CTLA-4 with CD86 and / or CD80.

[0022] In one embodiment, the anti-glycCTLA-4 antibody competes for specific binding to glycosylated CTLA-4 with an antibody comprising a V H and V L Preferably, these antibodies have human framework regions, i.e., are humanized versions of STC1807, and optionally contain human constant domains, e.g., human constant domains from human IgG1, IgG2, IgG3, or IgG4. One skilled in the art will appreciate that one or more amino acid substitutions can be made in the CDRs or framework regions of the humanized antibodies to improve binding affinity or other parameters. In embodiments, the anti-glycCTLA-4 antibody binds to the K of glycosylated CTLA-4. d Less than the K compared to unglycosylated CTLA-4 d In embodiments, the anti-glycCTLA-4 antibody binds to the K of glycosylated CTLA-4. d Less than the K compared to unglycosylated CTLA-4 d In one embodiment, the anti-glycCTLA-4 antibody binds to the K of glycosylated CTLA-4 protein. d is the K expressed by the binding of the antibody to unglycosylated CTLA-4d In one embodiment, the anti-glycCTLA-4 antibody binds to a glycosylated CTLA-4 protein with a K of at most one-fifth. d is the K expressed by the binding of the antibody to the unglycosylated CTLA-4 protein d In one embodiment, the antibody exhibits binding to cells expressing WT CTLA-4 (expressed as green counts / mm) in a flow cytometry binding assay. 2 ) is the green count of objects bound to cells expressing unglycosylated CTLA-4 / mm 2 In one embodiment, the antibody is detectable directly or indirectly by a fluorescent marker or a marker. In one embodiment, the antibody is directly labeled with a fluorescent marker or a marker (such as FITC). In one embodiment, the binding affinity of STC1807 MAb or its binding domain or humanized or chimeric form to glycosylated CTLA-4 is 0.1-13nM or 0.1-10nM or 0.1-5nM, including lower and upper limits. In one embodiment, the antibody inhibits the interaction of CTLA-4 with CD86, and specifically inhibits the interaction of glycosylated CTLA-4 expressed by effector T cells with CD86 expressed by antigen presenting cells. In one embodiment, the antibody inhibits the interaction of CTLA-4 with CD80, and specifically inhibits the interaction of glycosylated CTLA-4 expressed by effector T cells with CD80 expressed by antigen presenting cells.

[0023] In some aspects, the antibody is recombinant. In some aspects, the antibody is IgG, IgM, IgA, or an antigen-binding fragment thereof. In other aspects, the antibody is Fab', F(ab')2, F(ab')3, a monovalent scFv, a bivalent scFv, a bispecific antibody, a bispecific scFv, or a single domain antibody. In some aspects, the antibody is a human or humanized antibody. In other aspects, the antibody is conjugated to an imaging agent, a chemotherapeutic agent, a toxin, or a radionuclide.

[0024] In another embodiment, provided herein is a composition comprising an antibody of the embodiments (eg, the antibody selectively binds to glycosylated CTLA-4 relative to unglycosylated CTLA-4) in a pharmaceutically acceptable carrier.

[0025] In another embodiment, an isolated polypeptide is provided, which comprises a fragment of at least 7 (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) consecutive amino acids of human CTLA-4, the fragment comprising at least one amino acid corresponding to position N113 or N145 of human CTLA-4. In other aspects, the isolated polypeptide of the embodiment comprises a fragment of at least 7 (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) consecutive amino acids of human CTLA-4, the fragment comprising at least one amino acid corresponding to position N113 or N145 of human CTLA-4, and wherein at least one of the amino acids corresponding to position N113 or N145 of human CTLA-4 is glycosylated. In certain aspects, the polypeptide of the embodiment is fused or conjugated to an immunogenic polypeptide (e.g., a keyhole limpet murine leukocyte antigen). In some aspects, the polypeptide further comprises a Cys residue at the C- or N-terminus. For example, in some aspects, the polypeptide is conjugated to the immunogenic polypeptide via a disulfide bond at the Cys residue.

[0026] In another embodiment, a composition comprising a polypeptide is provided, the polypeptide comprising a fragment of at least 7 (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) consecutive amino acids of human CTLA-4, the fragment comprising at least one amino acid corresponding to position N113 or N145 of human CTLA-4, wherein at least one of the amino acids corresponding to position N113 or N145 of human CTLA-4 is glycosylated, wherein the polypeptide is formulated in a pharmaceutically acceptable carrier. In some aspects, the composition is an immunogenic composition. In some aspects, the immunogenic composition further comprises an adjuvant, such as alum or Freund's adjuvant.

[0027] In another embodiment, there is provided herein a method for treating a subject with cancer, the method comprising administering an effective amount of an antibody or isolated polypeptide of the embodiment to the subject. In some aspects, a method for treating cancer comprises administering an effective amount of a polypeptide (e.g., a glycosylated CTLA-4 polypeptide) to the subject. In other aspects, a method for treating cancer comprises administering an effective amount of an antibody of the embodiment to the subject (e.g., an antibody that selectively binds to glycosylated CTLA-4 relative to unglycosylated CTLA-4), such as, but not limited to, a humanized or chimeric form of STC1807, or an antibody that competes with STC1807 for binding to glycosylated CTLA-4. In some aspects, the cancer is breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, skin cancer, brain cancer, liver cancer, bladder cancer, gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer. In certain aspects, the cancer is adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain / CNS tumors in adults, brain / CNS tumors in children, breast cancer, male breast cancer, cancer in adolescents, cancer in children, cancer in young adults, cancer of unknown primary, Castleman's disease, cervical cancer, colon / rectal cancer, endometrial cancer, esophageal cancer, Ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), gestational trophoblastic disease, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, laryngeal or hypopharyngeal cancer, leukemia (e.g., acute lymphocytic leukemia (ALL) in adults, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CML), or In some aspects, the antibody is administered in a pharmaceutically acceptable composition. In other aspects, the antibody is administered systemically. In specific aspects, the antibody is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or topically.

[0028] In some aspects, the method further comprises administering to the subject at least a second anti-cancer therapy. In some aspects, wherein the second anti-cancer therapy is surgery, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy.

[0029] In another embodiment, provided herein is a method for assessing CTLA-4 glycosylation, N-linked glycosylation, or N-glycosylation, the method comprising contacting a sample containing CTLA-4 with an antibody of the embodiments (e.g., an antibody that selectively binds to glycosylated CTLA-4 relative to unglycosylated CTLA-4). In certain aspects, the method is an in vitro method. In certain aspects, the sample is a cell sample.

[0030] In another embodiment, a method for preparing an antibody is provided, comprising: administering a polypeptide according to the embodiments (e.g., a polypeptide having a fragment of at least 7 consecutive amino acids of human CTLA-4, the fragment comprising at least one amino acid corresponding to position N113 or N145 of human CTLA-4, wherein at least one of the amino acids corresponding to position N113 or N145 of human CTLA-4 is glycosylated) to an animal, and isolating the antibody from the animal. For example, the animal can be a mouse, rat, rabbit, or human. In certain aspects, a method further comprises identifying the CDRs of the antibody and humanizing the sequences surrounding the CDRs to produce a humanized antibody. In other aspects, the method comprises recombinantly expressing the humanized antibody. Thus, in another embodiment, provided herein is an isolated antibody produced by the aforementioned method. Thus, in certain embodiments, provided herein are isolated antibodies that selectively bind to a polypeptide of the embodiments (e.g., a polypeptide comprising a fragment of at least 7 contiguous amino acids of human CTLA-4, said fragment comprising at least one amino acid corresponding to position N113 or N145 of human CTLA-4, wherein at least one of the amino acids corresponding to position N113 or N145 of human CTLA-4 is glycosylated) relative to unglycosylated CTLA-4. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figures 1A-1CCTLA-4 binding to CD80 is glycosylation-specific. Time-lapse microscopy and quantification of the dynamic interaction between green fluorescently labeled CD80-Fc and CTLA-4. (A) Time-lapse microscopy images (at the 20-hour time point) showing the dynamic interaction between CTLA-4 and 293T cells expressing wild-type CLTA-4 and the CLTA-4 2NQ mutant (i.e., unglycosylated CTLA-4). Merged images (20x) of green fluorescence (green fluorescently labeled CTLA-4 / Fc protein) of cells expressing CTLA-4 WT (A) or the 2NQ CTLA-4 mutant (B) are shown. (C) This figure shows the quantitative binding of CTLA-4 / Fc protein to HEK293T cells expressing CLTA-4 WT or CTLA-4 2NQ at hourly time points.

[0032] Figure 2A -C. CTLA-4 binding to CD86 is glycosylation-specific. Time-series microscopy and quantification of the dynamic interaction between green fluorescently labeled CD86-Fc and CTLA-4. (A) Time-series microscopy images showing the interaction between CTLA-4 and 293T cells expressing wild-type CLTA-4 and the CLTA-4 2NQ mutant (unglycosylated form) at the last time point (at the 20-hour time point). Merged images (20x) of green fluorescence (green fluorescently labeled CTLA-4 / Fc protein) of cells expressing CTLA-4 WT (A) or 2NQ CTLA-4 mutant (B) are shown. (C) This figure shows the quantitative binding of CTLA-4 / Fc protein to HEK293T cells expressing CLTA-4 WT or CTLA-4 2NQ at each hourly time point.

[0033] Figure 3A and 3B Development of monoclonal antibodies specific for glycosylated CTLA4. Dot blot analysis of CTLA4 antibodies using purified CTLA4 or PNGase F-treated CTLA4. (A) Dot blot membrane depicts the carbohydrate-specific binding activity of several antibodies, including STC1807 and STC1810. (B) Sample layout of the corresponding 96-well dot blot assay plate.

[0034] Figure 4 Neutralizing activity of anti-glycCTLA-4-1 antibodies. The blocking activity of 65 purified monoclonal antibodies against CD86-Fc protein binding to CTLA-4-expressing cells was measured over time. Antibodies were used at 10 μg / mL.

[0035] Figure 5Sensorgram of anti-CTLA4 antibody analyzed by Octet. From high-throughput K D Summary of screened data. Data were fit to a 1:1 binding model to extract association and dissociation rates. KD was calculated using the ratio kd:ka. The graph shows the response versus time, indicating the progression of the interaction.

[0036] Figure 6 Binding analysis of STC1807 and control antibodies on 293T cells expressing wild-type and mutant CTLA-4 proteins. Binding of the anti-glycCTLA-4 antibody STC1807 to 293T cells expressing marker-tagged wild-type and mutant CTLA-4 proteins and control 293T cells. STC1807 recognizes N113 glycosylation but not N145 or 2NQ. N113, with Q replacing N at position 113 of CTLA-4 (SEQ ID NO: 1); N145, with Q replacing N at position 145; and 2NQ, with Q replacing N at each of positions 113 and 145, or control 293T cells. Anti-marker is shown as a loading control.

[0037] Figure 7A -D. Neutralizing activity and EC of the anti-glycCTLA-4 antibody STC1807 50 (A) Activity of STC1807 in blocking the binding of CD86-Fc protein to cells expressing CTLA-4 as a function of antibody concentration. (B) Inhibition of CTLA-4-CD86 binding as a function of STC1807 concentration. 50 The concentration of STC1808 was 2.189 μg / mL. (C) The activity of STC1808 in blocking the binding of CD86-Fc protein to cells expressing CTLA-4, as a function of antibody concentration. (D) The activity of STC1813 in blocking the binding of CD86-Fc protein to cells expressing CTLA-4, as a function of antibody concentration.

[0038] Figure 8A -D. Neutralizing activity and EC of the anti-glycCTLA-4 antibody hSTC1807 and the FDA-approved anti-CTLA4 ipilimumab 50 (A) Activity of human chimeric STC1807 (hSTC1807) in blocking the binding of CD86-Fc protein to cells expressing CTLA-4 as a function of antibody concentration. (B) Inhibition of CTLA-4-CD86 binding as a function of STC1807 concentration. EC 50is 0.3313 μg / mL. (C) Activity of ipilimumab in blocking the binding of CD86-Fc protein to cells expressing CTLA-4 as a function of antibody concentration. (D) Inhibition of CTLA-4-CD86 binding as a function of ipilimumab concentration. EC 50 It is 0.3068μg / mL.

[0039] Figure 9A A and B. Different binding sites between ipilimumab and STC1807. Competitive binding between STC1807 and ipilimumab was assessed using an epitope binning experiment. Additional binding of the secondary antibody indicates an unoccupied epitope (non-competitor), and no binding indicates epitope blocking (competitor). (A) STC1807 loading. (B) Ipilimumab loading.

[0040] Figure 10A A. and B. STC1807 exhibits increased binding affinity compared to ipilimumab. The binding affinities of STC1807 and ipilimumab (reduced equilibrium dissociation constant [KD] values) were compared using a Biacore binding assay. The graph depicts the response versus time, showing the progression of the interaction for (A) STC1807 (KD 0.47 nM) and (B) ipilimumab (KD 13.4 nM).

[0041] Figure 11A A and B. Increased IFN-γ and IL-2 secretion in the presence of STC1807. The effect of hSTC1807 on T cell proliferation (T) in response to stimulatory cells (DCs, dendritic cells). The graph shows IFN-γ (A) and IL-2 (B) cytokine levels in the presence of STC1807 and control mouse IgG. Cytokines in the supernatant were quantified by ELISA on day 5. DETAILED DESCRIPTION

[0042] N-glycosylation is a post-translational modification that is initiated in the endoplasmic reticulum (ER) and subsequently processed in the Golgi apparatus (Schwarz and Aebi, Current Opinion in Structural Biology 21, 576-582 (2011)). This type of modification is first catalyzed by the membrane-associated oligosaccharyltransferase (OST) complex, which transfers preformed glycans composed of oligosaccharides to asparagine (Asn) side chain acceptors located within the NXT motif (-Asn-X-Ser / Thr-) (Cheung and Reithmeier, Methods 41(4):451-59 (2007); Helenius and Aebi, Science 291(5512):2364-69 (2001)). The addition or removal of sugars from preformed glycans is mediated by a group of glycotransferases and glycosidases, respectively, which tightly regulate the N-glycosylation cascade in a cell-dependent and location-dependent manner.

[0043] The extracellular interaction between CTLA-4 and CD86 and CD80 has a significant impact on tumor-associated immune evasion. N-linked glycosylation of CTLA-4 can enhance its binding to CD80 and / or CD86, leading to the inhibition of T cell-mediated immune responses. Therefore, anti-CTLA-4 antibodies can exhibit enhanced inhibitory effects relative to more general CTLA-4 antibodies.

[0044] As used herein, and unless otherwise indicated, the term "cytotoxic T-lymphocyte-associated protein 4" or "CTLA-4" refers to CTLA-4 from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys (cynomolgus), dogs, and rodents (e.g., mice and rats). Unless otherwise indicated, CTLA-4 also includes various CTLA-4 isoforms, related CTLA-4 polypeptides, including SNP variants thereof, and differently modified forms of CTLA-4, including, but not limited to, phosphorylated CTLA-4, glycosylated CTLA-4, and ubiquitinated CTLA-4.

[0045] An exemplary amino acid sequence of human CTLA-4 is provided below, with the N-linked glycosylation sites indicated in bold and underlined (N113 and N145):

[0046]

[0047] As shown in Table 1 below, both N-glycosylation sites are located in the extracellular domain of CTLA-4.

[0048] Key Features Location length describe Topological domain 36-161 126 Extracellular transmembrane 162-182 21 spiral Topological domain 183-233 41 cytoplasmic

[0049] The specific glycosylation site of a particular CTLA-4 isoform or variant may differ from the amino acid at position 113 or 145 of that particular CTLA-4 isoform or variant.

[0050] In those cases, based on sequence alignments and other common knowledge in the art, one of ordinary skill in the art will be able to determine the glycosylation sites of any particular CTLA-4 isoform or variant corresponding to N113 and N145 of the exemplary human CTLA-4 described above. Thus, antibodies that selectively bind to glycosylated forms of CTLA-4 isoforms or variants relative to unglycosylated CTLA-4 isoforms or variants are also provided herein. The glycosylation sites of a CTLA-4 isoform or variant can be the sites corresponding to N113 and N145 of the human CTLA-4 sequence provided above. Also provided herein are polypeptides comprising a fragment of at least 7 (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) consecutive amino acids of a CTLA-4 isoform or variant, the fragment comprising at least one amino acid corresponding to position N113 or N145 of the exemplary human CTLA-4 sequence provided above.

[0051] As used herein, and unless otherwise indicated, the articles "a", "an", and "the" refer to one or more than one of the grammatical object of the article. By way of example, an antibody refers to one antibody or more than one antibody.

[0052] As used herein, and unless otherwise indicated, the terms "or" and "and / or" are used interchangeably, unless expressly indicated to refer to only alternatives, or the alternatives are mutually exclusive. As used herein, and unless otherwise indicated, "another" means at least a second or more.

[0053] As used herein, and unless otherwise indicated, the term "about" indicates that a value includes the inherent variation of error for the device, method being employed to determine the value, or the variation that exists among the study subjects.

[0054] As used herein, and unless otherwise indicated, the term "antibody" refers to a polypeptide product of a B cell in the immunoglobulin (or "Ig") class of polypeptides that is capable of binding to a specific molecular antigen, such as IgG, IgM, IgA, IgD, IgE, and other molecules having antigen-binding fragments thereof. Antibodies can be composed of two identical pairs of polypeptide chains, each pair having one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), and each amino-terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids, and each carboxyl-terminal portion of each chain includes a constant region (see Borrebaeck (ed.) (1995) Antibody Engineering, 2nd edition, Oxford University Press.; Kuby (1997) Immunology , 3rd edition, WH Freeman and Company, New York). Specific molecular antigens herein include glycosylated human CTLA-4. Antibodies provided herein include, but are not limited to, polyclonal antibodies, monoclonal antibodies, synthetic antibodies, recombinantly produced antibodies, bispecific antibodies, multispecific antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, intrabodies, and anti-idiotypic (anti-Id) antibodies.

[0055] As used herein, and unless otherwise noted, the term "isolated" when used to refer to an antibody, Fab or polynucleotide means that the molecule referred to does not contain at least one component found in nature. The term includes antibodies, Fab or polynucleotides extracted from some or all of the other components found in their natural environment. The components of the antibody's natural environment include, for example, red blood cells, white blood cells, thrombocytes, plasma, proteins, nucleic acids, salts and nutrients. The components of the Fab or polynucleotide's natural environment include, for example, lipid membranes, cell organelles, proteins, nucleic acids, salts and nutrients. The antibodies, Fab or polynucleotides of the present invention may also contain or be completely free of or substantially free of all of these components or any other components of the cell from which they are isolated or recombinantly produced.

[0056] As used herein, and unless otherwise indicated, the term "monoclonal antibody" refers to an antibody that is the product of a single cell clone or hybridoma or a cell population derived from a single cell. Monoclonal antibody also means an antibody produced by recombinant methods from immunoglobulin genes encoding heavy and light chains to produce a single molecule immunoglobulin species. The amino acid sequence of the antibody in a monoclonal antibody preparation is essentially homogeneous, and the binding activity of the antibodies in such a preparation exhibits essentially the same antigen-binding activity. In contrast, polyclonal antibodies are obtained from different B cells in a population, which are combinations of immunoglobulin molecules that bind to a specific antigen. Each immunoglobulin of a polyclonal antibody can bind to a different epitope of the same antigen. Methods for producing monoclonal and polyclonal antibodies are well known in the art (Harlow and Lane, 1996). Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press (1989) and Borrebaeck (editor), Antibody Engineering:A Practical Guide , WH Freeman and Co., Publishers, New York, pp. 103-120 (1991)).

[0057] As used herein, and unless otherwise indicated, the term "human antibody" refers to antibodies having human variable regions and / or human constant regions corresponding to human germline immunoglobulin sequences, or portions thereof. Such human germline immunoglobulin sequences are described in Kabat et al. (1991). Sequences ofProteins ofImmunological Interest , Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242. Herein, the human antibody can include an antibody that binds to glycosylated human CTLA-4 and is encoded by a nucleic acid sequence that is a naturally occurring somatic variant of a human germline immunoglobulin nucleic acid sequence.

[0058] As used herein, and unless otherwise indicated, the term "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

[0059] As used herein, and unless otherwise noted, the term "humanized antibody" refers to a chimeric antibody comprising a human immunoglobulin (e.g., a receptor antibody), wherein natural complementarity determining region ("CDR") residues are replaced with residues from corresponding CDRs with desired specificity, affinity and ability from non-human species (e.g., donor antibody), such as mouse, rat, rabbit or non-human primate. In some cases, one or more FR region residues of a human immunoglobulin are replaced with corresponding non-human residues. In addition, a humanized antibody can have residues not found in a receptor antibody or a donor antibody. These modifications are made to further improve antibody performance. A humanized antibody heavy chain or light chain can have substantially all at least one or more variable regions, wherein all or substantially all CDRs correspond to the CDRs of a non-human immunoglobulin, and all or substantially all FRs are FRs of human immunoglobulin sequences. A humanized antibody can have at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a constant region of a human immunoglobulin. For additional details, see Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992); Carter et al., Proc. Natl. Acd. Sci. USA 89:4285-4289 (1992); and U.S. Pat. Nos. 6,800,738, 6,719,971, 6,639,055, 6,407,213, and 6,054,297.

[0060] As used herein, and unless otherwise indicated, the term "recombinant antibody" refers to an antibody prepared, expressed, established or separated by a recombinant manner. Recombinant antibodies can be antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies separated from a recombinant combinatorial antibody library, antibodies separated from animals (such as mice or cattle) that are transgenic and / or transchromosomal for human immunoglobulin genes (see, e.g., Taylor, LD et al., Nucl. Acids Res. 20: 6287-6295 (1992)), or antibodies prepared, expressed, established or separated by any other means, the other means comprising splicing immunoglobulin gene sequences to other DNA sequences. Such recombinant antibodies can have variable and constant regions, including those derived from human germline immunoglobulin sequences (see Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). The recombinant antibody can also be subjected to in vitro mutagenesis (or, when using transgenic animals for human Ig sequences, in vivo somatic mutagenesis), and the V of the recombinant antibody can be H and V L The amino acid sequence of the region may be a sequence that, although derived from human germline V H and V L sequences that are not naturally present in the human antibody germline repertoire in vivo.

[0061] As used herein, and unless otherwise indicated, the term "antigen-binding fragment" and similar terms refer to a portion of an antibody that includes the amino acid residues that immunospecifically bind to an antigen and confer specificity and affinity on the antibody for that antigen. An antigen-binding fragment may be referred to as a functional fragment of an antibody. An antigen-binding fragment may be monovalent, divalent, or multivalent.

[0062] Molecules with antigen binding fragments include, for example, Fd, Fv, Fab, F(ab'), F(ab)2, F(ab')2, F(ab)3, F(ab')3, single-chain Fv (scFv), diabodies, triabodies, tetrabodies, minibodies, or single domain antibodies. ScFv can be a monovalent scFv or a divalent scFv. Other molecules with antigen binding fragments can include, for example, heavy or light chain polypeptides, variable region polypeptides, or CDR polypeptides, or portions thereof, as long as such antigen binding fragments retain binding activity. Such antigen binding fragments can be found described, for example, in Harlow and Lane, Antibodies:A Laboratory Manual , Cold Spring Harbor Laboratory, New York (1989); Myers (ed.), Molec.Biology and Biotechnology: A Comprehensive DeskReference , New York: VCH Publisher, Inc.; Huston et al., Cell Biophysics, 22: 189-224 (1993); Plückthun and Skerra, Meth. Enzymol., 178: 497-515 (1989) and Day, ED, Advanced Immunochemistry, 2nd ed., Wiley-Liss, Inc., New York, NY (1990). An antigen-binding fragment can be a polypeptide having an amino acid sequence of at least 5 consecutive amino acid residues, at least 10 consecutive amino acid residues, at least 15 consecutive amino acid residues, at least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino acid residues, at least 60 consecutive amino acid residues, at least 70 consecutive amino acid residues, at least 80 consecutive amino acid residues, at least 90 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least 125 consecutive amino acid residues, at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues, at least 200 consecutive amino acid residues, or at least 250 consecutive amino acid residues.

[0063] The heavy chain of an antibody represents a polypeptide chain of approximately 50-70 kDa, wherein the amino-terminal portion includes a variable region of approximately 120 to 130 or more amino acids, and the carboxyl-terminal portion includes a constant region. Based on the amino acid sequence of the heavy chain constant region, the constant region can be one of five different types called alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ). Different heavy chains have different sizes: α, δ, and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When combined with a light chain, these different types of heavy chains produce the five well-known classes of antibodies: IgA, IgD, IgE, IgG, and IgM, respectively, including the four subclasses of IgG, namely IgG1, IgG2, IgG3, and IgG4. The heavy chain can be a human heavy chain.

[0064] The light chain of an antibody represents a polypeptide chain of about 25 kDa, wherein the amino terminal portion includes a variable region of about 100 to about 110 or more amino acids, and the carboxyl terminal portion includes a constant region. The approximate length of the light chain is 211-217 amino acids. Based on the amino acid sequence of the constant domain, there are two different types, referred to as kappa (κ) or lambda (λ). Light chain amino acid sequences are well known in the art. The light chain can be a human light chain.

[0065] The variable domain or variable region of an antibody represents a portion of the light or heavy chain of an antibody, generally located at the amino terminus of the light or heavy chain, and having a length of about 120-130 amino acids in the heavy chain and about 100-110 amino acids in the light chain, and is used for the binding and specificity of each specific antibody to its specific antigen. The sequence of the variable domains varies greatly between different antibodies. The variability of the sequence is concentrated in the CDRs, while the less variable parts of the variable domains are called framework regions (FRs). The CDRs of the light and heavy chains are primarily responsible for the interaction between the antibody and the antigen. The numbering of the amino acid positions used herein is according to the EU index, as in Kabat et al. (1991). Sequences of proteins of immunological interest (USDepartment of Health and Human Services, Washington, DC) 5th Edition. The variable region can be a human variable region.

[0066] CDR stands for immunoglobulin (Ig or antibody) V H One of the three hypervariable regions (H1, H2 or H3) within the non-framework region of the β-sheet framework, or in the antibody V L One of the three hypervariable regions (L1, L2 or L3) within the non-framework region of the β-pleated framework. Therefore, CDR is the variable region sequence interspersed in the framework region sequence. CDR region is well known to those skilled in the art and has been defined by, for example, Kabat as the region with the highest variability within the antibody variable domain (Kabat et al., J.Biol.Chem.252:6609-6616 (1977); Kabat, Adv.Prot.Chem.32:1-75 (1978)). CDR region sequences are also structurally defined by Chothia as those residues that are not part of the conserved β-pleated framework and are therefore able to adapt to different conformations (Chothia and Lesk, J.Mol.Biol.196:901-917 (1987)). Both terms are recognized in the art. The positions of the CDRs within canonical antibody variable domains have been determined by comparison of numerous structures (Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); Morea et al., Methods 20:267-279 (2000)). Since the number of residues in the hypervariable regions varies among different antibodies, the additional residues associated with the canonical positions are typically numbered a, b, c, etc. next to the residue numbers in the canonical variable domain numbering scheme (Al-Lazikani et al., supra (1997)). Such nomenclature is also well known to those skilled in the art.

[0067] A universal numbering system has been developed and widely adopted, the ImMunoGeneTics (IMGT) Information (Lafranc et al., 2003, Dev. Comp. Immunol., 27(1):55-77). IMGT is a comprehensive information system that specializes in immunoglobulins (Ig), T cell receptors (TRs), and major histocompatibility complexes (MHCs) of humans and other vertebrates. In this article, CDRs are represented by amino acid sequence and position in the light chain or heavy chain. Since the "position" of CDRs within the structure of the immunoglobulin V domain is conserved between species and exists in structures called loops, a numbering system is used that aligns the variable domain sequences based on structural features, CDRs, and framework residues, and is easily identified. This information can be used to transplant and replace CDR residues from an immunoglobulin from one species into a receptor framework, typically from a human antibody. Honegger et al., 2001, J. Mol. Biol., 309:657-670 have developed another numbering system (AHon). The correspondence between numbering systems, including, for example, Kabat numbering and the IMGT unique numbering system, is well known to those skilled in the art (see, for example, Kabat, supra; Chothia et al., supra; Martin, 2010, Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag; and Lefranc et al., 1999, Nuc. Acids Res., 27:209-212).

[0068] AbM and Contact method have also defined CDR region sequence.AbM hypervariable region represents the compromise between Kabat CDR and Chothia structure loop, and is used by the AbM antibody modeling software of Oxford Molecular (referring to, for example, Martin, 2010, Antibody Engineering, volume 2, chapter 3, Springer Verlag). " contact " hypervariable region is based on the analysis to available complex crystal structure.The residue from each in these hypervariable regions or CDR is as described below.

[0069] An exemplary description of the CDR region sequences is shown in Table 2 below. The positions of the CDRs within canonical antibody variable regions have been determined by comparing numerous structures (Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948; Morea et al., 2000, Methods, 20:267-279). Because the number of residues in the hypervariable regions varies among different antibodies, additional residues associated with canonical positions are typically numbered a, b, c, etc. next to the residue numbers in the canonical variable domain numbering scheme (Al-Lazikani et al., supra). This nomenclature is also well known to those skilled in the art.

[0070] Table 2. Exemplary descriptions of CDR region sequences

[0071] IMGT Kabat AbM Chothia Contact <![CDATA[V H CDR1]]> 27-38 31-35 26-35 26-32 30-35 <![CDATA[V H CDR2]]> 56-65 50-65 50-58 53-55 47-58 <![CDATA[V H CDR3]]> 105-117 95-102 95-102 96-101 93-101 <![CDATA[V L CDR1]]> 27-38 24-34 24-34 26-32 30-36 <![CDATA[V L CDR2]]> 56-65 50-56 50-56 50-52 46-55 <![CDATA[V L CDR3]]> 105-117 89-97 89-97 91-96 89-96

[0072] One or more CDRs can also be covalently or non-covalently incorporated into a molecule to make it an immunoadhesin. An immunoadhesin can incorporate one or more CDRs as part of a larger polypeptide chain, one or more CDRs can be covalently linked to another polypeptide chain, or one or more CDRs can be non-covalently incorporated. The CDRs allow the immunoadhesin to bind to a specific target antigen.

[0073] As used herein and unless otherwise indicated, the term "binding" refers to an interaction between molecules. The interaction can be, for example, a non-covalent interaction, including hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions. The strength of the total non-covalent interactions between an antibody and a single epitope of a target molecule (such as glycosylated human CTLA-4) is the affinity of the antibody for that epitope. "Binding affinity" generally refers to the strength of the sum of the non-covalent interactions between a single binding site of a molecule (e.g., a binding protein, such as an antibody) and its binding partner (e.g., an antigen).

[0074] The affinity of a binding molecule X (such as an antibody) for its binding partner Y (such as the antibody's cognate antigen) can often be expressed in terms of the dissociation constant (K d ) or equilibrium dissociation constant (K D ) represents. Low-affinity antibodies generally bind antigen slowly and dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods for measuring binding affinity are known in the art, any of which may be used for the purposes of the present disclosure. "K" may be measured by assays known in the art, such as by binding assays. D ” or “K D K can be measured in a radiolabeled antigen binding assay (RIA). D, for example, using a Fab form of the antibody of interest and its antigen (Chen, et al., (1999) J. Mol. Biol. 293:865-881). K can also be measured as follows D or K D Value: As measured by surface plasmon resonance using Biacore, e.g., a BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ), or by biolayer interferometry, e.g., using an Octet QK384 system (ForteBio, Menlo Park, CA). As used herein, and unless otherwise indicated, an antibody is considered to "selectively bind" a first antigen relative to a second antigen if it binds the first antigen with a higher affinity than the second antigen. An antibody generally does not bind to a completely unrelated antigen.

[0075] As used herein, and unless otherwise indicated, the term "polypeptide" as used herein includes oligopeptides having 2-30 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25 or 30 amino acids) as well as longer amino acid chains, for example, more than 30 amino acids, more than 50 amino acids, more than 100 amino acids, more than 150 amino acids, more than 200 amino acids, more than 300 amino acids, more than 400 amino acids, more than 500 amino acids or more than 600 amino acids. Polypeptides can be produced, for example, by recombinant expression or by chemical synthesis. The polypeptides of the present disclosure can be post-translationally modified or chemically modified (e.g., glycosylation, carbamylation, phosphorylation, biotinylation, attachment of fluorescent dyes, etc.). Polypeptides can be glycosylated at specific sites. Polypeptides can include non-natural amino acids that are not encoded by the natural genetic code. For example, the polypeptide may include a methylated backbone structure, a peptoid backbone structure (poly-N-substituted glycine), L-amino acids, R-amino acids, etc. The polypeptide may have a wild-type sequence, a naturally occurring variant sequence, a mutant sequence (e.g., a point mutant, a deletion mutant), etc.

[0076] Anti-glycCTLA-4 antibodies

[0077] Provided herein are isolated antibodies that selectively bind to glycosylated CTLA-4 relative to unglycosylated CTLA-4. CTLA-4 can be human CTLA-4. Glycosylated CTLA-4 can be a specific N-glycan structure of CTLA-4 or a glycopeptide of CTLA-4. In certain embodiments, the antibodies provided herein are antigen-binding fragments that selectively bind to glycosylated CTLA-4 relative to unglycosylated CTLA-4.

[0078] In certain embodiments, the isolated antibodies provided herein selectively bind to human CTLA-4 glycosylated at N113, N1145, or N113 and N145 relative to unglycosylated CTLA-4. In certain embodiments, the isolated antibodies selectively bind to human CTLA-4 glycosylated at N113. In certain embodiments, the isolated antibodies selectively bind to human CTLA-4 glycosylated at N145. In certain embodiments, the isolated antibodies selectively bind to human CTLA-4 glycosylated at N113 and N145.

[0079] In certain aspects, the anti-glycCTLA-4 antibodies inhibit the interaction of glycosylated CTLA-4 expressed by effector T cells with CD86 or CD80 expressed by antigen-presenting cells. In certain aspects, the anti-glycCTLA-4 antibodies bind to CTLA-4 and mask or screen one or more glycosylation motifs to block binding or other interactions of molecules with the motifs and can block glycosylation of CTLA-4 at the glycosylation sites. In specific embodiments, the anti-glycCTLA-4 antibodies mask glycosylation sites at one or more of N113 and N145.

[0080] In certain embodiments, the antibodies provided herein selectively bind to one or more glycosylation motifs of CTLA-4. In certain embodiments, the antibodies selectively bind to glycopeptides and adjacent peptides having a glycosylation motif. In certain embodiments, the antibodies selectively bind to the K of glycosylated CTLA-4. d Compared with the K of unglycosylated PD-1 d In certain embodiments, the antigen-binding fragment binds to the K of glycosylated CTLA-4. d Compared with the K of unglycosylated CTLA-4 d In certain embodiments, the antibody binds to a K of glycosylated CTLA-4 that is less than 50%. d Compared with the K of unglycosylated CTLA-4 d In other aspects, the antibody binds to the K of glycosylated CTLA-4. d is relative to the K of unglycosylated CTLA-4. d at most one tenth.

[0081] Provided herein are monoclonal antibodies that preferentially bind to glycosylated CTLA-4, specifically STC1807. Also provided are humanized and chimeric forms of STC1807 and antibodies that compete for binding to STC1807. The heavy and light chain variable domains of STC1807 are provided in Table 3 below.

[0082] In a specific aspect, an anti-glycCTLA-4 monoclonal antibody STC1807 is provided, which has the amino acid sequences of SEQ ID NOs: 3 and 5, respectively (mature V H and V L Provided herein are heavy and light chain variable domains comprising the amino acid sequence of the SEQ ID NO: 1 region, and antigen-binding portions thereof, as well as humanized and chimeric forms thereof. Provided herein are anti-glycCTLA-4 antibodies that compete with STC1807 MAb for binding to CTLA-4 and / or bind to the same epitope as STC1807.

[0083] The nucleic acid (DNA) and corresponding amino acid sequences of the heavy and light chain variable (V) domains of the monoclonal antibody, STC1807 mAb, are shown in Table 3 below. SEQ ID NOs: 2 and 3 are STC1807 V H The nucleotide and amino acid sequences of the domain, and SEQ ID NO: 4 and 5 are STC1807κV L Nucleotide and amino acid sequences of the mature forms of the domains. Table 4 provides the Chothia, AbM, Kabat, and Contact heavy and light chain V domain CDRs of STC1807.

[0084] In one embodiment, an anti-glycCTLA-4 antibody that specifically and preferentially binds to glycosylated CTLA-4 comprises a V having the amino acid sequence of SEQ ID NO: 3. H domain and / or a V domain having an amino acid sequence of SEQ ID NO: 5 L In one embodiment, the anti-glycCTLA-4 antibody competes for specific binding to glycosylated CTLA-4 with an antibody comprising V domain of SEQ ID NO: 3. H Domain and V of SEQ ID NO:5 L In other embodiments, the anti-glycCTLA-4 antibody comprises a V H domain and / or V L domain, the V H The V domain has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 3, wherein the VL The domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 5. These anti-glycCTLA-4 antibodies can be chimeric antibodies and comprise human constant domains, e.g., human constant domains from human IgG1, IgG2, IgG3, or IgG4.

[0085] In one embodiment, an anti-glycCTLA-4 antibody that specifically and preferentially binds to glycosylated CTLA-4 comprises V H domain, the V H The domain comprises Chothia CDRs 1-3 having the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively; AbM CDRs 1-3 having the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 8, respectively; Kabat CDRs 1-3 having the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 8, respectively; or Contact CDRs 1-3 having the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively, or a combination thereof. In one embodiment, the anti-glycCTLA-4 antibody competes for specific binding to glycosylated CTLA-4 with a specific antibody comprising V H domain, the V H The domain comprises Chothia CDRs 1-3 having the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively; AbM CDRs 1-3 having the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 8, respectively; Kabat CDRs 1-3 having the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 8, respectively; or Contact CDRs 1-3 having the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively, or a combination thereof. In one embodiment, an anti-glycCTLA-4 antibody that specifically and preferentially binds to glycosylated CTLA-4 comprises V L domain, the V LThe domain comprises Chothia, AbM, or Kabat CDRs 1-3 having the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively; or comprises Contact CDRs 1-3 having the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively, or a combination thereof. In one embodiment, the anti-glycCTLA-4 antibody competes for specific binding to glycosylated CTLA-4 with a specific antibody comprising V L domain, the V L The domain comprises Chothia, AbM, or Kabat CDRs 1-3 having the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively; or comprises Contact CDRs 1-3 having the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively, or a combination thereof. In one embodiment, the anti-glycCTLA-4 antibody comprises or competes for binding to a specific antibody comprising V H domain and contains V L domain, the V H The domain comprises Chothia CDR1-3 having the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively; comprises AbM CDR 1-3 having the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 8, respectively; comprises Kabat CDR 1-3 having the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 8, respectively; or comprises Contact CDR 1-3 having the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively; the V L The domain comprises Chothia, AbM or Kabat CDR1-3 having the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively; or comprises Contact CDR 1-3 having the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively. Preferably, V H and V LThe domains have CDRs of the same class, ie, both have Chothia, AbM, Kabat, or Contact CDRs.

[0086] In other embodiments, the anti-glycCTLA-4 antibody has a V region comprising CDR H1, CDR H2, and CDR H3. H The anti-glycCTLA-4 antibody may have a V domain, wherein the CDR H1, CDR H2, and CDR H3 have an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions in one, two, or three of the following CDRs: a CDR having an amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively, or a CDR having an amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 8, respectively, or a CDR having an amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 8, respectively, or a CDR having an amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively. L A domain comprising CDR L1, CDR L2, and CDR L3 having an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions in 1, 2, or 3 CDRs: CDRs having the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively, or CDRs having the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively. The anti-glycCTLA-4 antibody may have a CDR L1, CDR L2, and CDR L3 having an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions in 1, 2, or 3 CDRs. H and V L In certain embodiments, the amino acid substitutions are conservative substitutions.

[0087] Preferably, the aforementioned antibody has human framework regions, ie, is a humanized form of STC1807, and optionally comprises a human constant domain, for example, a human constant domain from human IgG1, IgG2, IgG3 or IgG4.

[0088] One skilled in the art will appreciate that one or more amino acid substitutions can be made in the CDRs and / or framework regions of a humanized antibody to improve binding affinity or other parameters. In one embodiment, the anti-glycCTLA-4 antibody competes for specific binding to glycosylated CTLA-4 with an antibody comprising the V H and V LIn one embodiment, the anti-glycCTLA-4 antibody binds to the K domain of glycosylated CTLA-4. d Less than the K compared to unglycosylated CTLA-4 d In embodiments, the anti-glycCTLA-4 antibody binds to the K of glycosylated CTLA-4. d Less than the K compared to unglycosylated CTLA-4 d In one embodiment, the anti-glycCTLA-4 antibody binds to the K of glycosylated CTLA-4 protein. d is the K expressed by the binding of the antibody to unglycosylated CTLA-4 d In one embodiment, the anti-glycCTLA-4 antibody binds to a glycosylated CTLA-4 protein with a K of at most one-fifth. d is the K expressed by the binding of the antibody to the unglycosylated CTLA-4 protein d In one embodiment, the antibody exhibits binding to cells expressing WT CTLA-4 (expressed as green counts / mm) in a flow cytometry binding assay. 2 ) is the green count of objects bound to cells expressing unglycosylated CTLA-4 / mm 2 In one embodiment, the antibody is detectable directly or indirectly by a fluorescent marker or a marker. In one embodiment, the antibody is directly labeled with a fluorescent marker or a marker (such as FITC). In one embodiment, the binding affinity of STC1807 MAb or its binding domain or humanized or chimeric form to glycosylated CTLA-4 is 0.1-13nM or 0.1-5nM, including lower and upper limits. In one embodiment, the antibody inhibits the interaction of CTLA-4 with CD86, and specifically inhibits the interaction of glycosylated CTLA-4 expressed by effector T cells with CD86 expressed by antigen presenting cells. In one embodiment, the antibody inhibits the interaction of CTLA-4 with CD80, and specifically inhibits the interaction of glycosylated CTLA-4 expressed by effector T cells with CD80 expressed by antigen presenting cells.

[0089] In one embodiment, the antibody inhibits the interaction of CD86 and CTLA-4, and specifically inhibits the interaction of glycosylated CTLA-4 expressed by effector T cells with CD86 expressed by antigen-presenting cells. In one embodiment, the antibody inhibits the interaction of CD80 and CTLA-4, and specifically inhibits the interaction of glycosylated CTLA-4 expressed by effector T cells with CD80 expressed by antigen-presenting cells.

[0090] Another embodiment provides an isolated nucleic acid molecule encoding an anti-glycCTLA-4V H domain and / or encodes an anti-glycCTLA-4 antibody V L domain, encoding anti-glycCTLA-4V H The nucleic acid of the domain comprises a nucleotide sequence having at least 90-98% identity to SEQ ID NO: 2, encoding an anti-glycCTLA-4 antibody V L The nucleic acid encoding the V domain comprises a nucleotide sequence that is at least 90-98% identical to SEQ ID NO: 4. H and / or V L The nucleotide sequence of the domain is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 2 or SEQ ID NO: 4, respectively.

[0091] Table 3 below provides the nucleotide and amino acid sequences of the heavy and light chain variable domains of STC1807.

[0092] Table 3. Nucleotide and amino acid sequences of the heavy and light chain variable domains of STC1807

[0093]

[0094] The CDR sequences of the STC1807 antibody according to the Chothia, AbM, Kabat, and Contact CDRs are provided below in Table 4. Thus, a humanized form of STC1807 is provided that preferentially binds to glycosylated CTLA-4 compared to unglycosylated CTLA-4 comprising the CDRs in Table 4 below grafted into human framework regions.

[0095] Table 4. CDR sequences of STC1807

[0096]

[0097]

[0098] In certain embodiments, the anti-glycCTLA-4-1 antibodies provided herein can be IgG, IgM, IgA, IgD, or IgE. The anti-glycCTLA-4-1 antibodies can also be chimeric antibodies, affinity matured antibodies, humanized antibodies, or human antibodies. The anti-glycCTLA-4 antibodies can also be camelized antibodies, intracellular antibodies, or anti-idiotypic (anti-Id) antibodies. In certain embodiments, the anti-glycCTLA-4 antibodies can be polyclonal antibodies or monoclonal antibodies.

[0099] In certain embodiments, the antibodies provided herein are antigen-binding fragments that selectively bind to glycosylated CTLA-4 relative to unglycosylated CTLA-4. The antigen-binding fragment can be Fd, Fv, Fab, F(ab'), F(ab)2, F(ab')2, F(ab)3, F(ab')3, single-chain Fv (scFv), diabody, triabody, tetrabody, minibody, or single-domain antibody. scFv can be a monovalent scFv or a divalent scFv.

[0100] Polyclonal or monoclonal antibodies, antigen-binding fragments and binding domains and CDRs (including engineered forms of any of the foregoing) that are specific for glycosylated CTLA-4, one or more of the various epitopes thereof, or conjugates of any of the foregoing, whether such antigens or epitopes are isolated from a natural source or are synthetic derivatives or variants of natural compounds, can be generated by known means and as described herein.

[0101] Antibodies can be produced from any animal source, including birds and mammals. In certain embodiments, the antibody is a sheep, mouse (e.g., mouse and rat), rabbit, goat, guinea pig, camel, horse or chicken antibody. In addition, newer technology allows the development and screening of human antibodies from human combinatorial antibody libraries. For example, bacterial phage antibody expression technology allows the production of specific antibodies in the absence of animal immunization, as described in U.S. Patent number 6,946,546, which is hereby incorporated by reference in its entirety. These techniques are further described in Marks et al., Bio / Technol., 10:779-783 (1992); Stemmer, Nature, 370:389-391 (1994); Gram et al., Proc. Natl. Acad. Sci. USA, 89:3576-3580 (1992); Barbas et al., Proc. Natl. Acad. Sci. USA, 91:3809-3813 (1994); and Schier et al., Gene, 169(2):147-155 (1996); which are hereby incorporated by reference in their entireties.

[0102] Methods for producing polyclonal antibodies in various animal species, as well as methods for producing various types of monoclonal antibodies (including humanized, chimeric and fully human monoclonal antibodies) are well known in the art. For example, the following U.S. patents provide useful descriptions of such methods and are incorporated herein by reference: U.S. Patent Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,196,265, 4,275,149, 4,277,437, 4,366,241, 4,469,797, 4,472,509 、4,606,855、4,703,003、4,742,159、4,767,720、4,816,567、4,867,973、4,938,948、4,946,778、5,021,236、5,164,296、5,196,066、5,223,409、5,403,484、5,420,2 53, 5,565,332, 5,571,698, 5,627,052, 5,656,434, 5,770,376, 5,789,208, 5,821,337, 5,844,091, 5,858,657, 5,861,155, 5,871,907, 5,969,108, 6,054,297, 6,165 ,464, 6,365,157, 6,406,867, 6,709,659, 6,709,873, 6,753,407, 6,814,965, 6,849,259, 6,861,572, 6,875,434, 6,891,024, 7,407,659 and 8,178,098, which are hereby incorporated by reference in their entirety.

[0103] In certain embodiments, the anti-glycCTLA-4 antibody may be a monoclonal antibody. In certain embodiments, the anti-glycCTLA-4 antibody may be a polyclonal antibody. An animal may be inoculated with an antigen, such as a glycosylated CTLA-4 polypeptide, to produce antibodies specific for the glycosylated CTLA-4 polypeptide. Antigens are often bound or conjugated to another molecule to enhance the immune response. A conjugate can be any peptide, polypeptide, protein, or non-proteinaceous substance that is bound to an antigen for eliciting an immune response in an animal. The antibodies produced by an animal in response to antigen inoculation comprise a plurality of different molecules (polyclonal antibodies) produced by a plurality of individual antibody-producing B lymphocytes. Under the correct conditions for the production of polyclonal antibodies in an animal, the majority of the antibodies in the animal's serum recognize collective epitopes on the antigenic compound to which the animal has been immunized.

[0104] In certain embodiments, monoclonal antibodies are produced by the method of producing monoclonal antibodies. In certain embodiments, monoclonal antibodies are produced by the method of producing polyclonal antibodies. In certain embodiments, monoclonal antibodies are produced by the method of producing monoclonal antibodies. In certain embodiments, monoclonal antibodies are produced by the method of producing monoclonal antibodies. In certain embodiments, monoclonal antibodies are produced by the method of producing monoclonal antibodies. In certain embodiments, monoclonal antibodies are produced by the method of producing monoclonal antibodies. In certain embodiments, monoclonal antibodies are produced by the method of producing monoclonal antibodies. In certain embodiments, monoclonal antibodies are produced by the method of producing monoclonal antibodies. The application of rats is well-known and can provide some advantages. Routine use of mice (for example, BALB / c mice) and the stable fusion of high percentage is usually provided.

[0105] Hybridoma technology involves fusing a single B lymphocyte from a mouse previously immunized with a glycosylated CTLA-4 polypeptide with an immortalized myeloma cell (often a mouse myeloma). This technology provides a means to propagate a single antibody-producing cell indefinitely, thereby producing an unlimited number of structurally identical antibodies with the same antigen or epitope specificity (monoclonal antibodies).

[0106] Anti-glycCTLA-4 antibodies can be produced by any method known in the art for producing polypeptides, e.g., in vitro synthesis, recombinant DNA production, and the like. Humanized antibodies can be produced by recombinant DNA technology. Recombinant immunoglobulin expression technology can also be used to produce the antibodies described herein. Recombinant production of immunoglobulin molecules, including humanized antibodies, is described in U.S. Pat. No. 4,816,397 (Boss et al.), U.S. Pat. Nos. 6,331,415 and 4,816,567 (both issued to Cabilly et al.), British Patent GB 2,188,638 (Winter et al.), and British Patent GB 2,209,757; which are hereby incorporated by reference in their entirety. Recombinant expression technology for immunoglobulins, including humanized immunoglobulins, can also be found in Goeddel et al., Gene Expression Technology Methods in Enzymology Volume 185 Academic Press (1991), and Borreback, Antibody Engineering , WH Freeman (1992); which are hereby incorporated by reference in their entirety. Additional information on the production, design and expression of recombinant antibodies can be found in Mayforth, Designing Antibodies , Academic Press, San Diego (1993).

[0107] Developed a method for replacing the light chain and heavy chain constant domains of monoclonal antibodies with similar domains of human origin and keeping the variable region of foreign antibodies intact. Alternatively, fully human monoclonal antibodies are produced in transgenic mice or rats of human immunoglobulin genes. Also developed is a method for converting the variable domains of monoclonal antibodies into a more human form by recombinantly constructing antibody variable domains with rodent and human amino acid sequences. In humanized monoclonal antibodies, only the hypervariable CDRs are derived from non-human (e.g., mouse, rat, chicken, llama) monoclonal antibodies, and the framework regions are derived from human amino acid sequences. It is believed that replacing the rodent-specific amino acid sequence in the antibody with the amino acid sequence found in the corresponding position of the human antibody will reduce the possibility of adverse immune reactions during treatment. The hybridoma or other cells producing the antibody may also undergo genetic mutations or other changes, which may or may not change the binding specificity of the antibody produced by the hybridoma.

[0108] By using monoclonal antibodies and other antibodies and recombinant DNA technology to produce other antibodies or chimeric molecules that retain the antigen or epitope specificity of the original antibody, that is, the molecule has a binding domain, it is possible to produce engineered antibodies. Such technology can involve introducing DNA encoding the CDRs of immunoglobulin variable regions or antibodies into the genetic material of the framework regions, constant regions or constant regions plus framework regions of different antibodies. See, for example, U.S. Patent Nos. 5,091,513 and 6,881,557, which are incorporated herein by reference.

[0109] In certain embodiments, the anti-glycCTLA-4 antibody is a human antibody. Human antibodies can be prepared by various methods known in the art, including the above-mentioned phage display method, which uses an antibody library derived from human immunoglobulin sequences (see U.S. Patent Nos. 4,444,887 and 4,716,111; and International Publication Nos. WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735, and WO 91 / 10741). Human antibodies can be produced using transgenic mice that cannot express functional endogenous immunoglobulins but can express human immunoglobulin genes. For example, human heavy and light chain immunoglobulin gene complexes can be introduced into mouse embryonic stem cells randomly or by homologous recombination. Alternatively, in addition to human heavy and light chain genes, human variable regions, constant regions, and diversity regions can be introduced into mouse embryonic stem cells. Mouse heavy and light chain immunoglobulin genes can be rendered inoperable individually or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletions in the JH region prevent endogenous antibody production. Modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. Chimeric mice are then bred to produce homozygous offspring expressing human antibodies. Transgenic mice are immunized with a selected antigen (e.g., all or part of a glycosylated CTLA-4 polypeptide) using conventional methods. Monoclonal antibodies against the antigen can be obtained from immunized transgenic mice using conventional hybridoma technology (see, e.g., U.S. Patent No. 5,916,771). The human immunoglobulin transgenes carried by the transgenic mice are rearranged during B cell differentiation, and subsequently undergo class switching and somatic mutations. Therefore, using such technology, therapeutically useful IgG, IgA, IgM, and IgE antibodies can be produced. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93, which is incorporated herein by reference in its entirety). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., International Publication Nos. WO 98 / 24893, WO 96 / 34096, and WO 96 / 33735; and U.S. Patent Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598, which are incorporated herein by reference in their entirety. Additionally, companies such as Abgenix, Inc. (Freemont, Calif.) and Medarex (Princeton, NJ) are working to provide human antibodies to selected antigens using technology similar to that described above.

[0110] In one embodiment, the antibody is a chimeric antibody, for example, an antibody comprising an antigen-binding sequence from a non-human donor, wherein the antigen-binding sequence is transplanted to a heterologous non-human, human or humanized sequence (for example, a framework and / or constant domain sequence). In one embodiment, the non-human donor is a rat. In one embodiment, the antigen-binding sequence is synthesized, for example, by mutagenesis (for example, phage display screening of a human phage library, etc.). In one embodiment, the chimeric antibody provided herein has a mouse V region and a human C region. In one embodiment, the mouse light chain V region is fused to a human kappa light chain. In one embodiment, the mouse heavy chain V region is fused to a human IgG1 C region.

[0111] Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., J. Immunol. Methods 125:191-202 (1989); and U.S. Patent Nos. 6,311,415, 5,807,715, 4,816,567, and 4,816,397, all of which are hereby incorporated by reference in their entirety. Chimeric antibodies comprising one or more CDRs from a non-human species and framework regions from a human immunoglobulin molecule can be produced using a variety of techniques known in the art, including, for example, CDR grafting (EP 239,400; International Publication No. WO 91 / 09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), covering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4 / 5):489-498 (1991); Studnicka et al., Protein Engineering 7:805 (1994); and Roguska et al., Proc. Natl. Acad. Sci. USA 91:969 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332), all of which are hereby incorporated by reference in their entireties.

[0112] An exemplary method for producing a recombinant chimeric anti-glycCTLA-4 antibody can comprise the following: a) constructing an expression vector by conventional molecular biology methods that encodes and expresses an antibody heavy chain in which the CDRs and variable regions of a murine anti-glycCTLA-4 monoclonal antibody are fused to an Fc region derived from a human immunoglobulin, thereby producing a vector for expressing the chimeric antibody heavy chain; b) constructing an expression vector by conventional molecular biology methods that encodes and expresses the antibody light chain of a murine anti-glycCTLA-4 monoclonal antibody, thereby producing a vector for expressing the chimeric antibody light chain; c) transferring the expression vector into a host cell by conventional molecular biology methods to produce a transfected host cell for expressing the chimeric antibody; and d) culturing the transfected cells by conventional cell culture techniques to produce the chimeric antibody.

[0113] An exemplary method for producing a recombinant humanized anti-glycCTLA-4 antibody can include the following: a) constructing an expression vector by conventional molecular biology methods that encodes and expresses an antibody heavy chain, wherein the minimal portion of the CDRs and variable region framework required to maintain the binding specificity of the donor antibody is derived from a non-human immunoglobulin, such as a murine anti-glycCTLA-4 monoclonal antibody, and the remainder of the antibody is derived from a human immunoglobulin, thereby producing a vector for expressing the humanized antibody heavy chain; b) constructing an expression vector by conventional molecular biology methods that encodes and expresses an antibody light chain, wherein the minimal portion of the CDRs and variable region framework required to maintain the binding specificity of the donor antibody is derived from a non-human immunoglobulin, such as a murine anti-glycCTLA-4 monoclonal antibody, and the remainder of the antibody is derived from a human immunoglobulin, thereby producing a vector for expressing the humanized antibody light chain; c) transferring the expression vector into a host cell by conventional molecular biology methods to produce a transfected host cell for expressing the humanized antibody; and d) culturing the transfected cells by conventional cell culture techniques to produce the humanized antibody.

[0114] For any exemplary method, host cells can be co-transfected with such expression vectors, which can contain different selection markers, but are preferably identical except for the heavy chain and light chain coding sequences. This program provides equal expression of heavy and light chain polypeptides. Alternatively, a single vector encoding heavy and light chain polypeptides can be used. The coding sequences of heavy and light chains can comprise cDNA or genomic DNA or both. The host cell for expressing the recombinant antibody can be a bacterial cell such as Escherichia coli, or more preferably a eukaryotic cell (e.g., Chinese hamster ovary (CHO) cell or HEK-293 cell). The selection of expression vectors depends on the selection of host cells, and can be selected to have desired expression and regulatory characteristics in the selected host cells. Other cell lines that can be used include, but are not limited to, CHO-K1, NSO and PER.C6 (Crucell, Leiden, the Netherlands). In addition, when selecting host cells to consider species-specific codon selection preferences and enhanced protein expression, codon usage can be optimized. For example, for CHO cell expression, DNA encoding the antibody can incorporate codons preferentially used by Chinese hamsters (Cricetulus griseus) (Chinese hamster ovary cells are derived from this). Codon optimization methods can be used to promote improved expression in the desired host cells (see, for example, Wohlgemuth et al., Philos. Trans. R. Soc. Lond. B Biol. Sci. 366 (1580): 2979-2986 (2011); Jestin et al., J. Mol. Evol. 69 (5): 452-457 (2009); Bollenbach et al., Genome Res. 17 (4): 401-404 (2007); Kurland et al., Prog. Nucleic Acid Res. Mol. Biol. 31: 191-219 (1984); Grosjean et al., Gene 18 (3): 199-209 (1982)).

[0115] In one embodiment, the antibody is an immunoglobulin single variable domain derived from a camelid antibody, preferably a heavy chain camelid antibody, without a light chain, which is referred to as a V H H domain sequences or Nanobodies TM Nanobody TM (Nb) is the smallest functional fragment or single variable domain (V HH), and are known to those skilled in the art. They are derived from heavy chain antibodies only seen in camelids (Hamers-Casterman et al., Nature 363: 446-448 (1993); Desmyter et al., Nat. Struct. Biol., 803-811 (1996)). In the "Camelidae" family, immunoglobulins lacking polypeptide light chains were found. "Camelidae" include Old World Camelidae (Bactrianus (Camelus bactrianus) and Dromedary (Camelus dromedarius)) and New World Camelidae (e.g., Lama paccos (Lama paccos), Lama glama (Lama glama), Lama guanicoe (Lama guanicoe) and Lamavicugna (Lama vicugna)). Single variable domain heavy chain antibodies are named Nanobody herein. TM or V H Nb antibodies. The small size and unique biophysical properties of Nb antibodies outperform conventional antibody fragments in recognizing unusual or hidden epitopes and binding to the cavity or active site of protein targets. Furthermore, Nb antibodies can be designed as multispecific and multivalent antibodies, attached to reporter molecules, or humanized. Nb antibodies are stable, tolerate the gastrointestinal system, and can be easily manufactured. Such embodiments can include a single variable domain antibody that binds glyc-CTLA-4 comprising a heavy chain comprising CDR H1, CDR H2, and CDR H3 having an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions in 1, 2, or 3 of the following CDRs: a CDR having the amino acid sequence of SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively, or a CDR having the amino acid sequence of SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:8, respectively, or a CDR having the amino acid sequence of SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:8, respectively, or a CDR having the amino acid sequence of SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively.

[0116] By unifying the antigen binding sites of two different specificities into a single construct, bispecific antibodies have the ability to bind two discrete antigens with extremely high specificity and therefore have great potential as therapeutic agents. Bispecific antibodies can initially be prepared by fusing two hybridomas, each of which is capable of producing different immunoglobulins. Bispecific antibodies can also be produced by connecting two scFv antibody fragments while omitting the Fc portion present in complete immunoglobulins. Each scFv unit in such a construct can be composed of an antibody heavy chain (V H ) and light chain (V L ) are connected to each other by a synthetic polypeptide linker that is often genetically engineered to minimize immunogenicity while maintaining maximum resistance to proteolysis. The individual scFv units can be linked by a variety of techniques, including the incorporation of a short (usually less than 10 amino acids) polypeptide spacer that bridges the two scFv units, thereby generating bispecific single-chain antibodies. The resulting bispecific single-chain antibody is thus a single polypeptide chain containing two V domains with different specificities. H / V L The substance of each scFv unit V H and V L The domains are separated by a polypeptide linker that is sufficiently long to allow intramolecular binding between the two domains, and wherein the scFv units thus formed are continuously linked to each other by a polypeptide spacer that is kept short enough to prevent unwanted associations, e.g., within the V domain of one scFv unit. H domain and another scFv unit V L The association between.

[0117] Examples of antigen-binding fragments include, but are not limited to: (i) Fab fragments, which consist of V L 、V H 、C L and C H1 domain; (ii) composed of V H and C H1 (iii) an "Fv" fragment consisting of the VL and VH domains of a single antibody; (iv) a "dAb" fragment consisting of the VH domain; (v) isolated CDR regions; (vi) a F(ab')2 fragment, a bivalent fragment comprising two linked Fab fragments; (vii) a single-chain Fv molecule ("scFv"), in which the V H domain and V LThe domains are connected by a peptide linker that allows the two domains to associate to form a binding domain; (viii) bispecific single-chain Fv dimers (U.S. Patent No. 5,091,513); and (ix) diabodies, multivalent or multispecific fragments constructed by gene fusion (U.S. Patent Application Publication No. 20050214860). Fv, scFv or diabody molecules can be stabilized by incorporating disulfide bonds connecting the VH and VL domains. Microbodies with scFv linked to the CH3 domain can also be prepared (Hu et al., Cancer Res., 56:3055-3061 (1996)).

[0118] Antibody-like binding peptide mimetics are also encompassed in the embodiments. Liu et al., Cell Mol. Biol., 49:209-216 (2003) describe "antibody-like binding peptide mimetics" (ABiPs), which are peptides that act as pared-down antibodies and have some of the advantages of longer serum half-lives and less cumbersome synthesis methods.

[0119] Glycosylated CTLA-4 peptide

[0120] In another embodiment, a composition is provided comprising a polypeptide comprising a fragment of at least 7 (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) consecutive amino acids of human CTLA-4, the fragment comprising at least one amino acid corresponding to position N113 or N145 of human CTLA-4, wherein at least one of the amino acids corresponding to position N113 or N145 of human CTLA-4 is glycosylated, wherein the polypeptide is formulated in a pharmaceutically acceptable carrier.

[0121] In certain embodiments, the present invention also provides a polypeptide of at least 7 consecutive amino acids of human CTLA-4, wherein the polypeptide has at least one amino acid corresponding to position N113 or N145 of human CTLA-4, wherein at least one of the amino acids corresponding to position N113 or N145 of human CTLA-4 is glycosylated. In certain embodiments, the polypeptide has at least 7 consecutive amino acids of human CTLA-4, wherein the amino acids have a glycosylated amino acid corresponding to position N113. In certain embodiments, the polypeptide has at least 7 consecutive amino acids of human CTLA-4, wherein the amino acids have a glycosylated amino acid corresponding to position N145.

[0122] For example, the polypeptide may be a fragment of amino acids 107-114 or 110-116 of human CTLA-4, wherein N113 is glycosylated. For another example, the polypeptide may be a fragment of amino acids 140-146 or 143-149 of human CTLA-4, wherein N145 is glycosylated. For another example, the polypeptide may be a fragment of amino acids 112-146 of human CTLA-4, wherein N113 and N145 are glycosylated.

[0123] In certain embodiments, the polypeptide comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 consecutive amino acids of human CTLA-4. In certain embodiments, the polypeptide comprises at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260 or 270, 280 consecutive amino acids of human CTLA-4. In certain embodiments, provided herein is a composition comprising at least two polypeptides provided herein. The at least two polypeptides can be separate molecules or linked into one molecule. In certain embodiments, the composition has at least 3 polypeptides, at least 4 polypeptides, or at least 5 polypeptides. In certain embodiments, the composition has 2 polypeptides, 3 polypeptides, 4 polypeptides, or 5 polypeptides.

[0124] In certain embodiments, the polypeptides provided herein include non-natural amino acids. In certain embodiments, the non-natural amino acids are methylated at the α-amino group to produce peptides with a methylated backbone. In certain embodiments, the non-natural amino acids are R-amino acids. In certain embodiments, the non-natural amino acids can include dyes (e.g., fluorescent dyes) or affinity tags. In certain embodiments, the polypeptides provided herein include chemical modifications. Chemical modifications include, for example, chemical modifications with biotin or fluorescent dyes. Those skilled in the art will recognize that methods for introducing non-natural amino acids into polypeptides and chemically modifying polypeptides are well known in the art.

[0125] In certain embodiments, a polypeptide of the embodiments is fused or conjugated to an immunogenic polypeptide (e.g., a keyhole limpet In some aspects, the polypeptide further comprises a Cys residue at the C- or N-terminus. For example, in some aspects, the polypeptide is conjugated to the immunogenic polypeptide via a disulfide bond at the Cys residue.

[0126] In another embodiment, provided herein is an immunogenic composition comprising a polypeptide comprising a fragment of at least 7 consecutive amino acids of human CTLA-4, wherein the fragment comprises at least one amino acid corresponding to position N113 or N145 of human CTLA-4, wherein at least one of the amino acids corresponding to position N113 or N145 of human CTLA-4 is glycosylated, wherein the polypeptide is formulated in a pharmaceutically acceptable carrier. In certain aspects, the immunogenic composition further comprises an adjuvant, such as alum or Freund's adjuvant.

[0127] In certain embodiments, a method for preparing an antibody is provided, comprising administering a polypeptide to an animal and isolating the antibody from the animal, wherein the polypeptide comprises a fragment of at least 7 consecutive amino acids of human CTLA-4, the fragment comprising at least one amino acid corresponding to position N113 or N145 of human CTLA-4, and wherein at least one of the amino acids corresponding to positions N113 and N145 of human CTLA-4 is glycosylated. The animal can be a mouse, rat, rabbit, or human. In certain aspects, a method further comprises identifying CDRs of the antibody and humanizing the sequences surrounding the CDRs to produce a humanized antibody. In other aspects, the method comprises recombinantly expressing the humanized antibody. Thus, in another embodiment, an isolated antibody produced by the aforementioned method is provided. Thus, in certain embodiments, provided herein is an isolated antibody that selectively binds a polypeptide of the embodiments (e.g., a polypeptide comprising a fragment of at least 7 contiguous amino acids of human CTLA-4, said fragment comprising at least one amino acid corresponding to position N113 or N145 of human CTLA-4, wherein at least one of the amino acids corresponding to position N113 or N145 of human CTLA-4 is glycosylated) relative to unglycosylated CTLA-4.

[0128] The polypeptides provided herein can be prepared by any method known in the art. For example, polypeptides can be prepared by chemical synthesis or recombinant production. Exemplary methods for expressing and purifying recombinant polypeptides can be found in, for example, Scopes R.K., Protein Purification-Principles and Practice, Springer Advanced Texts in Chemistry, 3rd edition (1994); Simpson RJ et al., Basic Methods in Protein Purification and Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1st edition (2008); Green MR and Sambrook J., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 4th edition (2012); Jensen KJ et al., Peptide Synthesis and Applications (Methods in Molecular Biology), Humana Press, 2nd edition (2013). Chemical synthesis of polypeptides can be accomplished using methods well known in the art (see Kelley and Winkler, 1990, see: GeneticEngineering Principles and Methods , Setlow JK, ed., Plenum Press, NY, Vol. 12, pp. 1-19; Stewart et al., 1984, JM Young, JD, Solid Phase Peptide Synthesis , Pierce Chemical Co., Rockford, Ill; Marglin and Merrifield, Ann. Rev. Biochem, 39: 841-866, 862 (1970). Merrifield, RB, 1963, J. Am. Chern. Soc. 85: 2149-2154; Chemical Approaches to the Synthesis of Peptides and Proteins ,Williams et al., eds., 1997, CRC Press, Boca Raton Fla.; Solid Phase Peptide Synthesis: A Practical Approach , Atherton and Sheppard, eds., 1989, IRL Press, Oxford, England; see also USPNs. 4,105,603; 3,972,859; 3,842,067; and 3,862,925).

[0129] Modifications and derivatives

[0130] Antibodies against glycosylated CTLA-4 can have the ability to neutralize or counteract the effects of glycosylated CTLA-4, regardless of the animal species, monoclonal cell line, or other source of the antibody. Certain animal species may be less preferred for generating therapeutic antibodies because they may be more likely to cause an allergic response due to activation of the complement system by the Fc portion of the antibody. However, intact antibodies can be enzymatically digested into Fc (complement binding) fragments, as well as antibody fragments having binding domains or CDRs. Removal of the Fc portion reduces the likelihood that the antibody fragment will elicit an undesirable immunological response, and thus, antibodies without the Fc portion can be used for prophylactic or therapeutic treatments. As described above, antibodies can also be constructed to be chimeric, partially, or fully human to reduce or eliminate adverse immunological consequences of administering antibodies to animals that have been produced in other species or have sequences from other species.

[0131] The binding properties of anti-glycated CTLA-4 antibodies can be further improved by screening for variants that exhibit the desired properties. For example, such improvements can be achieved using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequences encoding them. In a specific embodiment, such phage can be used to display antigen-binding fragments expressed from a library or combinatorial antibody library (e.g., human or murine), such as Fab and Fv or disulfide-stabilized Fv. Phage expressing antigen-binding fragments that bind to the antigen of interest can be selected or identified using antigen, for example, using labeled antigen or antigen bound or captured to a solid surface or beads. Phage used in these methods are typically filamentous phage, including fd and M13. Antigen-binding fragments are expressed as recombinant fusion proteins fused to the phage gene III or gene VIII protein. Examples of phage display methods that can be used to prepare antibodies or polypeptides as described herein include those disclosed in Brinkman et al., J Immunol Methods, 182:41-50 (1995); Ames et al., J. Immunol. Methods, 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol., 24:952-958 (1994); Persic et al., Gene, 187:9-18 (1997); Burton et al., Adv. Immunol. 57:191-280 (1994); PCT Publications WO 92 / 001047, WO 90 / 02809, WO 91 / 10737, WO 92 / 01047, WO 92 / 18619, WO 93 / 11236, WO 95 / 15982, WO 95 / 20401; and U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108; all of which are hereby incorporated by reference in their entirety.

[0132] As described in the above references, after phage selection, the antibody coding regions can be isolated from the phage and used to produce complete antibodies, including humanized antibodies, or any other desired fragments, and expressed in any desired host (including mammalian cells, insect cells, plant cells, yeast and bacteria), for example, as described in detail below. For example, techniques for recombinant production of Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art, such as those disclosed in PCT Publication WO 92 / 22324; Mullinax, RL et al., BioTechniques, 12(6):864-869 (1992); and Sawai et al., Am. J. Reprod. Immunol. 34:26-34 (1995); and Better, M. et al. Science 240:1041-1043 (1988); all of which are hereby incorporated by reference in their entirety. Examples of techniques that can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston, JS et al., Methods in Enzymology 203:46-88 (1991); Shu, L. et al., Proc. Natl. Acad. Sci. (USA) 90:7995-7999; and Skerra. A. et al., Science 240:1038-1040 (1988); all of which are hereby incorporated by reference in their entirety.

[0133] As described herein, phage display technology can be used to increase the affinity of anti-glycCTLA-4 antibodies. This technology can be used to obtain high-affinity antibodies that can be used in the combination methods described herein. This technique, known as affinity maturation, employs mutagenesis or CDR walking and reselection, wherein such a receptor or ligand (or its extracellular domain) or antigenic fragment thereof is used to identify antibodies that bind to the antigen with higher affinity than the original or parent antibody (see, e.g., Glaser, SM et al., J. Immunol. 149: 3903-3913 (1992)). Mutagenesis of entire codons rather than individual nucleotides results in a semi-random library of amino acid mutations. A library can be constructed from a set of variant clones, each differing by a single amino acid change in a single CDR, and containing variants representing every possible amino acid substitution for each CDR residue. By contacting the immobilized mutants with a labeled antigen, mutants with increased binding affinity for the antigen can be screened. Any screening method known in the art can be used to identify mutant antibodies with increased avidity for the antigen (e.g., ELISA) (see, e.g., Wu, H. et al., Proc. Natl. Acad. Sci. (USA) 95(11):6037-6042 (1998); Yelton, DE et al., J. Immunol. 155:1994-2004 (1995). CDR walking of randomized light chains can also be used (see Schier et al., J. Mol. Biol. 263:551-567 (1996)).

[0134] Random mutagenesis can be used together with phage display method, to identify improved CDR and / or variable region.Phage display technology can alternatively be used to increase (or reduce) CDR affinity by directed mutagenesis (e.g., affinity maturation or "CDR walking").This technology uses target antigen or its antigen fragment to identify such antibody: it has a CDR (see, e.g., Glaser, SM et al., J.Immunol.149:3903-3913 (1992)) with higher (or lower) affinity binding antigen compared with initial antibody or parent antibody.

[0135] Methods for achieving such affinity maturation are described, for example, in: Krause, JC et al., MBio. 2(1) pii:e00345-10. doi: 10.1128 / mBio.00345-10 (2011); Kuan, CT et al., Int. J. Cancer 10.1002 / ijc.25645; Hackel, BJ et al., J. Mol. Biol. 401(1):84-96 (2010); Montgomery, DL et al., MAbs 1(5):462-474 (2009); Gustchina, E. et al., Virology 393(1):112-119 (2009); Finlay, WJ et al., J. Mol. Biol. 388(3):541-558 (2009); Bostrom, J. et al., Methods Mol. Biol. 525:353-376 (2009); Steidl, S. et al., Mol. Immunol. 46(1):135-144 (2008); and Barderas, R. et al., Proc. Natl. Acad. Sci. (USA) 105(26):9029-9034 (2008); all of which are hereby incorporated by reference in their entirety.

[0136] Also provided herein are derivatives of anti-glycCTLA-4 antibodies or glycosylated CTLA-4 polypeptides having 1, 2, 3, 4, 5 or more amino acid substitutions, additions, deletions or modifications relative to the "parent" (or wild-type) molecule. Such amino acid substitutions or additions can introduce naturally occurring (i.e., DNA-encoded) or non-naturally occurring amino acid residues. Such amino acids can be glycosylated (e.g., with altered levels of mannose, 2-N-acetylglucosamine, galactose, fucose, glucose, sialic acid, 5-N-acetylneuraminic acid, 5-hydroxyacetylneuraminic acid, etc.), acetylated, pegylated, phosphorylated, amidated, derivatized with known protecting / blocking groups, proteolytically cleaved, linked to cellular ligands or other proteins, etc. In certain embodiments, the altered carbohydrate modifications modulate one or more of the following: antibody solubility, promotion of antibody subcellular trafficking and secretion, promotion of antibody assembly, conformational integrity, and antibody-mediated effector functions. In certain embodiments, the altered carbohydrate modifications enhance antibody-mediated effector function relative to an antibody lacking the carbohydrate modifications. Carbohydrate modifications that result in altered antibody-mediated effector function are well known in the art (e.g., see Shields, RL et al., J. Biol. Chem. 277(30):26733-26740 (2002); Davies J. et al. Biotechnology & Bioengineering 74(4):288-294 (2001); both of which are hereby incorporated by reference in their entirety). Methods for altering carbohydrate content are known to those skilled in the art, see, for example, Wallick, SC et al., J. Exp. Med. 168(3):1099-1109 (1988); Tao, MH et al., J. Immunol. 143(8):2595-2601 (1989); Routledge, EG et al., Transplantation 60(8):847-53 (1995); Elliott, S. et al., Nature Biotechnol. 21:414-21 (2003); Shields, RL et al., J. Biol. Chem. 277(30):26733-26740 (2002); all of which are hereby incorporated by reference in their entirety.

[0137] Substitution variants can contain an exchange of one amino acid with another amino acid at one or more sites within the antibody or polypeptide provided herein, and can be designed to modulate one or more properties of the antibody or polypeptide, with or without loss of other functions or performance. The substitution can be conservative, i.e., an amino acid is replaced by an amino acid with a similar shape and charge. Conservative substitutions are well known in the art and include, for example, the following changes: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamic acid to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Alternatively, the substitution may be non-conservative, thereby affecting the function or activity of the polypeptide.Non-conservative changes typically involve replacing one residue with a chemically different residue, such as replacing a non-polar or uncharged amino acid with a polar or charged amino acid, or vice versa.

[0138] In certain embodiments, the antibody may comprise a first set of CDRs as listed in Table 4, but may have substitutions (e.g., conservative substitutions) at residues that are not conserved in another or all other CDR groups. For example, the antibody may comprise CDRs from AbM, Kabat, or Contact groups, but may have one or more substitutions at residues that do not correspond to those in a Chothia-type CDR2 sequence (SEQ ID NO: 7) within a CDR2 sequence (SEQ ID NO: 10, 12, or 14). Similarly, the trailing residues on SEQ ID NO: 18 may be substituted, e.g., with conservative substitutions. Similarly, residues 1-4 on SEQ ID NO: 20 may be substituted, e.g., with conservative substitutions. These are just a few examples, but one of ordinary skill will appreciate which substitutions may be made based on what is shown in Table 4.

[0139] In certain embodiments, an antibody having a first set of CDRs (e.g., Chothia, AbM, Kabat, and Contact) may comprise a framework region having one or more amino acid substitutions such that the one or more substituted residues are identical at the corresponding position to a second set of CDRs (e.g., by sequence alignment and / or according to Kabat numbering), the second set of CDRs having a leading (i.e., N-terminal) or trailing (i.e., C-terminal) residue that is not present in the first set of CDRs. For example, the framework region adjacent to the C-terminal end of the Contact-type CDR2 of the heavy chain has substituted residues corresponding to one or more of trailing residues 12-18 of the amino acid sequence of SEQ ID NO: 12 (Kabat-type CDR2). Similarly, in certain embodiments, the framework region adjacent to the C-terminal end of the AbM-type CDR2 of the heavy chain has substituted residues corresponding to one or more of trailing residues 11-18 of the amino acid sequence of SEQ ID NO: 12 (Kabat-type CDR2). In certain embodiments, the framework region adjacent to the C-terminal end of the Chothia-type CDR2 of the heavy chain has substituted residues corresponding to one or more of the trailing residues 8-18 of the amino acid sequence of SEQ ID NO: 12 (Kabat-type CDR2). In certain embodiments, the framework region adjacent to the C-terminal end of the Chothia-, AbM-, or Kabat-type CDR1 of the light chain has substituted residues corresponding to one or more of the trailing residues 5-6 of the amino acid sequence of SEQ ID NO: 19 (Contact-type CDR1). In certain embodiments, the framework region adjacent to the N-terminal end of the Kabat- or AbM-type CDR2 of the heavy chain has substituted residues corresponding to one or more of the leading residues 1-3 of the amino acid sequence of SEQ ID NO: 14 (Contact-type CDR2). In certain embodiments, the framework region adjacent to the N-terminal end of the Chothia-type CDR2 of the heavy chain has substituted residues corresponding to one or more of the leading residues 1-5 of the amino acid sequence of SEQ ID NO: 14. In certain embodiments, the framework region adjacent to the N-terminal end of the Chothia-, AbM-, or Kabat-type CDR3 of the heavy chain has substituted residues corresponding to one or more of the lead residues 1-2 of the amino acid sequence of SEQ ID NO: 15 (Contact-type CDR3). In certain embodiments, the framework region adjacent to the N-terminal end of the Chothia-, AbM-, or Kabat-type CDR2 of the light chain has substituted residues corresponding to one or more of the lead residues 1-4 of the amino acid sequence of SEQ ID NO: 20 (Contact-type CDR2).

[0140] In certain embodiments, humanized antibodies are derivative antibodies. Such humanized antibodies are included in the amino acid residue replacement, deletion or addition in one or more non-human CDRs. Compared with non-derivative humanized antibodies, humanized antibody derivatives can have substantially the same combination, better combination or poorer combination. In certain embodiments, one, two, three, four or five amino acid residues of CDR have been mutated, such as replaced, deleted or added.

[0141] In certain embodiments, the polypeptide is a derivatized polypeptide. Such polypeptides include amino acid residue substitutions, deletions, or additions compared to wild-type human CTLA-4. Derivatized polypeptides can have substantially the same binding, better binding, or worse binding to anti-glycCTLA-4 antibodies compared to non-derivatized polypeptides. In certain embodiments, one, two, three, four, or five amino acid residues of human CTLA-4 have been mutated, such as by substitution, deletion, or addition.

[0142] Antibodies or polypeptides as described herein can be modified by chemical modification using techniques known to those skilled in the art, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, and the like. In one embodiment, the derivative polypeptide or derivative antibody has a function similar to or identical to that of the parent polypeptide or antibody. In another embodiment, the derivative polypeptide or derivative antibody exhibits an altered activity relative to the parent polypeptide or parent antibody. For example, the derivative antibody (or fragment thereof) may bind more tightly to its epitope or be more resistant to proteolysis than the parent antibody.

[0143] The substitution, addition or deletion in the derivatized antibody can be in the Fc region of the antibody and can thereby be used to change the binding affinity of the antibody to one or more FcγRs. Methods for modifying antibodies (which have altered binding to one or more FcγRs) are known in the art, see, for example, PCT Publication Nos. WO 04 / 029207, WO 04 / 029092, WO04 / 028564, WO 99 / 58572, WO 99 / 51642, WO 98 / 23289, WO 89 / 07142, WO 88 / 07089 and U.S. Patent Nos. 5,843,597 and 5,642,821; they are all hereby incorporated by reference in their entirety. In certain embodiments, the antibody or other molecule may have altered affinity for activating FcγRs (e.g., FcγRIIIA). Preferably, such modifications also have altered Fc-mediated effector functions. Modifications that affect Fc-mediated effector functions are well known in the art (see U.S. Pat. No. 6,194,551 and WO 00 / 42072). In certain embodiments, modifications of the Fc region result in antibodies with altered antibody-mediated effector functions, altered binding to other Fc receptors (e.g., Fc activating receptors), altered antibody-dependent cell-mediated cytotoxicity (ADCC) activity, altered C1q binding activity, altered complement-dependent cytotoxicity activity (CDC), phagocytic activity, or any combination thereof.

[0144] Derived antibodies or polypeptides can also have a changed half-life (e.g., serum half-life) of the parent molecule or antibody in a mammal, preferably a human. In certain embodiments, such changes result in a half-life of greater than 15 days, preferably greater than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months. The increased half-life of the humanized antibody or polypeptide in a mammal, preferably a human, results in a higher serum titer of the antibody or polypeptide in a mammal, and thus reduces the frequency of administration of the antibody or polypeptide and / or reduces the concentration of the antibody or polypeptide to be administered. By techniques known to those skilled in the art, antibodies or polypeptides with increased in vivo half-life can be produced. For example, by modifying (e.g., replacing, deleting, or adding) amino acid residues identified as participating in the interaction between the Fc domain and the FcRn receptor, antibodies or polypeptides with increased in vivo half-life can be produced. Humanized antibodies as described herein can be engineered to increase biological half-life (see, e.g., U.S. Patent No. 6,277,375).For example, humanized antibodies as described herein can be engineered in the Fc-hinge domain to have increased in vivo or serum half-life.

[0145] By connecting polymer molecules such as high molecular weight polyethylene glycol (PEG) to the antibody or polypeptide, antibodies or polypeptides with increased in vivo half-life as described herein can be produced. PEG can be connected to the antibody or polypeptide with or without a multifunctional linker, whether by site-specific conjugation of PEG to the N- or C-terminal of the molecule or antibody, or by the ε-amino group present on a lysine residue. Straight or branched polymer derivatizations causing minimal bioactivity loss can be used. The degree of conjugation can be closely monitored by SDS-PAGE and mass spectrometry to ensure the appropriate conjugation of the PEG molecule to the antibody. Unreacted PEG can be separated from the antibody-PEG conjugate by, for example, size exclusion or ion exchange chromatography.

[0146] Antibodies or polypeptides as described herein can also be modified by the methods and coupling agents described by Davis et al. (see U.S. Patent No. 4,179,337) to provide compositions that can be injected into the circulatory system of mammals without substantially any immunogenic response. Removal of the Fc portion can reduce the likelihood that the antibody fragment will elicit an undesirable immunological response, and therefore, antibodies without Fc can be used for prophylactic or therapeutic treatments. As described above, antibodies can also be constructed to be chimeric, partially or fully human, thereby reducing or eliminating the adverse immunological consequences of administering antibodies to animals that have been produced in other species or have sequences from other species.

[0147] Fusions and conjugates

[0148] The anti-glycCTLA-4 antibodies or glycosylated CTLA-4 polypeptides provided herein also can be expressed as a fusion protein with other proteins or chemically conjugated to another moiety.

[0149] In certain embodiments, provided herein are antibodies or polypeptides having an Fc portion, wherein the Fc portion may differ by isotype or subclass, may be chimeric or hybrid, and / or may be modified, for example, to improve effector function, half-life control, tissue accessibility, enhance biophysical characteristics (such as stability), and improve production efficiency (and at a lower cost). Many modifications that can be used to construct disclosed fusion proteins and methods for preparing them are known in the art, see, for example, Mueller, JP et al., Mol. Immun. 34(6): 441-452 (1997), Swann, PG, Curr. Opin. Immun. 20: 493-499 (2008), and Presta, LG, Curr. Opin. Immun. 20: 460-470 (2008). In certain embodiments, the Fc region is a native IgG1, IgG2, or IgG4 Fc region. In certain embodiments, the Fc region is a hybrid, for example, a chimera with an IgG2 / IgG4 Fc constant region. Modifications to the Fc region include, but are not limited to, modifications to IgG4 to prevent binding to Fcγ receptors and complement, modifications to IgG1 to improve binding to one or more Fcγ receptors, modifications to IgG1 to minimize effector function (amino acid changes), IgG1 with altered glycans / no glycans (usually by changing the expression host), and IgG1 with altered pH-dependent binding to FcRn. The Fc region may include the entire hinge region, or less than the entire hinge region.

[0150] Another embodiment includes IgG2-4 hybrids and IgG4 mutants that have reduced binding to FcRs, thereby increasing their half-life. Representative IgG2-4 hybrids and IgG4 mutants are described in Angal et al., Molec. Immunol. 30(1):105-108 (1993); Mueller et al., Mol. Immun. 34(6):441-452 (1997); and U.S. Patent No. 6,982,323; all of which are hereby incorporated by reference in their entirety. In certain embodiments, the IgG1 and / or IgG2 domains are deleted, for example, Angal et al. describe IgG1 and IgG2 in which serine 241 is replaced by proline.

[0151] In certain embodiments, provided herein are fusion proteins or polypeptides having at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acids.

[0152] In certain embodiments, provided herein are anti-glycated CTLA-4 antibodies or glycosylated CTLA-4 polypeptides linked to, covalently bound to, or complexed with at least one moiety. Such moieties can be, but are not limited to, moieties that increase the efficacy of the molecule as a diagnostic or therapeutic agent. In certain embodiments, the moiety can be an imaging agent, a toxin, a therapeutic enzyme, an antibiotic, a radiolabeled nucleotide, or the like.

[0153] In certain embodiments, the moiety can be an enzyme, a hormone, a cell surface receptor, a toxin (such as abrin, ricin A, Pseudomonas exotoxin (i.e., PE-40), diphtheria toxin, ricin, gelonin, or pokeweed antiviral protein), a protein (such as tumor necrosis factor, an interferon (e.g., alpha-interferon, beta-interferon), a nerve growth factor, a platelet-derived growth factor, a tissue-type plasminogen activator, or an apoptotic agent (e.g., tumor necrosis factor-alpha, tumor necrosis factor-beta)), a biological response modifier (such as, for example, a lymphokine (e.g., interleukin-1 ("IL-1"), interleukin-2 ("IL-3"), or a combination thereof). -2", interleukin-6 ("IL-6")), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF") or macrophage colony stimulating factor, ("M-CSF")) or growth factors (e.g., growth hormone ("GH"))), cytotoxins (e.g., cytostatic or cytocidal agents such as paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione (dihydroxy anthracin dione), mitoxantrone, plicamycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE; e.g., vedotin), and puromycin and its analogs or homologs), antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., dichloromethane, thioepa chlorambucil, melphalan, (carmustine; BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, plicamycin, and anthramycin (AMC)), or antimitotic agents (e.g., vincristine and vinblastine).

[0154] Techniques for conjugating such therapeutic moieties to antibodies are well known; see, e.g., Amon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,” in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Reisfeld et al. (eds.), 1985, pp. 243-56, Alan R. Liss, Inc.); Hellstrom et al., “Antibodies For Drug Delivery,” in CONTROLLED DRUG DELIVERY (2nd ed.), Robinson et al. (eds.), 1987, pp. 623-53, Marcel Dekker, Inc.); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in MONOCLONAL ANTIBODIES '84: BIOLOGICAL AND CLINICAL APPLICATIONS, Pinchera et al. (eds.), 1985, pp. 475-506); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibodies In Cancer Therapy", in MONOCLONALANTIBODIES FOR CANCER DETECTION AND THERAPY, Baldwin et al. (eds.), 1985, pp. 303-16, Academic Press; Thorpe et al., Immunol. Rev. 62: 119-158 (1982); Carter et al., Cancer J. 14(3): 154-169 (2008); Alley et al., Curr. Opin. Chem. Biol. 14(4): 529-537 (2010); Carter et al., Amer. Assoc. Cancer Res. Educ. Book. 2005(1):147-154(2005); Carter et al., Cancer J. 14(3):154-169(2008); Chari, Acc. Chem Res. 41(1):98-107(2008); Doronina et al., Nat. Biotechnol.21(7):778-784 (2003); Ducry et al., Bioconjug Chem. 21(1):5-13 (2010); Senter, Curr. Opin. Chem. Biol. 13(3):235-244 (2009); and Teicher, Curr Cancer Drug Targets. 9(8):982-1004 (2009). Auristatin E) (MMAE), for example, vedotin; or a combination thereof.

[0155] In a preferred embodiment, the antibody is conjugated to maytansine, a benzobridged macrolide originally isolated from the bark of the Ethiopian shrub Maytenus ovatus. This cytotoxic agent and its derivatives (e.g., pseudomaytansine) bind to tubulin near the vinca alkaloid binding site. They are thought to have a high affinity for tubulin located at the ends of microtubules, while having a lower affinity for sites distributed throughout the microtubules. Inhibition of microtubule dynamics causes cells to arrest in the G2 / M phase of the cell cycle, ultimately leading to cell death by apoptosis (Oroudjev et al., Mol. Cancer Ther., 10L2700-2713 (2010)). Two maytansine derivatives (thiol-containing pseudomaytansines) include DM1 and DM4 (ImmunoGen, Inc., Waltham, MA), which have been widely used in combination with irreversible and reversible linkers. Specifically, DM1 linked to an antibody with a thioether linker is called "emtansine"; DM1 linked to an antibody with an SPP linker is called "mertansine." DM4 linked with an SPDB linker is called "ravtansine"; and DM4 linked with an sSPDB linker is called "soravtansine" (ImmunoGen, Inc., Waltham, MA). In one embodiment, the anti-glyc CTLA-4 antibody-ADC comprises a mertansine-mimetic payload DM1 that acts on tubulin. In one embodiment, the anti-glyc CTLA-4 antibody-ADC comprises a mertansine-mimetic payload DM4 that acts on tubulin. In one embodiment, the anti-glyc CTLA-4 antibody-ADC comprises a payload that acts on DNA, e.g., DGN462 (ImmunoGen, Inc., Waltham, MA). In one embodiment, the anti-glycCTLA-4 antibody component of the anti-glycCTLA-4 antibody-ADC is a chimeric or humanized form of STC1807, or a binding portion thereof. In one embodiment, the anti-glycCTLA-4 antibody component of the anti-glycCTLA-4 antibody-ADC is a chimeric or humanized form of STC1807, or a binding portion thereof.

[0156] In a specific embodiment, the cytotoxic agent conjugated to the anti-glycCTLA-4 antibody is MMAE (monomethyl auristatin E (or demethyl-auristatin E)), a highly toxic anti-tumor agent whose antimitotic activity involves inhibiting cell division by blocking the polymerization of tubulin. Vedotin (an international nonproprietary name) refers to the structure of MMAE in the MMAE-antibody conjugate and its connection to the antibody. In more specific embodiments, the ADC is STC1807 (chimeric or humanized form)-MMAE or STC1807 (chimeric or humanized form)-MMAE.

[0157] Many chemical linkers are known and used to conjugate cytotoxic or DNA-acting drug payloads to antibodies to generate ADCs. Certain linkers (used alone or in combination) that have been included for the production of ADCs comprising anti-glycCTLA-4 antibodies (particularly those that are internalized upon binding to their targets as described herein) include SMCC (4-(N-maleimidomethyl)cyclohexanecarboxylic acid N-hydroxysuccinimide ester); SPDB (N-succinimidyl 3-(2-pyridyldithio)butyrate); SPP (N-succinimidyl 4-(2-pyridyldithio)pentanoate); sulfo-SPDB or sSPDB (N-succinimidyl-4-(2-pyridyldithio)-2-sulfobutyrate); the thioether linker succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (MCC); and vc (valine-citrulline dipeptide linker). As an example, engineered linkers (e.g., SMCC, SPDB, S-SPDB) (Immunogen, Inc.) have been designed to stabilize the ADC before it binds to the tumor and then optimize payload efficacy after the ADC is internalized into the cancer cell. Other linkers, such as the dipeptide vc linker (which is a cathepsin-cleavable linker), can be used to conjugate antibodies to cytotoxic agents, such as auristatin, a mitotic inhibitor derived from dolastatin 10, e.g., monomethyl auristatin E (MMAE), e.g., vitin. Cytotoxins can be conjugated to antibodies such that more than one toxin molecule is attached to each antibody molecule, e.g., on average, 2, 3, 4, 5, 6, 7, or 8 toxin molecules per antibody.

[0158] In one specific embodiment, MMAE is indirectly linked to the antibody cysteine ​​via a maleimidocaproyl (MC) linker, which is coupled to valine-citrulline-p-aminobenzyloxycarbonyl-MMAE (MC-vc-PAB-MMAE). In the linear structure of "MC-vc-PAB-MMAE," "MC" consists of maleimide and caproic acid and is the portion that is linked to the antibody, typically through a cysteine ​​group on the H chain. In turn, "MC" is linked to a "vc" linker consisting of valine (Val) and citrulline (Cit), which is a cathepsin-cleavable linker that is cleaved by cathepsins within tumors or cancer cells. "vc" is linked to a spacer, p-aminobenzoic acid, to which the MMAE cytotoxin is attached. MC-vc-PAB-MMAE ADC releases free, membrane-permeable MMAE upon cleavage by a protease, such as cathepsin B. In one embodiment, the linker to the antibody is stable in extracellular fluids but is cleaved by cathepsins once the ADC enters the tumor or cancer cell, thereby activating the anti-mitotic mechanism of MMAE or other toxin drugs. In another embodiment, monomethyl auristatin F (MMAF) is linked to the antibody cysteine ​​via a maleimidocaproyl group (MC-MMAF). Compared to the MC-vc-PAB-MMAE ADC, the MC-MMAF ADC, like the MCC-DM1 ADC, is not cleavable and must be internalized and degraded intracellularly, releasing the cysteine-MC-MMAF as the active drug within the cell.

[0159] In one embodiment, the cytotoxic payload is released in the lysosome after the ADC is internalized into the cell. In the lysosome, lysosomal enzymes digest the antibody component of the ADC. After lysosomal degradation, the drug (and drug-linker) payload is released into the cytoplasm, where the drug binds to the intracellular target, ultimately leading to cell death. Optimally, the released payload is fully active while the linker is still connected. In other embodiments where the target bound to the ADC results in poor transport to the lysosome, a linker that is stable outside the target cell but cleaves the payload from the antibody component once it enters the cell provides an alternative mode of releasing the payload within the cell but outside the lysosome. In other embodiments, the linker is stable in the extracellular fluid, but is cleaved by cathepsins once the ADC enters the tumor or cancer cell, thereby activating the anti-mitotic or other cytotoxic mechanisms of the toxin drug. In other embodiments, the payload released by the action of the cleavable linker can enter adjacent cancer cells and kill them through the bystander effect, thereby enhancing the targeting and tumor killing activity of the ADC.

[0160] In certain embodiments, the antibodies and polypeptides described herein can be conjugated to a marker, such as a peptide, to facilitate purification. In certain embodiments, the marker is: a hexa-histidine peptide; a hemagglutinin "HA" tag (SEQ ID NO: 22: YPYDVPDYA), which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, IA et al., Cell, 37: 767-778 (1984)); or a "flag" tag (Knappik, A. et al., Biotechniques 17 (4): 754-761 (1994)).

[0161] In certain embodiments, the moiety can be an imaging agent detectable in an assay. Such an imaging agent can be an enzyme, a prosthetic group, a radiolabel, a non-radioactive paramagnetic metal ion, a hapten, a fluorescent label, a phosphorescent molecule, a chemiluminescent molecule, a chromophore, a luminescent molecule, a bioluminescent molecule, a photoaffinity molecule, a colored particle or a ligand, such as biotin.

[0162] In certain embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; the prosthetic group complex includes, but is not limited to, streptavidin / biotin and avidin / biotin; the fluorescent material includes, but is not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; the luminescent material is, but is not limited to, luminol; the bioluminescent material includes, but is not limited to, luciferase, luciferin, and aequorin; the radioactive material includes, but is not limited to, bismuth ( 213 Bi), carbon ( 14 C), chromium ( 51 Cr), cobalt ( 57 Co), fluorine ( 18 F), gadolinium ( 153 Gd, 159 Gd), gallium ( 68 Ga, 67 Ga), germanium ( 68 Ge), holmium ( 166 Ho), indium ( 115 In, 113 In, 112 In, 111 In), iodine ( 131 I. 125 I. 123 I. 121 I), lanthanum ( 140 La), Lutetium ( 177 Lu), manganese ( 54 Mn), molybdenum ( 99 Mo), palladium ( 103Pd), phosphorus ( 32 P), praseodymium ( 142 Pr), promethium ( 149 Pm), rhenium ( 186 Re、 188 Re), rhodium ( 105 Rh), ruthenium ( 97 Ru), Samarium ( 153 Sm), Scandium ( 47 Sc), selenium ( 75 Se), strontium ( 85 Sr), sulfur ( 35 S), technetium ( 99 Tc), thallium ( 201 Ti), tin ( 113 Sn, 117 Sn), tritium ( 3 H), xenon ( 133 Xe), Ytterbium ( 169 Yb, 175 Yb), yttrium ( 90 Y), zinc ( 65 Zn); positron-emitting metals and non-radioactive paramagnetic metal ions using various positron emission tomography techniques.

[0163] Imaging agents can be conjugated directly or indirectly to antibodies or polypeptides provided herein through intermediates (e.g., linkers known in the art) using techniques known in the art. Regarding metal ions that can be conjugated to antibodies and other molecules described herein for use as diagnostic agents, see, for example, U.S. Patent No. 4,741,900. Some conjugation methods involve the use of metal chelate complexes, using, for example, organic chelating agents such as diethylenetriaminepentaacetic anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-toluenesulfonamide; and / or tetrachloro-3-6α-diphenyl glycoluril-3 connected to the antibody. Monoclonal antibodies can also react with enzymes in the presence of coupling agents such as glutaraldehyde or periodate. In the presence of these coupling agents or by reacting with isothiocyanates, conjugates with fluorescein markers can be prepared.

[0164] In certain embodiments, an antibody or polypeptide as described herein can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980. Such a heteroconjugate antibody can additionally bind a hapten (e.g., fluorescein) or a cell marker (e.g., 4-1-BB, B7-H4, CD4, CD8, CD14, CD25, CD27, CD40, CD68, CD163, CTLA4, GITR, LAG-3, OX40, TIM3, TIM4, TLR2, LIGHT, ICOS, B7-H3, B7-H7, B7-H7CR, CD70, CD47) or a cytokine (e.g., IL-7, IL-15, IL-12, IL-4 TGF-β, IL-10, IL-17, IFNγ, Flt3, BLys) or a chemokine (e.g., CCL21).

[0165] In certain embodiments, the anti-glycated CTLA-4 antibodies or glycosylated CTLA-4 polypeptides described herein can also be attached to a solid support, which can be used for immunoassays or purification of target antigens or other molecules that can bind to target antigens that have been immobilized on the support (via binding to the antibodies or antigen-binding fragments described herein). Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0166] Protein purification

[0167] Protein purification techniques are well known to those skilled in the art. These techniques relate to homogenization of cells, tissues or organs and crude separation into polypeptide and non-polypeptide fractions on one level. Unless otherwise noted, chromatography and electrophoresis techniques can be used to further purify the protein or polypeptide of interest to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suitable for preparing pure peptides are ion exchange chromatography, size exclusion chromatography, reversed-phase chromatography, hydroxyapatite chromatography, polyacrylamide gel electrophoresis, affinity chromatography, immunoaffinity chromatography and isoelectric focusing. A particularly effective method for purifying peptides is fast liquid chromatography (FPLC) or even high performance liquid chromatography (HPLC). As known in the art, it is believed that the order of carrying out various purification steps can be changed, or some steps can be omitted, and still produce a suitable method for preparing a substantially purified polypeptide.

[0168] Purified polypeptide is intended to represent a composition that can be separated from other components, wherein the polypeptide is purified to any degree relative to its naturally obtainable state. Therefore, isolated or purified polypeptide also represents a polypeptide that is separated from its possible naturally occurring environment. Usually, "purified" represents a polypeptide composition that has been subjected to fractionation to remove various other components, and said composition has retained the biological activity of its expression substantially. When using the term "substantially purified", the name will represent a composition in which the polypeptide forms the main component of the composition, for example, accounting for about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the protein in the composition.

[0169] In view of the present disclosure, various methods for quantifying the degree of polypeptide purification are known to those skilled in the art. These include, for example, determining the specific activity of the active fraction, or analyzing and assessing the amount of polypeptide in the fraction by SDS / PAGE. A preferred method for assessing fraction purity is to calculate the specific activity of the fraction, compare it with the specific activity of the initial extract, and thereby calculate the purity thereof, which is assessed by the "purification factor." Of course, the actual unit used to express the amount of activity will depend on the specific assay technique selected for use after purification, and whether the expressed polypeptide exhibits detectable activity.

[0170] It is not usually required that polypeptide is always provided in its most purified state. In fact, it is considered that the product with a lower degree of basic purification may have practicality in certain embodiments. By combining still fewer purification steps, or by using different forms of the same general purification scheme, partial purification can be completed. For example, it should be understood that the cation exchange column chromatography method used by HPLC equipment usually results in a larger "multiple" purification than the same technology using a low-pressure chromatography system. The method showing a lower relative degree of purification can have an advantage in the total recovery of the protein product or in the activity of the protein maintained to be expressed.

[0171] Affinity chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be separated and a molecule to which it can specifically bind. This is a receptor-ligand type of interaction. Column materials are synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb substances from a solution. Elution occurs by changing the conditions to conditions where binding does not occur (e.g., pH, ionic strength, temperature, etc., which are changed). The matrix should be a substance that does not adsorb molecules to any significant degree and has a wide range of chemical, physical, and thermal stability. The ligand should be coupled in a manner that does not affect its binding characteristics. The ligand should also provide relatively tight binding. It should be possible to elute the substance without destroying the sample or the ligand.

[0172] Size exclusion chromatography (SEC) is a chromatographic method in which molecules in a solution are separated based on their size, or in more technical terms, their hydrodynamic volume. It is commonly applied to macromolecules or macromolecular complexes, such as proteins and industrial polymers. Typically, when an aqueous solution is used to transport the sample through the chromatographic column, the technique is called gel filtration chromatography, while when an organic solvent is used as the mobile phase, the name gel permeation chromatography is used. The basic principle of SEC is that particles of different sizes will elute (filter) through the stationary phase at different rates. This results in a separation of a solution of particles based on size. If all particles are loaded simultaneously or nearly simultaneously, particles of the same size should elute together.

[0173] High-performance liquid chromatography (or high-pressure liquid chromatography, HPLC) is a form of column chromatography commonly used in biochemistry and analytical chemistry to separate, identify, and quantify compounds. HPLC uses a column filled with a chromatographic filler (stationary phase), a pump to move one or more mobile phases through the column, and a detector to display the retention time of molecules. Retention time varies depending on the interaction between the stationary phase, the analyte, and the solvent or solvents used.

[0174] Also provided herein is a method for assessing CTLA-4 glycosylation, N-linked glycosylation, or N-glycosylation, comprising contacting a sample containing CTLA-4 with an antibody of the embodiments (e.g., an antibody that selectively binds to glycosylated CTLA-4 relative to unglycosylated CTLA-4). In certain aspects, the method is an in vitro method. In certain aspects, the sample is a cell sample.

[0175] Nucleic Acids

[0176] The present disclosure also encompasses nucleic acid molecules (DNA or RNA) encoding any of the anti-glycCTLA-4 antibodies or glycosylated CTLA-4 polypeptides described herein. Also provided herein are vector molecules (such as plasmids) that are constructed to transport or replicate such nucleic acid molecules. Nucleic acids can be single-stranded, double-stranded, and can contain single-stranded and double-stranded portions.

[0177] pharmaceutical preparations

[0178] When pursuing clinical applications of pharmaceutical compositions containing antibodies, it is often beneficial to prepare pharmaceutical or therapeutic compositions that are suitable for the intended application. Generally, pharmaceutical compositions can have an effective amount of an anti-glycated CTLA-4 antibody or glycosylated CTLA-4 polypeptide described herein, or another agent dissolved or dispersed in a pharmaceutically acceptable carrier.

[0179] Also provided herein are compositions comprising an anti-glycCTLA-4 antibody or glycosylated CTLA-4 polypeptide as described herein. In certain embodiments, the composition may comprise at least 0.1% by weight of the antibody or polypeptide. In certain embodiments, the composition may comprise at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more of the anti-glycCTLA-4 antibody or glycosylated CTLA-4 polypeptide by weight. In other embodiments, for example, the anti-glycCTLA-4 or glycosylated CTLA-4 polypeptide may comprise about 2% to about 75%, about 25% to about 60%, about 30% to about 50%, or any range therein, of the weight of the composition. The amount of the active compound(s) in each therapeutically useful composition can be prepared in such a manner that a suitable dosage will be obtained in any given unit dose of the compound. Those skilled in the art who prepare such pharmaceutical formulations will consider a variety of factors, such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations, and therefore, a variety of dosages and treatment regimens may be desirable.

[0180] The composition can be a pharmaceutical composition comprising an anti-glycated CTLA-4 antibody or glycosylated CTLA-4 polypeptide as an active ingredient and a pharmaceutically acceptable carrier. The pharmaceutical composition can further include one or more additional active ingredients. The pharmaceutically acceptable carrier can be one that is approved by a regulatory agency of the U.S. federal or state government or listed in the U.S. Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopeias for use in animals, and more particularly humans.

[0181] As used herein, and unless otherwise indicated, the term "carrier" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), excipient, stabilizer, or vehicle administered with a therapeutic agent. A "pharmaceutically acceptable carrier" is a carrier that is nontoxic to the cells or mammals exposed to it at the dosage and concentration employed, and can be a sterile liquid, such as water and oil, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. A pharmaceutically acceptable molecular entity or composition does not produce adverse, allergic, or other adverse reactions when properly administered to an animal, such as a human. In view of the present disclosure, the preparation of pharmaceutical compositions with antibodies or additional active ingredients is known to those skilled in the art, as exemplified by Remington's Pharmaceutical Sciences, 18th edition, 1990, which is incorporated herein by reference. In addition, for animal (e.g., human) administration, it should be understood that the formulation should meet the sterility, pyrogenicity, general safety, and purity standards required by the FDA Office of Biological Standards.

[0182] It is contemplated that the composition includes from about 0.001 mg to about 10 mg of total antibody or polypeptide per milliliter (ml). Thus, the concentration of the antibody or polypeptide in the composition can be about, at least about, or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml or more (or any range derivable therein). wherein about, at least about, or at most about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, %, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% can be an anti-glyc CTLA-4 antibody or a glycosylated CTLA-4 polypeptide.

[0183] In view of the present disclosure, the preparation of pharmaceutical compositions having antibodies or other polypeptides as described herein as active ingredients is known to those skilled in the art, as exemplified by Remington's Pharmaceutical Sciences, 18th edition, 1990, which is incorporated herein by reference. In addition, for animal (including human) administration, it should be understood that the preparations should meet the sterility, pyrogenicity, general safety and purity standards required by the FDA Office of Biological Standards.

[0184] Pharmaceutically acceptable carriers include liquid, semisolid (i.e., paste) or solid carriers. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, adhesives, fillers, etc. or combinations thereof. Pharmaceutically acceptable carriers may include aqueous solvents (e.g., water, alcohol / water solutions, ethanol, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils and injectable organic esters such as ethyl oleate), dispersion media, coating agents (e.g., lecithin), surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, inert gases, parabens (e.g., methyl paraben, propyl paraben), trichloroethane, methyl paraben ... In some embodiments, the present invention provides a pharmaceutical composition comprising a pharmaceutical composition comprising a pharmaceutically acceptable salt, ... Such procedures are routine to those skilled in the art.

[0185] In certain embodiments, the pharmaceutically acceptable carrier can be an aqueous pH buffered solution. Examples include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (e.g., less than about 10 amino acid residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN®. , polyethylene glycol (PEG) and PLURONICS TM .

[0186] In certain embodiments, pharmaceutically acceptable carrier can be sterile liquid, for example water and oil, including those oils from petroleum, animal, plant or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil etc. Water can be a carrier, particularly when intravenously administering the pharmaceutical composition. Saline solution and dextrose aqueous solution and glycerol solution can also be used as liquid carriers, especially liquid carriers for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, polysorbate 80 etc. The composition can also contain a small amount of wetting agent or emulsifier, or pH buffer. These compositions can take the form of solution, suspension, emulsion, tablet, pill, capsule, powder, sustained-release preparation etc.

[0187] Certain embodiments of the present disclosure may have different types of carriers, depending on whether it is administered in solid, liquid, or aerosol form, and whether it needs to be sterile for the route of administration (such as injection). The composition can be formulated for administration: intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, intramuscularly, subcutaneously, mucosally, orally, topically, topically, topically, by inhalation (e.g., aerosol inhalation), by injection, by infusion, by continuous infusion, by local perfusion directly bathing target cells, by catheter, by lavage, in a lipid composition (e.g., liposomes), or by other methods or any combination of the foregoing known to those of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th edition, 1990, incorporated herein by reference). Generally, such compositions can be prepared as liquid solutions or suspensions; solid forms can also be prepared, which are suitable for preparing solutions or suspensions after adding a liquid before injection; and the preparations can also be emulsified.

[0188] The anti-glycCTLA-4 antibody or glycosylated CTLA-4 polypeptide can be formulated into a composition in free base, neutral or salt form. Pharmaceutically acceptable salts include acid addition salts, such as those formed with the free amino groups of the proteinaceous composition, or formed with inorganic acids (such as hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid or mandelic acid). Salts formed with free carboxyl groups can also be derived from inorganic bases, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide; or organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine or procaine.

[0189] In other embodiments, pharmaceutical compositions with lipids are provided herein. Lipids can include a class of materials that are characteristically insoluble in water and that can be extracted with organic solvents. Examples include compounds containing long-chain aliphatic hydrocarbons and derivatives thereof. Lipids can be naturally occurring or synthetic (i.e., designed or produced by people). Lipids can be biological substances. Biological lipids are well known in the art and include, for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, sphingolipids, glycolipids, sulfatides, lipids with ether- and ester-connected fatty acids, polymerizable lipids and combinations thereof. Compounds that are understood to be lipids by those skilled in the art except those specifically described herein can also be used.

[0190] Those of ordinary skill in the art will be familiar with a range of techniques that can be used to disperse compositions in lipid vehicles. For example, antibodies or polypeptides can be dispersed in solutions containing lipids, dissolved with lipids, emulsified with lipids, mixed with lipids, combined with lipids, covalently bound to lipids, contained in lipids as suspensions, contained in micelles or liposomes, or complexed with micelles or liposomes, or in addition associated with lipids or lipid structures by any means known to those of ordinary skill in the art. Dispersions may or may not result in the formation of liposomes.

[0191] Typically, the ingredients of the composition are supplied separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or anhydrous concentrate in a hermetically sealed container, such as an ampoule or sachet indicating the amount of active agent. When the composition is to be administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is to be administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed before administration.

[0192] The amount of active ingredient in each therapeutically useful composition can be prepared in such a manner that a suitable dosage will be obtained in any given unit dose of the compound. Those skilled in the art who prepare such pharmaceutical formulations may consider a variety of factors, such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations, and therefore a variety of dosages and treatment regimens may be desirable.

[0193] Unit dose or dosage represent the physically discrete unit that is suitable for experimenter, and each unit contains the pharmaceutical composition of predetermined amount, and described amount is calculated to produce the expectation response relevant with its administration (i.e. suitable approach and treatment scheme) discussed above.According to treatment number and unit dose, the amount to be used depends on the effect of expectation.The actual dosage of the composition of the embodiment of the present invention that is applied to patient or experimenter can be determined by physical and physiological factors, such as experimenter's weight, age, health and sex, the type of the disease being treated, the degree of disease penetration, previous or simultaneous treatment intervention, patient's idiopathic disease, route of administration and the effectiveness, stability and toxicity of specific treatment substance. In other non-limiting examples, the dosage can have a range of from about 1 μg / kg / body weight, about 5 μg / kg / body weight, about 10 μg / kg / body weight, about 50 μg / kg / body weight, about 100 μg / kg / body weight, about 200 μg / kg / body weight, about 350 μg / kg / body weight, about 500 μg / kg / body weight, about 1 mg / kg / body weight, about 5 mg / kg / body weight, about 10 mg / kg / body weight, about 50 mg / kg / body weight, about 100 mg / kg / body weight, about 200 mg / kg / body weight, about 350 mg / kg / body weight, about 500 mg / kg / body weight, to about 1000 mg / kg / body weight or more, and any range derivable therein. In non-limiting examples of ranges derivable from the numbers listed herein, ranges from about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 μg / kg / body weight to about 500 mg / kg / body weight, etc. can be administered based on the above numbers. In any event, the practitioner responsible for administration will determine the concentration of the active ingredient(s) in the composition and the appropriate dosage(s) for an individual subject.

[0194] Those of ordinary skill in the art will appreciate that the compositions described herein are not limited to the specific properties of therapeutic preparations. For example, such compositions can be provided in a preparation together with a physiologically tolerable liquid, gel or solid carrier, diluent and excipient. These therapeutic preparations can be administered to mammals for veterinary purposes, such as for domestic animals, and for the clinical use of the human race, in a manner similar to other therapeutic agents. In general, the dosage required for the therapeutic effect can vary according to the specific requirements of the type of use and mode of administration and individual subjects. The actual dosage of the composition administered to animal patients (including human patients) can be determined by physical and physiological factors, such as body weight, the severity of the disease, the type of the disease being treated, previous or simultaneous therapeutic interventions, the patient's idiopathic disease, and route of administration. Depending on dosage and route of administration, the number of administrations of preferred dosage and / or effective dose can vary according to the response of the subject. In any case, the practitioner responsible for administration will determine the concentration of one or more active ingredients in the composition and one or more suitable dosages of the individual subject.

[0195] Treatment of diseases

[0196] As used herein, and unless otherwise indicated, the term "subject" refers to an animal that is the object of treatment, observation, and / or experimentation. "Animal" includes vertebrates and invertebrates, such as fish, shellfish, reptiles, birds, and especially mammals. "Mammals" include, but are not limited to, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, horses, primates, such as monkeys, chimpanzees, apes, and humans. In certain embodiments, the subject is a human.

[0197] As used herein, and unless otherwise indicated, the terms "cancer" or "cancerous" refer to the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, hematological cancers and solid tumors.

[0198] As used herein, and unless otherwise indicated, the term "treat" means administering or applying a therapeutic agent to a subject or performing a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit for a disease or health-related condition. For example, treatment can include administering a therapeutically effective amount of an anti-glycCTLA-4 antibody to a subject. When used in reference to a cancer patient, the term "treat" means an action that may reduce the severity of the cancer, or delay or slow the progression of the cancer, including (a) inhibiting cancer growth, reducing cancer growth rate, arresting progression, reducing cancer invasiveness, or preventing cancer metastasis, and (b) causing cancer regression, delaying or minimizing one or more symptoms associated with the presence of cancer, or prolonging the survival of a cancer patient.

[0199] As used herein, and unless otherwise indicated, the term "therapeutically effective amount" refers to an amount of an agent (e.g., an antibody or polypeptide described herein or any other agent described herein) sufficient to reduce and / or ameliorate the severity and / or duration of a given disease, disorder, or condition and / or symptoms associated therewith. A therapeutically effective amount of an agent (including a therapeutic agent) can be an amount necessary to: (i) reduce or ameliorate the progression or development of a given disease, disorder, or condition, (ii) reduce or ameliorate the recurrence, development, or onset of a given disease, disorder, or condition, and / or (iii) improve or enhance the prophylactic or therapeutic effect of another therapy (e.g., a therapy other than administration of an antibody provided herein). A therapeutically effective amount of a substance / molecule / agent of the present disclosure (e.g., an anti-glycCTLA-4 antibody or glycosylated CTLA-4 polypeptide) can vary depending on factors such as the disease state, the age, sex, and weight of the individual, and the ability of the substance / molecule / agent to elicit a desired response in the individual. A therapeutically effective amount encompasses an amount in which any toxic or deleterious effects of the substance / molecule / agent are outweighed by the therapeutically beneficial effects.

[0200] As used herein, and unless otherwise indicated, the term "administering" refers to the act of injecting or otherwise physically delivering a substance present outside the body into a patient's body, such as by mucosal, intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art. When a disease, disorder, or condition, or its symptoms, is being treated, administration of the substance typically occurs after the onset of the disease, disorder, or condition, or its symptoms. When a disease, disorder, or condition, or its symptoms, is being prevented, administration of the substance typically occurs before the onset of the disease, disorder, or condition, or its symptoms.

[0201] Also provided herein are therapeutic uses of anti-glycCTLA-4 antibodies and glycosylated CTLA-4 polypeptides. These antibodies or polypeptides can be used to modulate the activity of CTLA-4 / CD86 signaling. These antibodies or polypeptides can be used to modulate the activity of CTLA-4 / CD80 signaling. These antibodies or polypeptides can also be used to treat diseases by inhibiting the inhibitory activity of CTLA-4 in T cell activation or proliferation. Therefore, provided herein are uses of such antibodies or polypeptides for upregulating a subject's immune system by inhibiting or blocking CTLA-4 signaling. In certain embodiments, provided herein are uses of antibodies or polypeptides for blocking CTLA-4 binding to CD86. In certain embodiments, provided herein are uses of antibodies or polypeptides for blocking CTLA-4 binding to CD80.

[0202] In certain embodiments, the present invention also provides therapeutic uses of anti-glycated CTLA-4 antibodies and glycosylated CTLA-4 polypeptides in the treatment of cancer. Upregulation of the immune system is particularly desirable in the treatment of cancer, and thus methods of treating cancer are also provided herein. Cancer refers to a neoplasm or tumor caused by abnormal, uncontrolled growth of cells. Cancer can be primary or metastatic. In specific embodiments, the cancer cells are CD86 or CD80 positive.

[0203] In certain aspects, the polypeptides or antibodies of the embodiments (e.g., glycosylated CTLA-4 polypeptides or antibodies that bind to glycosylated CTLA-4) can be administered to treat cancer. In specific embodiments, the anti-glycCTLA-4 antibody is a chimeric or humanized form of STC1807. Cancers for which the present treatment methods are applicable include any malignant cell type, such as those found in solid tumors or hematological tumors. Exemplary solid tumors may include, but are not limited to, tumors of organs selected from the group consisting of pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. Exemplary hematological tumors include tumors of the bone marrow, T or B cell malignancies, leukemias, lymphomas, blastomas, myelomas, and the like. Other examples of cancers that can be treated using the methods provided herein include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia, squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, stomach cancer or gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer or renal cancer. , prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer, melanoma, superficial spreading melanoma, lentigo malignant melanoma, acral lentiginous melanoma, nodular melanoma, and B-cell lymphomas (including low-grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, multiple myeloma, acute myeloid leukemia (AML), and chronic myeloblastic leukemia.

[0204] Cancer may specifically be of the following histological types, but is not limited to: malignant neoplasms; carcinoma; undifferentiated carcinoma; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatricoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; bile duct carcinoma; hepatocellular carcinoma; combined hepatocellular and bile duct carcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma, familial polyposis coli; solid carcinoma; malignant carcinoid tumor; bronchiolar-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe cell carcinoma; oncocytic carcinoma; oncocytic adenocarcinoma; basophilic cell carcinoma; clear cell adenocarcinoma; granulosa cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-cystic sclerosing carcinoma; adrenocortical carcinoma; endometrioid carcinoma carcinoma); skin appendage carcinoma; apocrine gland carcinoma; sebaceous gland carcinoma; cervical gland carcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous transformation; malignant thymoma; malignant ovarian stromal tumor; malignant thecoma cell tumor; malignant granulosa cell tumor; malignant testicular blastoma (androblastoma) ma, malignant); Sertoli cell carcinoma; malignant Leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extramammary paraganglioma; pheochromocytoma; glomus sarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma within giant nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; mesenchymal Sarcoma; malignant mixed tumor; mixed Müllerian tumor; Wilms' tumor; hepatoblastoma; carcinosarcoma; malignant mesenchymal tumor; malignant Brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma; malignant goiter; choriocarcinoma; malignant mesonephroblastoma; angiosarcoma; malignant hemangioendothelioma; Kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; malignant odontoma ameloblastoma; ameloblastic odontosarcoma; malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pinealoma; chordoma; malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectodermal tumor; cerebellar sarcoma; ganglioblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant neurilemoma; malignant granular cell tumor;Malignant lymphoma; Hodgkin's disease; Hodgkin's; paragranuloma; small lymphocytic malignant lymphoma; diffuse large cell malignant lymphoma; follicular malignant lymphoma; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small bowel disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0205] In certain embodiments, the antibodies or polypeptides provided herein can be used to treat cancer, which is breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, brain cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer or skin cancer.

[0206] The polypeptides or antibodies can be used herein as anti-tumor agents in a variety of ways. Provided herein are methods of using polypeptides or antibodies as anti-tumor agents and thus comprise contacting a tumor cell population with a therapeutically effective amount of the polypeptide or antibody for a period of time sufficient to inhibit tumor cell growth.

[0207] Various delivery systems are also known and can be used to administer anti-glycCTLA-4 antibodies or related molecules of glycosylated CTLA-4 polypeptides, or related pharmaceutical compositions, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibody or fusion protein, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), constructs as part of a retroviral or other vector, and the like.

[0208] Methods of administration as provided herein include, but are not limited to, injection, such as by parenteral administration (e.g., intradermal administration, intramuscular administration, intraperitoneal administration, intravenous administration, and subcutaneous administration), epidural and mucosal (e.g., intranasal and oral routes). In certain embodiments, the antibodies, other molecules, or pharmaceutical compositions provided herein are administered intramuscularly, intravenously, subcutaneously, intravenously, intraperitoneally, orally, intramuscularly, subcutaneously, intracavitary, transdermally, or dermally. The composition can be administered by any convenient route, for example, by infusion or rapid bolus injection, by absorption through the epithelium or mucocutaneous lining (e.g., oral mucosa, rectal mucosa, and intestinal mucosa, etc.), and can be administered together with other bioactive agents. Administration can be systemic or local. In addition, pulmonary administration can also be adopted, for example, by using an inhaler or nebulizer and formulating with an atomizing agent. See, e.g., U.S. Patent Nos. 6,019,968, 5,985,20, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078 and PCT Publication Nos. WO 92 / 19244, WO 97 / 32572, WO 97 / 44013, WO 98 / 31346, and WO 99 / 66903, all of which are hereby incorporated by reference in their entirety. In certain embodiments, the antibodies, other molecules, or pharmaceutical compositions provided herein are administered topically to the area in need of treatment, which can be achieved by, e.g., local infusion, by injection, or by means of an implant, which is a porous, non-porous, or gel-like material, including a membrane, e.g., a silicone rubber membrane, or a fiber. In certain embodiments, when administering an antibody or other molecule as described herein, care is taken to use a material that the antibody or other molecule does not absorb.

[0209] In certain embodiments, the antibodies or polypeptides provided herein are formulated in liposomes for targeted delivery. Liposomes are vesicles composed of concentric, ordered phospholipid bilayers that encapsulate an aqueous phase. Liposomes typically have various types of lipids, phospholipids, and / or surfactants. The components of the liposomes are arranged in a bilayer configuration, similar to the lipid arrangement of biological membranes. Liposomes can be useful delivery vehicles, in part due to their biocompatibility, low immunogenicity, and low toxicity. Methods for preparing liposomes are known in the art and are provided herein, see, for example, Epstein et al., 1985, Proc. Natl. Acad. Sci. USA, 82: 3688; Hwang et al., 1980 Proc. Natl. Acad. Sci. USA, 77: 4030-4; U.S. Patent Nos. 4,485,045 and 4,544,545; all of which are hereby incorporated by reference in their entirety.

[0210] Also provided herein is a method for preparing liposomes with extended serum half-life (i.e., extended circulation time), such as those disclosed in U.S. Patent number 5,013,556. In certain embodiments, the liposomes used in the method provided herein will not be quickly cleared from the circulation, i.e., will not be absorbed into the mononuclear phagocyte system (MPS). Also provided herein is a sterically stabilized liposome, which is prepared using conventional methods known to those skilled in the art. The sterically stabilized liposome can contain a lipid component with a large and highly flexible hydrophilic portion, which reduces the reaction of undesirable liposomes with serum proteins, reduces opsonization with serum components, and reduces the recognition of MPS. Polyethylene glycol can be used to prepare sterically stabilized liposomes. For the preparation of liposomes and sterically stabilized liposomes, see, e.g., Bendas et al., 2001 BioDrugs, 15(4):215-224; Allen et al., 1987 FEBS Lett. 223:42-6; Klibanov et al., 1990 FEBS Lett., 268:235-7; Blum et al., 1990, Biochim. Biophys. Acta., 1029:91-7; Torchilin et al., 1996, J. Liposome Res. 6:99-116; Litzinger et al., 1994, Biochim. Biophys. Acta, 1190:99-107; Maruyama et al., 1991, Chem. Pharm. Bull., 39:1620-2; Klibanov et al., 1991, Biochim Biophys Acta, 1062; 142-8; Allen et al., 1994, Adv. Drug Deliv. Rev, 13: 285-309, which are hereby incorporated by reference in their entirety.

[0211] Also provided herein are liposomes suitable for specific organ targeting, see, e.g., U.S. Patent No. 4,544,545, or liposomes suitable for specific cell targeting, see, e.g., U.S. Patent Application Publication No. 2005 / 0074403, which are hereby incorporated by reference as a whole. Using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE), a particularly useful liposome for the compositions and methods provided herein can be produced by reverse phase evaporation. Liposomes can be extruded through a filter with a defined pore size to produce liposomes with a desired diameter. In certain embodiments, molecules with antigen-binding fragments such as F (ab') can be conjugated to liposomes using previously described methods, see, e.g., Martin et al., 1982, J.Biol.Chem.257:286-288, which are hereby incorporated by reference as a whole.

[0212] Humanized or chimeric antibodies as described herein can also be formulated into immunoliposomes. Immunoliposomes refer to liposome compositions in which antibodies or fragments thereof are covalently or non-covalently attached to the surface of the liposomes. Chemical methods for attaching antibodies to the surface of liposomes are known in the art, see, for example, U.S. Patent No. 6,787,153; Allen et al., 1995, Stealth Liposomes, Boca Rotan: CRC Press, 233-44; Hansen et al., 1995, Biochim.Biophys.Acta, 1239: 133-144, which are hereby incorporated by reference in their entirety. In certain embodiments, the immunoliposomes for the methods and compositions provided herein are further spatially stabilized. In certain embodiments, humanized antibodies as described herein are covalently or non-covalently attached to a hydrophobic anchor that is stably rooted in the lipid bilayer of the liposome. Examples of hydrophobic anchors include, but are not limited to, phospholipids, such as phosphatidylethanolamine (PE), phosphatidylinositol (PI). To achieve covalent linkage between the antibody and the hydrophobic anchor, any biochemical strategy known in the art can be used, see, for example, J. Thomas August, ed., 1997, Gene Therapy: Advances inPharmacology, Vol. 40, Academic Press, San Diego, Calif., pp. 399-435, which are hereby incorporated by reference in their entirety. For example, functional groups on antibody molecules can react with reactive groups on hydrophobic anchors associated with liposomes, e.g., amino groups of lysine side chains on antibodies can be coupled to liposome-associated N-glutaryl-phosphatidylethanolamine activated with a water-soluble carbodiimide; or thiol groups of reduced antibodies can be coupled to liposomes via thiol-reactive anchors such as pyridylthiopropionylphosphatidylethanolamine. See, e.g., Dietrich et al., 1996, Biochemistry, 35: 1100-1105; Loughrey et al., 1987, Biochim. Biophys. Acta, 901: 157-160; Martin et al., 1982, J. Biol. Chem. 257: 286-288; Martin et al., 1981, Biochemistry, 20: 4429-38, which are hereby incorporated by reference in their entirety. Immunoliposome formulations with anti-glycosylated CTLA-4 antibodies can be particularly effective as therapeutic agents because they deliver the active ingredient to the cytoplasm of target cells (i.e., cells containing the receptor to which the antibody is to bind). In certain embodiments, the immunoliposomes can have an increased half-life in the blood, particularly in target cells, and can be internalized into the cytoplasm of target cells, thereby avoiding loss of the therapeutic agent or degradation by the endolysosomal pathway.

[0213] The immunoliposome compositions provided herein can have one or more vesicle-forming lipids, antibodies of the present invention or other molecules or fragments or derivatives thereof, and optional hydrophilic polymers. The vesicle-forming lipids can be lipids having two hydrocarbon chains (such as acyl chains and polar head groups). Examples of vesicle-forming lipids include phospholipids, for example, phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol, sphingomyelin and glycolipids, for example, cerebrosides, gangliosides. Other lipids that can be used for the preparations provided herein are known to those skilled in the art and are included in this specification. In certain embodiments, the immunoliposome compositions further include hydrophilic polymers, for example, polyethylene glycol and ganglioside GM1, which increase the serum half-life of the liposomes. Methods for conjugating hydrophilic polymers to liposomes are well known in the art and are included in this specification. Other exemplary immunoliposomes and methods for their preparation can be found in, for example, U.S. Patent Application Publication No. 2003 / 0044407; PCT International Publication No. WO 97 / 38731, Vingerhoeads et al., 1994, Immunomethods, 4:259-72; Maruyama, 2000, Biol. Pharm. Bull. 23(7):791-799; Abra et al., 2002, Journal of Liposome Research, 12(1&2):1-3; Park, 2002, Bioscience Reports, 22(2):267-281; ​​Bendas et al., 2001 BioDrugs, 14(4):215-224, J. Thomas August, ed., 1997, Gene Therapy:Advances in Pharmacology 40, Academic Press, San Diego, Calif., pp. 399-435; both of which are hereby incorporated by reference in their entirety.

[0214] Also provided herein are methods for treating cancer patients by administering to the patient a unit dose of an anti-glycated CTLA-4 antibody. Also provided herein are methods for treating cancer patients by administering to the patient a unit dose of a glycosylated CTLA-4 polypeptide. A unit dose represents a physically discrete unit suitable as a unit dosage for a subject, each unit containing a predetermined quantity of active agent calculated to produce the desired therapeutic effect in combination with the required diluent (i.e., carrier or vehicle).

[0215] The antibody, polypeptide or composition is administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The amount to be administered depends on the ability of the subject to be treated, the subject's system to utilize the active ingredient, and the degree of the desired therapeutic effect. The precise amount of the active ingredient to be administered depends on the practitioner's judgment and is unique to each individual subject. However, suitable dosage ranges for systemic application are disclosed herein and depend on the route of administration. Suitable regimens for initial administration and booster administration are also contemplated, and typically include an initial administration followed by repeated doses of subsequent injections or other administrations at intervals of one or more hours. Exemplary multiple administrations are described herein and can be used to maintain continuous high serum and tissue levels of a polypeptide or antibody. Alternatively, continuous intravenous infusions sufficient to maintain the concentration in the blood within the range specified for in vivo treatment are contemplated.

[0216] A therapeutically effective amount is a predetermined amount calculated to achieve the desired effect. Generally, the dosage will vary with the patient's age, condition, sex, and degree of disease, and can be determined by one skilled in the art. If any complications arise, the dosage can be adjusted by the individual physician.

[0217] In certain embodiments, provided herein are antibodies, polypeptides or pharmaceutical compositions packaged in airtight sealed containers such as ampoules or sachets. In one embodiment, provided herein are antibodies, polypeptides or pharmaceutical compositions in the form of dry sterile lyophilized powder or anhydrous concentrates in airtight sealed containers, and can be reconstituted to an appropriate concentration, for example, with water or saline, to administer to a subject. In certain embodiments, provided herein are antibodies, polypeptides or pharmaceutical compositions in the form of dry sterile lyophilized powders of at least 5 mg, more preferably at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg or at least 75 mg, in airtight sealed containers. Freeze-dried antibodies, polypeptides or pharmaceutical compositions provided herein should be stored in initial containers at 2 to 8 ° C and should be administered within 12 hours, preferably within 6 hours, within 5 hours, within 3 hours or within 1 hour after reconstruction. In an alternative embodiment, provided herein are antibodies, polypeptides or pharmaceutical compositions in liquid form in airtight sealed containers, the container indicating the amount and concentration of the antibody, polypeptide or pharmaceutical composition. In certain embodiments, a liquid form of an antibody, polypeptide, or pharmaceutical composition provided herein is provided in a hermetically sealed container at at least 1 mg / ml, more preferably at least 2.5 mg / ml, at least 5 mg / ml, at least 8 mg / ml, at least 10 mg / ml, at least 15 mg / ml, at least 25 mg / ml, at least 50 mg / ml, at least 100 mg / ml, at least 150 mg / ml, at least 200 mg / ml.

[0218] The precise dose to be employed in the formulation also depends on the route of administration and the severity of the condition, and should be determined according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems. For the anti-glycated CTLA-4 antibody or glycosylated CTLA-4 polypeptide, the dose administered to the patient is generally 0.01 mg / kg to 100 mg / kg of the patient's body weight. In certain embodiments, the dosage administered to the patient is between 0.01 mg / kg to 20 mg / kg, 0.01 mg / kg to 10 mg / kg, 0.01 mg / kg to 5 mg / kg, 0.01 to 2 mg / kg, 0.01 to 1 mg / kg, 0.01 mg / kg to 0.75 mg / kg, 0.01 mg / kg to 0.5 mg / kg, 0.01 mg / kg to 0.25 mg / kg, 0.01 to 0.15 mg / kg, 0.01 to 0.10 mg / kg, 0.01 to 0.05 mg / kg, or 0.01 to 0.025 mg / kg patient body weight. The dosage administered to the patient can be 0.2 mg / kg, 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 6 mg / kg, or 10 mg / kg. It is expected that a dosage as low as 0.01 mg / kg will show a noticeable pharmacodynamic effect. It is estimated that the dosage level of 0.10-1mg / kg is the most suitable. It is also possible to expect that higher dosage (for example, 1-30mg / kg) is active. Generally, due to the immune response to exogenous polypeptides, the half-life of human antibodies in the human body is longer than the antibodies from other species. Therefore, it is possible to implement lower dosages of human antibodies and lower frequency administration. In addition, by modification, for example, lipidation, by enhancing the uptake and tissue penetration of antibodies, it is possible to reduce the dosage and frequency of the antibody or polypeptide provided herein.

[0219] In another embodiment, the composition can be delivered in a controlled release or sustained release system. Any technology known to those skilled in the art can be used to produce a sustained release formulation having one or more antibodies, molecules or pharmaceutical compositions provided herein. See, for example, U.S. Patent No. 4,526,938; PCT Publication WO 91 / 05548; PCT Publication WO 96 / 20698; Ning et al., Radiotherapy & Oncology 39: 179-189 (1996), Song et al., PDA Journal of Pharmaceutical Science & Technology 50: 372-397 (1995); Cleek et al., Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24: 853-854 (1997); and Lam et al., Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24: 759-760 (1997); They are all hereby incorporated by reference in their entirety. In one embodiment, a pump can be used in a controlled release system (see Langer, supra; Sefton, 1987, CRC Crit. Ref Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery 88:507; and Saudek et al., 1989, N. Engl. J. Med. 321:574).In another embodiment, polymeric materials can be used to achieve controlled release of antibodies or polypeptides (see, e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Press., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J., Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 7 1:105); U.S. Patent No. 5,679,377; U.S. Patent No. 5,916,597; U.S. Patent No. 5,912,015; U.S. Patent No. 5,989,463; U.S. Patent No. 5,128,326; PCT Publication No. WO 99 / 15154; and PCT Publication No. WO 99 / 20253); all of which are hereby incorporated by reference in their entirety.

[0220] Examples of polymers that can be used in sustained-release formulations include, but are not limited to, poly(hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. In yet another embodiment, a controlled-release system can be placed near the therapeutic target (e.g., the lungs), thereby requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, Vol. 2, pp. 115-138 (1984)). In another embodiment, a polymer composition useful as a controlled-release implant is used according to Dunn et al. (see U.S. Pat. No. 5,945,155, which is hereby incorporated by reference in its entirety). Based on the therapeutic effect of the in situ controlled release of bioactive substances from polymeric systems, implantation can generally occur anywhere in the patient's body where therapeutic treatment is required.

[0221] In another embodiment, non-polymer sustained delivery system is used, and thus the non-polymer implant in the subject's body is used as a drug delivery system. After implantation in vivo, the organic solvent of the implant will dissipate, disperse or leach into the tissue fluid around from the composition, and the non-polymeric material will gradually condense or precipitate to form a solid microporous matrix (referring to U.S. Patent number 5,888,533). Controlled release system has also been discussed in the review of Langer (1990, Science 249: 1527-1533). Any technology well known to those skilled in the art can be used for producing the sustained release formulation comprising one or more therapeutic agents provided herein. See, e.g., U.S. Patent No. 4,526,938; International Publication Nos. WO 91 / 05548 and WO 96 / 20698; Ning et al., 1996, Radiotherapy & Oncology 39:179-189; Song et al., 1995, PDA Journal of Pharmaceutical Science & Technology 50:372-397; Cleek et al., 1997, Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854; and Lam et al., 1997, Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760; all of which are hereby incorporated by reference in their entirety.

[0222] Also provided herein are embodiments in which the composition has a nucleic acid encoding an antibody or polypeptide provided herein, wherein the nucleic acid can be administered in vivo to promote expression of the antibody or polypeptide encoded thereby: construct it as part of an appropriate nucleic acid expression vector and administer it so that it becomes intracellular, for example, by using a retroviral vector (see U.S. Patent No. 4,980,286), or by direct injection, or by using microparticle bombardment (e.g., a gene gun; Biolistics, Dupont), or by coating with lipids or cell-surface receptors or transfection agents, or by administering it in conjunction with a homeobox-like peptide known to enter the nucleus (see, e.g., Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88: 1864-1868). Alternatively, the nucleic acid can be introduced intracellularly and integrated into the host cell DNA for expression by homologous recombination.

[0223] Treatment of a subject with the antibody, polypeptide or pharmaceutical composition provided herein of a therapeutically effective amount can include single treatment or a series of treatments. It is contemplated that the antibody, polypeptide or pharmaceutical composition provided herein can be administered systemically or topically to treat a disease, such as suppressing tumor cell growth or killing cancer cells in a cancer patient suffering from locally advanced or metastatic cancer. They can be administered intravenously, intrathecally and / or intraperitoneally. They can be administered alone or in combination with antiproliferative drugs. In one embodiment, they are administered before surgery or other operations to reduce the cancer load in the patient. Alternatively, they can be administered postoperatively to ensure that any remaining cancer (for example, cancer that surgery does not eliminate) will not survive. In certain embodiments, they can be administered after the primary cancer subsides to prevent metastasis.

[0224] Combination therapy

[0225] In certain embodiments, the compositions and methods of the present invention involve administering a glycosylated CTLA-4 polypeptide or an antibody that selectively binds to glycosylated CTLA-4 in combination with a second or additional therapy. Such therapy can be used to treat any disease associated with CTLA-4 or glycosylated CTLA-4. For example, the disease can be cancer, and the second therapy is an anti-cancer or anti-hyperproliferative therapy.

[0226] The methods and compositions, including combined therapies, enhance the treatment or protective effect of another anti-cancer or anti-hyperproliferative therapy, and / or increase its therapeutic effect. Therapeutic and preventive methods and compositions can be provided in a combined amount that effectively achieves the desired effect, and the desired effect is, for example, killing cancer cells and / or inhibiting the excessive proliferation of cells. The method can involve administering a polypeptide or antibody and a second therapy. The second therapy may have or not have a direct cytotoxic effect. For example, the second therapy can be an agent that raises the immune system without a direct cytotoxic effect. Tissue, tumor or cell can be exposed to one or more compositions or pharmacological preparations comprising one or more agents (for example, antibodies or anticancer agents), or by exposing two or more different compositions or preparations to tissue, tumor and / or cell, wherein a composition provides 1) polypeptide or antibody, 2) anticancer agent, or 3) polypeptide or antibody and anticancer agent. In addition, it is contemplated that such combined therapies can be used in combination with chemotherapy, radiotherapy, surgical therapy or immunotherapy.

[0227] As applied to cells, the terms "contacting" and "exposing" are used herein to describe the process of delivering a therapeutic polypeptide or antibody and a chemotherapeutic or radiotherapeutic agent to or placing them in direct juxtaposition with a target cell. For example, to achieve cell killing, the two agents are delivered to the cell in a combined amount effective to kill the cell or prevent it from dividing.

[0228] The anti-glyc CTLA-4 antibody or glycosylated CTLA-4 polypeptide can be administered before, during, after, or in various combinations relative to the second or additional anticancer therapy. The time interval between administrations can range from simultaneous to a few minutes to several days to several weeks. In embodiments where the antibody or polypeptide is provided to the patient separately from the anticancer agent, it is generally ensured that a long period of time does not elapse between each delivery time, so that the two compounds can still exert a beneficial combined effect on the patient. In such cases, it is contemplated that the anti-glyc CTLA-4 antibody or glycosylated CTLA-4 polypeptide and the second therapy can be provided to the patient within about 12-24 or 72 hours of each other, and more specifically within about 6-12 hours of each other. In some cases, the treatment period can be significantly extended, with a few days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks) between each administration.

[0229] In specific embodiments, anti-glyc CTLA-4 antibodies are administered to a patient in combination with one or more other anti-CTLA-4 antibodies, including ipilimumab, for cancer treatment. In other embodiments, anti-glyc CTLA-4 antibodies are administered to a patient in combination with one or more anti-PD-1 antibodies, and in a specific embodiment, the anti-PD-1 antibody is pembrolizumab, nivolumab, or pidilizumab for cancer treatment. In other embodiments, anti-glyc CTLA-4 antibodies are administered with an agent that inhibits the activity of CTLA-4, PD-L1, or PD-1, such as an immunoadhesin having an extracellular receptor or ligand binding portion of a PD-1, PD-L1, or CTLA-4 protein fused to an Fc domain. In certain embodiments, anti-glyc CTLA-4 antibodies are administered in combination with durvalumab.

[0230] In a specific embodiment, an anti-glycCTLA-4 antibody is administered in combination with an antibody that preferentially binds to glycosylated PD-L1 relative to unglycosylated PD-L1. Specifically, an anti-glycCTLA-4 antibody can be administered in combination with a chimeric or humanized form of the anti-PD-L1 antibody STM004 or STM115, which preferentially binds to glycosylated PD-L1 relative to unglycosylated PD-L1, and the amino acid sequences (and encoding nucleotide sequences) of the heavy and light chain variable domains are disclosed in PCT publication WO 2016 / 160792, entitled "Antibodies Specific To Glycosylated PD-L1 And Methods Of Use Thereof," published on October 6, 2016, which is incorporated herein by reference. Anti-glyc-CTLA-4 antibodies are also administered in combination with chimeric or humanized forms of the anti-PD-L1 antibodies STM073 and SMT108, which preferentially bind to glycosylated PD-L1 relative to unglycosylated PD-L1, and the amino acid sequences (and encoding nucleotide sequences) of the heavy and light chain variable domains are disclosed in U.S. Provisional Application No. 62 / 314,652, filed March 29, 2016, entitled “Dual Function Antibodies Specific To Glycosylated PD-L1 And Methods Of Use Thereof,” which is incorporated herein by reference. In certain embodiments, anti-glyc CTLA-4 antibodies are administered in combination with atezolizumab or avelumab.

[0231] In certain embodiments, a course of treatment can last 1-90 days or longer (this such range includes the interval days). It is contemplated that one agent may be administered on any day from the 1st to the 90th day (this such range includes the interval days) or any combination thereof, and another agent may be administered on any day from the 1st to the 90th day (this such range includes the interval days) or any combination thereof. Within a day (24-hour period), one or more administrations of one or more agents may be administered to the patient. In addition, after the course of treatment, it is contemplated that there is a period of time when anticancer treatment is not administered. This period of time may last 1-7 days, and / or 1-5 weeks, and / or 1-12 months or longer (this such range includes the interval days), depending on the patient's condition, such as their prognosis, strength, health, etc. If necessary, the treatment cycle can be repeated.

[0232] Various combinations can be used. The following are some examples of treatments using an anti-glycated CTLA-4 antibody or glycosylated CTLA-4 polypeptide as "A" and a second anticancer therapy as "B": A / B / AB / A / BB / B / AA / B / BB / A / AA / B / B / BB / A / B / BB / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / AB / A / B / AB / A / A / B / AB / A / A / BA / A / A / BB / A / A / A / AA / B / A / AA / A / B / A

[0233] Any antibody, polypeptide or pharmaceutical composition provided herein and the co-administration of a second therapy to a patient will follow the general protocol for administering such a second therapy, taking into account the toxicity, if any, of the second therapy. Therefore, in certain embodiments, there is a step of monitoring the toxicity attributable to the co-therapy.

[0234] chemotherapy

[0235] According to embodiments of the present invention, multiple chemotherapeutic agents can be used as the second therapy.Chemotherapeutic agents can be compounds or compositions used in the treatment of cancer.These agents or medicines can be classified according to their intracellular activity patterns, for example, whether they affect the cell cycle and at what stage they affect the cell cycle.Alternatively, based on its direct cross-linking DNA, embedding DNA or by affecting the ability of nucleic acid synthesis to induce chromosome and mitotic aberrations, agents can be characterized.

[0236] Examples of chemotherapeutic agents include: alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquinone, meturedopa, and uredopa; ethyleneimines and methylmelamines including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; annonaceous lactones (particularly bratacin and bratacinone); camptothecins (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogue adolesine); , kazelesin and biselesin); Nostoc cyclic peptides (particularly Nostoc cyclic peptide 1 and Nostoc cyclic peptide 8); dolastatin; duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1); cypermethrin; sarcodictyin; spongestatin; nitrogen mustards such as chlorambucil, naphthiazolin, clofosamide, estramustine, ifosfamide, mechlorethamine, chlorambucil, chlorambucil oxide, melphalan, chlorambucil, phenylephrine, prednimustine, trofosfamide and uracil mustard; nitrosureas such as carmustine, chlorozolin, fotemustine, lomustine, nimustine and ranimustine; antibiotics such as enediyne antibiotics (e.g., carmustine, chlorambucil, chlorambucil, estramustine, ifosfamide, mechlorethamine, chlorambucil, chlorambucil, biselesin, prednimustine, trofosfamide and uracil mustard); nitrosureas such as carmustine, chlorozolin, fotemustine, lomustine, nimustine and ranimustine; Spectinomycins, particularly calicheamicin gamma and calicheamicin omega); daptomycins, including daptomycin A; bisphosphonates, such as clodronate; esperamicins; and the neocarcin chromophores and related chromoprotein enediyne antibiotic chromophores, aclacinomycins, actinomycins, authrarnycins, azaserine, bleomycins, actinomycin C, carabicin, carminomycins, carmomycins, chromomycins, dactinomycins, daunorubicin, detoxibacin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanopropionic acid), daunorubicin, ... morpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, mexilomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycin, peplomycin, potfiromycin, puromycin, triferon-adriamycin, rhodorubicin, streptozocin, streptozocin, tuberculin, ubenimex, zinstatin and daurubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as leucovorin, pteropterin and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiabendazole and thioguanine;Pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as captestosterone, drotosterol propionate, cyclothiocarbamate, melastane, and testolactone; anti-adrenal drugs such as mitotane and trilostane; folic acid supplements such as frolinic acid; aceglucuronolide; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; colcemid; diazocone; elformithine; elliptonium acetate; epothilones; etoglucosamine; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansines such as maytansine and ansamitocin; mitoxantrone; mitoxantrone; mopidanmol; nitraerine; pentostatin; methambucil; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; sizolan; spirogermanium; tinuzzoic acid; triazoline; 2,2',2"-trichlorotriethylamine; trichothecenes (particularly T-2 toxin, verracurin A, rod- sporin A and serpentin); urethan; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulcitol; pipobroman; gacytosine; cytarabine ("Ara-C"); cyclophosphamide; taxanes, for example, paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; m Toxantrone; vincristine; vinorelbine; noxolin; teniposide; edatrexate; daunorubicin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, plicamycin, gemcitabine, navelbine, farnesyl-protein transferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

[0237] Radiation therapy

[0238] Another conventional anticancer therapy that can be used in combination with the methods and compositions described herein is radiotherapy or radiation therapy. Radiotherapy includes the use of gamma-rays, X-rays and / or directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors, such as microwaves, proton beam irradiation (U.S. Patent Nos. 5,760,395 and 4,870,287; they are all hereby incorporated by reference in their entirety) and ultraviolet irradiation are also contemplated. Most likely, all of these factors cause wide-ranging damage to DNA, DNA precursors, replication and repair of DNA, and assembly and maintenance of chromosomes.

[0239] The tumor microenvironment is inherently suppressive due to the presence of bone marrow-derived suppressor cells and regulatory T cells that infiltrate the tumor and act to suppress the immune response. In addition, the expression of certain inhibitory molecules on T cells and antigen-presenting cells (APCs) can limit an effective immune response. Radiation mediates anti-tumor effects by inducing tumor cell apoptosis, senescence, and autophagy, and in some cases, can stimulate a more effective immune response.

[0240] The abscopal effect is a physiological process in which targeted radiation of a primary tumor induces an anti-tumor response at distant sites outside the radiation field. The mechanism responsible for the abscopal effect is thought to be immune-mediated and involves enhanced presentation of tumor antigens to T cells and the release of cytokines and other pro-inflammatory factors that stimulate local and systemic immune responses. Because the abscopal effect affects tumors located distal to the primary tumor receiving radiation therapy, agents that can trigger the abscopal effect are particularly advantageous in treating metastatic tumors, which are often more difficult to treat once they have spread to secondary sites in the body.

[0241] The anti-glycCTLA-4 antibodies or glycosylated CTLA-4 polypeptides described herein can stimulate local and systemic immune responses. In certain embodiments, a therapeutically effective amount of an antibody, polypeptide, or pharmaceutical composition as described herein is administered before, simultaneously with, or after radiation therapy to achieve a synergistic, concomitant abscopal effect.

[0242] In certain embodiments, a therapeutically effective amount of an antibody, polypeptide, or pharmaceutical composition described herein is administered that effectively sensitizes a tumor in a host to radiation. The radiation may be ionizing radiation, particularly gamma radiation. In certain embodiments, the gamma radiation is emitted by a linear accelerator or a radionuclide. The irradiation of the tumor with a radionuclide may be external or internal.

[0243] In certain embodiments, administration of an antibody, polypeptide, or pharmaceutical composition described herein is initiated up to one month, particularly up to 10 days or one week, prior to tumor irradiation. In addition, tumor irradiation is segmented, with administration of an antibody, polypeptide, or pharmaceutical composition described herein maintained during the interval between the first and last irradiation periods.

[0244] Irradiation can also be X-ray radiation. The dosage range for X-rays is from daily doses of 50-200 roentgens for a long period of time (3 to 4 weeks) to a single dose of 2000-6000 roentgens. The dosage range for radioisotopes varies widely and depends on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.

[0245] Immunotherapy

[0246] The skilled artisan will appreciate that immunotherapy can be used in conjunction or in combination with the methods of the embodiments. In the context of cancer treatment, immunotherapeutics generally rely on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab It is such an example. Check point inhibitors such as ipilimumab (ipilumimab), pembrolizuman (pembrolizuman), nivolumab and atezolizumab are other examples. Immune effectors can be antibodies that are, for example, specific for some markers on the surface of tumor cells. A single antibody can serve as the effector of therapy, or it can recruit other cells to actually affect cell killing. Antibodies can also be conjugated to drugs or toxins (e.g., chemotherapeutic agents, radionuclides, ricin A chain, cholera toxin, pertussis toxin) and are used only as targeting agents. Alternatively, effectors can be lymphocytes carrying surface molecules that interact directly or indirectly with tumor cell targets. Various effector cells include cytotoxic T cells and NK cells.

[0247] In one aspect of immunotherapy, tumor cells carry some markers that are easy to target, that is, they are not present on most other cells. There are many tumor markers, and any of these can be suitable for targeting in the context of embodiments of the present invention. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B and p155. An alternative aspect of immunotherapy is to combine anticancer effects with immunostimulatory effects. There are also immunostimulatory molecules, including: cytokines, such as IL-2, IL-4, IL-12, GM-CSF, γ-IFN, chemokines, such as MIP-1, MCP-1, IL-8, and growth factors, such as FLT3 ligand.

[0248] Examples of immunotherapies currently under investigation or in use are immune adjuvants, e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Pat. Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, Infect Immun., 66(11):5329-36 (1998); Christodoulides et al., Microbiology, 66(11):5329-36 (1998)); cytokine therapies, e.g., interferon α, β, and γ, IL-1, GM-CSF, and TNF (Bukowski et al., Clin Cancer Res., 4(10):2337-47 (1998); Davidson et al., J. Immunol., 20(1):1147-115 (1998)); Immunother., 21(5):389-98 (1998); Hellstrand et al., Acta Oncol. 37(4):347-53 (1998)); gene therapy, e.g., TNF, IL-1, IL-2, and p53 (Qin et al., Proc Natl Acad Sci USA, 95(24):14411-6 (1998); Austin-Ward and Villaseca, Rev Med Chil, 126(7):838-45 (1998); U.S. Patent Nos. 5,830,880 and 5,846,945); and monoclonal antibodies, e.g., anti-PD1, anti-PDL1, anti-CD20, anti-ganglioside GM2, and anti-p185 (Topalian et al., The New England journal of medicine, 366:2443-2454 (2012); Brahmer et al., The New England journal of medicine 366:2455-2465 (2012); Hollander, Front Immunol (2012):3:3. doi:10.3389 / fimmu.2012.00003; Hanibuchi et al., Int J Cancer, 78(4):480-5 (1998); U.S. Patent No. 5,824,311); all of which are hereby incorporated by reference in their entireties. It is contemplated that one or more anti-cancer therapies can be used with the therapies described herein that include the use of anti-glyc CTLA-4 antibodies or glycosylated CTLA-4 polypeptides.

[0249] Operation

[0250] Approximately 60% of cancer patients will undergo some type of surgery, including preventive, diagnostic or staging, curative and palliative surgery. Curative surgery includes resection, in which all or part of the cancerous tissue is physically removed, excised and / or destroyed, and may be used in conjunction with other therapies such as the treatment according to the embodiments of the present invention, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy and / or alternative therapies. Tumor resection refers to the physical removal of at least a portion of a tumor. In addition to tumor resection, surgical treatments also include laser surgery, cryosurgery, electrosurgery and surgery controlled under a microscope (Mohs surgery).

[0251] After removing part or all of the cancerous cells, tissues or tumors, a cavity may form in the body. Treatment can be accomplished by perfusing the area, directly injecting or topically applying additional anticancer therapies. Such treatments can be repeated, for example, every 1, 2, 3, 4, 5, 6 or 7 days, or every 1, 2, 3, 4 and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. These treatments can also have different dosages.

[0252] Other medicines

[0253] It is contemplated that other agents may be used in combination with certain aspects of the embodiments of the present invention to improve the therapeutic effect of the treatment. These additional agents include agents that influence the upward regulation of cell surface receptors and GAP connections, cell growth inhibitors and differentiation agents, cell adhesion inhibitors, agents that increase the sensitivity of hyperproliferative cells to apoptosis inducers, or other biological agents. By increasing the number of GAP connections to increase intercellular signaling, the anti-hyperproliferative effect on adjacent hyperproliferative cell colonies can be increased. In other embodiments, cell growth inhibitors or differentiation agents may be used in combination with certain aspects of the embodiments of the present invention to improve the anti-hyperproliferative efficacy of the treatment. It is contemplated that cell adhesion inhibitors are used in combination with the efficacy of the embodiments of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents (such as antibody c225) that increase the sensitivity of hyperproliferative cells to apoptosis may be used in combination with certain aspects of the embodiments of the present invention to improve therapeutic efficacy.

[0254] Kits and diagnostic agents

[0255] In various aspects, provided herein are kits containing therapeutic agents and / or other therapeutic agents and delivery agents. In certain embodiments, kits for preparing and / or administering the therapies provided herein are contemplated. The kits can comprise one or more sealed vials containing any of the pharmaceutical compositions provided herein. The kits can include, for example, at least an anti-glycCTLA-4 antibody or a glycosylated CTLA-4 polypeptide, and reagents for preparing, formulating, and / or administering the components provided herein or performing one or more steps of the methods provided herein.

[0256] In certain embodiments, the kit can include an anti-glycCTLA-4 antibody and at least one auxiliary agent. In certain embodiments, the kit can include a glycosylated CTLA-4 polypeptide and at least one auxiliary agent.

[0257] In certain embodiments, the kit further comprises a second anticancer agent. The second anticancer agent can be a chemotherapeutic agent, an immunotherapeutic agent, a hormonal therapeutic agent, or a cytokine.

[0258] In certain embodiments, the test kit may further comprise suitable container means, which is a container that does not react with the components of the test kit, such as an eppendorf tube, an assay plate, a syringe, a bottle or a test tube. The container may be made of a sterilizable material such as plastic or glass.

[0259] The kit may further include an instruction chart outlining the procedural steps of the methods described herein, and will essentially follow the same procedures as described herein or known to those of ordinary skill. The instruction information may be in a computer-readable medium containing machine-readable instructions that, when executed by a computer, causes a display of a real or virtual program for delivering a pharmaceutically effective amount of an antibody or polypeptide provided herein. The kit may also include a notice in the form of a governmental agency regulating the manufacture, use, or sale of a drug or biological product, reflecting approval by the agency for production, use, or sale for human administration.

[0260] Example

[0261] It should be understood that modifications that do not significantly change the nature and spirit of the various embodiments described herein are also contemplated.Accordingly, the following examples are intended to be illustrative and not limiting in any way.

[0262] Materials and methods

[0263] Immunoblotting analysis, immunocytochemistry and immunoprecipitation.Immunoblotting analysis was performed as previously described (Lim et al., 2008, Gastroenterology, 135: 21 28-40; and Lee et al., 2007, Cell, 130: 440-455). Image acquisition and band intensity quantification were performed using the Bio-Rad ChemiDoc imaging system (Bio-Rad, Hercules, CA. USA). For immunocytochemistry, cells were fixed at room temperature in 4% paraformaldehyde for 15 minutes, permeabilized in 5% Triton X-100 for 5 minutes, and then stained with primary antibodies. The secondary antibodies used were anti-mouse Alexa Fluor 488 or 594 dye conjugates and / or anti-rabbit Alexa Fluor 488 or 594 dye conjugates (Life Technologies). Cell nuclei were stained with 4', 6-diamidino-2-phenylindole (DAPI blue) (Life Technologies). After sealing, cells were observed using a multiphoton confocal laser scanning microscope (Nikon A1+, Melville, NY, USA).

[0264] CTLA-4 and CD80 / CD86 (CD80 / CTLA-4 or CD86 / CTLA-4) interaction assay. To measure the interaction between CTLA-4 protein and CD80 or CD86 protein, cells expressing CTLA-4 were fixed in 4% paraformaldehyde at room temperature for 15 minutes and then incubated with recombinant human CD80-Fc or CD86-Fc chimeric protein (R&D Systems) for 1 hour. The secondary antibody used was an anti-human Alexa Fluor 488 dye conjugate (Life Technologies). The fluorescence intensity of the Alexa Fluor 488 dye was then monitored using a real-time microscope IncuCyte (Essen BioScience, Ann Arbor, Michigan, USA).

[0265] K D Determine and bin by Octet. For high-throughput K D For screening, the antibody ligand was loaded onto the sensor at 20 nM solution. A baseline was established in PBS containing 1 mg / ml bovine serum albumin (assay buffer), and the association step was performed by immersing the sensor in a single concentration of analyte in assay buffer. Dissociation was performed and monitored in fresh assay buffer. All experiments were performed with 1,000 rpm sensor shaking. Data were fitted to a 1:1 binding model using ForteBio's data analysis software to extract association and dissociation rates. The ratio k was used. d / ka Calculate K D . In a typical epitope binning assay, the antigen CTLA-4-His (10 nM) was preincubated with the secondary antibody (10 nM) at room temperature for 1 hour. A control antibody (20 nM) was loaded onto the AMC sensor (ForteBio) and the remaining Fc binding sites on the sensor were blocked with a whole mouse IgG antibody (Jackson ImmunoResearch). The sensor was exposed to the preincubated antigen-secondary antibody mixture. The raw data was processed using ForteBio's Data Analysis Software 7.0, and the competitive binding of the antibody pairs was evaluated. Additional binding of the secondary antibody indicates an unoccupied epitope (non-competitor), while no binding indicates epitope blocking (competitor).

[0266] Glycosylation analysis of CTLA-4. To confirm the glycosylation of the CTLA-4 protein, cell lysates were treated with the enzymes PNGase F, Endo H, and β-glycosidase (New England BioLabs, Ipswich, MA, USA) as described by the manufacturer.

[0267] Statistical Analysis. Data in the bar graphs represent the mean fold change relative to the untreated or control group and the standard deviation of three independent experiments. Statistical analysis was performed using SPSS (Ver. 20, SPSS, Chicago, IL). Correlations between protein expression and BLBC subsets were analyzed using Spearman correlation and Mann-Whitney test. Experimental data were subjected to Student's t test. AP values ​​< 0.05 were considered statistically significant.

[0268] Example 1: Glycosylated CTLA-4 binds to CD80 / 86

[0269] To measure CTLA-4 / CD80 or CD86 interactions, cells expressing CTLA-4 were incubated with recombinant human CD80-Fc or CD86-Fc chimeric proteins (R&D Systems) for 1 hour and then incubated with anti-human Alexa Fluor 488 dye conjugate (Life Technologies). The fluorescence intensity of the Alexa Fluor 488 dye was then monitored using a real-time microscope IncuCyte (Essen BioScience, Ann Arbor, Michigan, USA) according to the manufacturer's instructions. To investigate whether the CTLA-4 glycan structure is important for its binding to CD80 or CD86, we incubated purified CD80- or CD86-Fc with lysates from 293T cells expressing Flag-tagged CTLA-4WT or CTLA-4 2NQ (unglycosylated form) and then analyzed CTLA-4 / CD80 ( Figure 1A -C) and CTLA-4 / CD86 ( Figure 2A As shown in Figures 1 and 2, only CTLA-4WT ( Figure 1A and C, Figure 2A and C) bind to CD80 and CD86, while CTLA4 2NQ ( Figure 1B and Figure 2B ) does not bind to CD80 and CD86. Thus, these results suggest that the integrity of the glycan structure of CTLA-4 is crucial for its interaction with CD80 and CD86.

[0270] Example 2: Production of anti-CTLA-4 antibodies

[0271] A monoclonal antibody specific for glycosylated CTLA-4 was developed. CTLA-4-His was purified from 293F cells that overexpress heavily glycosylated CTLA-4. Hybridomas producing monoclonal antibodies against glycosylated human CTLA-4 were obtained by fusing SP2 / 0 murine myeloma cells with spleen cells isolated from human CTLA-4-immunized BALB / c mice (n=4; Antibody Solutions, Inc., Sunnyvale, CA, USA) according to standard protocols. Prior to fusion, serum from immunized mice was verified for binding to the CTLA-4 immunogen using FACS analysis. More than 3,000 hybridomas producing monoclonal antibodies (mAbs) were generated. The specificity of the antibody-producing hybridomas was again tested.

[0272] Among them, 65 candidate hybridomas producing mAbs were selected by FACS using 293T cells expressing CTLA-4 WT or 2NQ (unglycosylated form) and grown in DCGF medium (Antibody Solutions), and the supernatant containing the monoclonal antibody was concentrated and purified. CTLA-4 was purified from 293T cells overexpressing heavily glycosylated CTLA-4, and more than 39 hybridoma supernatants were screened in a dot blot assay (Figure 3). Some of them, including STC1807 and STC1810, showed sugar-specific binding activity.

[0273] Using the live cell imaging assay Incucyte (Essen Bioscience), the ability of purified mAbs to neutralize or inhibit the interaction between CTLA-4 and CD86 (CTLA4 / CD86 binding interaction) was tested. For this purpose, 293T cells expressing CTLA-4 were seeded in 96-well plates and incubated with CTLA-4 antibodies, recombinant human CD86-Fc protein, and anti-human-Fc Alexa Fluor 488 dye conjugate (Life Technologies). The green fluorescence signal was measured every 2 hours and quantified using the IncuCyte Zoom system (Essen BioScience). The results of this assay showed that of the 65 mAbs tested, only one, called STC1807, completely blocked the binding of CTLA-4 to CD86 ( Figure 4 The sequences of the heavy and light chain variable domains of STC1807 are provided in Table 3.

[0274] To test the specificity of STC1807 for glycosylation of the CTLA-4 antigen, Western blot analysis was performed using fully glycosylated human CTLA-4 protein and non-glycosylated or mono-glycosylated CTLA-4 (i.e., N113Q, N145Q, and 2NQ). STC1807 recognized N113 glycosylation but neither N145 nor 2NQ ( Figure 5 ).

[0275] To determine the K of CTLA-4 antibodies in a high-throughput format DThe antibody ligand was loaded onto the Octet sensor at a 20 nM solution to obtain a specific binding value. A baseline was established in PBS containing 1 mg / mL bovine serum albumin (assay buffer), and the association step was performed by immersing the sensor in a single concentration of analyte in assay buffer. Dissociation was performed and monitored in fresh assay buffer. All experiments were performed while shaking the sensor at 1,000 rpm. The data were fitted to a 1:1 binding model using ForteBio (Menlo Park, CA, USA) data analysis software to extract association and dissociation rates. K was calculated using the ratio kd:ka. D The data are summarized in Figure 6 and Table 5.

[0276] Table 5. Kinetic parameters of anti-CTLA-4 antibodies determined by Octet.

[0277]

[0278]

[0279] Example 3. Neutralizing Activity of Anti-GlycCTLA-4 Antibodies

[0280] To measure the inhibitory effect of antibodies on the interaction between CTLA-4 and CD86, 293T cells expressing CTLA-4 were seeded in 96-well plates and incubated with CTLA-4 antibodies (STC1807, STC1808, or STC1813), recombinant human CD86-Fc protein, and anti-human-Fc Alexa Fluor 488 dye conjugate (Life Technologies). The green fluorescence signal was measured every 2 hours and quantified using the IncuCyte Zoom system (Essen BioScience). Figure 7A Binding of CD86-Fc to cells expressing CTLA-4 in the presence of the anti-glycCTLA-4 antibody STC1807 at concentrations ranging from 0.125 μg / mL to 8 μg / mL is shown. Figure 7B STC1807 was shown to effectively inhibit the CTLA4 / CD86 interaction in this assay, with neutralizing activity (reduced half-maximal effective concentration EC 50 ) was 2.189 μg / mL. Figure 7C Binding of CD86-Fc to cells expressing CTLA-4 in the presence of the anti-glycCTLA-4 antibody STC1808 at concentrations ranging from 0.125 μg / mL to 8 μg / mL is shown. Figure 7DBinding of CD86-Fc to cells expressing CTLA-4 in the presence of the anti-glycCTLA-4 antibody STC1813 at concentrations ranging from 0.125 μg / mL to 8 μg / mL is shown.

[0281] When the human chimera of STC1807 (hSTC1807) was compared with the FDA-approved antibody ipilimumab, hSTC1807 showed comparable neutralizing effects. Figure 8A Binding of CD86-Fc to cells expressing CTLA-4 over 24 hours in the presence of human chimeric antibody hSTC1807 at concentrations of 0.125 μg / mL to 8 μg / mL is shown. Figure 8B STC1807 was shown to be a potent inhibitor of CTLA-4 / CD86 interaction in this assay, with a neutralizing activity of 0.3313 μg / mL found. Figure 8C Binding of CD86-Fc to cells expressing CTLA-4 over 24 hours in the presence of ipilimumab at concentrations ranging from 0.125 μg / mL to 8 μg / mL is shown. Figure 8D Ipilimumab was shown to effectively inhibit the CTLA-4 / CD86 interaction in this assay, with a neutralizing activity of 0.3068 μg / mL found.

[0282] Example 4. Antibody Binding Assay of STC1807 and Ipilimumab

[0283] In a typical epitope binning assay, the antigen CTLA-4-His (10 nM) was pre-incubated with the secondary antibody (10 nM) at room temperature for 1 hour. A control antibody (20 nM) was loaded onto an AMC sensor (ForteBio), and the remaining Fc-binding sites on the sensor were blocked with intact mouse IgG antibodies (Jackson ImmunoResearch, West Grove, PA, USA). The sensor was exposed to the pre-incubated antigen-secondary antibody mixture. Raw data was processed using ForteBio data analysis software 7.0, and competitive binding of the antibody pairs was assessed. Additional binding of the second antibody indicated an unoccupied epitope (non-competitor), and no binding indicated epitope blocking (competitor). Figure 9A Additional binding of Ipilimumab (secondary antibody pre-incubated with CD86-His) on the STC1807 loaded sensor is shown. Figure 9B Additional binding of STC1807 (secondary antibody pre-incubated with CD86-His) on the ipilimumab-loaded sensor is shown, suggesting a different binding epitope. These results suggest that STC1807 and ipilimumab bind to different epitopes of CTLA-4.

[0284] Example 5. Determination of KD by Biocore Assay

[0285] The binding affinity (reduced equilibrium dissociation constant [KD] value) of STC1807 was compared to that of the FDA-approved anti-CTLA4 antibody ipilimumab using a Biacore binding assay. KD determinations were performed by surface plasmon resonance using a Biacore X100 instrument (GE Healthcare, Uppsala, Sweden). Mouse IgG1 was immobilized on a research-grade CM5 chip using standard procedures and the antibody was plated on an HBS-EP + Buffer was passed over the chip at 2 μg / mL. Next, six concentrations of CTLA4 (each diluted 2-fold) were passed over the chip. Sensorgram data were analyzed using Biacore X100 Evaluation Software, version 2.0.1, using a 1:1 binding kinetics analysis. STC1807 was found to have a KD of 0.47 nM, indicating very strong binding affinity, while ipilimumab exhibited lower affinity for the CTLA4 protein (KD of 13.4 nM) ( FIG10 and Table 6 ).

[0286] Table 6. BIACORE assay of anti-glyc-CTLA-4 antibody binding to CTLA-4.

[0287] Antibody antigen <![CDATA[k a (1 / Ms)]]> <![CDATA[k d (1 / s)]]> <![CDATA[K D (M)]]> STC1807 CTLA-4-His <![CDATA[4.63X10 5 ]]> <![CDATA[2.17X 10 -4 ]]> <![CDATA[4.69X10 -10 ]]> Ipilimumab CTLA-4-His <![CDATA[2.04X10 5 ]]> <![CDATA[2.73X10 -3 ]]> <![CDATA[1.34X10 -8 ]]>

[0288] Example 6. Effect of anti-Glyc antibodies on T cell proliferation

[0289] To evaluate the efficacy of STC1807 in vitro, mixed lymphocyte reactions (MLRs) were performed using dendritic cells (DCs, induced from PBMCs of allogeneic donors (Immunospot #CTL-CP1)) cultured for 7 days in the presence of IL-4 (500 U / mL) and GM-CSF (250 U / mL). DCs were isolated using a human pan-DC enrichment kit (Miltenyi Biotech #130-100-777) according to the manufacturer's recommendations and used to stimulate allogeneic memory or naive CD4 + T-cells were also enriched from PBMCs of another allogeneic donor (Immunospot #CTL-CP1) using CD4 microbeads (Miltenyi Biotech #130-045-101). + T cells. DC (1×10 4 DC / well) in a 96-well flat-bottom plate (Nunc) with 1×10 5T-cells / well were co-cultured in culture medium in the presence of STC1807. After 5 days of culture, the concentrations of IFN-γ and IL-2 in the culture supernatant were determined by cytokine ELISA kits (BioLegend) according to the manufacturer's instructions. As shown in Figure 11, in the presence of STC1807, the secretion of T cell proliferation markers IFN-γ and IL-2 was significantly increased.

[0290] Example 7: Antibody Humanization - Framework Region

[0291] As noted above, for some purposes, including, for example, for the in vivo treatment of human diseases, it is preferred to adopt humanized derivatives of mouse monoclonal antibodies. In order to form such humanized antibodies, first the framework sequence (" parent (Parental) " sequence) of mouse monoclonal antibodies is compared with the framework sequence of one group of " acceptor (Acceptor) " human antibodies to identify the difference in the framework sequence. Humanization is achieved by replacing the mismatched framework residues between the parent and the acceptor. About future back mutations, analysis is performed at the displacement of potential important positions, such as those in Vernier district, VH / VL interchain interface or CDR specification category determination position (see, Foote, J. et al., J.Molec.Biol.224:487-499 (1992)).

[0292] The Conserved Domain Database (COD) (Marchler-Bauer, et al. (2011) Nucleic Acids Res. 39: D225-D229) can be used to determine the domain content of each amino acid chain and the approximate boundaries of each domain. Variable domain boundaries can be precisely determined along with the boundaries of the CDRs according to several commonly used definitions (Kabat, E.A., et al. (1991) "Sequences of Proteins of Immunological Interest," 5th ed. NIH Publication No. 91-3242; Chothia, C., et al., J. Mol. Biol. 196: 901-917 (1987); Honegger, A., et al., J. Molec. Biol. 309(3): 657-670 (2001)).

[0293] Use MAFFT (Katoh, K. et al., Nucleic Acids Res.30:3059-3066 (2002)) to produce the multiple alignment of parental sequence and mouse and people's germline sequence, and the entries in each alignment are sorted according to the sequence identity with the parental sequence.By with 100% sequence identity clustering and excluding redundant entries, the reference set is simplified to the unique set of sequence.

[0294] Optimal receptor framework selection is based on the entire parent antibody sequence identity for the receptor in the framework of the two chains; However, the positions constituting the VH / VL interchain interface are of particular interest. In addition, the CDR loop lengths and CDR positions responsible for the discrete canonical structure sets defined for the five CDRs (Chothia, C. et al., J. Mol. Biol. 196: 901-917 (1987); Martin, AC et al., J. Molec. Biol 263: 800-815 (1996); Al-Laziniki, B. et al., J. Molec. Biol. 273: 927-948 (1997)) were compared with the germline to determine which germline frameworks have identical interface residues and are known to support similar CDR loop conformations.

[0295] Based on the sequence alignment of the parent antibody with the human germline, the closest matching entry was identified. The preferred human germline was selected based on the following criteria: (1) sequence identity throughout the framework; (2) identical or compatible interchain interface residues; (3) support for loops with the canonical conformation of the parent CDRs; (4) the combination of heavy and light germlines found in the expressed antibody; and (5) the presence of N-glycosylation sites that must be removed.

[0296] A structural model of the Fv region of a humanized antibody was generated. Candidate structural template fragments of the FR and CDR and complete Fv were aligned based on their sequence identity to the target and qualitative crystallographic measurements of the template structure (such as resolution in angstroms). (units) scoring, ranking, and selection from the antibody database.

[0297] In order to structurally align the CDR with the FR template, the 5 residues on either side of the CDR are included in the CDR template. Based on the overlapping segments and the generated structural sequence alignment, the alignment of the fragments is generated. By MODELLER (Sall, A. et al.; J.Molec.Biol.234:779-815 (1993)), the template fragments and the alignment are processed. This scheme creates the conformational constraints derived from a set of aligned structural templates. By conjugate gradient and simulated annealing optimization procedures, a set of structures that meet the constraints is created. Model structures are selected from this set based on energy scores, which are derived from the scoring of the protein structure and the satisfaction of the conformational constraints. The model is checked and the side chains of the different positions between the target and the template are optimized using a side chain optimization algorithm and minimized energy. A set of visualization and computational tools is used to evaluate CDR conformational variability, local stacking, and surface analysis to select one or more preferred models.

[0298] The structural model of the parental antibody is built and defects are checked, such as stress, bond angle or dihedral angle in the bad, bond length of atomic stacking. These defects may indicate the potential problem of antibody structure stability. Modeling scheme is intended to minimize such defects. The initial structural model of humanized Fv contains all safe displacements (i.e., displacement that will not affect binding affinity or stability) and cautious displacement (i.e., carrying out position displacement, but the position may be important for binding affinity). Do not change the displacement at the risk-related position that is considered to reduce with binding affinity or stability reduction. Template search and selection are performed separately with parental template search, so as to create good independent model, rather than the close matching variant model of parent. Along with the assessment of carrying out potential displacement, the model is updated to reflect the influence of preferred displacement and back mutation.

[0299] Example 8: Antibody humanization-constant region:

[0300] The variable regions of the STC1807 heavy chain (VH) and its kappa light chain (VL) were modified by replacing the mouse constant regions with human IgG1 constant regions (CH1-CH3) in pFUSEss-CHIg-hG1 and pFUSEss-CLIg-hK vectors (Invivogen), respectively. The heavy and light chimeric constructs were transfected into 293F suspension cells at a 1:1 ratio for 5 days. The chimeric antibody (hSTC1807) was purified by protein A affinity column on HPLC.

[0301] Throughout this application, reference has been made to various publications. The disclosures of these publications are hereby incorporated herein by reference in their entirety for a more comprehensive description of the prior art to which this disclosure relates. Although examples of certain specific embodiments are provided herein, it will be apparent to those skilled in the art that various changes and modifications may be made. Such modifications are also intended to fall within the scope of the appended claims.

Claims

1. An isolated monoclonal antibody that selectively binds to glycosylated CTLA-4 relative to the unglycosylated form of CTLA-4, the monoclonal antibody comprising: V H domain, the V H The domain comprises: CDR H1 shown in SEQ ID NO: 6, CDR H2 shown in SEQ ID NO: 7, and CDR H3 shown in SEQ ID NO: 8 according to the Chothia numbering system, and V L domain, the V L The domain comprises: CDR L1 as set forth in SEQ ID NO: 16, CDR L2 as set forth in SEQ ID NO: 17, and CDR L3 as set forth in SEQ ID NO: 18 according to the Chothia numbering system; or V H domain, the V H The domain comprises: CDR H1 shown in SEQ ID NO: 9, CDR H2 shown in SEQ ID NO: 10, and CDR H3 shown in SEQ ID NO: 8 according to the AbM numbering system, and V L domain, the V L The domain comprises: CDR L1 shown in SEQ ID NO: 16, CDR L2 shown in SEQ ID NO: 17, and CDR L3 shown in SEQ ID NO: 18 according to the AbM numbering system; or V H domain, the V H The domain comprises: CDR H1 shown in SEQ ID NO: 11, CDR H2 shown in SEQ ID NO: 12, and CDR H3 shown in SEQ ID NO: 8 according to the Kabat numbering system, and V L domain, the V L The domain comprises: CDR L1 as shown in SEQ ID NO: 16, CDR L2 as shown in SEQ ID NO: 17, and CDR L3 as shown in SEQ ID NO: 18 according to the Kabat numbering system; or V H domain, the V H The domain comprises: CDR H1 shown in SEQ ID NO: 13, CDR H2 shown in SEQ ID NO: 14, and CDR H3 shown in SEQ ID NO: 15 according to the Contact numbering system, and V L domain, the V L The domain comprises: CDR L1 shown in SEQ ID NO: 19 according to the Contact numbering system, CDR L2 shown in SEQ ID NO: 20, and CDR L3 shown in SEQ ID NO:

21.

2. The isolated monoclonal antibody of claim 1, wherein the antibody selectively binds to CTLA-4 glycosylated at position N113 relative to unglycosylated CTLA-4.

3. The isolated monoclonal antibody of claim 1, wherein the antibody has a binding affinity for glycosylated CTLA-4 of 0.1-10 nM, inclusive.

4. The isolated monoclonal antibody of claim 1, wherein the antibody binds to the K of glycosylated CTLA-4. d is relative to the K of ipilimumab d One tenth of.

5. The isolated monoclonal antibody of claim 1, wherein the antibody blocks the binding of CTLA-4 to CD86 with a neutralizing activity that is 2-10 times greater than the EC50 exhibited by the antibody binding to cells expressing unglycosylated CTLA-4 in a live cell imaging system.

6. The isolated monoclonal antibody of claim 1, wherein the antibody masks glycosylation of CTLA-4 at N113.

7. The isolated monoclonal antibody according to claim 1, wherein V H The domain has the amino acid sequence of SEQ ID NO: 3, and V L Having the amino acid sequence of SEQ ID NO:

5.

8. The isolated monoclonal antibody according to claim 1, wherein V H The domain has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

3.

9. The isolated monoclonal antibody according to claim 1, wherein V L The domain has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

5.

10. The isolated monoclonal antibody of claim 1, which has human framework regions.

11. The isolated monoclonal antibody of claim 1 , which has a heavy or light chain human framework region comprising 1, 2, 3, 4, 5, or 6 amino acid substitutions.

12. The isolated monoclonal antibody of claim 1, comprising a human constant domain.

13. The isolated monoclonal antibody of claim 1, wherein the antibody is IgG, IgM, or IgA.

14. The isolated monoclonal antibody of claim 1, wherein the antibody is a Fab', a F(ab')2, a monovalent scFv, or a bivalent scFv.

15. The isolated monoclonal antibody of claim 1, wherein the antibody is a human antibody or a humanized antibody.

16. A conjugate, wherein the isolated monoclonal antibody according to any one of claims 1 to 15 is conjugated to an imaging agent or a radionuclide.

17. A composition comprising the isolated monoclonal antibody of any one of claims 1 to 15 or the conjugate of claim 16 and a pharmaceutically acceptable carrier.

18. Use of the isolated monoclonal antibody of any one of claims 1 to 15 or the conjugate of claim 16 in the preparation of a pharmaceutical composition for treating a subject having cancer, wherein the cancer is breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, brain cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer.

19. The use according to claim 18, wherein the isolated monoclonal antibody or the conjugate is formulated for topical administration.

20. The use of claim 18, wherein the isolated monoclonal antibody or the conjugate is formulated for intravenous, intradermal, intratumoral, intramuscular, intraperitoneal, or subcutaneous administration.

21. A method for assessing CTLA-4 glycosylation for non-diagnostic and non-therapeutic purposes, the method comprising contacting a sample containing CTLA-4 with the isolated monoclonal antibody of any one of claims 1-15 or the conjugate of claim 16.

22. The method of claim 21, wherein the sample is a cell sample.

23. Use of the isolated monoclonal antibody of any one of claims 1 to 15 or the conjugate of claim 16 in the preparation of a kit for assessing CTLA-4 glycosylation.

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