Antibodies specific for glycosylated LAG3 and methods of use thereof

Abstract

Antibodies that selectively bind to glycosylated LAG3 are provided compared to non-glycosylated LAG3. In some embodiments, LAG3 polypeptides that include glycosylated amino acid positions are also provided. Methods for producing and using such antibodies and polypeptides (e.g., for cancer treatment) are also provided.

Description

[Technical Field]

[0001] Array List This application contains a Sequence Listing which has been filed electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on September 22, 2020, is named 24258_0014P1_Sequence_Listing.txt, and is 12,288 bytes in size.

[0002] The present invention relates generally to the fields of medicine, molecular biology, and oncology. More specifically, the present invention relates to antibodies for treating cancer. [Background technology]

[0003] Perpetuating T cell activation has dramatically transformed the treatment of a wide range of malignancies, including the development of ipilimumab, a CTLA-4-specific antibody and the first FDA-approved checkpoint blocker targeting T cell responses, enabling the treatment of metastatic melanoma (Hodi et al., The New England Journal of Medicine 363, 711-723 (2010)).

[0004] Despite the impressive impact of CTLA-4 and PD1-PDL1-targeted cancer immunotherapy, the majority of patients with many tumor types fail to respond. Consequently, focus has shifted to targeting alternative inhibitory receptors (IRs) and suppressive mechanisms within the tumor microenvironment.

[0005] Upregulated expression of IR is essential for balancing costimulatory receptor activity and limiting T cell activation, thereby preventing autoimmunity, autoinflammation, and tissue damage. However, tumors can hijack these so-called immune checkpoint mechanisms to protect against antitumor immune responses elicited by CD4+ and CD8+ T cells. Initially described in the context of chronic viral infections, tolerized antigen-specific T cells exhibit elevated expression of IR within the tumor microenvironment, corresponding to functional unresponsiveness measured as reduced proliferation and cytokine release. The resulting immune tolerance, along with the recruitment of regulatory T cells, poses multiple barriers to effective tumor elimination. Thus, recent cancer immunotherapy approaches have aimed to reverse this exhaustion by targeting IR to "release the brakes" and allow cytotoxic T cells to be reactivated to attack tumors.

[0006] Lymphocyte-activation gene-3 (LAG3; CD223) suppresses T cell activation and cytokine secretion, thereby potentially acting as a co-IR to ensure immune homeostasis. LAG-3 exerts differential inhibitory effects on various types of lymphocytes. Meanwhile, LAG-3 can effectively prevent the pathogenesis of autoimmune disorders. The precise molecular mechanisms underlying LAG-3 signaling and its interaction with other immune checkpoints remain largely unknown. However, LAG-3 exhibits significant synergy with PD-1 in multiple settings to inhibit immune responses. LAG3 can also be extensively glycosylated. Long et al., “The promising immune checkpoint LAG-3: from tumor microenvironment to cancer immunotherapy,” Genes & Cancer, Vol. 9 (5-6), May 2018.

[0007] On this basis, glycosylated LAG3-specific antibodies may be useful in cancer therapy. Summary of the Invention [Means for solving the problem]

[0008] Provided herein are isolated monoclonal antibodies that selectively bind to glycosylated LAG3 (anti-glycLAG3 antibodies herein). In some embodiments, the antibodies selectively bind to LAG3 glycosylated at positions N188, N250, N256, and / or N343 compared to unglycosylated LAG3. In some embodiments, the antibodies increase the secretion of IFN-γ and / or IL-2. In some embodiments, the antibodies increase T cell proliferation. In some embodiments, the antibodies block LAG3 binding to one or more of Gal-3, MHCII, LSECtin, and CD3.

[0009] In some embodiments, the isolated antibody selectively binds to human LAG3 with N188 glycosylation. In some embodiments, the isolated antibody selectively binds to human LAG3 with N250 glycosylation. In some embodiments, the isolated antibody selectively binds to human LAG3 with N256 glycosylation. In some embodiments, the isolated antibody selectively binds to human LAG3 with N343 glycosylation. In some embodiments, the isolated antibody selectively binds to human LAG3 with N188 and N250 glycosylation. In some embodiments, the isolated antibody selectively binds to human LAG3 with N188 and N256 glycosylation. In some embodiments, the isolated antibody selectively binds to human LAG3 with N188 and N343 glycosylation. In some embodiments, the isolated antibody selectively binds to human LAG3 with N250 and N256 glycosylation. In some embodiments, the isolated antibody selectively binds to human LAG3 with N250 and N343 glycosylation. In some embodiments, the isolated antibody selectively binds to human LAG3 with N256 and N343 glycosylation. In some embodiments, the isolated antibody selectively binds to human LAG3 with N188, N250, and N256 glycosylation. In some embodiments, the isolated antibody selectively binds to human LAG3 with N188, N250, and N343 glycosylation. In some embodiments, the isolated antibody selectively binds to human LAG3 with N188, N256, and N343 glycosylation. In some embodiments, the isolated antibody selectively binds to human LAG3 with N250, N256, and N343 glycosylation. In some embodiments, the isolated antibody selectively binds to human LAG3 having N188, N250, N256 and N343 glycosylation.

[0010] In certain aspects, an anti-glycoLAG3 antibody binds to LAG3 and can mask or screen one or more glycosylation motifs, blocking binding or other interaction of the molecule with the motifs and blocking glycosylation of LAG3 at those glycosylation sites. In particular embodiments, the anti-glycoLAG3 antibody masks the glycosylation sites at one or both of N188, N250, N256, and N343.

[0011] In some embodiments, the antibody selectively binds to one or more glycosylation motifs. In some embodiments, the antibody binds to glycopeptides containing glycosylation motifs and adjacent peptides. In some embodiments, the antibody binds to peptide sequences located three-dimensionally near one or more of the glycosylation motifs.

[0012] In certain embodiments, the binding affinity of the anti-glycolyzed LAG3 antibody for glycosylated LAG3 is 0.1 to 13 nM, or 0.1 to 10 nM, or 0.1 nM to 5 nM, inclusive. In certain embodiments, the antibody exhibits a K d K smaller than half of d In a further embodiment, the K d K less than one-tenth of d The antibody binds to glycosylated LAG3.

[0013] In particular embodiments, heavy and light chain variable domains (mature V without any signal sequence) having the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 5, respectively. H and V LThe anti-glycoLAG3 monoclonal antibody STC1317 having the amino acid sequence of the STC1317 region, as well as antigen-binding portions thereof, and humanized chimeric forms thereof, is presented herein. Anti-glycoLAG3 antibodies that compete with the STC1317 MAb for binding to glycosylated LAG3 and / or bind to the same epitope as STC1317 are presented herein.

[0014] The nucleic acid (DNA) sequences and corresponding amino acid sequences of the heavy and light chain variable (V) domains of the STC1317 MAb are presented and shown in Table 3 below. SEQ ID NO: 2 and SEQ ID NO: 3 represent STC1317V H SEQ ID NO: 4 and SEQ ID NO: 5 are the nucleotide and amino acid sequences of the mature form of the STC1317 kappa light chain variable domain. Table 4 presents the heavy and light chain V domain CDRs of STC1317 according to Chothia, AbM, Kabat, and Contact.

[0015] In one embodiment, the anti-glycLAG3 antibody that specifically and preferentially binds to glycosylated LAG3 is VGA having the amino acid sequence of SEQ ID NO:3. H domain, and / or V having the amino acid sequence of SEQ ID NO: 5 L In one embodiment, the anti-glycOLAG3 antibody comprises the V domain of SEQ ID NO: 3 for specific binding to glycosylated LAG3. H Domain and V of SEQ ID NO:5 L In other embodiments, the anti-glycLAG3 antibody competes with an antibody comprising 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 V that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:5. L These anti-glycLAG3 antibodies may be chimeric but also contain human constant domains, for example, derived from human IgG1, IgG2, IgG3, or IgG4.

[0016] In one embodiment, an anti-glycLAG3 antibody that specifically and preferentially binds glycosylated LAG3 is a V antibody that comprises CDRs 1-3 of Chothia having the amino acid sequences of SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively; CDRs 1-3 of AbM having the amino acid sequences of SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:8, respectively; CDRs 1-3 of Kabat having the amino acid sequences of SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:8, respectively; or CDRs 1-3 of Contact having the amino acid sequences of SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively, or a combination thereof. H In one embodiment, the anti-glycolyzed LAG3 antibody comprises a V domain that, for specific binding to glycosylated LAG3, comprises CDRs 1-3 of Chothia having the amino acid sequences of SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively; CDRs 1-3 of AbM having the amino acid sequences of SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:8, respectively; CDRs 1-3 of Kabat having the amino acid sequences of SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:8, respectively; or CDRs 1-3 of Contact having the amino acid sequences of SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively, or a combination thereof. H 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-glycLAG3 antibody comprises CDRs H1, H2, and H3 having amino acid sequences with 1, 2, 3, 4, or 5 amino acid substitutions in one, two, or three of the CDRs of Chothia having the amino acid sequences of SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively; or the CDRs of AbM having the amino acid sequences of SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:8, respectively; or the CDRs of Cabat having the amino acid sequences of SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:8, respectively; or the CDRs of Contact having the amino acid sequences of SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively. H The anti-glycLAG3 antibody has the V domain. H and V L Both domains may have amino acid substitutions within the CDRs. In some embodiments, the amino acid substitutions are conservative substitutions.

[0018] Preferably, the antibody has human framework regions, ie is a humanized form of STC1317, and optionally includes human constant domains, for example from human IgG1, IgG2, IgG3, or IgG4.

[0019] It will be appreciated by those skilled in the art that one or more amino acid substitutions may be made in the CDR and / or framework regions of the humanized antibody to improve binding affinity or other parameters. In some embodiments, the anti-glycoLAG3 antibody has the V-type or V-type amino acid substitutions described above for specific binding to glycosylated LAG3. H and V L In one embodiment, the anti-glycoLAG3 antibody competes with glycosylated LAG3 with a K of 0.1-10 nM or 1-20 nM, inclusive. d In some embodiments, the anti-glycolLAG3 antibody binds at a K, as indicated by the antibody binding to unglycosylated LAG3. d K is less than half of dIn one embodiment, the anti-glycoLAG3 antibody binds to glycosylated LAG3 at a K d K less than one-fifth of d In one embodiment, the anti-glycolyzed LAG3 antibody binds to glycosylated LAG3 protein at a K d K less than one-tenth of d It binds to glycosylated LAG3 protein at

[0020] In one embodiment, the antibody is directly or indirectly detectable by a fluorescent label or marker. In one embodiment, the antibody is directly labeled with a fluorescent label or marker, such as FITC, or is detected by a fluorescently labeled secondary antibody. In one embodiment, the binding affinity of STC1317 MAb, or a chimeric or humanized form thereof, for glycosylated LAG3 is 0.1-13 nM or 1-5 nM, inclusive.

[0021] In embodiments, the anti-glycOLAG3 antibody is selected from the group consisting of the antibodies V, ... H and V L The antibodies compete with antibodies comprising the K domain and CDRs therein. Preferably, these antibodies have human framework regions, i.e., are humanized forms of STC1317, and optionally include human constant domains, e.g., from human IgG1, IgG2, IgG3, or IgG4. Those skilled in the art will recognize that one or more amino acid substitutions can be made within the CDR or framework regions of the humanized antibody to improve binding affinity or other parameters. In some embodiments, the anti-glycosylated LAG3 antibody has a K β -binding domain exhibited against non-glycosylated LAG3. d K smaller than half of d In some embodiments, the anti-glycoLAG3 antibody binds to glycosylated LAG3 at a K d K smaller than half of dIn one embodiment, the anti-glycoLAG3 antibody binds to glycosylated LAG3 at a K, as indicated by the antibody binding to unglycosylated LAG3. d K less than one-fifth of d In one embodiment, the anti-glycolyzed LAG3 antibody binds to glycosylated LAG3 protein at a K d K less than one-tenth of d In one embodiment, the antibody binds to glycosylated LAG3 protein at 1 mm for binding to cells expressing unglycosylated LAG3 in a cell flow cytometry binding assay. 2 3-fold, 5-fold, 10-fold, 20-fold, or 50-fold greater than the green counts per mm for cells expressing wild-type LAG3 2 The binding affinity is expressed as green counts per 1000 ribonucleotides. In one embodiment, the antibody is directly or indirectly detectable by a fluorescent label or marker. In one embodiment, the antibody is directly labeled with a fluorescent label or marker, such as FITC. In one embodiment, the binding affinity of the STC1317 MAb, or a binding domain thereof, or a humanized or chimeric form thereof, for glycosylated STC1317 is 0.1-13 nM, or 0.1-10 nM, or 0.1-5 nM, inclusive.

[0022] In some embodiments, the antibody is recombinant. In certain embodiments, the antibody is IgG, IgM, IgA, or an antigen-binding fragment thereof. In other embodiments, the antibody is a Fab', F(ab')2, F(ab')3, monovalent scFv, bivalent scFv, bispecific antibody, bispecific scFv, or single domain antibody. In some embodiments, the antibody is a human or humanized antibody. In further embodiments, the antibody is conjugated to an imaging agent, a chemotherapeutic agent, a toxin, or a radionuclide.

[0023] In further embodiments, provided herein are compositions comprising an antibody of multiple embodiments (e.g., an antibody that selectively binds to glycosylated LAG3 compared to non-glycosylated LAG3) in a pharmaceutically acceptable carrier.

[0024] In yet a further embodiment, an isolated polypeptide is provided comprising a fragment of at least seven (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) consecutive amino acids of human LAG3, including at least one amino acid corresponding to positions N188, N250, N256, or N343 of human LAG3. In further aspects, the isolated polypeptide of several embodiments comprises a fragment of at least seven (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) consecutive amino acids of human LAG3, including at least one amino acid corresponding to positions N188, N250, N256, or N343 of human LAG3, wherein at least one of the amino acids corresponding to positions N188, N250, N256, or N343 of human LAG3 is glycosylated. In some aspects, the polypeptide of several embodiments is fused or conjugated to an immunogenic polypeptide (e.g., keyhole limpet hemocyanin, KLH). In certain aspects, the polypeptide further comprises a Cys residue at the C-terminus or N-terminus. For example, in some aspects, the polypeptide is conjugated to the immunogenic polypeptide by a disulfide bond at the Cys residue.

[0025] In yet a further embodiment, a composition is provided comprising a polypeptide comprising a fragment of at least seven (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) consecutive amino acids of human LAG3, including at least one amino acid corresponding to positions N188, N250, N256, or N343 of human LAG3, wherein at least one of the amino acids corresponding to positions N188, N250, N256, or N343 of human LAG3 is glycosylated, and 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.

[0026] In still further embodiments, provided herein are methods for treating a subject having cancer, the methods comprising administering to the subject an effective amount of an antibody or isolated polypeptide of embodiments. In certain aspects, the method for treating cancer comprises administering to the subject an effective amount of a polypeptide (e.g., a glycosylated LAG3 polypeptide). In a further aspect, the method for treating cancer comprises administering to the subject an effective amount of an antibody of embodiments (e.g., an antibody that selectively binds to glycosylated LAG3 compared to non-glycosylated LAG3), such as, but not limited to, a humanized or chimeric form of STC1317, or an antibody that competes with STC1317 for binding to glycosylated LAG3. In some aspects, the cancer is breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, skin cancer, brain tumor, 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 embodiments, the cancer is selected from the group consisting of adrenal gland cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, adult brain / CNS tumors, pediatric brain / CNS tumors, 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's family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), gestational trophoblastic disease, Hodgkin's disease, Kaposi's sarcoma, renal cancer, laryngeal or hypopharyngeal cancer, leukemia (e.g., adult acute lymphoblastic (ALL), acute myeloid (AML), chronic lymphocytic (CLL), chronic myeloid (CML), chronic myelomonocytic (CMML), childhood leukemia), The cancer may be liver cancer, lung cancer (e.g., non-small cell, small cell), pulmonary carcinoid tumor, lymphoma, cutaneous lymphoma, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity cancer, paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, pediatric non-Hodgkin's lymphoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma (e.g., adult soft tissue cancer), skin cancer (e.g., basal and squamous cell, melanoma, Merkel cell), small intestine cancer, gastric cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, or Wilms' tumor. In certain embodiments, the antibody is in a pharmaceutically acceptable composition. In further embodiments, the antibody is administered systemically. In particular embodiments, the antibody is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or topically.

[0027] In some embodiments, the method further comprises administering at least a second anti-cancer therapy to the subject. In certain embodiments, the second anti-cancer therapy is surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy.

[0028] In still further embodiments, provided herein are methods for assessing LAG3 glycosylation, N-linked glycosylation, or N-glycosylation, comprising contacting a LAG3-containing sample with an antibody of several embodiments (e.g., the antibody selectively binds glycosylated LAG3 compared to non-glycosylated LAG3). In some aspects, the method is an in vitro method. In certain aspects, the sample is a cell sample.

[0029] In yet a further embodiment, a method of making an antibody is provided, comprising administering to an animal a polypeptide according to embodiments (e.g., a polypeptide having a fragment of at least 7 consecutive amino acids of human LAG3, comprising at least one amino acid corresponding to positions N188, N250, N256, or N343 of human LAG3, wherein at least one of the amino acids corresponding to positions N188, N250, N256, or N343 of human LAG3 is glycosylated) and isolating the antibody from the animal. For example, the animal may be a mouse, rat, rabbit, or human. In certain aspects, the method further comprises identifying CDRs of the antibody and humanizing sequences surrounding the CDRs to generate a humanized antibody. In yet a further aspect, the method comprises recombinantly expressing the humanized antibody. Thus, in a further embodiment, provided herein is an isolated antibody generated by the above method. Thus, in some embodiments, the present specification provides isolated antibodies that selectively bind to a polypeptide of multiple embodiments (e.g., a polypeptide comprising a fragment of at least 7 consecutive amino acids of human LAG3, including at least one amino acid corresponding to positions N188, N250, N256, or N343 of human LAG3, wherein at least one of the amino acids corresponding to positions N188, N250, N256, or N343 of human LAG3 is glycosylated) compared to unglycosylated LAG3. [Brief explanation of the drawings]

[0030] [Figure 1] Figure 1 shows that LAG3 is highly glycosylated. SDS-PAGE analysis of histidine-tagged LAG3 protein. Protein samples (0.5 μg) were pretreated with or without PNGase F for 1 hour. Proteins were separated on a 4-12% NuPAGE gel and stained with Coomassie Brilliant Blue. [Figure 2] (Figures 2A and 2B) Development of monoclonal antibodies specific for glycosylated LAG3. Dot blot analysis of anti-LAG3 monoclonal antibodies using LAG3-Fc or PNGase F-treated LAG3-Fc (0.5 μg per well). (A) Dot blot membrane illustrating the glycospecific binding activity of several antibodies, including STC1317. LAG3-Fc and PNGase F-treated LAG3-Fc were blotted onto a nitrocellulose membrane. Anti-LAG3 mAb (0.5 μg purified) and an anti-mouse IgG secondary antibody were used to detect Ag-Ab interactions. For controls, anti-LAG3 antibodies purified from the culture supernatants of fusion hybridomas LAG3B (Balb / c-derived) and LAG3N (NZW-derived), respectively, were used. (B) Sample layout of the corresponding 96-well dot blot assay plate. [Figure 3-1] (FIG. 3A) Sensorgrams of anti-LAG3 antibodies analyzed by Octet. Summary of data obtained from high-throughput KD screening. Data were fit to a 1:1 binding model to extract association and dissociation rates. KD was calculated using the ratio of kd:ka. The graph shows the response over time, representing the progression of the interaction. Sensorgram of anti-LAG3 antibody STC1301-1316 (A). [Figure 3-2](Figure 3B) Sensorgrams of anti-LAG3 antibodies analyzed by Octet. Summary of data obtained from high-throughput KD screening. Data were fit to a 1:1 binding model to extract association and dissociation rates. KD was calculated using the ratio of kd:ka. The graph shows the response over time, representing the progression of the interaction. Sensorgrams of anti-LAG3 antibodies STC1317-STC1322 (B). [Figure 4] Figure 1 shows the KD determination of STC1317 by Biacore. Determination of the binding affinity (reduced equilibrium dissociation constant [KD] value) of STC1317 using a Biacore binding assay. Six concentrations of LAG3, each a two-fold dilution, were passed over the chip. The graph depicts the response over time, showing the progression of the interaction for STC1317. [Figure 5] (FIGS. 5A and 5B) Increased secretion of IFN-γ and IL-2 in the presence of STC1317. Effect of STC1317 on T cell proliferation (T) in response to stimulator cells (DCs, dendritic cells). Graphs represent IL-2 (A) and IFN-γ (B) cytokine levels in the presence of STC1317 and control mouse IgG. Cytokines were quantified in supernatants by ELISA on day 5. DETAILED DESCRIPTION OF THE INVENTION

[0031] N-glycosylation is a post-translational modification that is initiated in the endoplasmic reticulum (ER) and subsequently processed in the Golgi (Schwarz & Aebi, Current Opinion in Structural Biology 21, 576-582 (2011)). This type of modification is primarily catalyzed by membrane-bound oligosaccharyltransferase (OST) complexes that transfer 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 groups of glycotransferases and glycosidases, respectively, that tightly control the N-glycosylation cascade in a cell- and location-dependent manner.

[0032] Galectin-3 (Gal-3) functions as a regulator of antigen-specific T cell activation. LAG3 expression correlates with Gal-3, and functional LAG3 is essential for Gal-3-mediated inhibition of cytotoxic T lymphocyte immune responses. LAG3 can be extensively glycosylated. Glycosylation is considered a target for Gal-3 binding. See page 182 of Long et al. (2018). Therefore, anti-glycoLAG3 antibodies can exert enhanced Gal-3 inhibitory effects compared to more general LAG3 antibodies.

[0033] As used herein, and unless otherwise specified, the term "lymphocyte-activation gene-3," or "LAG3," refers to LAG3 of any vertebrate origin, including mammals, such as primates (e.g., humans, cynomolgus monkeys (cynos)), dogs, and rodents (e.g., mice and rats). Unless otherwise specified, LAG3 also includes various LAG3 isoforms, related LAG3 polypeptides, including SNP variants thereof, as well as different modified forms of LAG3, including, but not limited to, phosphorylated LAG3, glycosylated LAG3, and ubiquitinated LAG3.

[0034] A representative amino acid sequence of human LAG3 is presented below, with sites for N-linked glycosylation underlined and in bold (N188, N250, N256 and N343):

[0035] [ka]

[0036] As shown in Table 1 below, both N-glycosylation sites are located in the extracellular domain of LAG3.

[0037] The specific glycosylation site of a particular LAG3 isoform or variant may be different from amino acid positions 188, 250, 256, or 343 of that particular LAG3 isoform or variant, which are sites located in the extracellular domain of LAG3. For any particular LAG3 isoform or variant, those skilled in the art can determine the glycosylation sites corresponding to N188, N250, N256, and N343 of the above-exemplified human LAG3 based on sequence alignment and other general knowledge in the art. Thus, antibodies that selectively bind to glycosylated forms of LAG3 isoforms or variants compared to unglycosylated LAG3 isoforms or variants are also provided herein. The glycosylation sites of a LAG3 isoform or variant may be the sites corresponding to N188, N250, N256, and N343 of the human LAG3 sequence provided above. Also provided herein are polypeptides comprising fragments of at least seven (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) consecutive amino acids of a LAG3 isoform or variant that includes at least one amino acid corresponding to positions N188, N250, N256, or N343 of the representative human LAG3 sequences provided above.

[0038] As used herein, and unless otherwise specified, the articles "a," "an," and "the" refer to one or to more than one of the grammatical object of the article. By way of example, an antibody refers to one antibody or to more than one antibody.

[0039] As used herein, and unless otherwise specified, the term "or" is used interchangeably with "and / or," unless it is expressly implied that the alternatives refer only to alternatives or that the alternatives are mutually exclusive. As used herein, and unless otherwise specified, "another" refers to at least a second or more.

[0040] As used herein, and unless otherwise specified, the term "about" indicates that a numerical value includes the inherent variation of error for the device, the method being employed to determine the numerical value, or the variation that exists in the testing subjects.

[0041] As used herein, and unless otherwise specified, the term "antibody" refers to a polypeptide product of B cells that belongs to the immunoglobulin (or "Ig") class of polypeptides and is capable of binding to a specific molecular antigen, such as IgG, IgM, IgA, IgD, IgE, etc., as well as other molecules having antigen-binding fragments thereof. Antibodies can be composed of a pair of two identical polypeptide chains, each pair having one heavy chain (approximately 50-70 kDa) and one light chain (approximately 25 kDa), with the amino-terminal portion of each chain containing a variable region of about 100 to about 130 or more amino acids and the carboxy-terminal portion of each chain containing a constant region (see Borrebaeck (ed.) (1995) Antibodies Engineering, Second Edition, Oxford University Press; Kuby (1997) Immunology, Third Edition, W.H. Freeman and Company, New York). The specific molecular antigen herein includes glycosylated human LAG3. 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.

[0042] As used herein, and unless otherwise specified, the term "isolated" when used in connection with an antibody, antigen-binding fragment, or polynucleotide means that the referenced molecule is free of at least one component found in nature. The term includes antibodies, antigen-binding fragments, or polynucleotides from which some or all of the other components found in their natural environment have been removed. Components of an antibody's natural environment include, for example, red blood cells, white blood cells, platelets, plasma, proteins, nucleic acids, salts, and nutrients. Components of an antigen-binding fragment or polynucleotide's natural environment include, for example, lipid membranes, cellular organelles, proteins, nucleic acids, salts, and nutrients. An antibody, antigen-binding fragment, or polynucleotide of the present invention may be free of, or ultimately substantially free from, all such components or any other components of the cells from which it was isolated or recombinantly produced.

[0043] As used herein, and unless otherwise specified, the term "monoclonal antibody" refers to an antibody that is the product of a single cell clone, or a hybridoma, or a cell population derived from a single cell. Monoclonal antibody is also intended to refer to an antibody produced by recombinant methods from heavy and light chain encoding immunoglobulin genes that generate a single immunoglobulin species. The amino acid sequences for antibodies within a monoclonal antibody preparation are substantially homogeneous, and the binding activity of antibodies within such a preparation exhibits substantially identical antigen-binding activity. In contrast, a polyclonal antibody is a combination of multiple immunoglobulin molecules obtained from different B cells within a population and that bind to a specific antigen. Each immunoglobulin in a polyclonal antibody can bind to a different epitope of the same antigen. Methods for producing both monoclonal and polyclonal antibodies are well known in the art (Harlow and Lane., Antibody: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989) and Borrebaeck (ed.), Antibody Engineering: A Practical Guide, W.H. Freeman and Co., Publishers, New York, pp. 103-120 (1991)).

[0044] As used herein, and unless otherwise specified, the term "human antibody" refers to an antibody having human variable and / or human constant regions, or portions thereof, corresponding to human germline immunoglobulin sequences. Such human germline immunoglobulin sequences are described in Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242. Herein, a human antibody can include antibodies that bind glycosylated human LAG3 and are encoded by a nucleic acid sequence that is a naturally occurring somatic variant of a human germline immunoglobulin nucleic acid sequence.

[0045] As used herein, and unless otherwise specified, the term "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies, so long as the desired biological activity is exhibited (see U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

[0046] As used herein, and unless otherwise specified, the term "humanized antibody" refers to a chimeric antibody comprising a human immunoglobulin (e.g., recipient antibody) in which native complementarity-determining region ("CDR") residues are replaced by residues from a corresponding CDR (e.g., donor antibody) of a non-human species, such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and capacity. In some cases, one or more FR region residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may possess residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. The heavy or light chain of a humanized antibody may possess substantially all of at least one or more variable regions, in which all or substantially all of the CDRs correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. A humanized antibody can comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further 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. Acad. Sci. US 89:4285-4289 (1992), and U.S. Patent Nos. 6,800,738, 6,719,971, 6,639,055, 6,407,213, and 6,054,297.

[0047] As used herein, and unless otherwise specified, the term "recombinant antibody" refers to an antibody that is prepared, expressed, created, or isolated by recombinant means. A recombinant antibody can be an antibody expressed using a recombinant expression vector transfected into a host cell, an antibody isolated from a recombinant, combinatorial antibody library, an antibody isolated from an animal (e.g., a mouse or a cow) that is transgenic and / or transchromosomal for human immunoglobulin genes (see, e.g., Taylor, LD et al., Nucl. Acids Res. 20:6287-6295 (1992)), or an antibody prepared, expressed, created, or isolated by any other means involving splicing immunoglobulin gene sequences into 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). Recombinant antibodies can also be subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis), thus improving the V and constant regions of the recombinant antibodies. H and V L The amino acid sequence of the region is human germline V H and V L The sequence may be derived from and related to a sequence, but may not naturally occur within the human antibody germline repertoire in vivo.

[0048] As used herein, and unless otherwise specified, the term "antigen-binding fragment" and similar terms refer to a portion of an antibody that immunospecifically binds to an antigen and contains amino acid residues that confer its specificity and affinity for the antigen to the antibody. An antigen-binding fragment may also be referred to as a functional fragment of an antibody. An antigen-binding fragment may be monovalent, bivalent, or multivalent.

[0049] Examples of molecules having an antigen-binding fragment include 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, and single-domain antibodies. An scFv can be a monovalent or divalent scFv. Other molecules having an antigen-binding fragment can include, for example, heavy or light chain polypeptides, variable region polypeptides, or CDR polypeptides, or portions thereof, so long as such antigen-binding fragments retain binding activity. Such antigen-binding fragments can be found, 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 Desk Reference, New York: VCH Publishers, Inc., Huston et al., Cell Biophysics, 22:189-224 (1993), Pluckthun and Skerra, Meth. Enzymol., 178:497-515 (1989), and Day, ED, Advanced Immunochemistry, Second Ed., Wiley-Liss, Inc., New York, NY (1990).An antigen-binding fragment can be a polypeptide having an amino acid sequence consisting of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues.

[0050] The heavy chain of an antibody refers to a polypeptide chain of approximately 50 to 70 kDa, the amino-terminal portion of which contains a variable region of approximately 120 to 130 or more amino acids, and the carboxy-terminal portion of which contains a constant region. The constant region can be one of five different types, termed alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the heavy chain constant region. Different heavy chains vary in size: α, δ, and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When combined with light chains, these different types of heavy chains give rise to the five well-known classes of antibodies: IgA, IgD, IgE, IgG, and IgM, each of which includes four subclasses of IgG: IgG1, IgG2, IgG3, and IgG4. The heavy chain can be a human heavy chain.

[0051] The light chain of an antibody refers to a polypeptide chain of approximately 25 kDa, with an amino-terminal portion containing a variable region of about 100 to about 110 or more amino acids and a carboxy-terminal portion containing a constant region. The approximate length of a light chain is 211 to 217 amino acids. There are two different types, called kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. The light chain may be a human light chain.

[0052] The variable domain, or variable region, of an antibody is generally located at the amino terminus of the light or heavy chain, approximately 120-130 amino acids in length in the heavy chain and approximately 100-110 amino acids in the light chain, and refers to the portion of the light or heavy chain antibody that determines the binding and specificity of each particular antibody for its specific antigen. Variable domains vary widely in sequence between different antibodies. Sequence variability is concentrated in the CDRs, while the less variable portions of the variable domain are called framework regions (FRs). The CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with the antigen. The numbering of amino acid positions used herein is based on the EU index as set forth in Kabat et al. (1991) Sequences of proteins of immunological interest (U.S. Department of Health and Human Services, Washington, DC), 5th ed. The variable region may be a human variable region.

[0053] CDRs are the amino acid sequences of immunoglobulin (Ig or antibody) V H One of the three hypervariable regions (H1, H2, or H3) within the non-framework regions of the β-sheet framework, or antibody V LCDR refers to one of the three hypervariable regions (L1, L2, or L3) in the non-framework region of the β-sheet framework. Thus, CDR is a variable region sequence interspersed within the framework region sequence. CDR regions are well known to those skilled in the art and have been defined, for example, by Kabat as the most hypervariable region in an antibody variable domain (Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat, Adv. Prot. Chem. 32:1-75 (1978)). CDR region sequences have also been structurally defined by Chothia as residues that are not part of the conserved β-sheet framework and therefore can adopt different conformations (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). Both terms are well recognized in the art. The positions of 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)). Because the number of residues within hypervariable regions varies among different antibodies, additional residues relative to the canonical position are conventionally numbered by adding the letter a, b, c, etc., next to the residue number in the canonical variable domain numbering scheme (Al-Lazikani et al., supra (1997)). Such nomenclature is also well known to those of skill in the art.

[0054] A universal numbering system has been developed and widely adopted: the ImMunoGeneTics (IMGT) Information System® (Lafranc et al., 2003, Dev. Comp. Immunol., 27(1):55-77). IMGT is an integrated information system specialized for immunoglobulins (Igs), T cell receptors (TRs), and major histocompatibility complexes (MHCs) of humans and other vertebrates. As used herein, the term CDR refers to both the amino acid sequence and location within a light or heavy chain. Because the "location" of CDRs within the immunoglobulin V domain structure is conserved across species and resides within structures called loops, CDR and framework residues can be readily identified using a numbering system that aligns variable domain sequences based on structural features. This information can be used to graft or replace CDR residues from one species' immunoglobulin into or with acceptor frameworks, typically derived from human antibodies. An additional numbering system (AHon) was developed by Honegger et al., 2001, J. Mol. Biol., 309: 657-670. The correspondence between numbering systems, including, for example, the Kabat numbering system and the IMGT specific numbering system, is well known to those skilled in the art (see, e.g., Kabat, Id; Chothia et al., Id., Martin, 2010, Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag, and Lefranc et al., 1999, Nuc. Acids Res., 27:209-212).

[0055] The sequences of the CDR regions have also been defined by the AbM and Contact methods. The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM modeling software (see, e.g., Martin, 2010, Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag). The "contact" hypervariable regions are based on an analysis of available complex crystal structures. Residues from each of these hypervariable regions or CDRs are listed below.

[0056] Representative representations of CDR region sequences are illustrated in Table 2 below. The positions of CDRs within canonical antibody variable regions have been determined by comparison of 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 within hypervariable regions varies between different antibodies, additional residues relative to the canonical position are conventionally numbered by adding a, b, c, and so forth, next to the residue number in the canonical variable region numbering scheme (Al-Lazikani et al., supra (1997)). Such nomenclature is also well known to those of skill in the art.

[0057] [Table 1]

[0058] One or more CDRs can be incorporated into a molecule, either covalently or noncovalently, to make it an immunoadhesin. An immunoadhesin can incorporate the CDR(s) as part of a larger polypeptide chain, the CDR(s) can be covalently linked to another polypeptide chain, or the CDR(s) can be incorporated noncovalently. The CDRs enable the immunoadhesin to bind to a specific antigen of interest.

[0059] As used herein, and unless otherwise specified, the terms "bind" or "binding" refer to intermolecular interactions. The interactions may be non-covalent interactions, including, for example, hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions. The strength of all non-covalent interactions between an antibody and a single epitope on a target molecule, such as glycosylated human LAG3, is the affinity of the antibody for that epitope. "Binding affinity" generally refers to the combined strength of non-covalent interactions that occur 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).

[0060] The affinity of a binding molecule X, e.g., an antibody, for its binding partner Y, e.g., the antibody's cognate antigen, is determined by the dissociation constant (K d ) or equilibrium dissociation constant (K D ) Low affinity antibodies generally bind to antigens slowly and tend to dissociate easily, while high affinity antibodies generally bind to antigens more rapidly and tend to remain bound for longer periods of time. Various methods for measuring binding affinity are known in the art, any of which can be used for purposes of this disclosure. D " or "K D The "K value" can be measured by assays known in the art, for example, by binding assays. D can be measured, for example, in a radiolabeled antigen binding assay (RIA) performed using the Fab version of the antibody of interest and its antigen (Chen, et al., (1999) J. Mol. Biol. 293:865-881). D or K DThe value can also be measured by using a surface plasmon resonance assay from Biacore, for example, using BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ), or by biolayer interferometry, for example, using the OctetQK384 system (ForteBio, Menlo Park, CA). As used herein, and unless otherwise specified, an antibody is said to be able to "selectively bind" to a first molecular antigen compared to a second molecular antigen if the antibody binds to the first molecular antigen with higher affinity than to the second molecular antigen. Antibodies generally do not bind to completely unrelated antigens.

[0061] As used herein, and unless otherwise specified, the term "polypeptide" includes oligopeptides having between 2 and 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, e.g., 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 chemical synthesis. Polypeptides of the present disclosure can be post-translationally or chemically modified (e.g., glycosylation, carbamylation, phosphorylation, biotinylation, attachment of fluorescent dyes, etc.). Polypeptides can be glycosylated at specific sites. Polypeptides can include unnatural amino acids not encoded by the natural genetic code. For example, polypeptides can include methylated backbone structures, peptoid backbone structures (poly-N-substituted glycines), L-amino acids, R-amino acids, etc. Polypeptides can have wild-type sequences, naturally occurring variant sequences, mutated sequences (e.g., point mutants, deletion mutants), etc.

[0062] Anti-glycLAG3 antibody Provided herein is an isolated antibody that selectively binds to glycosylated LAG3 compared to non-glycosylated LAG3. The LAG3 can be human LAG3. The glycosylated LAG3 can be a specific N-glycan structure of LAG3 or a glycopeptide of LAG3. In some embodiments, the antibody provided herein is an antigen-binding fragment that selectively binds to glycosylated LAG3 compared to non-glycosylated LAG3.

[0063] In some embodiments, the isolated antibodies provided herein selectively bind to human LAG3 glycosylated at N188, N250, N256, N343, or any combination thereof, relative to unglycosylated LAG3. In some embodiments, the isolated antibodies selectively bind to human LAG3 with only N188 glycosylation. In some embodiments, the isolated antibodies selectively bind to human LAG3 with only N250 glycosylation. In some embodiments, the isolated antibodies selectively bind to human LAG3 with only N256 glycosylation. In some embodiments, the isolated antibodies selectively bind to human LAG3 with only N343 glycosylation. In some embodiments, the isolated antibodies selectively bind to human LAG3 with only N188 and N250 glycosylation. In some embodiments, the isolated antibodies selectively bind to human LAG3 with only N188 and N256 glycosylation. In some embodiments, the isolated antibody selectively binds to human LAG3 with only N188 and N343 glycosylation. In some embodiments, the isolated antibody selectively binds to human LAG3 with only N250 and N256 glycosylation. In some embodiments, the isolated antibody selectively binds to human LAG3 with only N250 and N343 glycosylation. In some embodiments, the isolated antibody selectively binds to human LAG3 with only N256 and N343 glycosylation. In some embodiments, the isolated antibody selectively binds to human LAG3 with only N188, N250, and N256 glycosylation. In some embodiments, the isolated antibody selectively binds to human LAG3 with only N188, N250, and N343 glycosylation. In some embodiments, the isolated antibody selectively binds to human LAG3 with only N188, N256, and N343 glycosylation. In some embodiments, the isolated antibody selectively binds to human LAG3 with only N250, N256, and N343 glycosylation, hi some embodiments, the isolated antibody selectively binds to human LAG3 with N188, N250, N256, and N343 glycosylation.

[0064] In certain aspects, an anti-glycoLAG3 antibody binds to LAG3 and can mask or shield one or more glycosylation motifs to block binding or other interaction of molecules bearing those motifs and block glycosylation of LAG3 at those glycosylation sites. In particular embodiments, the anti-glycoLAG3 antibody masks the glycosylation sites at one or more of N188, N250, N256, and N343.

[0065] In some embodiments, provided herein are antibodies that selectively bind to one or more glycosylation motifs of LAG3. In some embodiments, the antibodies selectively bind to glycopeptides having a glycosylation motif and an adjacent peptide. In some embodiments, the antibodies selectively bind to a glycopeptide having a K α -receptor peptide (K α -receptor peptide) that is expressed against unglycosylated LAG3. d K that is at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% smaller than d In certain embodiments, the antigen-binding fragment selectively binds to glycosylated LAG3 at a K d K less than 50% of d In some embodiments, the antibody binds to glycosylated LAG3 at a K d K smaller than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50% of d In a further embodiment, the antibody binds to glycosylated LAG3 at a K d K less than one-tenth of d It binds to glycosylated LAG3 at

[0066] Monoclonal antibodies that preferentially bind to the glycosylated LAG3 described herein, particularly STC1317, are presented. Humanized and chimeric forms of STC1317, as well as antibodies that compete for binding to STC1317, are also presented. The heavy and light chain variable domains of STC1317 are presented in Table 3 below.

[0067] In particular embodiments, heavy and light chain variable domains having the amino acid sequences of SEQ ID NOs: 3 and 5 (mature V without any signal sequence) are H and V L Presented herein are anti-glycLAG3 monoclonal antibodies STC1317 and antigen-binding portions thereof, each having the amino acid sequence (region) of LAG3. Presented herein are anti-glycLAG3 antibodies that compete with STC1317 MAb for binding to LAG3 and / or that bind to the same epitope as STC1317.

[0068] The heavy and light chain variable (V) domains of STC1317mAb were analyzed using its monoclonal antibody. The null nucleic acid (DNA) and corresponding amino acid sequences are presented and shown in Table 3 below. Numbers 2 and 3 are STC 1317 V H The nucleotide and amino acid sequences of the domains and SEQ ID NOs: 4 and 5 are STC1317 kappa V L Nucleus of the mature form of the domain The nucleotide and amino acid sequences of STC1317 are shown in Table 4. The heavy and light chain V domain CDRs of M, Kabat, and Cotact are presented.

[0069] In one embodiment, the anti-glycLAG3 antibody that specifically and preferentially binds to glycosylated LAG3 is VGA having the amino acid sequence of SEQ ID NO:3. H domain, and / or V having the amino acid sequence of SEQ ID NO: 5 LIn one embodiment, the anti-glycOLAG3 antibody comprises the V domain of SEQ ID NO: 3 for specific binding to glycosylated LAG3. H Domain and V of SEQ ID NO:5 L In other embodiments, the anti-glycLAG3 antibody competes with an antibody comprising 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 V that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:5. L These anti-glycLAG3 antibodies may be chimeric antibodies, but may also contain human constant domains, for example, derived from human IgG1, IgG2, IgG3, or IgG4.

[0070] In one embodiment, an anti-glycLAG3 antibody that specifically and preferentially binds glycosylated LAG3 is a V antibody that comprises CDRs 1-3 of Chothia having the amino acid sequences of SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively; CDRs 1-3 of AbM having the amino acid sequences of SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:8, respectively; CDRs 1-3 of Kabat having the amino acid sequences of SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:8, respectively; or CDRs 1-3 of Contact having the amino acid sequences of SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively, or a combination thereof. H In one embodiment, the anti-glycolyzed LAG3 antibody comprises a V domain that, for specific binding to glycosylated LAG3, comprises CDRs 1-3 of Chothia having the amino acid sequences of SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively; CDRs 1-3 of AbM having the amino acid sequences of SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:8, respectively; CDRs 1-3 of Kabat having the amino acid sequences of SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:8, respectively; or CDRs 1-3 of Contact having the amino acid sequences of SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively, or a combination thereof. HIn one embodiment, an anti-glycolyzed LAG3 antibody that specifically and preferentially binds to glycosylated LAG3 competes with an antibody comprising a V domain comprising CDRs 1-3 of Chothia, AbM, or Kabat having the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively; or CDRs 1-3 of Contact having the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively, or a combination thereof. L In one embodiment, the anti-glycolyzed LAG3 antibody comprises a V domain that contains CDRs 1-3 of Chothia, AbM, or Kabat having the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively, for specific binding to glycosylated LAG3; or CDRs 1-3 of Contact having the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively, or a combination thereof. L In one embodiment, the anti-glycLAG3 antibody competes with an antibody comprising a V domain comprising CDRs 1-3 of Chothia having the amino acid sequences of SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively; CDRs 1-3 of AbM having the amino acid sequences of SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:8, respectively; CDRs 1-3 of Kabat having the amino acid sequences of SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:8, respectively; or CDRs 1-3 of Contact having the amino acid sequences of SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively. H domain and comprising CDR1-3 of Chothia, AbM, or Kabat having the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively; or comprising CDR1-3 of Contact having the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively. L Preferably, the antibody comprises a V domain, or competes for binding thereto. H and V L The domains have CDRs of the same class, ie, both have Chothia, AbM, Kabat, or Contact CDRs.

[0071] In other embodiments, the anti-glycLAG3 antibody comprises CDRs H1, H2, and H3 having amino acid sequences with 1, 2, 3, 4, or 5 amino acid substitutions in one, two, or three of the CDRs having the amino acid sequences of SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively; or the CDRs having the amino acid sequences of SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:8, respectively; or the CDRs having the amino acid sequences of SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:8, respectively; or the CDRs having the amino acid sequences of SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively. H The anti-glycLAG3 antibody has a V domain including CDRs L1, L2, and L3 having amino acid sequences with 1, 2, 3, 4, or 5 amino acid substitutions in one, two, or three of the CDRs having the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively, or the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively. L The anti-glycLAG3 antibody may have a V domain. H and V L Both domains may have amino acid substitutions within the CDRs. In some embodiments, the amino acid substitutions are conservative substitutions.

[0072] Preferably, the antibody has human framework regions, ie is a humanized form of STC1317, and optionally includes human constant domains, for example from human IgG1, IgG2, IgG3, or IgG4.

[0073] It will be appreciated by those skilled in the art that one or more amino acid substitutions may be made within the CDR and / or framework regions of the humanized antibody to improve binding affinity or other parameters. In some embodiments, the anti-glycoLAG3 antibody comprises a humanized antibody having a CDR and / or framework region as defined above for specific binding to glycosylated LAG3. H and V LIn some embodiments, the anti-glycosylated LAG3 antibody competes with antibodies comprising the K domain and CDRs therein. d K is less than half of d In some embodiments, the anti-glycoLAG3 antibody binds to glycosylated LAG3 at a K d K is less than half of d In one embodiment, the anti-glycoLAG3 antibody binds to glycosylated LAG3 at a K, as indicated by the antibody binding to unglycosylated LAG3. d K less than one-fifth of d In one embodiment, the anti-glycolyzed LAG3 antibody binds to glycosylated LAG3 protein at a K d K less than one-tenth of d In one embodiment, the antibody binds to glycosylated LAG3 protein at 1 mm for binding to cells expressing unglycosylated LAG3 in a cell flow cytometry binding assay. 2 3-, 5-, 10-, 20-, or 50-fold greater green counts per mm for cells expressing WT LAG3 2 The binding affinity is expressed as green counts per 1000 ribonucleotides. In one embodiment, the antibody is directly or indirectly detectable by a fluorescent label or marker. In one embodiment, the antibody is directly labeled with a fluorescent label or marker, such as FITC. In one embodiment, the binding affinity of STC1317 MAb, or a binding domain thereof, or a humanized or chimeric form thereof, for glycosylated LAG3 is 0.1-13 nM or 0.1-5 nM, inclusive.

[0074] Yet another embodiment is an anti-glycolytic antibody comprising a nucleotide sequence that is at least 90-98% identical to SEQ ID NO:2. H domain, and / or a nucleotide sequence that is at least 90-98% identical to SEQ ID NO:4. LIn some embodiments, isolated nucleic acid molecules encoding each of the V domains are provided. H and / or V L The nucleotide sequence encoding 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.

[0075] Table 3 below provides the nucleotide and amino acid sequences of the heavy and light chain variable domains of STC1317.

[0076] [Table 2]

[0077] The CDR sequences of the STC1317 antibody, based on the CDRs of Chothia, AbM, Kabat, and Contact, are presented below in Table 4. Thus, a humanized form of STC1317 comprising the CDRs of Table 4 below grafted into human framework regions is presented, which preferentially binds to glycosylated LAG3 compared to non-glycosylated LAG3.

[0078] TIFF0007811544000004.tif115170

[0079] In some embodiments, the anti-glycLAG3 antibody provided herein may be IgG, IgM, IgA, IgD, or IgE. The anti-glycLAG3 antibody may also be a chimeric antibody, an affinity-matured antibody, a humanized antibody, or a human antibody. The anti-glycLAG3 antibody may also be a camelized antibody, an intrabody, or an anti-idiotype (anti-Id) antibody. In some embodiments, the anti-glycLAG3 antibody may be a polyclonal antibody or a monoclonal antibody.

[0080] In some embodiments, the antibodies provided herein are antigen-binding fragments that selectively bind to glycosylated LAG3 compared to non-glycosylated LAG3. The antigen-binding fragment may be an 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. The scFv may be a monovalent scFv or a bivalent scFv.

[0081] By known means and as described herein, polyclonal or monoclonal antibodies, antigen-binding fragments, and binding domains, and CDRs (including engineered forms of any of the above) can be created that are specific for glycosylated LAG3, one or more of its epitopes, or any of the conjugates described above, whether such antigens or epitopes are isolated from natural sources or are synthetic derivatives or variants of the natural compounds.

[0082] Antibodies can be produced from any animal origin, including birds and mammals. In some embodiments, the antibodies are ovine, murine (e.g., mouse and rat), rabbit, goat, guinea pig, camel, horse, or chicken. In addition, newer technologies allow for the development and screening of human antibodies from human combinatorial antibody libraries. For example, bacteriophage antibody expression technology allows for the production of specific antibodies without immunizing animals, as described in U.S. Patent No. 6,946,546, the entire contents of which are incorporated herein by reference. 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 incorporated herein by reference in their entireties.

[0083] Methods for producing polyclonal antibodies in various animal species, as well as for producing various types of monoclonal antibodies, including humanized, chimeric, and fully human types, are well known in the art. See, for example, the following U.S. patents, which are incorporated herein by reference in their entireties: 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, Specification No. 003; Specification No. 4,742,159; Specification No. 4,767,720; Specification No. 4,816,567; Specification No. 4,867,973; Specification No. 4,938,948; Specification No. 4,946,778; Specification No. 5, Specification No. 021,236; Specification No. 5,164,296; Specification No. 5,196,066; Specification No. 5,223,409; Specification No. 5,403,484; Specification No. 5,420,253; Specification No. 5,565,332; Specification No. 5,571,698; Specification No. 5,627,052; Specification No. 5,656,434; Specification No. 5,770,376; Specification No. 5,789,208; Specification No. 5,821,337; Specification No. 5,844,091 Specification; Specification No. 5,858,657; Specification No. 5,861,155; Specification No. 5,871,907; Specification No. 5,969,108; Specification No. 6,054,297; Specification No. 6,165,464; Specification No. 6,365, Nos. 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 provide descriptions of such methods and are incorporated herein by reference.

[0084] In some embodiments, the anti-glycLAG3 antibody can be a monoclonal antibody. In some embodiments, the anti-glycLAG3 antibody can be a polyclonal antibody. To produce antibodies specific to glycosylated LAG3 polypeptides, animals can be inoculated with an antigen, such as a glycosylated LAG3 polypeptide. Often, the antigen is bound or conjugated to another molecule to enhance the immune response. The conjugate can be any peptide, polypeptide, protein, or non-protein substance bound to the antigen used to elicit an immune response in the animal. Antibodies produced in an animal in response to antigen inoculation comprise a variety of non-identical molecules (polyclonal antibodies) produced by various individual antibody-producing B lymphocytes. If conditions are appropriate for intraanimal polyclonal antibody production, most antibodies in the animal's serum will recognize a common epitope on the antigenic compound with which the animal was immunized.

[0085] This specificity can be further enhanced by affinity purification, which selects for only those antibodies that recognize the antigen or epitope of interest. Methods for generating monoclonal antibodies (MAbs) can begin along the same lines as those for preparing polyclonal antibodies. In some embodiments, rodents, such as mice and rats, are used in generating monoclonal antibodies. In some embodiments, rabbit, sheep, or frog cells are used in generating monoclonal antibodies. The use of rats is well known and may offer certain advantages. Mice (e.g., BALB / c mice) are routinely used and generally result in a high percentage of stable fusions.

[0086] Hybridoma technology involves fusing a single B lymphocyte from a mouse previously immunized with glycosylated LAG3 polypeptide with an immortal myeloma cell (usually a mouse myeloma). This technology provides a method for propagating a single antibody-producing cell for an indefinite number of generations so that unlimited quantities of structurally identical antibodies (monoclonal antibodies) with the same antigen or epitope specificity can be produced.

[0087] Anti-glycLAG3 antibodies can be produced by any method known in the art useful for producing polypeptides, such as in vitro synthesis, recombinant DNA production, etc. Humanized antibodies can be produced by recombinant DNA technology. The antibodies described herein can also be produced using recombinant immunoglobulin expression technology. Recombinant production of immunoglobulin molecules, including humanized antibodies, is described in U.S. Pat. Nos. 4,816,397 (Boss et al.), 6,331,415 and 4,816,567 (all to Cabilly et al.), GB 2,188,638 (Winter et al.), and GB 2,209,757, which are incorporated herein by reference in their entireties. Techniques for recombinantly expressing immunoglobulins, including humanized immunoglobulins, can also be found in Goeddel et al., Gene Expression Technology Methods in Enzymology Vol. 185, Academic Press (1991), and Borreback, Antibody Engineering, W. H. Freeman (1992), which are incorporated herein by reference in their entireties. Additional information regarding the generation, design, and expression of recombinant antibodies can be found in Mayforth, Designing Antibodies, Academic Press, San Diego (1993).

[0088] Methods have been developed to replace the light and heavy chain constant domains of a monoclonal antibody with similar domains of human origin, while leaving the variable regions of the foreign antibody intact. Alternatively, fully human monoclonal antibodies have been generated in mice or rats transgenic for human immunoglobulin genes. Methods have also been developed to convert the variable domains of monoclonal antibodies to a more human-like form by recombinantly constructing antibody variable domains with both rodent and human amino acid sequences. In humanized monoclonal antibodies, only the hypervariable CDRs are derived from non-human (e.g., mouse, rat, chicken, or llama) monoclonal antibodies, and the framework regions are derived from human amino acid sequences. Replacing amino acid sequences within an antibody that are characteristic of rodents with amino acid sequences found in the corresponding positions in human antibodies is believed to reduce the likelihood of adverse immune reactions during therapeutic use. Hybridomas or other cells that produce antibodies can be subject to genetic mutations or other modifications that may or may not alter the binding specificity of the antibodies produced by the hybridoma.

[0089] Engineered antibodies can be created using monoclonal and other antibodies, as well as recombinant DNA technology, to generate other antibodies or chimeric molecules (i.e., molecules that contain a binding domain) that retain the antigen or epitope specificity of the original antibody. Such technology can involve introducing DNA encoding an immunoglobulin variable region or the CDRs of an antibody into genetic material corresponding to the framework regions, constant regions, or constant regions plus framework regions of a different antibody. See, e.g., U.S. Patent Nos. 5,091,513 and 6,881,557, incorporated herein by reference.

[0090] In certain embodiments, anti-glycLAG3 antibody is a human antibody.Human antibody can be produced by various methods known in the art, including the above-mentioned phage display method using 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 antibody can also be produced using transgenic mice that are unable to express functional endogenous immunoglobulins but can express human immunoglobulin genes. For example, human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, human variable, constant, and diversity regions can be added to human heavy and light chain genes and introduced into mouse embryonic stem cells. Separately from or simultaneously with the introduction of human immunoglobulin loci by homologous recombination, the mouse heavy and light chain immunoglobulin genes can be rendered nonfunctional. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to generate chimeric mice. The chimeric mice are then bred to generate homozygous offspring expressing human antibodies. The transgenic mice are immunized using conventional methods with a selected antigen, such as all or a portion of a glycosylated LAG3 polypeptide. Monoclonal antibodies targeting the antigen can be obtained from the immunized transgenic mice using conventional hybridoma technology (see, e.g., U.S. Pat. No. 5,916,771). The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation and subsequently undergo class switching and somatic mutation. Thus, using such a technique, therapeutically useful IgG, IgA, IgM, and IgE antibodies can be generated.For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93, incorporated herein by reference in its entirety). For a detailed discussion of such 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. Pat. 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 by reference in their entireties. Additionally, companies such as Abgenix, Inc. (Freemont, Calif.) and Medarex (Princeton, NJ) may be involved in providing human antibodies targeted to a selected antigen using technology similar to that described above.

[0091] In one embodiment, the antibody is a chimeric antibody, e.g., an antibody comprising an antigen-binding sequence derived from a non-human donor grafted onto heterologous non-human, human, or humanized sequences (e.g., framework and / or constant domain sequences). In one embodiment, the non-human donor is a rat. In one embodiment, the antigen-binding sequence is synthetic, e.g., obtained by mutagenesis (e.g., phage display screening of a human phage library). In one embodiment, the chimeric antibody provided herein has a mouse V region and a human C region. In one embodiment, a mouse light chain V region is fused to a human kappa light chain. In one embodiment, a mouse heavy chain V region is fused to a human IgG1 C region.

[0092] Methods for producing chimeric antibodies are known in the art (see, e.g., 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 incorporated herein by reference in their entireties). Chimeric antibodies, comprising one or more CDRs derived from a species other than human and a framework region derived from a human immunoglobulin molecule, have been developed using techniques such as, for example, CDR grafting (EP 239,400; WO 91 / 09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering, 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 incorporated herein by reference in their entireties.

[0093] A typical method for producing a recombinant chimeric anti-glycLAG3 antibody may include the following steps: a) constructing, by conventional molecular biology methods, an expression vector that encodes and expresses an antibody heavy chain in which the CDRs and variable region of a mouse anti-glycLAG3 monoclonal antibody are fused to an Fc region derived from human immunoglobulin, thereby generating a vector for expressing the chimeric antibody heavy chain; b) constructing, by conventional molecular biology methods, an expression vector that encodes and expresses the antibody light chain of the mouse anti-glycLAG3 monoclonal antibody, thereby generating a vector for expressing the chimeric antibody light chain; c) transferring the expression vector into a host cell by conventional molecular biology methods to generate a transfected host cell for expressing the chimeric antibody; and d) culturing the transfected cell by conventional cell culture techniques to produce the chimeric antibody.

[0094] A typical method for producing a recombinant humanized anti-glycLAG3 antibody comprises the following steps: a) constructing, by conventional molecular biology methods, an expression vector encoding and expressing an antibody heavy chain in which the CDRs and the minimum portion of the variable region framework required to retain the binding specificity of the donor antibody are derived from a non-human immunoglobulin, such as a mouse anti-glycLAG3 monoclonal antibody, and the remaining portion of the antibody is derived from a human immunoglobulin, thereby producing a vector for expressing a humanized antibody heavy chain; b) constructing, by conventional molecular biology methods, an expression vector encoding and expressing an antibody heavy chain in which the CDRs and the minimum portion of the variable region framework required to retain the binding specificity of the donor antibody are derived from a non-human immunoglobulin, such as a mouse anti-glycLAG3 monoclonal antibody, and the remaining portion of the antibody is derived from a human immunoglobulin; The method may include constructing an expression vector encoding and expressing an antibody light chain, wherein a minimal portion of the variable region framework to be expressed is derived from a non-human immunoglobulin, such as a mouse anti-glycLAG3 monoclonal antibody, and the remainder of the antibody is derived from a human immunoglobulin, thereby producing a vector for expressing a 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 cell by conventional cell culture techniques to produce the humanized antibody.

[0095] For any of the exemplary methods, host cells can be co-transfected with such expression vectors (which may contain different selectable markers, but which, with the exception of the heavy and light chain coding sequences, are preferably identical). This procedure achieves equal expression of heavy and light chain polypeptides. Alternatively, a single vector encoding both heavy and light chain polypeptides can be used. The coding sequences for the heavy and light chains can comprise cDNA or genomic DNA, or both. Host cells used to express recombinant antibodies can be bacterial cells, such as Escherichia coli, or more preferably eukaryotic cells (e.g., Chinese hamster ovary (CHO) cells or HEK-293 cells). The choice of expression vector will depend on the choice of host cell and can also be selected to have the desired expression and regulatory characteristics in the selected host cell. Other cell lines that can be used include, but are not limited to, CHO-K1, NSO, and PER.C6 (Crucell, Leiden, The Netherlands). Furthermore, codon usage can be optimized when a host cell is selected to offset species-specific codon usage bias and enhance protein expression. For example, for CHO cell expression, the antibody-encoding DNA can incorporate codons preferentially used by Chinese hamsters (Cricetulus griseus) (the source of Chinese hamster ovary cells).Codon optimization methods can be employed to facilitate improved expression by desired host cells (see, e.g., 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)).

[0096] In one embodiment, the antibody is a camelid antibody, preferably a heavy chain camelid antibody (light chain deficient and V H A Nanobody™ (Nb) is an immunoglobulin single variable domain derived from a H domain sequence or known as a Nanobody™. A Nanobody™ (Nb) is the smallest functional fragment or single variable domain (VH domain) of a naturally occurring single-chain antibody. H H), and are well known to those skilled in the art. Nanobodies™ (Nbs) are derived from heavy chain-only antibodies found in camelids (Hamers-Casterman et al., Nature 363: 446-448 (1993); Desmyter et al., Nat. Struct. Biol., 803-811 (1996)). Immunoglobulins lacking light chain polypeptides are found in the "camelidae" family. "Camelidae" includes Old World camelids (Camelus bactrianus and Camelus dromedarius) and New World camelids (e.g., Lama paccos, Lama glama, Lama guanicoe, and Lama vicugna). Single variable domain heavy chain antibodies are known as Nanobodies™, or V HNbs are herein designated H antibodies. Due to their small size and unique biophysical properties, Nbs are superior to conventional antibody fragments in recognizing rare or hidden epitopes and binding to the cavities or active sites of protein targets. Furthermore, Nbs can be designed as multispecific and multivalent antibodies linked to reporter molecules or humanized. Nbs are stable, persist in the gastrointestinal system, and are easily produced.

[0097] By combining two antigen-binding sites with different specificities in a single construct, bispecific antibodies have the ability to combine two distinct antigens with superior specificity and therefore have superior potential as therapeutic agents. Bispecific antibodies can primarily be generated by fusing two hybridomas, each capable of producing a different immunoglobulin. Bispecific antibodies can also be generated by linking two scFv antibody fragments, while omitting the Fc portion present in intact immunoglobulins. Each scFv unit in such a construct consists of an antibody heavy chain (V) linked to each other via a synthetic polypeptide linker. H ) and light chain (V L ), although synthetic polypeptide linkers are often genetically engineered to minimize immunogenicity while maximizing resistance to proteolysis. Individual scFv units can be linked by several techniques, including the incorporation of a short (usually less than 10 amino acids) polypeptide spacer that bridges two scFv units, thereby creating a bispecific single-chain antibody. The resulting bispecific single-chain antibody thus contains two sets of Vs with different specificities on a single polypeptide chain. H / V L In this case, the V within each scFv unit is a species containing H and V L The domains are separated by a polypeptide linker long enough to allow intramolecular association between these two domains, and the scFv unit so formed can be, for example, the V of one scFv unit. Hdomain and the V of the other scFv unit L are sequentially tethered to one another through a polypeptide spacer that is kept short enough to prevent undesired association between the two.

[0098] Examples of antigen-binding fragments include, but are not limited to: (i) V L , V H , C L , and C H1 (ii) a Fab fragment consisting of the V domain; H and C H1 (iii) an "Fd" fragment consisting of a single antibody V domain; L and V H (iv) an "Fv" fragment consisting of a V domain; H (v) isolated CDR regions; (vi) F(ab')2 fragments, bivalent fragments containing two linked Fab fragments; (vii) single-chain Fv molecules ("scFv"), V domains linked by a peptide linker that allows the two domains to associate to form a binding domain. H Domains and V L (viii) bispecific single-chain Fv dimers (U.S. Pat. No. 5,091,513); and (ix) diabodies, multivalent or multispecific fragments constructed by gene fusion (U.S. Pat. App. Pub. No. 20050214860). Fv, scFv, or diabody molecules include V H and V L It can be stabilized by the incorporation of disulfide bridges linking the domains. Minibodies can also be made with scFvs linked to the CH3 domain (Hu et al., Cancer Res., 56:3055-3061 (1996)).

[0099] Antibody-like binding peptidomimetics are also contemplated in some embodiments. Liu et al., Cell Mol. Biol., 49:209-216 (2003) describe "antibody-like binding peptidomimetics" (ABiPs), which are peptides that act as paired-down antibodies and have certain advantages, such as longer serum half-lives and less cumbersome synthesis methods.

[0100] Glycosylated LAG3 Polypeptide In yet a further embodiment, a composition is provided comprising a polypeptide comprising a fragment of at least seven (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) consecutive amino acids of human LAG3, including at least one amino acid corresponding to positions N188, N250, N256, or N343 of human LAG3, wherein at least one of the amino acids corresponding to positions N188, N250, N256, or N343 of human LAG3 is glycosylated, and the polypeptide is formulated in a pharmaceutically acceptable carrier.

[0101] Also provided herein in some embodiments are polypeptides consisting of at least seven contiguous amino acids of human LAG3, having at least one amino acid corresponding to positions N188, N250, N256, or N343 of human LAG3, wherein at least one of the amino acids corresponding to positions N188, N250, N256, or N343 of human LAG3 is glycosylated. In some embodiments, the polypeptide has at least seven contiguous amino acids of human LAG3, having an amino acid corresponding to position N188, that is glycosylated. In some embodiments, the polypeptide has at least seven contiguous amino acids of human LAG3, having an amino acid corresponding to position N250, that is glycosylated. In some embodiments, the polypeptide has at least seven contiguous amino acids of human LAG3, having an amino acid corresponding to position N256, that is glycosylated. In some embodiments, the polypeptide has at least seven contiguous amino acids of human LAG3, having an amino acid corresponding to position N343, that is glycosylated.

[0102] For example, the polypeptide can be a fragment of amino acids 182-190 or 187-194 of human LAG3 in which N188 is glycosylated. In another example, the polypeptide can be a fragment of amino acids 247-254 or 248-256 of human LAG3 in which N250 is glycosylated, or a fragment of amino acids 252-259 or 255-265 of human LAG3 in which N256 is glycosylated. In yet another example, the polypeptide can be a fragment of amino acids 250-260 or 249-257 of human LAG3 in which N250 and N256 are glycosylated. In another example, the polypeptide can be a fragment of amino acids 340-347 or 342-349 of human LAG3 in which 343 is glycosylated. Those skilled in the art will understand that the polypeptides contemplated herein include any and all polypeptides having at least seven consecutive amino acids of human LAG3, including at least one amino acid corresponding to positions N188, N250, N256 or N343 of human LAG3, wherein at least one of said amino acids corresponding to positions N188, N250, N256 or N343 of human LAG3 is glycosylated.

[0103] In some 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 LAG3. In some 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 LAG3. Provided herein are compositions comprising at least two polypeptides provided herein in some embodiments. The at least two polypeptides can be separate molecules or can be linked as one molecule. In some embodiments, the composition has at least three polypeptides, at least four polypeptides, or at least five polypeptides. In some embodiments, the composition has two polypeptides, three polypeptides, four polypeptides, or five polypeptides.

[0104] In some embodiments, the polypeptides presented herein comprise unnatural amino acids. In some embodiments, the unnatural amino acids are methylated at the alpha amino group to generate peptides with a methylated backbone. In some embodiments, the unnatural amino acids are R-amino acids. In some embodiments, the unnatural amino acids may comprise dyes (e.g., fluorescent dyes) or affinity tags. In some embodiments, the polypeptides presented herein comprise chemical modifications. Chemical modifications include, for example, chemical modifications with biotin, fluorescent dyes. Those skilled in the art will recognize that methods for introducing unnatural amino acids into polypeptides and chemically modifying polypeptides are well known in the art.

[0105] In some embodiments, the polypeptides of several embodiments are fused to or conjugated to an immunogenic polypeptide (e.g., keyhole limpet hemocyanin, KLH). In certain aspects, the polypeptide further comprises a Cys residue at the C-terminus or N-terminus. For example, in some aspects, the polypeptide is conjugated to the immunogenic polypeptide by a disulfide bond at the Cys residue.

[0106] In yet a further embodiment, provided herein is an immunogenic composition comprising a polypeptide comprising a fragment of at least seven consecutive amino acids of human LAG3, the fragment comprising at least one amino acid corresponding to positions N188, N250, N256, or N343 of human LAG3, wherein at least one of the amino acids corresponding to positions N188, N250, N256, or N343 of human LAG3 is glycosylated, wherein the polypeptide is formulated in a pharmaceutically acceptable carrier. In some aspects, the immunogenic composition further comprises an adjuvant, such as alum or Freund's adjuvant.

[0107] In some embodiments, a method for producing an antibody is provided, comprising administering a polypeptide to an animal and isolating an antibody from the animal, wherein the polypeptide comprises a fragment of at least 7 consecutive amino acids of human LAG3, having at least one amino acid corresponding to positions N188, N250, N256, or N343 of human LAG3, and wherein at least one of the amino acids corresponding to positions N188, N250, N256, or N343 of human LAG3 is glycosylated. The animal may be a mouse, rat, rabbit, or human. In certain aspects, the method further comprises identifying CDRs of the antibody and humanizing the sequences surrounding the CDRs to produce a humanized antibody. In yet a further aspect, the method includes recombinantly expressing the humanized antibody. Thus, in a further embodiment, an isolated antibody produced by the above method is provided. Thus, in some embodiments, provided herein are isolated antibodies that selectively bind to a polypeptide of multiple embodiments (e.g., a polypeptide comprising a fragment of at least 7 consecutive amino acids of human LAG3, including at least one amino acid corresponding to positions N188, N250, N256, or N343 of human LAG3, wherein at least one of the amino acids corresponding to positions N188, N250, N256, or N343 of human LAG3 is glycosylated) compared to unglycosylated LAG3.

[0108] The polypeptides presented herein can be prepared by any method known in the art.For example, polypeptides can be prepared by chemical synthesis or recombinant production.Representative methods for expressing and purifying recombinant polypeptides can be found, for example, in Scopes RK, 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 achieved by using methods well known in the art (Kelley and Winkler, 1990, In: Genetic Engineering Principles and Methods, Setlow J. K, ed., Plenum Press, NY, Vol. 12, pp. 1-19; Stewart et al., 1984, J. M. Young, J. D., Solid Phase Peptide Synthesis, Pierce Chemical Co., Rockford, IL; Marglin and Merrifield, Ann. Rev. Biochem, 39:841-866, at 862 (1970); Merrifield, R. B., 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 See Approach, Atherton & Sheppard, Eds., 1989, IRL Press, Oxford, England; see also U.S. Patent Nos. 4,105,603, 3,972,859, 3,842,067, and 3,862,925).

[0109] Modifications and Derivatives Antibodies against glycosylated LAG3 may be capable of neutralizing or counteracting the effects of glycosylated LAG3, regardless of the animal species, monoclonal cell line, or other antibody source. Certain animal species are considered less desirable for generating therapeutic antibodies because they are more likely to cause allergic reactions due to activation of the complement system through the Fc portion of the antibody. However, whole antibodies can be enzymatically digested into Fc (complement-binding) fragments and antibody fragments containing the binding domain or CDR. Removal of the Fc portion reduces the likelihood that the antibody fragment will induce an undesired immune response, and thus Fc-free antibodies can be used for prophylactic or therapeutic treatment. As described above, antibodies may also be constructed to be chimeric, partially, or fully human to reduce or eliminate adverse immunological consequences resulting from administering to animals antibodies with sequences generated in or derived from other species.

[0110] The binding properties of anti-glycLAG3 antibodies can be further improved by screening for variants exhibiting 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 carrying the polynucleotide sequences encoding them. In particular embodiments, such phage can be used to display antigen-binding fragments, such as Fabs and Fvs, or disulfide-stabilized Fvs, expressed from repertoire or combinatorial antibody libraries (e.g., human or murine). Phage expressing antigen-binding fragments that bind to the antigen of interest can be selected or identified using the antigen, for example, labeled antigen, or antigen bound or captured to a solid surface or bead. The phages used in these methods are generally filamentous phages, including fd and M13. Antigen-binding fragments are expressed as recombinant fusion proteins to phage gene III or gene VIII proteins.Examples of phage display methods that can be used to generate antibodies or polypeptides as described herein include those described 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), and 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; and 5,403,484. and 5,969,108, all of which are incorporated herein by reference in their entireties.

[0111] After phage selection, as described in the above references, the antibody coding region from the phage can be isolated and used to produce whole antibodies, including humanized antibodies or any other desired fragment, or can be expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, as described in more detail below. For example, techniques for recombinantly producing 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, R.L. et al., BioTechniques, 12(6):864-869 (1992); 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 incorporated herein by reference in their entireties. 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, J.S. 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 incorporated by reference in their entirety.

[0112] Phage display technology can be used to increase the affinity of anti-glycLAG3 antibodies as described herein. This technology can be used to obtain high-affinity antibodies that can be used in the combinatorial methods described herein. This technique, called affinity maturation, employs mutagenesis or CDR walking and reselection using such receptors or ligands (or their extracellular domains), or antigenic fragments thereof, to identify antibodies that bind to antigens with higher affinity than the initial or parent antibody (see, e.g., Glaser, SM et al., J. Immunol. 149:3903-3913 (1992)). Mutagenesis of entire codons, rather than single nucleotides, results in a semi-randomized repertoire of amino acid mutations. Libraries can be constructed consisting of a pool of variant clones, each of which differs by a single amino acid change in a single CDR and contains variants representing each possible amino acid substitution for each CDR residue. Mutants with increased binding affinity to antigen can be screened by contacting the immobilized mutants with labeled antigen. Any screening method known in the art can be used to identify mutant antibodies with increased avidity for an 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, which randomizes the light chain, can also be used (see Schier et al., J. Mol. Biol. 263:551-567(1996)).

[0113] Random mutagenesis can be used in conjunction with phage display methods to identify improved CDRs and / or variable regions. Alternatively, phage display techniques can be used to increase (or decrease) the affinity of CDRs by directed mutagenesis (e.g., affinity maturation or "CDR walking"). This technique uses a target antigen or an antigenic fragment thereof to identify antibodies with CDRs that bind to the antigen with higher (or lower) affinity compared to the initial or parent antibody (see, e.g., Glaser, SM et al., J. Immunol. 149:3903-3913 (1992)).

[0114] 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 incorporated herein by reference in their entireties.

[0115] Also provided herein are derivatives of anti-glycLAG3 antibodies or glycosylated LAG3 polypeptides having one, two, three, four, five, or more amino acid substitutions, additions, deletions, or modifications compared to the "parent" (or wild-type) molecule. Such amino acid substitutions or additions allow for the introduction of naturally occurring (i.e., DNA-encoded) or non-naturally occurring amino acid residues. Such amino acids may be glycosylated (e.g., altered in content with mannose, 2-N-acetylglucosamine, galactose, fucose, glucose, sialic acid, 5-N-acetylneuraminic acid, 5-glycolneuraminic acid, etc.), acetylated, pegylated, phosphorylated, amidated, derivatized with known protecting / blocking groups, proteolytic cleavage, linked to cellular ligands or other proteins, etc. In some embodiments, the altered carbohydrate modification modulates one or more of the following: antibody solubility, enhanced intracellular trafficking and antibody secretion, enhanced antibody assembly, conformational integrity, and antibody-mediated effector function. In some embodiments, the altered carbohydrate modification enhances antibody-mediated effector function compared to an antibody lacking the carbohydrate modification. Carbohydrate modifications that result in altered antibody-mediated effector function are well known in the art (see, e.g., Shields, R.L. et al., J. Biol. Chem. 277(30): 26733-26740 (2002); Davies J. et al. Biotechnology & Bioengineering 74(4): 288-294 (2001), all of which are incorporated herein by reference in their entireties).Methods for altering carbohydrate content are known to those skilled in the art, see, e.g., 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 incorporated herein by reference in their entireties.

[0116] Substitutional variants can involve the exchange of one amino acid for another at one or more sites within an antibody or polypeptide as provided herein, and can be designed to modulate one or more properties of the antibody or polypeptide, with or without loss of other function or property. Substitutions can be conservative, i.e., one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art, and include, for example, 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, substitution can be non-conservative, so that the function or activity of polypeptide is affected. Non-conservative changes generally involve substituting a residue with a chemically different residue, eg, a polar or charged amino acid for a non-polar or uncharged amino acid, and vice versa.

[0117] In some embodiments, an antibody may comprise a first set of CDRs as set forth in Table 4, but with substitutions (e.g., conservative substitutions) at residues that are not conserved in one or all of the other CDR set(s). For example, an antibody may comprise a Chothia, Abm, or Contact-type set of CDRs, but with one or more substitutions in the heavy chain CDR1 sequence (SEQ ID NO: 6, 9, or 13) at residues that do not correspond to residues in the Kabat-type CDR1 sequence (SEQ ID NO: 11). Similarly, residues 1-4 on SEQ ID NO: 20 may be substituted, such as with conservative substitutions. Similarly, tailing residues on SEQ ID NO: 18 and / or SEQ ID NO: 8 may be substituted, such as with conservative substitutions. These are just a few examples, but one of skill in the art will understand what substitutions can be made based on the substitutions shown in Table 4.

[0118] In some embodiments, an antibody having a first set of CDRs (e.g., Chothia, AbM, Kabat, and Contact) may contain framework regions with one or more amino acid substitutions that match one or more substituted residues with a second set of CDRs with leading (i.e., N-terminal) or tailing (i.e., C-terminal) residues that are not present in the first set of CDRs at corresponding positions (as assessed by sequence alignment and / or numbering according to Kabat). For example, the framework region adjacent to the C-terminus of Contact-type CDR2 of the light chain has a substituted residue corresponding to the tailing residue of the amino acid sequence of SEQ ID NO: 17 (Kabat-type CDR2). In some embodiments, the framework region adjacent to the N-terminus of AbM-, Kabat-, or Chothia-type CDR2 of the light chain has a substituted residue corresponding to one or more of leading residues 1-4 of the amino acid sequence of SEQ ID NO: 20 (Contact-type CDR1). Similarly, in some embodiments, the framework region adjacent to the C-terminus of the Contact-type CDR1 of the light chain has a substituted residue corresponding to one or more of leading residues 1-6 of the amino acid sequence of SEQ ID NO: 16 (Kabat-type CDR1). In some embodiments, the framework region adjacent to the C-terminus of the Chothia-type CDR2 of the heavy chain has a substituted residue corresponding to one or more of tailing residues 9-17 of the amino acid sequence of SEQ ID NO: 12 (Kabat-type CDR2). In some embodiments, the framework region adjacent to the N-terminus of the Chothia-, AbM-, or Kabat-type CDR3 of the heavy chain has a substituted residue corresponding to one or more of leading residues 1-2 of the amino acid sequence of SEQ ID NO: 15 (Contact-type CDR3). In some embodiments, the framework region adjacent to the N-terminus of the Kabat-type CDR1 of the heavy chain has a substituted residue corresponding to one or more of leading residues 1-5 of the amino acid sequence of SEQ ID NO: 9 (AbM-type CDR1). These are just a few examples, but one of skill in the art will be able to understand what framework substitutions can be made based on the substitutions shown in Table 4.

[0119] In some embodiments, a humanized antibody is a derivative antibody. Such a humanized antibody contains substitutions, deletions, or additions of amino acid residues in one or more non-human CDRs. Derivatives of humanized antibodies may have substantially the same binding activity, better binding activity, or poorer binding activity compared to non-derivative humanized antibodies. In some embodiments, one, two, three, four, or five amino acid residues in the CDRs are mutated, e.g., substituted, deleted, or added.

[0120] In some embodiments, the polypeptide is a derivative polypeptide. Such polypeptides contain substitutions, deletions, or additions of amino acid residues compared to wild-type human LAG3. Compared to non-derivative polypeptides, derivative polypeptides may have substantially the same, better, or poorer binding to anti-glycoLAG3 antibodies. In some embodiments, one, two, three, four, or five amino acid residues of human LAG3 are mutated, e.g., substituted, deleted, or added.

[0121] Antibodies or polypeptides as described herein may 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, etc. In one embodiment, the derivative polypeptide or antibody has a similar or identical function as the parent polypeptide or antibody. In another embodiment, the derivative polypeptide or antibody exhibits altered activity compared to the parent polypeptide or antibody. For example, the derivative antibody (or fragment thereof) may bind to its epitope more tightly than the parent antibody or may be more resistant to proteolysis.

[0122] Substitutions, additions, or deletions in the derived antibody can be in the Fc region of the antibody and can also serve to alter the binding affinity of the antibody to one or more FcγRs. Methods for modifying antibodies to have altered binding to one or more FcγRs are known in the art; see, e.g., PCT Publication Nos. WO 04 / 029207, WO 04 / 029092, WO 04 / 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, all of which are incorporated herein by reference in their entireties. In some embodiments, antibodies or other molecules may have altered affinity for an activating FcγR, e.g., FcγRIIIA. Preferably, such modifications also have altered Fc-mediated effector function. Modifications that affect Fc-mediated effector function are well known in the art (see U.S. Pat. No. 6,194,551 and WO 00 / 42072). In some embodiments, modifications of the Fc region result in antibodies with altered antibody-mediated effector function, 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 (CDC), altered phagocytic activity, or any combination thereof.

[0123] A derivative antibody or polypeptide may have a half-life (e.g., serum half-life) in a mammal, preferably a human, that is altered from that of the parent molecule or antibody. In some embodiments, such an alteration results 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. An increased half-life of a humanized antibody or polypeptide in a mammal, preferably a human, would result in a higher serum titer of the antibody or polypeptide in the mammal, thus allowing for less frequent administration of the antibody or polypeptide and / or for lower concentrations of the antibody or polypeptide to be administered. Antibodies or polypeptides with increased in vivo half-lives can be generated by techniques known to those skilled in the art. For example, antibodies or polypeptides with increased in vivo half-lives can be generated by modifying (e.g., substituting, deleting, or adding) amino acid residues identified as being involved in the interaction between the Fc domain and the FcRn receptor. 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 increase in vivo or serum half-life.

[0124] Antibodies or polypeptides as described herein that have extended in vivo half-lives can be generated by linking the antibody or polypeptide to a polymer molecule, such as high molecular weight polyethylene glycol (PEG). PEG can be linked to the antibody or polypeptide through site-specific conjugation of PEG to the N- or C-terminus of the molecule or antibody, or through the ε-amino group present in lysine residues, with or without a multifunctional linker. Linear or branched polymer derivatization that minimally impairs biological activity can be used. The degree of conjugation can be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to the antibody. Unreacted PEG can be separated from the antibody-PEG conjugate by, for example, size exclusion or ion exchange chromatography.

[0125] Antibodies or polypeptides as described herein can also be modified by the methods and coupling agents described by Davis et al. (see U.S. Pat. No. 4,179,337) to provide compositions that can be injected into the circulatory system of a mammal without substantial immunogenic response. Removal of the Fc portion can reduce the likelihood that an antibody fragment will elicit an unwanted immune response, and thus, Fc-less antibodies can be used for prophylactic or therapeutic treatments. As noted above, antibodies can also be constructed to be chimeric, partially or fully human, to reduce or eliminate adverse immunological consequences resulting from administering to animals antibodies with sequences generated in or derived from other species.

[0126] Fusions and conjugates The anti-glycLAG3 antibodies or glycosylated LAG3 polypeptides presented herein can also be expressed as fusion proteins with other proteins or chemically conjugated to other moieties.

[0127] In some embodiments, provided herein are antibodies or polypeptides having an Fc portion, which may vary 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 reduce costs). Many modifications useful in constructing the disclosed fusion proteins and methods for making 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 some embodiments, the Fc region is a native IgG1, IgG2, or IgG4 Fc region. In some embodiments, the Fc region is a hybrid, e.g., a chimera having an IgG2 / IgG4 Fc constant region. Modifications to the Fc region include, but are not limited to, an IgG4 modified to block binding to Fcγ receptors and complement, an IgG1 modified to improve binding to one or more Fcγ receptors, an IgG1 modified (amino acid changes) to minimize effector function, an IgG1 with altered / absent glycans (typically by changing the expression host), and an IgG1 with altered pH-dependent binding to FcRn. The Fc region may include the entire hinge region or less than the entire hinge region.

[0128] Another embodiment includes IgG2-4 hybrids and IgG4 mutants that have reduced FcR binding and extended half-lives. 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 incorporated by reference in their entireties. In some embodiments, the IgG1 and / or IgG2 domains are deleted; for example, Angal et al. describe IgG1 and IgG2 mutants in which serine 241 is replaced with proline.

[0129] In some embodiments, 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 are provided herein.

[0130] In some embodiments, the present invention provides an anti-glycolytic LAG3 antibody or glycosylated LAG3 polypeptide that is linked to, covalently bound to, or conjugated with at least one moiety. Such a moiety may be, but is not limited to, a moiety that increases the effectiveness of the molecule as a diagnostic or therapeutic agent. In some embodiments, the moiety may be an imaging agent, a toxin, a therapeutic enzyme, an antibiotic, a radiolabeled nucleotide, or the like.

[0131] In some embodiments, the moiety is an enzyme, a hormone, a cell surface receptor, a toxin (e.g., abrin, ricin A, Pseudomonas exotoxin (i.e., PE-40), diphtheria toxin, ricin, gelonin, or pokeweed antiviral protein, etc.), a protein (e.g., tumor necrosis factor, interferon (e.g., alpha interferon, beta interferon), nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, etc.). beta, or apoptotic agents (e.g., tumor necrosis factor alpha, tumor necrosis factor beta), etc.), biological response modifiers (e.g., lymphokines (e.g., interleukin-1 ("IL-1"), interleukin-2 ("IL-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"), etc.), 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, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracycl ... caine, lidocaine, propranolol, monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE; e.g., vedotin), and puromycin, and analogs or homologs thereof), antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, melphalan, BiCNU® (carmustine;The anticoagulant may be an anticoagulant such as cis-dichlorodiammineplatinum(II) (DDP) (cisplatin), an anthracycline (e.g., daunorubicin (formerly daunomycin) and doxorubicin), an antibiotic (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), or an antimitotic (e.g., vincristine and vinblastine);

[0132] Techniques for conjugating such therapeutic moieties to antibodies are well known and are described, for example, in 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 Antibody In Cancer Therapy”, in MONOCLONAL ANTIBODIES 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, Ducrry 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), e.g., vedotin, or combinations thereof.

[0133] In a preferred embodiment, the antibody is conjugated to maytansine, a benzanthamocyanide originally isolated from the bark of the Ethiopian shrub Maytenus ovatus. This cytotoxic agent and its derivatives (e.g., maytansinoids) bind to tubulin near the vinca alkaloid binding site. It is believed to have high affinity for tubulin located at the ends of microtubules but lower affinity for sites distributed throughout the microtubule. Inhibition of microtubule dynamics causes cell arrest in the G2 / M phase of the cell cycle and ultimately cell death by apoptosis (Oroudjev et al., Mol. Cancer Ther., 10L2700-2713 (2010)). Two maytansine derivatives (thiol-containing maytansinoids), including DM1 and DM4 (ImmunoGen, Inc., Waltham, MA), are widely used in combination with both irreversible and reversible linkers. In particular, DM1 linked to an antibody via a thioether linker is referred to as "emtansine," and DM1 linked to an antibody via an SPP linker is referred to as "mertansine." DM4 linked to an SPDB linker is referred to as "ravtansine," and DM4 linked to an sSPDB linker is referred to as "soravtansine" (ImmunoGen, Inc., Waltham, MA). In one embodiment, an anti-glycLAG3 antibody-ADC comprises a tubulin-active maytansinoid payload DM1. In one embodiment, an anti-glycLAG3 antibody-ADC comprises a tubulin-active maytansinoid payload DM4. In one embodiment, an anti-glycLAG3 antibody-ADC comprises a DNA-active payload, e.g., DGN462 (ImmunoGen, Inc., Waltham, MA). In one embodiment, the anti-glycLAG3 antibody component of an anti-glycLAG3 antibody-ADC is a chimeric or humanized form of STC1807, or a binding portion thereof. In one embodiment, the anti-glycLAG3 antibody component of the anti-glycLAG3 antibody-ADC is a chimeric or humanized form of STC1317, or a binding portion thereof.

[0134] In particular embodiments, the cytotoxic agent conjugated to the anti-glycLAG3 antibody is the highly toxic antitumor agent, MMAE (monomethylauristatin E (or desmethylauristatin E)), whose antimitotic activity is associated with blocking tubulin polymerization, thereby inhibiting cell division. The International Nonproprietary Name (INO) vedotin refers to the linking structure to the antibody in the MMAE+MMAE-antibody conjugate. In more particular embodiments, the ADC is STC1317 (chimeric or humanized form)-MMAE, or STC1317 (chimeric or humanized form)-MMAE.

[0135] Several chemical linkers are known and used to conjugate cytotoxic or DNA-acting drug payloads to antibodies to produce ADCs. To produce ADCs containing anti-glycLAG3 antibodies, particularly those antibodies that internalize after binding to their targets as described herein, certain linkers that can be incorporated, either alone or in combination, 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-sulfobutanoate); the thioether linker succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (MCC); and vc (valine-citrulline dipeptide linker). For example, engineered linkers (e.g., SMCC, SPDB, S-SPDB) (Immunogen, Inc.) are designed to be stable before the ADC binds to the tumor and then optimize payload efficacy once the ADC is internalized inside the cancer cell. Other linkers, such as the dipeptide vc linker, a cathepsin-cleavable linker, can be used to conjugate antibodies to cytotoxic agents, such as auristatins, which are mitotic inhibitors derived from dolastatin 10, such as monomethyl auristatin E (MMAE), e.g., vedotin. Cytotoxins can be conjugated to antibodies such that more than one toxin molecule is linked to each antibody molecule, although there can be, for example, an average of 2, 3, 4, 5, 6, 7, or 8 toxin molecules per antibody.

[0136] In a specific embodiment, MMAE is indirectly linked to a cysteine ​​on an antibody via a maleimidocaproyl (MC) linking group, which in turn is coupled to valine-citrulline-p-aminobenzyloxycarbonyl-MMAE (MC-vc-PAB-MMAE). In the linear structure of "MC-vc-PAB-MMAE," "MC" is composed of maleimide and caproic acid and is the moiety that typically links to the antibody via a cysteine ​​group on the heavy chain. "MC" is then linked to a "vc" linker, which is composed of valine (Val) and citrulline (Cit) and is a cathepsin-cleavable linker that is cleaved by cathepsins inside tumor or cancer cells. "vc" links to a spacer "PAB," i.e., para-aminobenzoic acid, to which the MMAE cytotoxin is linked. MC-vc-PAB-MMAE ADCs release free, membrane-permeable MMAE upon cleavage by proteases, such as cathepsin B. In one embodiment, the linker to the antibody is stable in extracellular fluids but is cleaved by cathepsins when the ADC enters tumor or cancer cells, thus activating the antimitotic mechanism of the MMAE or other toxin drug. In another embodiment, monomethylauristatin F (MMAF) is linked to a cysteine ​​on the antibody by a maleimidocaproyl group (MC-MMAF). In contrast to the MC-vc-PAB-MMAE ADC, the MC-MMAF ADC, like the MMC-DMI ADC, is non-cleavable and must be internalized and degraded to release cysteine-MC-MMAF as the active drug inside the cell.

[0137] In one embodiment, the cytotoxic payload is released into the lysosome after internalization of the ADC 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 and ultimately causes cell death. Optimally, the released payload is fully active with the linker still attached. In other embodiments, where target binding to the ADC causes poor lysosomal transport, a linker that is stable outside the target cell but cleaves the payload from the antibody component inside the cell provides an alternative mode for releasing the payload intracellularly but outside the lysosome. In other embodiments, the linker is stable in extracellular fluids but is cleaved by cathepsins upon entry of the ADC into tumor or cancer cells, thereby activating the antimitotic or other cytotoxic mechanisms of the toxin drug. In other embodiments, the payload released by the action of the cleavable linker can enter and kill neighboring cancer cells via bystander action, thus enhancing the targeting and tumor-killing activity of the ADC.

[0138] In some embodiments, antibodies and polypeptides as described herein may be conjugated to a marker, such as a peptide, to facilitate purification. In some embodiments, the marker is a hexahistidine peptide, the hemagglutinin "HA" tag (SEQ ID NO: 22: YPYDVPDYA) corresponding to an epitope derived from the influenza hemagglutinin protein (Wilson, I. A. et al., Cell, 37:767-778 (1984)), or a "Flag" tag (Knappik, A. et al., Biotechniques 17(4):754-761 (1994)).

[0139] In some embodiments, the moiety can be an imaging agent that is 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.

[0140] In some embodiments, enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; prosthetic group complexes include, but are not limited to, streptavidin / biotin and avidin / biotin; fluorescent materials include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; luminescent materials include, but are not limited to, luminol; bioluminescent materials include, but are not limited to, luciferase, luciferin, and aequorin; and radioactive materials include, but are 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 ( 103 Pd), phosphorous acid ( 32P), praseodymium ( 142 Pr), promethium ( 149 Pm), rhenium ( 186 Re, 188 Re), rhodium (105Rh), ruthenium ( 97 Ru), samarium ( 153 Sm), Scandium ( 47 Sc), selenium ( 75 Se), strontium ( 85 Sr), sulfur ( 35 S), technitium ( 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); various positron-emitting metals using positron emission tomography, and non-radioactive paramagnetic metal ions.

[0141] Imaging agents can be conjugated to the antibodies or polypeptides presented herein directly or indirectly through intermediates (e.g., linkers known in the art) using techniques known in the art. For metal ions that can be conjugated to antibodies and other molecules as described herein for use as diagnostic agents, see, e.g., U.S. Pat. No. 4,741,900. Some conjugation methods involve the use of metal chelate complexes utilizing organic chelators such as diethylenetriaminepentaacetic anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and / or tetrachloro-3-6α-diphenylglycouril-3 linked to the antibody. Monoclonal antibodies can also be reacted with enzymes in the presence of coupling agents such as glutaraldehyde or periodate. Conjugates containing fluorescein markers can be prepared in the presence of such coupling agents or by reaction with isothiocyanates.

[0142] In some embodiments, an antibody or polypeptide as described herein may be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980. Such heteroconjugate antibodies may further bind to 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, LIGH, 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).

[0143] In some embodiments, the anti-glycolyzed LAG3 antibodies or glycosylated LAG3 polypeptides described herein can also be linked to a solid support (which may be useful for immunoassays or purification of target antigens or other molecules capable of binding to target antigens immobilized on a support via conjugation with antibodies or antigen-binding fragments as described herein). Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0144] Protein purification Protein purification techniques are well known to those skilled in the art. At one level, these techniques involve the homogenization of cells, tissues, or organs and their crude fractionation into polypeptide and non-polypeptide fractions. Unless otherwise specified, the protein or polypeptide of interest can be further purified using chromatographic and electrophoretic techniques 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, reverse-phase chromatography, hydroxyapatite chromatography, polyacrylamide gel electrophoresis, affinity chromatography, immunoaffinity chromatography, and isoelectric focusing. A particularly efficient method for purifying peptides is fast performance liquid chromatography (FPLC) or high performance liquid chromatography (HPLC). As is generally known in the art, the order in which the various purification steps are performed can be varied, or certain steps can be omitted, while still providing a suitable method for preparing a substantially purified polypeptide.

[0145] A purified polypeptide is intended to refer to a composition that can be isolated from other components, and the polypeptide is purified to any degree compared to its naturally obtainable state. An isolated or purified polypeptide therefore also refers to a polypeptide that is free from the environment in which it may naturally occur. Generally, "purified" refers to a polypeptide composition that has been fractionated to remove various other components, and the composition substantially retains its expressed biological activity. When the term "substantially purified" is used, this designation refers to a composition in which the polypeptide constitutes the major component of the composition, for example, accounting for about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, etc., of the protein in the composition, or more.

[0146] Various methods for quantifying the degree of purification of a polypeptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction or assessing the amount of polypeptide within a fraction by SDS / PAGE analysis. A preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, compare it to the specific activity of the initial extract, and calculate the purity therein (assessed by the "fold purification"). The actual units used to express the amount of activity will, of course, depend on the specific assay technique chosen to track purification, whether or not the expressed polypeptide exhibits detectable activity.

[0147] There is no general requirement that a polypeptide always be provided in its most purified state. Indeed, it is believed that products of substantially less purity may have utility in certain embodiments. Partial purification can be achieved by combining fewer purification steps or by utilizing different forms of the same general purification scheme. For example, it is recognized that cation exchange column chromatography performed using an HPLC apparatus generally results in a higher "fold" of purification than the same technique using a low-pressure chromatography system. Methods that exhibit a lower relative degree of purification may still have advantages in overall recovery of protein product or in maintaining the activity of the expressed protein.

[0148] Affinity chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule to which it can specifically bind. This is a receptor-ligand type interaction. The column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material can then specifically adsorb the substance from solution. Elution occurs by changing the conditions to those in which binding does not occur (e.g., changes in pH, ionic strength, temperature, etc.). The matrix should be a material that does not significantly adsorb molecules to any degree and has a wide range of chemical, physical, and thermal stability. The ligand should be coupled in a way that does not affect its binding properties. The ligand should also provide relatively tight binding. It should be possible to elute the substance without destroying the sample or the ligand.

[0149] 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 typically applied to large molecules or macromolecular complexes, such as proteins and industrial polymers. Generally, when an aqueous solution is used to transport the sample through the column, the technique is known as 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 elute (sieve) through the stationary phase at different rates. This causes the separation of the solution of particles based on size. If all particles are loaded at or near the same time, particles of the same size should elute together.

[0150] High-performance liquid chromatography (or high-pressure liquid chromatography, HPLC) is a form of column chromatography frequently used in biochemistry and analytical chemistry to separate, identify, and quantify compounds. HPLC utilizes a column that holds a chromatographic packing material (stationary phase), a pump that moves a mobile phase(s) through the column, and a detector that indicates the retention time of molecules. Retention time varies depending on the interactions between the stationary phase, the analyte molecule, and the solvent(s) used.

[0151] Also provided herein are methods for assessing glycosylation, N-linked glycosylation, or N-glycosylation of LAG3, comprising contacting a LAG3-containing sample with an antibody of several embodiments (e.g., the antibody selectively binds to glycosylated LAG3 compared to non-glycosylated LAG3). In some aspects, the method is an in vitro method. In certain aspects, the sample is a cell sample.

[0152] nucleic acid The present disclosure also contemplates nucleic acid molecules (DNA or RNA) encoding any of the anti-glycolyzed LAG3 antibodies or glycosylated LAG3 polypeptides described herein. Also provided herein are vector molecules (e.g., plasmids, etc.) configured to transmit or replicate such nucleic acid molecules. Nucleic acids can be single-stranded, double-stranded, or contain both single- and double-stranded portions.

[0153] Pharmaceutical preparations When clinical applications of pharmaceutical compositions containing antibodies are carried out, it is generally beneficial to prepare pharmaceutical or therapeutic compositions suitable for the intended use. Generally, pharmaceutical compositions can have an effective amount of an anti-glycolyzed LAG3 antibody or glycosylated LAG3 polypeptide as described herein, or can be dissolved or dispersed in a pharmaceutically acceptable carrier together with additional agents.

[0154] Also provided herein are compositions comprising an anti-glycLAG3 antibody or glycosylated LAG3 polypeptide as described herein. In some embodiments, the composition may comprise at least 0.1% by weight of the antibody or polypeptide. In some 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 by weight of the anti-glycLAG3 antibody or glycosylated LAG3 polypeptide. In other embodiments, for example, the anti-glycLAG3 or glycosylated LAG3 polypeptide may comprise about 2% to about 75%, about 25% to about 60%, about 30% to about 50%, or any range therein by weight of the composition. The amount of active compound(s) in each therapeutically useful composition can be adjusted so that a suitable dosage is obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product expiration date, and other pharmacological considerations will be considered by those skilled in the art of preparing such pharmaceutical formulations, and various dosages and treatment regimens may be desired accordingly.

[0155] The composition can be a pharmaceutical composition having an anti-glycolyzed LAG3 antibody or glycosylated LAG3 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 a carrier approved by a federal or state government regulatory agency, or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias for use in animals, and more particularly in humans.

[0156] As used herein, and unless otherwise specified, the term "carrier" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), excipient, stabilizer, or vehicle with which a therapeutic is administered. A "pharmaceutically acceptable carrier" is a carrier that, at the dosage and concentration employed, is nontoxic to cells or mammals exposed thereto, and can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. A pharmaceutically acceptable molecular entity or composition does not produce harmful, allergic, or other untoward reactions when administered to an animal, such as a human, as appropriate. The preparation of pharmaceutical compositions with antibodies or additional active ingredients will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed., 1990, incorporated herein by reference. Additionally, for animal (e.g., human) administration, it is understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

[0157] It is contemplated that the compositions contain between about 0.001 mg and about 10 mg total antibody or polypeptide per ml. Thus, the concentration of antibody or polypeptide in the composition can be approximately at least 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). Of these, approximately at least 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 anti-glycLAG3 antibodies or glycosylated LAG3 polypeptides.

[0158] The preparation of pharmaceutical compositions having as active ingredients antibodies or other polypeptides as described herein will be known to those of skill in the art in light of the present disclosure, and as exemplified by Remington's Pharmaceutical Sciences, 18th Ed., 1990, incorporated herein by reference. Additionally, it will be understood that for animal (including human) administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by the FDA Office of Biological Standards.

[0159] Pharmaceutically acceptable carriers include liquid, semi-solid i.e., paste, or solid carriers. Examples of carriers or diluents include fats, oils, water, saline solution, lipids, liposomes, resins, binders, fillers, etc., or combinations thereof. Pharmaceutically acceptable carriers include those known to those skilled in the art, such as aqueous solvents (e.g., water, alcoholic / aqueous solutions, ethanol, saline solution, 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, etc.), dispersion media, coatings (e.g., lecithin), surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, inert gases, parabens (e.g., methylparaben, propylparaben), etc. , chlorobutanol, phenol, sorbic acid, thimerosal), isotonicity agents (e.g., sugars, sodium chloride), absorption delaying agents (e.g., aluminum monostearate, gelatin), salts, drugs, drug stabilizers (e.g., buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.), gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, liquid and nutrient replenishers, such materials, and combinations thereof, may be included. Except insofar as any conventional media, agents, diluents, or carriers are deleterious to the recipient or to the therapeutic effectiveness of the composition contained therein, their use in administrable compositions used in practicing the methods is appropriate. The pH and precise concentration of the various components in the pharmaceutical composition are adjusted according to well-known parameters. In accordance with certain aspects of the present disclosure, the composition may be combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, mixing, encapsulation, absorption, grinding, etc. Such procedures are routine to those skilled in the art.

[0160] In some embodiments, the pharmaceutically acceptable carrier may be an aqueous pH-buffered solution. Examples include buffers such as phosphate, citric acid, 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 dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.

[0161] In some embodiments, pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water can be a suitable carrier, particularly when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions are also useful as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, polysorbate-80, and the like. The compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like.

[0162] Certain embodiments of the present disclosure may have different types of carriers depending on whether the carrier is to be administered in solid, liquid, or aerosol form, and whether it needs to be sterile for the route of administration, e.g., injection, etc. The compositions may be formulated for administration intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, intramuscularly, subcutaneously, mucosally, orally, topically, locally, by inhalation (e.g., aerosol inhalation), by injection, by infusion, by continuous infusion, by local perfusion directly bathing target cells, via a catheter, via a lavage system, in a lipid composition (e.g., liposomes), or by any other method or combination of the above, as known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed., 1990, incorporated herein by reference). Generally, such compositions can be prepared as liquid solutions or suspensions; solid forms suitable for preparing solutions or suspensions upon addition of a liquid prior to injection can also be prepared; and emulsified preparations can also be prepared.

[0163] Anti-glycoLAG3 antibodies or glycosylated LAG3 polypeptides can be formulated into compositions in free base, neutral, or salt form. Pharmaceutically acceptable salts include acid addition salts, such as salts formed with free amino groups of proteinaceous compositions, or with inorganic acids such as hydrochloric 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 such as sodium, potassium, ammonium, calcium, or ferric hydroxide; or organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, or procaine.

[0164] In a further embodiment, a pharmaceutical composition comprising a lipid is provided herein. Lipids can broadly encompass a class of substances that are characterized by their insolubility in water and can be extracted using organic solvents. Some examples include compounds containing long-chain aliphatic hydrocarbons and their derivatives. Lipids can be naturally occurring or synthetic (i.e., engineered or produced by humans). Lipids can be biological materials. Biological lipids are well known in the art and include, for example, neutral lipids, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulfatides, lipids with ether- and ester-linked fatty acids, polymerizable lipids, and combinations thereof. Compounds other than those specifically described herein that are understood by those skilled in the art as lipids can also be used.

[0165] Those skilled in the art are familiar with the range of techniques that can be adopted to disperse compositions in lipid media.For example, antibody or polypeptide can be dispersed in a solution containing lipid, dissolved with lipid, emulsified with lipid, mixed with lipid, combined with lipid, covalently bound to lipid, contained in lipid as a suspension, contained in or complexed with micelle or liposome, or otherwise associated with lipid or lipid structure by any means known to those skilled in the art.Dispersion may or may not cause liposome formation.

[0166] Generally, the components of the composition are supplied individually or mixed together in unit dosage form, for example, as a lyophilized powder or water-free concentrate in a sealed container, such as an ampoule or sachet indicating the quantity of active ingredient. When the composition is administered by infusion, the composition can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed before administration.

[0167] The amount of active ingredient in each therapeutically useful composition can be adjusted so that a suitable dosage is obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product expiration date, and other pharmacological considerations will be considered by those skilled in the art of preparing such pharmaceutical formulations, and various dosages and treatment regimens may be desired accordingly.

[0168] A unit dose or dosage refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined amount of a pharmaceutical composition calculated to produce the desired response discussed above in conjunction with its administration, i.e., an appropriate route and treatment regimen. The amount administered depends on the desired effect, depending on both the number of treatments and the unit dose. The actual dosage of the composition of the present embodiment administered to a patient or subject can be determined by physical and physiological factors, such as the subject's weight, age, health, and sex, the type of disease being treated, the extent of disease penetration, previous or current therapeutic interventions, the patient's sudden illness, the route of administration, and the potency, stability, and toxicity of the specific therapeutic agent. In other non-limiting examples, the dose can be about 1 microgram / kg / body weight, about 5 micrograms / kg / body weight, about 10 micrograms / kg / body weight, about 50 micrograms / kg / body weight, about 100 micrograms / kg / body weight, about 200 micrograms / kg / body weight, about 350 micrograms / kg / body weight, about 500 micrograms / kg / body weight, about 1 milligram / kg / body weight, about 5 milligrams / kg / body weight, about 10 milligrams / kg / body weight, about 50 milligrams / kg / body weight, about 100 milligrams / kg / body weight, about 200 milligrams / kg / body weight, about 350 milligrams / kg / body weight, about 500 milligrams / kg / body weight to about 1000 milligrams / kg / body weight, or more, per administration, and any range derivable therein. Non-limiting examples of ranges derivable from the numbers provided herein include ranges from about 5 milligrams / kg / body weight to about 100 milligrams / kg / body weight, about 5 micrograms / kg / body weight to about 500 milligrams / kg / body weight, etc., that can be administered based on the numbers set forth above. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[0169] As those skilled in the art will appreciate, the compositions described herein are not limited by the specific nature of the therapeutic preparation. For example, such compositions can be formulated with physiologically tolerable liquid, gel, or solid carriers, diluents, and excipients. These therapeutic preparations can be administered to mammals in a manner similar to other therapeutic agents, for veterinary use, such as for domestic animals, and for clinical use in humans. Generally, the dosage required for therapeutic efficacy will vary depending on the type of use and mode of administration, as well as the individualized requirements of the individual subject. The actual dosage of a composition administered to an animal patient, including a human patient, can be determined by physical and physiological factors, such as body weight, severity of the condition, type of disease being treated, previous or current therapeutic interventions, any idiopathic illnesses of the patient, and the route of administration. Depending on the dosage and route of administration, the frequency of administration and / or effective amount of the preferred dosage may vary based on the subject's response. The practitioner responsible for administration will, in any event, determine the concentration of the active ingredient(s) in the composition and the appropriate dose(s) for the individual subject.

[0170] Disease Treatment As used herein, and unless otherwise specified, the term "subject" refers to an animal that is the object of treatment, observation, and / or experiment. "Animal" includes vertebrates and invertebrates, such as fish, crustaceans, reptiles, birds, etc., and particularly 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 some embodiments, the subject is a human.

[0171] As used herein, and unless otherwise specified, the term "cancer" or "cancerous" refers 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.

[0172] As used herein, and unless otherwise specified, the terms "treat," "treating," or "treatment" refer to administering or administering a therapeutic agent to a subject, or performing a procedure or therapy in a subject to obtain a therapeutic benefit from a disease or health-related condition. For example, treatment may include administering a therapeutically effective amount of an anti-glycLAG3 antibody to a subject. When used in connection with a cancer patient, the terms "treat," "treating," or "treatment" refer to measures that may reduce the severity of the cancer or delay or slow the progression of the cancer, including (a) inhibiting the growth of the cancer, reducing the rate of growth of the cancer, preventing the onset of the cancer, reducing the invasiveness of the cancer, or preventing the metastasis of the cancer, and (b) causing regression of the cancer, or delaying or minimizing one or more symptoms associated with the presence of the cancer, or prolonging the survival of the cancer patient.

[0173] As used herein, and unless otherwise specified, 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 its associated symptoms. A therapeutically effective amount of an agent, including a therapeutic agent, may be the amount necessary to (i) reduce or ameliorate the progression or progression of a given disease, disorder, or condition, (ii) reduce or ameliorate the recurrence, onset, or manifestation 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-glycLAG3 antibody or glycosylated LAG3 polypeptide) may vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the ability of the substance / molecule / agent to elicit a desired response in an individual. A therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of the substance / molecule / agent are outweighed by the therapeutically beneficial effects.

[0174] As used herein, and unless otherwise specified, the term "administering" or "administration" refers to the act of injecting or otherwise physically delivering a substance present outside the body into the patient, for example, by mucosal, intradermal, intravenous, intramuscular delivery methods, etc., and / or any other physical delivery method described herein or known in the art. When treating a disease, disorder, or condition, or a symptom thereof, administration of the substance generally occurs after the disease, disorder, or condition, or a symptom thereof, has manifested. When preventing a disease, disorder, or condition, or a symptom thereof, administration of the substance generally occurs before the disease, disorder, or condition, or a symptom thereof, has manifested.

[0175] Therapeutic uses of anti-glycoLAG3 antibodies and glycosylated LAG3 polypeptides are also provided herein. These antibodies or polypeptides can be used to regulate the activity of LAG3 / MHCII signaling. These antibodies or polypeptides can also be used to treat diseases by inhibiting the inhibitory activity of LAG3 on T cell activation or proliferation and cytokine secretion. Thus, the use of such antibodies or polypeptides in upregulating a subject's immune system by inhibiting or blocking LAG3 signaling is provided herein. In some embodiments, the use of antibodies or polypeptides to block the binding of LAG3 to Gal-3, MHCII, liver sinusoidal endothelial cell lectin (LSECtin), and / or CD3 is provided herein.

[0176] In some embodiments, the therapeutic use of anti-glycLAG3 antibodies and glycosylated LAG3 polypeptides in the treatment of cancer is also provided herein.Upregulation of the immune system is particularly desirable in the treatment of cancer, and therefore methods for treating cancer are also provided herein.Cancer refers to neoplasms or tumors caused by abnormal and uncontrolled cell growth.Cancer can be primary cancer or metastatic cancer.In particular embodiments, cancer cells are positive for MCHII.

[0177] In certain aspects, the polypeptides or antibodies of several embodiments (e.g., glycosylated LAG3 polypeptides or antibodies that bind glycosylated LAG3) can be administered to treat cancer. In particular embodiments, the anti-glycLAG3 antibody is a chimeric or humanized form of STC1317. Cancers for which the present treatment methods are useful include any malignant cell type, such as those found in solid tumors or hematological tumors. Exemplary solid tumors can 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, leukemia, lymphoma, blastoma, myeloma, and the like. Further examples of cancers that may be treated using the methods provided herein include carcinoma, lymphoma, blastoma, sarcoma, leukemia, squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma of the lung), cancer of the peritoneum, hepatocellular carcinoma, gastric or stomach 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 or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer, melanoma, superficial spreading melanoma, lentigo maligna melanoma, peripheral leukemia, and ovarian cancer. These include, but are not limited to, acral lentiginous melanoma, nodular melanoma, and B-cell lymphomas (including low-grade / follicular non-Hodgkin's 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 mass disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, multiple myeloma, acute myeloid leukemia (AML), and chronic myeloblastic leukemia.

[0178] Cancers include, specifically, the following histological types: neoplasms, malignant; carcinoma; undifferentiated carcinoma; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; calcifying cell carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma within adenomatous polyps; adenocarcinoma, familial polyposis coli; solid tumors; malignant carcinoid tumor; branchioloalveolar carcinoma; papillary adenocarcinoma; chromophobe carcinoma; eosinophilic carcinoma; oxophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular Adenocarcinoma; Papillary follicular carcinoma; Non-encapsulated sclerosing carcinoma; Adrenal cortical carcinoma; Endometrioid carcinoma; Adnexal carcinoma; Apocrine adenocarcinoma; Sebaceous gland carcinoma; Ceruminous adenocarcinoma; 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; Acinic cell carcinoma; Adenosquamous carcinoma; Adenocarcinoma with squamous metaplasia; Malignant thymic carcinoma; Malignant ovarian stromal tumor; Malignant theca cell tumor; Malignant granulosa cell tumor; Malignant androblastoma; Sertoli cell tumor; Malignant Leydig cell tumor; Malignant lipocytoma; Malignant paraganglioma; Malignant extramammary paraganglioma; Pheochromocytoma; Hemangioangiosarcoma; Malignant melanoma; Amelanotic malignant melanoma; Superficial spreading melanoma; Malignant melanoma in giant pigmented nevus; Epithelioid cell melanoma; Malignant blue nevus; Sarcoma; Fibrosarcoma; Malignant fibrous histiocytoma; Myxosarcoma; Liposarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Embryonic rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Stromal sarcoma; Malignant mixed tumor; Müllerian mixed tumor; Nephroblastoma; Hepatoblastoma; Carcinosarcoma; Malignant mesenchymal cell tumor; Malignant Brenner tumor; Malignant phyllodes tumor; Synovial sarcoma; Malignant mesothelioma; Dysgerminoma; Embryonal carcinoma; Malignant teratoma; Malignant ovarian goiter; Choriocarcinoma; Malignant mesonephroma; Hemangiosarcoma tumor; malignant hemangioendothelioma; Kaposi's sarcoma; malignant hemangiopericytoma; lymphosarcoma; osteosarcoma; parosteal osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; malignant odontogenic tumor; ameloblastic odontosarcoma; malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pinealoma; chordoma; malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectodermal; cerebellar sarcoma; gangliocytoma; neuroblastoma; retinoblastoma; olfactory nerve tumor; malignant meningioma; neurofibrosarcoma;This includes, but is not limited to, malignant schwannoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's; lateral granuloma; small lymphocytic lymphoma; large cell diffuse lymphoma; follicular lymphoma; mycosis fungoides; other defined non-Hodgkin's lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphocytic 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.

[0179] In some embodiments, the antibodies or polypeptides presented herein can be used to treat a cancer that 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.

[0180] The polypeptides or antibodies can be used herein as anti-tumor agents in a variety of modalities. Presented herein are methods of using the polypeptides or antibodies as anti-tumor agents, thus comprising contacting a population of tumor cells with a therapeutically effective amount of the polypeptide or antibody for a period of time sufficient to inhibit tumor cell growth.

[0181] Various delivery systems are also known and can be used to administer anti-glycLAG3 antibodies, or related molecules of glycosylated LAG3 polypeptides, or related pharmaceutical compositions, such as liposome encapsulation, 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 comprising nucleic acid as part of a retroviral or other vector, etc.

[0182] Methods of administration as provided herein include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural, and mucosal (e.g., intranasal and oral routes) injection. In some embodiments, antibodies, other molecules, or pharmaceutical compositions provided herein are administered intramuscularly, intravenously, subcutaneously, intravenously, intraperitoneally, orally, intramuscularly, subcutaneously, intracavity, transdermally, or cutaneously. Compositions can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered in conjunction with other biologically active agents. Administration can be systemic or local. In addition, pulmonary administration can be employed, for example, by use of an inhaler or nebulizer and a formulation containing an aerosolizing 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 incorporated herein by reference in their entireties. In some embodiments, antibodies, other molecules, or pharmaceutical compositions provided herein are administered locally to the area in need of treatment; local administration can be achieved, for example, by local infusion, by injection, or by implants, which can be porous, non-porous, or gelatinous materials, including membranes, such as sialastic membranes or fibers. In some embodiments, when administering antibodies or other molecules as described herein, care is taken to use materials to which the antibodies or other molecules do not adsorb.

[0183] In some embodiments, the antibodies or polypeptides provided herein are formulated in liposomes for targeted delivery. Liposomes are vesicles composed of concentrically arranged phospholipid bilayers (encapsulating an aqueous phase). Liposomes generally contain various types of lipids, phospholipids, and / or surfactants. The components of liposomes are arranged in a bilayer configuration similar to the lipid arrangement of biological membranes. Liposomes can be useful delivery vehicles due in part to their biocompatibility, low immunogenicity, and low toxicity. Methods for preparing liposomes are known in the art and are provided herein; see, e.g., 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. Pat. Nos. 4,485,045 and 4,544,545, all of which are incorporated herein by reference in their entireties.

[0184] Also provided herein are methods for preparing liposomes with extended serum half-lives, i.e., enhanced circulation times, such as those disclosed in U.S. Pat. No. 5,013,556. In some embodiments, the liposomes used in the methods provided herein are not rapidly cleared from the circulation, i.e., are not taken up by the mononuclear phagocyte system (MPS). Also provided herein are sterically stabilized liposomes, prepared using common methods known to those skilled in the art. Sterically stabilized liposomes may contain lipid components with bulky, highly flexible, hydrophilic portions (which reduce undesirable reactions of liposomes with serum proteins, reduce opsonization with serum components, and reduce recognition by the MPS). Sterically stabilized liposomes can be prepared using polyethylene glycol. For the preparation of liposomes and sterically stabilized liposomes, see, for example, 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.

[0185] Liposomes configured to target specific organs (see, e.g., U.S. Pat. No. 4,544,545) or specific cells (see, e.g., U.S. Patent Application Publication No. 2005 / 0074403) are also provided herein (both patents are incorporated herein by reference in their entireties). Particularly useful liposomes for use in the compositions and methods provided herein can be produced by reverse-phase deposition using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes can be extruded through filters of defined pore size to obtain liposomes with the desired diameter. In some embodiments, molecules having antigen-binding fragments, e.g., F(ab'), can be conjugated to liposomes using previously described methods; see, e.g., Martin et al., 1982, J. Biol. Chem. 257: 286-288, incorporated herein by reference in their entireties.

[0186] Humanized or chimeric antibodies as described herein can also be formulated as immunoliposomes. Immunoliposomes refer to liposomal compositions in which antibodies or fragments thereof are covalently or non-covalently linked to the surface of the liposome. Chemistry for linking antibodies to the surface of liposomes is 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, the entire contents of which are incorporated herein by reference. In some embodiments, the immunoliposomes for use in the methods and compositions provided herein are further sterically stabilized. In some embodiments, humanized antibodies as described herein are covalently or non-covalently linked to a hydrophobic anchor that is stably anchored within the lipid bilayer of the liposome. Examples of hydrophobic anchors include, but are not limited to, phospholipids, such as phosphatidylethanolamine (PE) and phosphatidylinositol (PI). To achieve covalent bonding between an antibody and a hydrophobic anchor, any biochemical strategy known in the art can be used, see, for example, J. Thomas August ed., 1997, Gene Therapy: Advances in Pharmacology, Volume 40, Academic Press, San Diego, Calif., pp. 399-435, the entire contents of which are incorporated herein by reference. For example, a functional group on an antibody molecule can react with an active group on a liposome-associated hydrophobic anchor; for example, the amino group of a lysine side chain on an antibody can be coupled with a liposome-associated N-glutaryl-phosphatidylethanolamine activated by a water-soluble carbodiimide; or the thiol group of a reduced antibody can be coupled with a liposome via a thiol-reactive anchor, such as pyridylthiopropionylphosphatidylethanolamine.See, for example, 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, the entire contents of which are incorporated herein by reference. Immunoliposome formulations containing anti-glycosylated LAG3 antibodies can be particularly effective as therapeutic agents because they deliver active ingredients to the cytoplasm of target cells, i.e., cells containing receptors to which the antibodies bind. In some embodiments, immunoliposomes can have an extended 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 via the endolysosomal pathway.

[0187] The immunoliposome compositions provided herein may comprise one or more vesicle-forming lipids, an antibody or other molecule of the present invention, or a fragment or derivative thereof, and optionally a hydrophilic polymer. The vesicle-forming lipid may be a lipid having two hydrocarbon chains, such as an acyl chain and a polar head group. Examples of vesicle-forming lipids include phospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol, sphingomyelin, and glycolipids, such as cerebrosides and gangliosides. Additional lipids useful in the formulations provided herein are known to those skilled in the art and are encompassed by this description. In some embodiments, the immunoliposome composition further comprises a hydrophilic polymer, such as polyethylene glycol and ganglioside GM1, that extends the serum half-life of the liposome. Methods for conjugating hydrophilic polymers to liposomes are well known in the art and are encompassed by this description. Additional representative immunoliposomes and methods of preparing such compositions are described, for example, in U.S. Patent Application Publication No. 2003 / 0044407, which is incorporated herein by reference in its entirety; PCT International Patent Application 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, Volume 40, Academic Press, San Diego, Calif., pp. 399-435.

[0188] Also provided herein is a method for treating cancer patients by administering anti-glycoLAG3 antibody to patients in a unit dose.Also provided herein is a method for treating cancer patients by administering glycosylated LAG3 polypeptide to patients in a unit dose.A unit dose refers to a physically separate unit that is suitable for single administration to a subject, and each unit contains a predetermined amount of active substance that is calculated to produce the desired therapeutic effect in association with the required diluent, i.e., carrier or vehicle.

[0189] The antibody, polypeptide, or composition is administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The amount administered will depend on the subject being treated, the capacity of the subject's system to utilize the active ingredient, and the degree of therapeutic effect desired. The precise amount of active ingredient required for administration depends on the judgment of the practitioner and is unique to each individual subject. However, suitable dosage ranges for systemic administration are disclosed herein and will depend on the route of administration. Suitable regimens for initial and booster administration are also contemplated and generally include an initial administration followed by subsequent repeated administrations spaced one or more hours apart by injection or other administration. Representative multiple administrations are described herein and are useful for maintaining continuously elevated serum and tissue levels of the polypeptide or antibody. Alternatively, continuous intravenous infusion sufficient to maintain blood concentrations within the range specified for in vivo therapeutic purposes is contemplated.

[0190] The therapeutically effective amount is a predetermined amount calculated to achieve the desired effect. Generally, the dosage varies according to the age, condition, sex and extent of the disease in the patient, and can be determined by those skilled in the art. If any complications occur, the dosage can be adjusted by the individual physician.

[0191] In some embodiments, the antibodies, polypeptides, or pharmaceutical compositions provided herein are contained in a sealed container, such as an ampoule or sachet. In one embodiment, the antibodies, polypeptides, or pharmaceutical compositions provided herein are supplied as a sterile, lyophilized powder or water-free concentrate in a sealed container and can be reconstituted, for example, with water or saline, to a concentration suitable for administration to a subject. In some embodiments, the antibodies, polypeptides, or pharmaceutical compositions provided herein are supplied as a sterile, lyophilized powder in a sealed container in a unit dosage 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. The lyophilized antibodies, polypeptides, or pharmaceutical compositions provided herein should be stored in their original container at 2-8°C and administered within 12 hours, preferably within 6 hours, 5 hours, 3 hours, or 1 hour, after reconstitution. In alternative embodiments, the antibodies, polypeptides, or pharmaceutical compositions provided herein are supplied in liquid form in a sealed container indicating the quantity and concentration of the antibody, polypeptide, or pharmaceutical composition. In some embodiments, the liquid form of the antibodies, polypeptides, or pharmaceutical compositions provided herein is supplied in a 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, or at least 200 mg / ml.

[0192] The precise dose to be employed in the formulation will also depend on the route of administration and the severity of the condition, and should be decided based on the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. For anti-glycolytic LAG3 antibodies or glycosylated LAG3 polypeptides, the dosage administered to a patient is generally 0.01 mg to 100 mg per kg of the patient's body weight. In some embodiments, the dosage administered to a patient is 0.01 mg to 20 mg, 0.01 mg to 10 mg, 0.01 mg to 5 mg, 0.01 mg to 2 mg, 0.01 mg to 1 mg, 0.01 mg to 0.75 mg, 0.01 mg to 0.5 mg, 0.01 mg to 0.25 mg, 0.01 mg to 0.15 mg, 0.01 mg to 0.10 mg, 0.01 mg to 0.05 mg, or 0.01 mg to 0.025 mg per kg of the patient's body weight. The dosage administered to a patient can be 0.2 mg / kg, 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 6 mg / kg, or 10 mg / kg. Doses as low as 0.01 mg / kg are expected to have significant pharmacodynamic effects. Dosage levels of 0.10 to 1 mg / kg are expected to be most suitable. Higher doses (e.g., 1 to 30 mg / kg) may also be expected to be active. Generally, human antibodies have a longer half-life in the human body than antibodies from other species due to the immune response to the foreign polypeptide. Therefore, lower dosages and less frequent administration are feasible for human antibodies. Furthermore, the dosage and frequency of administration of the antibodies or polypeptides presented herein can be reduced by enhancing antibody uptake and tissue penetration through modifications, such as lipidation.

[0193] In yet another embodiment, the compositions can be delivered in a controlled-release or sustained-release system. Any technique known to those skilled in the art can be used to produce sustained-release formulations with one or more of the antibodies, molecules, or pharmaceutical compositions presented herein. See, e.g., U.S. Pat. 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., Proc. 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), all of which are incorporated herein by reference in their entireties. 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 the antibody or polypeptide (see, e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., 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; 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 Nos. 5,679,377; 5,916,597; 5,912,015; 5,989,463; 5,128,326; see also PCT Publication No. WO 99 / 15154; and PCT Publication No. WO 99 / 20253); all of which are incorporated herein by reference in their entireties.

[0194] 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-vinylpyrrolidone), 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 in proximity to the therapeutic target (e.g., the lungs), thus 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, polymer compositions useful as controlled-release implants are used according to Dunn et al. (see U.S. Pat. No. 5,945,155), which is incorporated herein by reference in its entirety. Implantation can generally be performed anywhere within the body of a patient in need of therapeutic treatment, based on the therapeutic effect of in situ controlled release of biologically active substances from the polymer system.

[0195] In another embodiment, a non-polymeric sustained delivery system is used, whereby a non-polymeric implant in the subject's body is used as a drug delivery system. When implanted in the body, the organic solvent of the implant dissipates, diffuses, or leaches from the composition into the surrounding tissue fluid, and the non-polymeric material gradually coagulates or precipitates to form a solid microporous matrix (see U.S. Pat. No. 5,888,533). Controlled release systems are also discussed in the review by Langer (1990, Science 249:1527-1533). Any technique known to those skilled in the art can be used to produce sustained release formulations containing one or more therapeutic agents presented herein. See, e.g., U.S. Pat. No. 4,526,938; International Publication Nos. WO 91 / 05548 and WO 96 / 20698, all of which are incorporated herein by reference in their entireties; 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, Proc. 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.

[0196] Also provided herein are embodiments in which a composition comprises a nucleic acid encoding an antibody or polypeptide as provided herein, and the nucleic acid can be administered in vivo to promote expression of the antibody or polypeptide it encodes by constructing the nucleic acid as part of a suitable nucleic acid expression vector and administering it so that it becomes intracellular, for example, by using a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by using biolistic methods (e.g., a gene gun; Biolistic, 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 incorporated into host cell DNA for expression by homologous recombination.

[0197] Treatment of a subject with a therapeutically effective amount of an antibody, polypeptide, or pharmaceutical composition provided herein can include a single treatment or a series of treatments. It is contemplated that the antibody, polypeptide, or pharmaceutical composition provided herein can be administered systemically or locally to treat disease, such as to inhibit tumor cell growth or kill cancer cells in cancer patients with locally advanced or metastatic cancer. It can be administered intravenously, intrathecally, and / or intraperitoneally. It can be administered alone or in combination with an antiproliferative agent. In one embodiment, it is administered before surgery or other procedures to reduce the cancer burden in a patient. Alternatively, it can be administered after surgery to ensure that any remaining cancer (e.g., cancer that could not be removed by surgery) does not survive. In some embodiments, it can be administered after regression of the primary cancer to prevent metastasis.

[0198] Combination treatment In certain embodiments, the compositions and methods of some embodiments involve administering a glycosylated LAG3 polypeptide or an antibody that selectively binds to glycosylated LAG3 in combination with a second or additional therapy. Such therapy can be applied to the treatment of any disease associated with LAG3 or glycosylated LAG3. For example, the disease can be cancer, and the second therapy is an anti-cancer or anti-hyperproliferative therapy.

[0199] Methods and compositions, including combination therapies, enhance the therapeutic or protective effects and / or increase the therapeutic effect of another anti-cancer or anti-hyperproliferative therapy. Therapeutic and prophylactic methods and compositions can be provided in combined amounts effective to achieve the desired effect, such as killing cancer cells and / or inhibiting cell hyperproliferation. This process can involve administering a polypeptide or antibody and a second therapy. The second therapy may or may not have a direct cytotoxic effect. For example, the second therapy can be an agent that upregulates the immune system without a direct cytotoxic effect. Tissues, tumors, or cells can be exposed to one or more compositions or pharmacological formulations containing one or more agents (e.g., antibodies or anti-cancer agents), or by coexposing the tissues, tumors, and / or cells to two or more different compositions or formulations, where one composition provides 1) a polypeptide or antibody, 2) an anti-cancer agent, or 3) both a polypeptide or antibody and an anti-cancer agent. It is also contemplated that such combination therapy may be used in conjunction with chemotherapy, radiation therapy, surgery, or immunotherapy.

[0200] The terms "contacted" and "exposed," when applied to a cell, are used herein to describe the process of delivering a therapeutic polypeptide or antibody and a chemotherapeutic or radiotherapeutic agent to, or placing in direct juxtaposition with, a target cell. To achieve cell killing, for example, both agents are delivered to the cell in a combined amount effective to kill the cell or prevent it from dividing.

[0201] The anti-glycLAG3 antibody or glycosylated LAG3 polypeptide can be administered before, during, or after a second or additional anti-cancer treatment, or in various combinations. Administration can occur simultaneously or at intervals ranging from minutes to days to weeks. In embodiments in which the antibody or polypeptide is provided to the patient separately from the anti-cancer agent, it is generally ensured that the time between each delivery is sufficient to allow the two compounds to still exert their beneficial combined effect on the patient. In such cases, it is contemplated that the anti-glycLAG3 antibody or glycosylated LAG3 polypeptide and the second therapy can be provided to the patient within about 12 to 24 hours or 72 hours of each other, more specifically, within about 6 to 12 hours of each other. In some circumstances, the duration of treatment can be significantly extended if several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) lapse between each administration.

[0202] In particular embodiments, the anti-glycLAG3 antibody is administered to a patient with cancer in combination with one or more other anti-LAG3 antibodies, including in combination with relatlimab (BMS-986016), LAG525, REGN3767, MGD013, FS118, TSR-033, or IMP321. In other embodiments, the anti-glycLAG3 antibody is administered in combination with one or more anti-PD-1 antibodies, and in particular embodiments, the anti-PD-1 antibody is durvalumab, nivolumab, pembrolizumab, avelumab, atezolizumab, or cemiplimab, administered to the patient for the treatment of cancer. In other embodiments, the anti-glycLAG3 antibody is administered with an agent that inhibits the activity of LAG3, CTLA-4, PD-L1, or PD-1, such as an immunoadhesin comprising an extracellular receptor or ligand-binding portion of a PD-1, PD-L1, LAG3, or CTLA-4 protein fused to an Fc domain. In some embodiments, the anti-glycLAG3 antibody is administered in combination with atezolizumab or avelumab.

[0203] In particular embodiments, the anti-glycLAG3 antibody is administered in combination with an antibody that preferentially binds glycosylated PD-1 relative to non-glycosylated PD-1. In particular, the anti-glycLAG3 antibody may be administered in combination with a chimeric or humanized form of the anti-PD-1 antibody STM418 or STM432, which preferentially binds glycosylated PD-1 relative to non-glycosylated PD-1, and whose heavy and light chain variable domain amino acid sequences (and encoding nucleotide sequences) are disclosed in PCT Publication WO 2017 / 096026, published June 8, 2017, entitled "Antibodies Specific for Glycosylated PD-1 and Methods of Use Thereof," which is incorporated herein by reference.

[0204] In particular embodiments, the anti-glycLAG3 antibody is administered in combination with an antibody that preferentially binds glycosylated PD-L1 compared to non-glycosylated PD-L1. In particular, the anti-glycLAG3 antibody may be administered in combination with a chimeric or humanized form of the anti-PD-L1 antibody STM004 or STM115, which preferentially binds glycosylated PD-L1 compared to non-glycosylated PD-L1, and whose heavy and light chain variable domain amino acid sequences (and encoding nucleotide sequences) are disclosed in PCT Publication WO 2016 / 160792, published October 6, 2016, entitled "Antibodies Specific for Glycosylated PD-L1 and Methods of Use Thereof," which is incorporated herein by reference. The anti-glyc-LAG3 antibody may also be administered in combination with chimeric or humanized forms of the anti-PD-L1 antibodies STM073 and SMT108, which preferentially bind to glycosylated PD-L1 compared to non-glycosylated 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 Patent Application No. 62 / 314,652, filed March 29, 2016, entitled "Dual Function Antibodies Specific for Glycosylated PD-L1 and Methods of Use Thereof," which is incorporated herein by reference.

[0205] In certain embodiments, a course of treatment may last for 1 to 90 days or more (including intermediate days). It is contemplated that one agent may be given on any day between 1 and 90 (including intermediate days), or any combination thereof, and another agent may be given on any day between 1 and 90 (including intermediate days), or any combination thereof. One or more doses of agent(s) may be given to a patient within a single day (24 hours). In addition, it is contemplated that a period of time will follow the course of treatment during which no anti-cancer treatment is administered. This period may last for 1 to 7 days, and / or 1 to 5 weeks, and / or 1 to 12 months or more (including intermediate days), depending on the patient's condition, e.g., their prognosis, intensity, health, etc. The treatment cycle may be repeated as necessary.

[0206] Various combinations can be employed. Some examples of treatment with an anti-glycLAG3 antibody or glycosylated LAG3 polypeptide are listed below, with "A" and a second anti-cancer therapy as "B": A / B / AB / A / BB / B / AA / A / BA / 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 / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A

[0207] When administering any antibody, polypeptide, or pharmaceutical composition provided herein to a patient in combination with a second therapy, the general protocol for administering such a second therapy is followed, taking into account the toxicity, if any, of the second therapy.Thus, in some embodiments, there is a step of monitoring the toxicity resulting from the combination therapy.

[0208] chemotherapy A wide variety of chemotherapeutic agents can be used as second therapies in accordance with this embodiment. Chemotherapeutic agents can be compounds or compositions administered in the treatment of cancer. These agents or drugs can be classified by their mode of activity within cells, for example, whether and at what stage they affect the cell cycle. Alternatively, agents can be characterized based on their ability to directly crosslink DNA, intercalate into DNA, or induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis.

[0209] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenethiophosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (synthetic analogs, KW-2 189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosulphonates, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma 11 and calicheamicin omega 11); dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, authrannycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin (idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, queramycin, rhodrubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as thotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, pteropterin, and thorubicin. Rimetrexate, etc.; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine, etc.; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine, etc.; androgens, such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone, etc.; antiadrenal agents, such as mitotane and trilostane, etc.; folic acid supplements, such as floric acid, etc.; acegra Ton; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformitin; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidynin; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidamol; nitraelin; pentostatin; fenamet; pirarubicin; losoxantrone; podophyllic acid;2-Ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; schizofiran; spirogermanium; tenuazonic acid; triazicon; 2,2',2''-trichlorotriethylamine; trichothecines (especially T-2 toxin, veracrine A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel, gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, e.g., cisplatin, oxaliplatin, and carboplatin Examples of suitable anti-inflammatory drugs include vinblastine, platinum, etoposide (VP-16), ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, irinotecan (e.g., CPT-11), topoisomerase inhibitors RFS2000, difluoromethylornithine (DMFO), retinoids such as retinoic acid, capecitabine, carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl protein transferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0210] Radiation therapy Another conventional anti-cancer 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 the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents, such as microwaves, proton beam irradiation (see U.S. Patent Nos. 5,760,395 and 4,870,287, all of which are incorporated herein by reference in their entirety), and UV irradiation, are also contemplated. All of these agents most likely cause extensive damage to DNA, the precursors of DNA, DNA replication and repair, and chromosome assembly and maintenance.

[0211] The tumor microenvironment is inherently inhibitory due to the presence of myeloid-derived suppressor cells and regulatory T cells, which infiltrate tumors and function to suppress immune responses. Additionally, the expression of certain inhibitory molecules on T cells and antigen-presenting cells (APCs) can limit effective immune responses. Irradiation contributes to antitumor effects through the induction of tumor cell apoptosis, senescence, and autophagy, and may stimulate more effective immune responses in some circumstances.

[0212] The abscopal effect is a physiological process by which targeted irradiation of a primary tumor induces antitumor responses at distant sites beyond the radiation field. The mechanisms involved in the abscopal effect are immune-mediated and are thought to involve 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 far from the primary tumor receiving radiation treatment, agents that can trigger the abscopal effect may be particularly advantageous in the treatment of metastatic tumors, which are often difficult to treat once they have spread to secondary sites in the body.

[0213] The anti-glycol LAG3 antibodies or glycosylated LAG3 polypeptides described herein can stimulate local and systemic immune responses. In some embodiments, a therapeutically effective amount of an antibody, polypeptide, or pharmaceutical composition as described herein is administered before, concurrently with, or after radiation therapy to achieve a synergistic abscopal effect.

[0214] In some embodiments, a therapeutically effective amount of an antibody, polypeptide, or pharmaceutical composition described herein is administered to effectively sensitize a tumor in a host to radiation. The radiation can be ionizing radiation, and particularly gamma radiation. In some embodiments, gamma radiation is emitted by a linear accelerator or a radionuclide. Irradiation of a tumor with a radionuclide can be external or internal.

[0215] In some embodiments, administration of an antibody, polypeptide, or pharmaceutical composition described herein begins up to one month, particularly up to ten days or one week, before tumor irradiation. Furthermore, tumor irradiation is fractionated, but administration of an antibody, polypeptide, or pharmaceutical composition described herein is maintained in the interval between the first and last irradiation sessions.

[0216] Irradiation can be X-ray irradiation. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods (3 to 4 weeks), to single doses of 2,000 to 6,000 roentgens. Dosage ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.

[0217] immunotherapy Those skilled in the art will understand that immunotherapy can be used in conjunction or in conjunction with the methods of some 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 (RITUXAN™) is one such example. Checkpoint inhibitors, such as ipilumimab, pembrolizumab, nivolumab, and atezolizumab, are other examples. The immune effector can be, for example, an antibody specific for some marker on the surface of tumor cells. The antibody can act alone as an effector of therapy or can introduce other cells to actually affect cell killing. The antibody can also be conjugated to a drug or toxin (e.g., a chemotherapeutic agent, a radionuclide, ricin A chain, cholera toxin, or pertussis toxin) and simply act as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0218] In one aspect of immunotherapy, tumor cells carry several markers that are easy to target, i.e., not present on the majority of other cells. Many tumor markers exist, any of which may be suitable for targeting in the context of this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erbB, and p155. An alternative aspect of immunotherapy is to combine anti-cancer effects with immunostimulatory effects. There are also immunostimulatory molecules, including cytokines such as IL-2, IL-4, IL-12, GM-CSF, γ-IFN, etc., chemokines such as MIP-1, MCP-1, IL-8, etc., and growth factors such as FLT3 ligand.

[0219] Examples of immunotherapies currently under investigation or in use include immunoadjuvants such as 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 therapy such as 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. Immunother., 21(5):389-98 (1998); Hellstrand et al., Acta Oncol. 37(4):347-53 (1998)), gene therapy, such as 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. Pat. Nos. 5,830,880 and 5,846,945), and monoclonal antibodies, such as 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. Pat. No. 5,824,311, all of which are incorporated herein by reference in their entireties. It is contemplated that one or more anti-cancer therapies may be employed in conjunction with the therapies described herein involving the use of anti-glycLAG3 antibodies or glycosylated LAG3 polypeptides.

[0220] surgery Approximately 60% of people with cancer undergo some type of surgery, including preventative, 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 of the present embodiments, 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 include laser surgery, cryosurgery, electrosurgery, and microsurgery (Mohs procedure).

[0221] When cancerous cells, tissues, or tumors are partially or completely removed, a cavity may be formed in the body. Treatment can be achieved by perfusion, direct injection, or local application of additional anti-cancer therapy to the area. Such treatment 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. In these treatments, dosage may also vary.

[0222] Other drugs It is contemplated that other agents may be used in combination with certain aspects of the present embodiments to improve the therapeutic efficacy of the treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of hyperproliferative cells to apoptosis inducers, or other biological agents. Increasing the number of GAP junctions can increase intercellular signaling, thereby increasing the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cytostatic or differentiation agents may be used in combination with certain aspects of the present embodiments to improve the anti-hyperproliferative efficacy of the treatment. Cell adhesion inhibitors are contemplated to improve the efficacy of the present embodiments. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as the antibody c225, may be used in combination with certain aspects of the present embodiments to improve the efficacy of the treatment.

[0223] Kits and Diagnostics In various aspects, kits containing therapeutic agents and / or other treatments and delivery agents are provided herein. In some embodiments, kits are contemplated for use in preparing and / or administering the therapies provided herein. Kits may include one or more sealed vials containing any of the pharmaceutical compositions provided herein. Kits may include, for example, at least an anti-glycoLAG3 antibody or a glycosylated LAG3 polypeptide, as well as reagents for preparing, formulating, and / or administering the components provided herein or for performing one or more steps of the methods provided herein.

[0224] In some embodiments, the kit may include an anti-glycLAG3 antibody and at least one ancillary reagent. In some embodiments, the kit may include a glycosylated LAG3 polypeptide and at least one ancillary reagent.

[0225] In some embodiments, the kit further comprises a second anti-cancer agent, which can be a chemotherapeutic agent, an immunotherapeutic agent, a hormonal therapy agent, or a cytokine.

[0226] In some embodiments, the kits may also comprise suitable container means, which may be a container that will not react with the components of the kit, such as an Eppendorf tube, an assay plate, a syringe, a bottle, or a tube, etc. The container may be made from a sterilizable material, such as plastic or glass.

[0227] The kits may further include an instruction sheet outlining the procedural steps of the methods provided herein, and follow substantially the same procedures as those described herein or known to those of skill in the art. When implemented using a computer, the instruction information may be in a computer-readable medium containing machine-readable instructions that display actual or hypothetical procedures for delivering a pharmaceutically effective amount of an antibody or polypeptide provided herein. The kits may also include a notice in a form prescribed by a government agency regulating the production, use, or sale of pharmaceutical or biological products, which notice reflects approval by the agency having jurisdiction over the production, use, or sale for human administration. [Example]

[0228] It is understood that modifications that do not substantially alter the nature and spirit of the various embodiments described herein are contemplated. Accordingly, the following examples are intended to be illustrative and not limiting in any way.

[0229] material and method K by Octet D Determination and binning of high-throughput K DFor screening, antibody ligands were loaded onto the sensor via 20 nM solutions. 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 assay buffer containing a single concentration of analyte. Dissociation was performed and monitored in fresh assay buffer. All experiments were performed with the sensor oscillating at 1,000 rpm. ForteBio's data analysis software was used to fit the data to a 1:1 binding model and extract association and dissociation rates. KD was calculated using the ratio kd / ka. In a representative epitope binning assay, the antigen LAG3-His (10 nM) was preincubated with a secondary antibody (10 nM) for 1 hour at room temperature. A control antibody (20 nM) was loaded onto an 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 a pre-incubated antigen-secondary antibody mixture. Raw data were processed using ForteBio's data analysis software 7.0, and antibody pairs were evaluated for competitive binding. Additional binding of the secondary antibody indicates an unoccupied epitope (non-competitor), while no binding indicates epitope blocking (competitor).

[0230] Glycosylation analysis of LAG3. To confirm glycosylation of the LAG3 protein, cell lysates were treated with the enzymes PNGase F, Endo H, and O-glycosidase (New England BioLabs, Ipswich, MA, USA) according to the manufacturer's instructions.

[0231] [Example 1] LAG3 is highly glycosylated Based on the UniProt database (http: / / www.uniprot.org), human LAG3 is glycosylated at N188, N250, N256, and N343. To confirm the glycosylation of LAG3 protein, we treated it with PNGase F (New England BioLabs) according to the manufacturer's instructions. When LAG3 protein is expressed in mammalian cells, the size of the protein is larger (57.5 kDa) than the calculated molecular weight (46.41 kDa). Treatment with PNGase F removes oligosaccharide moieties from the protein, reducing the protein size to the expected 46.41 kDa, as shown in Figure 1. This result confirmed the glycosylation of LAG3 protein (Figure 1).

[0232] [Example 2] Generation of anti-LAG3 antibodies We obtained purified LAG3-His protein from 293F cells overexpressing heavily glycosylated LAG3 from Novoprotein. Hybridomas producing monoclonal antibodies against glycosylated human LAG3 were obtained by fusing SP2 / 0 mouse myeloma cells with spleen cells isolated from human LAG3-immunized BALB / c mice (n = 4) and NZW mice (n = 4; Antibody Solutions, Inc., Sunnyvale, CA, USA) according to standard protocols. Prior to fusion, sera from immunized mice were validated for binding to the LAG3 immunogen using FACS analysis. Over 3,000 monoclonal antibody (mAb)-producing hybridomas were generated. Antibody-producing hybridomas were retested for specificity. Among them, 22 mAb-producing candidate hybridomas were selected by FACS using LAG3 WT or 4NQ (deglycosylated form)-expressing 293T cells and grown in DCGF medium (Antibody Solutions). The monoclonal antibody-containing supernatants were concentrated and purified. Using dot blot analysis, the purified mAbs were tested for their ability to bind to glycosylated but not deglycosylated LAG3. The results of this assay showed that of the 22 mAbs tested, 3 mAbs specifically bound to glycosylated LAG3 (Figure 2).

[0233] To facilitate the purification of each antibody from hybridoma supernatant, the isotype of each antibody was determined by ELISA. Antibodies for each antibody isotype (Sigma-Aldrich, Cat. #ISO2) were used according to the manufacturer's instructions. The isotype determination results for the LAG3 antibody are listed in Table 5. This isotype information was used to select column resins for affinity chromatography purification. For fast protein liquid chromatography (FPLC), Protein G was used for IgG1 type, and Protein A was used for IgG2a and IgG2b (Table 5).

[0234] [Table 4]

[0235] High-throughput K D For screening, antibody ligands were loaded onto Octet sensors (anti-mouse IgG Fc capture (AMC) biosensors) via 20 nM solutions. 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 assay buffer containing a single concentration of analyte. Dissociation was performed and monitored in fresh assay buffer. All experiments were performed with the sensor oscillating at 1,000 revolutions per minute. Data analysis software from ForteBio (Menlo Park, CA, USA) was used to fit the data to a 1:1 binding model to extract association and dissociation rates. The ratio of kd:ka was used to calculate K. D The data are summarized in Figure 3 and Table 6. Among these, STC1317 had a 2.85 x 10 9 The strongest binding affinity was observed with the KD of M.

[0236] [Table 5]

[0237] [Example 3] Antibody sequencing Hybridoma cell pellets were frozen at -80°C, and total RNA was isolated using the RNeasy Plus Mini kit (Qiagen, Hilden, DE) and quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). cDNA synthesis and rapid amplification of cDNA ends (RACE) were performed using the SMARTer RACE 5' / 3' kit according to the manufacturer's instructions (Clontech, Mountain View, CA, USA). PCR primers for Ig-specific PCR amplification were purchased from the Novagen Mouse Ig Primer Set (Merck KGaA, Darmstadt, DE, Germany). The heavy chain primer set (A–F), light (kappa) chain primer set (A–G), and 0.5 μg of template RNA were used per 25 μL reaction volume. After gel separation, the PCR product was ligated into pCR 2.1 vector, transformed into competent cells, and selected on 100 μg / mL ampicillin and 50 μL X-gal / IPTG agar plates. White colonies were picked after 24 hours. After cultivation, plasmid DNA was isolated and subjected to sequencing using M13 forward and reverse primers. The sequences and CDRs of the STC1317 heavy and kappa light chain variable regions are listed in Table 3 and Table 4, respectively.

[0238] [Example 4] KD measurement by Biocore 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 flowed over the chip at 2 μg / mL in HBS-EP+ buffer. Six concentrations of LAG3, each diluted 2-fold, were then passed over the chip. Sensorgram data were analyzed using Biacore X100 Evaluation Software version 2.0.1 using 1:1 binding kinetics. STC1317 was found to have KDs of 0.021 nM and 0.406 nM for the Fc-tagged and His-tagged LAG3 proteins, respectively. This indicates the very strong binding affinity of STC1317 for LAG3 (Figure 4 and Table 7).

[0239] [Table 6]

[0240] [Example 5] Effect of anti-GlycLAG3 antibody on T cell proliferation To evaluate the in vitro efficacy of STC1317, a mixed lymphocyte reaction (MLR) was performed using dendritic cells (DCs) derived from PBMCs of an allogeneic donor (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 universal 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. CD4+ T cells were also enriched from PBMCs of another allogeneic donor (Immunospot #CTL-CP1) using CD4 microbeads (Miltenyi Biotech #130-045-101). DCs (1 × 10 4 DCs (number per well) were cultured in 96-well flat-bottom plates (Nunc) at 1 × 10 5The cells were co-cultured with 100 T cells / well in the presence of STC1317. After 5 days of culture, the concentrations of IFN-γ and IL-2 in the culture supernatant were determined using a cytokine ELISA kit (BioLegend) according to the manufacturer's instructions. As shown in Figure 5, the secretion of IFN-γ and IL-2, markers of T cell proliferation, was significantly increased in the presence of STC1317.

[0241] [Example 6] Antibody Humanization - Framework Regions As alluded to above, for certain purposes, including, for example, use in the in vivo treatment of human disease, it is preferable to employ humanized derivatives of murine monoclonal antibodies. To generate such humanized antibodies, the framework sequences of a murine monoclonal antibody (the "parent" sequence) are first aligned with the framework sequences of a series of "acceptor" human antibodies to identify differences in the framework sequences. Humanization is achieved by substituting non-matching framework residues between the parent and acceptor. Substitutions at potentially critical positions, such as positions within the Vernier zone, the VH / VL chain interface, or CDR canonical class-defining positions, can be analyzed for forward backmutation (see Foote, J. et al., J. Molec. Biol. 224:487-499 (1992)).

[0242] The Conserved Domain Database (COD) (Marchler-Bauer, et al. (2011) Nucleic Acids Res. 39:D225-D229) can be used to determine the interior and approximate boundaries of each amino acid chain domain. Variable domain boundaries can be precisely determined along with CDR boundaries according to several commonly used definitions (Kabat, EA et al. (1991) "Sequences of Proteins of Immunological Interest," Fifth Edition. NIH Publication No. 91-3242; Chothia, C. et al., J. Mol. Biol. 196:901-917 (1987); Honegger, A. et al., J. Mol. Biol. 309(3):657-670 (2001)).

[0243] Multiple alignments of parental sequences against mouse and human germline sequences were generated using MAFFT (Katoh, K. et al., Nucleic Acids Res. 30: 3059-3066 (2002)), and entries in each alignment were ordered according to sequence identity to the parental sequences. The reference set was reduced to a unique set of sequences by clustering at 100% sequence identity and removing redundant entries.

[0244] Selection of the optimal acceptor framework is based on overall parent antibody sequence identity to the acceptor, spanning both chain frameworks; however, positions constituting the VH / VL interchain interface are of particular interest. Additionally, the CDR loop lengths and CDR positions associated with each set of canonical structures defined for the five CDRs (Chothia, C. et al., J. Mol. Biol. 196:901-917 (1987); Martin, A. C. et al., J. Molec. Biol. 263:800-815 (1996); Al-Laziniki, B. et al., J. Molec. Biol. 273:927-948 (1997)) are compared with germline frameworks to determine which germline frameworks are known to share the same interface residues and support similar CDR loop conformations.

[0245] Based on the sequence alignment of the parent antibody to the human germline sequences, the entries showing the closest matches are identified. The selection of preferred human germlines is based on ordered criteria: (1) sequence identity across the framework; (2) identical or compatible interchain interface residues; (3) support loops with the parent CDR canonical conformation; (4) the combination of heavy and light chain germline sequences found in the expressed antibody; and (5) the presence of N-glycosylation sites that must be removed.

[0246] A structural model of the Fv region of a humanized antibody is generated. Candidate structural template fragments for the FRs and CDRs and complete Fv are scored, ranked, and selected from an antibody database based on their sequence identity to the target and qualitative crystallographic indices of the template structure, such as resolution in angstroms (Å).

[0247] To structurally align the CDR to the FR template, five residues on either side of the CDR are included in the CDR template. A fragment alignment is generated based on the overlapping segments and the generated structural sequence alignment. The template fragments along with the alignment are processed by MODELLER (SalI, A. et al.; J. Molec. Biol. 234:779-815(1993)). This protocol creates conformational constraints derived from a set of aligned structural templates. A set of structures that satisfy the constraints is created by simulating a conjugate gradient and annealing optimization procedure. A model structure is selected from this set based on the score of the protein structure and the energy score derived from the degree of satisfaction of the conformational constraints. The model is assessed, and the side chains at different positions between the target and the template are optimized using a side chain optimization algorithm, and energy is minimized. Visualization devices and computerized tools are used to assess CDR conformational variation, local packing, and surface analysis to select one or more preferred models.

[0248] A structural model of the parent antibody is constructed and assessed for imperfections, such as poor atom packing, distortions in bond lengths, bond angles, or dihedral angles. These imperfections may indicate potential problems with the structural stability of the antibody. The modeling protocol strives to minimize such imperfections. The initial structural model of the humanized Fv contains all safe substitutions (i.e., substitutions that should not affect binding affinity or stability) and conservative substitutions (i.e., positional substitutions, but where the position may be important for binding affinity). Substitutions at positions thought to be associated with risk of decreased binding affinity or decreased stability are left untouched. To create a good standalone model rather than a closest-matching parent variant model, template search and selection are performed separately from the parent template search. When evaluation of substitution possibilities is performed, the model is updated to reflect the effects of favorable substitutions and backmutations.

[0249] [Example 7] Antibody humanization - constant region The variable region of the STC1317 heavy chain (VH) and the variable region of its kappa light chain (VL) can be modified by replacing the mouse constant region with the human IgG1 constant region (CH1-CH3) in the pFUSEss-CHIg-hG1 and pFUSEss-CLIg-hK vectors (Invivogen), respectively. The heavy and light chain chimeric constructs can be transfected into 293F suspension cells at a 1:1 ratio for 5 days. The chimeric antibody (hSTC1317) can be purified by HPLC using a Protein A affinity column.

[0250] Throughout this application, various publications are cited. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this disclosure pertains. While examples of certain specific embodiments have been presented herein, it will be apparent to those skilled in the art that various changes and modifications can be made. Such modifications are also intended to fall within the scope of the appended claims. The present invention includes the following embodiments. [1] An isolated monoclonal antibody that selectively binds to glycosylated LAG3 relative to the non-glycosylated form of LAG3. [2] The isolated antibody according to [1] above, which increases the secretion of IFN-γ and / or IL-2, or increases T cell proliferation. [3] The isolated antibody according to [1] above, which blocks the binding of LAG3 to one or more of Gal-3, MHCII, LSECtin and CD3. [4] An isolated antibody described in [1] or [3] above, which selectively binds to LAG3 glycosylated at positions N188, N250, N256, N343, or any combination thereof, compared to unglycosylated LAG3. [5] The isolated antibody according to any one of [1] to [4] above, wherein the binding affinity of the anti-glycLAG3 antibody to glycosylated LAG3 is 0.1 to 10 nM, inclusive of the lower and upper limits. [6] An isolated antibody according to any one of [1] to [5] above, which masks glycosylation of LAG3 at one or more of N188, N250, N256 or N145. [7] The isolated antibody according to any one of [1] to [6] above, which competes or cross-competes with MAb STC1317 for specific binding to glycosylated LAG3. [8] V H The domain has the amino acid sequence of SEQ ID NO: 3, and V L The isolated antibody according to any one of [1] to [6] above, which has the amino acid sequence of SEQ ID NO: 5. [9] V H An isolated antibody according to any one of [1] to [6] above, wherein the domain has an amino acid sequence that is at least 90%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO:3.

[10] V L An isolated antibody according to any one of [1] to [6] or [9] above, wherein the domain has an amino acid sequence that is at least 90%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO: 5.

[11] A V comprising a Chothia CDR H1 having the amino acid sequence of SEQ ID NO: 6, a CDR H2 having the amino acid sequence of SEQ ID NO: 7, and a CDR H3 having the amino acid sequence of SEQ ID NO: 8. H The isolated antibody according to any one of [1] to [6] above, having a domain.

[12] V comprising AbM CDR H1 having the amino acid sequence of SEQ ID NO: 9, CDR H2 having the amino acid sequence of SEQ ID NO: 10, and CDR H3 having the amino acid sequence of SEQ ID NO: 8 H The isolated antibody according to any one of [1] to [6] above, having a domain.

[13] V comprising Kabat CDR H1 having the amino acid sequence of SEQ ID NO: 11, CDR H2 having the amino acid sequence of SEQ ID NO: 12, and CDR H3 having the amino acid sequence of SEQ ID NO: 8 H The isolated antibody according to any one of [1] to [6] above, having a domain.

[14] V comprising Contact CDR H1 having the amino acid sequence of SEQ ID NO: 13, CDR H2 having the amino acid sequence of SEQ ID NO: 14, and CDR H3 having the amino acid sequence of SEQ ID NO: 15 H The isolated antibody according to any one of [1] to [6] above, having a domain.

[15] V comprising CDR L1 having the amino acid sequence of SEQ ID NO: 16, CDR L2 having the amino acid sequence of SEQ ID NO: 17, and CDR L3 having the amino acid sequence of SEQ ID NO: 18 L An isolated antibody according to any one of [1] to [6] or

[11] to

[14] above, having a domain.

[16] V comprising Contact CDR L1 having the amino acid sequence of SEQ ID NO: 19, CDR L2 having the amino acid sequence of SEQ ID NO: 20, and CDR L3 having the amino acid sequence of SEQ ID NO: 21 L An isolated antibody according to any one of [1] to [6] or

[11] to

[14] above, having a domain.

[17] V comprising CDRs H1, H2, and H3 having amino acid sequences with 1, 2, 3, 4, or 5 amino acid substitutions in one, two, or three of the CDRs having the amino acid sequences of SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively, or having the amino acid sequences of SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:8, respectively, or having the amino acid sequences of SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:8, respectively, or having the amino acid sequences of SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively. H The isolated antibody according to any one of [1] to [6] above, having a domain.

[18] V comprising CDRs L1, L2 and L3 having the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively, or having 1, 2, 3, 4 or 5 amino acid substitutions in one, two or three of the CDRs having the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively. L The isolated antibody according to any one of [1] to [6] or

[17] above, having a domain.

[19] The isolated antibody according to any one of [1] to [6] or

[11] to

[18] above, which has a human framework region.

[20] An isolated antibody according to any one of [1] to [6] or

[11] to

[18] above, having a heavy or light chain human framework region with 1, 2, 3, 4, 5, or 6 amino acid substitutions.

[21] An isolated antibody according to any one of [1] to [6] or

[11] to

[20] above, which comprises a human constant domain.

[22] The isolated antibody according to any one of [1] to [6] or

[11] to

[21] above, which is IgG, IgM, IgA, or an antigen-binding fragment thereof.

[23] The isolated antibody according to any one of [1] to [6] or

[11] to

[22] above, which is a Fab', F(ab')2, F(ab')3, monovalent scFv, divalent scFv, or single domain antibody.

[24] The isolated antibody according to any one of [1] to [6] or

[11] to

[23] above, which is a human antibody or a humanized antibody.

[25] The isolated antibody according to any one of [1] to

[24] above, which is conjugated to an imaging agent, a chemotherapeutic agent, a toxin, or a radionuclide.

[26] A composition comprising the isolated antibody according to any one of [1] to

[25] above in a pharmaceutically acceptable carrier.

[27] A method for treating a subject having cancer, comprising the step of administering to the subject an effective amount of the isolated antibody according to any one of [1] to

[26] above in a pharmaceutically acceptable composition.

[28] The method according to

[27] above, wherein 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, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer.

[29] The method according to

[27] or

[28] above, wherein the isolated antibody is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or topically.

[30] The method according to

[27] or

[29] above, further comprising the step of administering at least one second anticancer therapy to the subject.

[31] The method according to

[30] above, wherein the second anticancer therapy is surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy.

[32] The method according to

[30] above, wherein the second anticancer therapy is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, or an anti-LAG3 antibody.

[33] The method according to

[30] above, wherein the second anticancer therapy is durvalumab, nivolumab, pembrolizumab, avelumab, atezolizumab, cemiplimab or ipilimumab.

[34] The method according to

[30] above, wherein the second anti-cancer therapy is an anti-LAG3 antibody that preferentially binds to glycosylated LAG3 compared to non-glycosylated LAG3.

[35] the second anti-cancer therapy is a humanized or chimeric anti-glycosylated LAG3 monoclonal antibody; H The domain has the amino acid sequence of SEQ ID NO: 3, and V L The method according to

[34] above, wherein the amino acid sequence of SEQ ID NO: 5 is

[36] A method for evaluating the glycosylation of LAG3, comprising the step of contacting a sample containing LAG3 with the antibody according to any one of [1] to

[25] above.

[37] The method described above in

[36] , which is further defined as an in vitro method.

[38] The method according to

[36] above, wherein the sample is a cell sample.

Claims

1. An isolated monoclonal antibody that selectively binds to glycosylated LAG3 compared to the non-glycosylated form of LAG3, The isolated monoclonal antibody comprises: i) a Chothia CDR H1 consisting of the amino acid sequence of SEQ ID NO:6, a Chothia CDR H2 consisting of the amino acid sequence of SEQ ID NO:7, and a Chothia CDR H3 consisting of the amino acid sequence of SEQ ID NO:8; ii) AbM CDR H1 consisting of the amino acid sequence of SEQ ID NO: 9, AbM CDR H2 consisting of the amino acid sequence of SEQ ID NO: 10, and AbM CDR H3 consisting of the amino acid sequence of SEQ ID NO: 8; iii) a Kabat CDR H1 consisting of the amino acid sequence of SEQ ID NO: 11, a Kabat CDR H2 consisting of the amino acid sequence of SEQ ID NO: 12, and a Kabat CDR H3 consisting of the amino acid sequence of SEQ ID NO: 8, or iv) CDR H1 of Contact consisting of the amino acid sequence of SEQ ID NO: 13, CDR H2 of Contact consisting of the amino acid sequence of SEQ ID NO: 14, and CDR H3 of Contact consisting of the amino acid sequence of SEQ ID NO: 15 V including Ｈ Contains the domain, and i) a Chothia, AbM or Kabat CDR L1 consisting of the amino acid sequence of SEQ ID NO: 16, a Chothia, AbM or Kabat CDR L2 consisting of the amino acid sequence of SEQ ID NO: 17, and a Chothia, AbM or Kabat CDR L3 consisting of the amino acid sequence of SEQ ID NO: 18, or ii) Contact CDR L1 consisting of the amino acid sequence of SEQ ID NO: 19, Contact CDR L2 consisting of the amino acid sequence of SEQ ID NO: 20, and Contact CDR L3 consisting of the amino acid sequence of SEQ ID NO: 21 V including Ｌ including the domain, The antibody.

2. 2. The isolated antibody of claim 1, which increases the secretion of IFN-γ and / or IL-2, or increases T cell proliferation.

3. 2. The isolated antibody of claim 1, which blocks binding of LAG3 to one or more of Gal-3, MHCII, LSECtin, and CD3.

4. An isolated antibody described in claim 1 or 3, which selectively binds to LAG3 glycosylated at positions N188, N250, N256, N343 or any combination thereof in the amino acid sequence of SEQ ID NO: 1 compared to unglycosylated LAG3.

5. The isolated antibody of any one of claims 1 to 4, wherein the binding affinity of the anti-glycLAG3 antibody to glycosylated LAG3 is 0.1 to 10 nM, inclusive of the lower and upper limits.

6. 6. The isolated antibody of any one of claims 1 to 5, which masks glycosylation of LAG3 at one or more of N188, N250, N256 or N343 in the amino acid sequence of SEQ ID NO:

1.

7. V Ｈ The domain has the amino acid sequence of SEQ ID NO: 3, and V Ｌ The isolated antibody of any one of claims 1 to 6, wherein said antibody has the amino acid sequence of SEQ ID NO:

5.

8. V Ｈ The isolated antibody of any one of claims 1 to 6, wherein the domain has an amino acid sequence that is at least 90%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO:

3.

9. V Ｌ 9. The isolated antibody of any one of claims 1 to 6, or 8, wherein the domain has an amino acid sequence that is at least 90%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO:

5.

10. The isolated antibody of any one of claims 1 to 6, which has human framework regions.

11. 7. The isolated antibody of any one of claims 1 to 6, having a heavy or light chain human framework region with 1, 2, 3, 4, 5, or 6 amino acid substitutions.

12. 12. The isolated antibody of any one of claims 1 to 6, 10 and 11, comprising a human constant domain.

13. 13. The isolated antibody of any of claims 1 to 6 and 10 to 12, which is an IgG, IgM, IgA, or an antigen-binding fragment thereof.

14. The isolated antibody of any one of claims 1 to 6 and 10 to 13, which is a Fab', F(ab')2, F(ab')3, monovalent scFv, or bivalent scFv.

15. The isolated antibody of any one of claims 1 to 6 and 10 to 14, which is a humanized antibody.

16. 16. The isolated antibody of any of claims 1-15, conjugated to an imaging agent, a chemotherapeutic agent, a toxin, or a radionuclide.

17. A composition comprising the isolated antibody of any one of claims 1 to 16 in a pharmaceutically acceptable carrier.

18. 17. A pharmaceutical composition for treating a subject with cancer, comprising an effective amount of the isolated antibody of any one of claims 1 to 16 and a pharmaceutically acceptable carrier.

19. 19. The pharmaceutical composition of claim 18, wherein 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, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer.

20. 20. The pharmaceutical composition of claim 18 or 19, formulated for intravenous, intradermal, intratumoral, intramuscular, intraperitoneal, subcutaneous, or topical administration.

21. The pharmaceutical composition of any one of claims 18 to 20, further comprising at least one second anti-cancer therapy.

22. 22. The pharmaceutical composition of claim 21, wherein the second anti-cancer therapy is an immunotherapy or cytokine therapy.

23. The pharmaceutical composition of claim 21, wherein the second anti-cancer therapy is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, or an anti-LAG3 antibody.

24. 22. The pharmaceutical composition of claim 21, wherein the second anticancer therapy is durvalumab, nivolumab, pembrolizumab, avelumab, atezolizumab, cemiplimab, or ipilimumab.

25. 22. The pharmaceutical composition of claim 21, wherein the second anti-cancer therapy is an anti-LAG3 antibody that preferentially binds to glycosylated LAG3 as compared to non-glycosylated LAG3.

26. the second anti-cancer therapy is a humanized or chimeric anti-glycosylated LAG3 monoclonal antibody; Ｈ The domain has the amino acid sequence of SEQ ID NO: 3, and V Ｌ 26. The pharmaceutical composition of claim 25, wherein said nucleotide sequence has the amino acid sequence of SEQ ID NO:

5.

27. A method for assessing the glycosylation of LAG3 in vitro, comprising contacting a LAG3-containing sample with an antibody according to any one of claims 1 to 16.

28. 28. The method of claim 27, wherein the sample is a cell sample.

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