Antibodies specific for glycosylated LAG3 and methods of use thereof

Abstract

The present application provides antibodies that selectively bind to glycosylated LAG3 relative to unglycosylated LAG3. In some aspects, the present application also provides LAG3 polypeptides comprising glycosylated amino acid positions. The present application also provides methods of making and using such antibodies and polypeptides (e.g., for treating cancer).

Description

[0001] Sequence Listing

[0002] This application contains a sequence listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on September 22, 2020, is named 24258_0014P1_Sequence_Listing.txt, and is 12,288 bytes in size. Technical Field

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

[0005] The persistence of T cell activation has greatly reshaped the treatment of a wide spectrum of malignant cancers. For example, the development of ipilimumab (a CTLA-4 specific antibody and the first FDA-approved checkpoint blocker targeting T cell responses) has made it possible to treat metastatic melanoma (Hodi et al., The New England Journal of Medicine 363, 711-723 (2010)).

[0006] Despite the impressive impact of CTLA-4 and PD1-PDL1-targeted cancer immunotherapies, a large proportion of patients across multiple tumor types do not respond. Consequently, focus has shifted to targeting alternative inhibitory receptors (IRs) and inhibitory mechanisms within the tumor microenvironment.

[0007] Upregulated expression of IRs is crucial for balancing co-stimulatory receptor activity and limiting T cell activation to prevent autoimmunity, autoinflammation, and tissue damage. However, tumors can hijack these so-called immune checkpoint mechanisms as a shield against anti-tumor immune responses initiated by CD4+ and CD8+ T cells. First described in the setting of chronic viral infection, tolerant antigen-specific T cells display elevated expression of IRs in the tumor microenvironment, corresponding to functional anergy as measured by reduced proliferation and cytokine release. Along with the recruitment of regulatory T cells, the resulting immune tolerance creates multiple obstacles to effective tumor elimination. Therefore, recent cancer immunotherapy approaches aim to reverse this exhausted state by targeting IRs to “release the brakes,” allowing cytotoxic T cells to reactivate and attack the tumor.

[0008] Lymphocyte activation gene-3 (LAG3; CD223) may be a promising co-IR, inhibiting T cell activation and cytokine secretion, thereby ensuring immune homeostasis. LAG-3 exerts different inhibitory effects on various types of lymphocytes. At the same time, LAG-3 can effectively prevent the occurrence of autoimmune diseases. The exact molecular mechanisms of LAG-3 signaling and interaction with other immune checkpoints are mostly unknown. However, LAG-3 has shown significant synergistic effects 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.

[0009] Based on this, glycosylated LAG3-specific antibodies may be valuable in cancer therapy. Summary of the Invention

[0010] Provided herein are isolated monoclonal antibodies that selectively bind to glycosylated LAG3 (herein, anti-glycLAG3 antibodies). In some aspects, the antibodies selectively bind to LAG3 glycosylated at positions N188, N250, N256, and / or N343 relative to unglycosylated LAG3. In certain aspects, the antibodies increase the secretion of IFN-γ and / or IL-2. In some aspects, the antibodies increase T cell proliferation. In some aspects, the antibodies block the binding of LAG3 to one or more of Gal-3, MHCII, LSECtin, and CD3.

[0011] In some embodiments, the isolated antibody selectively binds to human LAG3 with glycosylation at N188. In some embodiments, the isolated antibody selectively binds to human LAG3 with glycosylation at N250. In some embodiments, the isolated antibody selectively binds to human LAG3 with glycosylation at N256. In some embodiments, the isolated antibody selectively binds to human LAG3 with glycosylation at N343. In some embodiments, the isolated antibody selectively binds to human LAG3 with glycosylation at N188 and N250. In some embodiments, the isolated antibody selectively binds to human LAG3 with glycosylation at N188 and N256. In some embodiments, the isolated antibody selectively binds to human LAG3 with glycosylation at N188 and N343. In some embodiments, the isolated antibody selectively binds to human LAG3 with glycosylation at N250 and N256. In some embodiments, the isolated antibody selectively binds to human LAG3 with glycosylation at N250 and N343. In some embodiments, the isolated antibody selectively binds to human LAG3 glycosylated at N256 and N343. In some embodiments, the isolated antibody selectively binds to human LAG3 glycosylated at N188, N250, and N256. In some embodiments, the isolated antibody selectively binds to human LAG3 glycosylated at N188, N250, and N343. In some embodiments, the isolated antibody selectively binds to human LAG3 glycosylated at N188, N256, and N343. In some embodiments, the isolated antibody selectively binds to human LAG3 glycosylated at N250, N256, and N343. In some embodiments, the isolated antibody selectively binds to human LAG3 glycosylated at N188, N250, N256, and N343.

[0012] In certain aspects, the anti-glycLAG3 antibody binds to LAG3 and masks or screens one or more glycosylation motifs to block binding or other interactions of the molecule with the motif, and can block glycosylation of LAG3 at the glycosylation site. In specific embodiments, the anti-glycLAG3 antibody masks glycosylation sites at one or more of N188, N250, N256, and N343.

[0013] In some aspects, the antibody selectively binds to one or more glycosylation motifs. In some aspects, the antibody binds to a glycopeptide comprising a glycosylation motif and an adjacent peptide. In some aspects, the antibody binds to a peptide sequence that is three-dimensionally located near one or more glycosylation motifs.

[0014] In certain aspects, the anti-glycLAG3 antibody binds to glycosylated LAG3 with a K of 0.1-13 nM, or 0.1 to 10 nM, or 0.1 nM to 5 nM, inclusive. d Less than the K expressed relative to unglycosylated LAG3 d In a further aspect, the antibody binds to the K of glycosylated LAG3 d is relative to the K of unglycosylated LAG3 d at most one tenth.

[0015] In one specific aspect, anti-glycLAG3 monoclonal antibody STC1317, and antigen-binding portions thereof, as well as humanized and chimeric forms thereof, is provided, wherein the monoclonal antibody has heavy and light chain variable domains having the amino acid sequences of SEQ ID NOs: 3 and 5, respectively (mature V domains without any signal sequence). H and V L Provided herein are anti-glycLAG3 antibodies that compete with STC1317 MAb for binding to glycosylated LAG3 and / or bind to the same epitope as STC1317.

[0016] Table 3 below shows the nucleic acid (DNA) and corresponding amino acid sequences of the heavy and light chain variable (V) domains of STC1317 MAb. SEQ ID NOs: 2 and 3 are STC1317 V H SEQ ID NOs: 4 and 5 are the nucleotide and amino acid sequences of the mature form of the STC1317 kappa light chain variable domain. Table 4 provides the Chothia, AbM, Kabat, and Contact heavy and light chain V domain CDRs of STC1317.

[0017] In one embodiment, an anti-glycLAG3 antibody that specifically and preferentially binds to glycosylated LAG3 comprises a V having an amino acid sequence of SEQ ID NO: 3. H domain and / or V domain having the amino acid sequence of SEQ ID NO: 5 L In one embodiment, the anti-glycLAG3 antibody is combined with a V domain comprising SEQ ID NO: 3 H domain and V of SEQ ID NO: 5 L In other embodiments, the anti-glycLAG3 antibody comprises a V domain that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 3. HThe anti-glycLAG3 antibodies may be chimeric antibodies and comprise human constant domains, e.g., constant domains from human IgG1, IgG2, IgG3, or IgG4.

[0018] In one embodiment, an anti-glycLAG3 antibody that specifically and preferentially binds to glycosylated LAG3 comprises V H domain, the V H The domain comprises Chothia CDRs 1-3 having the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively; AbM CDRs 1-3 comprising the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 8, respectively; Kabat CDRs 1-3 comprising the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 8, respectively; or Contact CDRs 1-3 comprising the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively, or a combination thereof. In one embodiment, the anti-glycLAG3 antibody is conjugated to a V domain comprising ... H The V domain of the antibody competes for specific binding to glycosylated LAG3. H The domain comprises Chothia CDRs 1-3 having the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively; AbM CDRs 1-3 having the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 8, respectively; Kabat CDRs 1-3 having the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 8, respectively; or Contact CDRs 1-3 having the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively, or a combination thereof. Preferably, V H and V L The domains have CDRs of the same class, ie, both have Chothia, AbM, Kabat, or Contact CDRs.

[0019] In other embodiments, the anti-glycLAG3 antibody has a V region comprising CDR H1, CDR H2, and CDR H3. HThe CDR H1, CDR H2 and CDR H3 have amino acid sequences having 1, 2, 3, 4 or 5 amino acid substitutions in one, two or three of the following CDRs: Chothia CDRs having the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively, or AbMCDRs having the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 8, respectively, or Cabat CDRs having the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 8, respectively, or Contact CDRs having the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively. The anti-glycLAG3 antibody may have a V domain. H and V L In some embodiments, the amino acid substitutions are conservative substitutions.

[0020] Preferably, the aforementioned antibody has a human framework region, ie is a humanized form of STC1317, and optionally comprises a human constant domain, such as a constant domain from human IgG1, IgG2, IgG3 or IgG4.

[0021] Those skilled in the art will appreciate that one or more amino acid substitutions may be made in the CDR and / or framework regions of a humanized antibody to improve binding affinity or other parameters. H and V L In one embodiment, the anti-glycLAG3 antibody binds to glycosylated LAG3, and its K d In embodiments, the K of an anti-glycLAG3 antibody binding to glycosylated LAG3 is 0.1-10 nM or 1-20 nM, inclusive. d Smaller than the K of the antibody binding to unglycosylated LAG3 d In one embodiment, the anti-glycLAG3 antibody binds to the glycosylated LAG3 protein at a K of half. d is relative to the K expressed by unglycosylated LAG3 binding d In one embodiment, the anti-glycLAG3 antibody binds to the glycosylated LAG3 protein with a K of at most one-fifth. d The K of the antibody binding to the unglycosylated LAG3 protein d at most one tenth.

[0022] In one embodiment, the antibody is detectable directly or indirectly via 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 via a fluorescently labeled secondary antibody. In one embodiment, the binding affinity of the STC1317 MAb or a chimeric or humanized form thereof for glycosylated LAG3 is 0.1-13 nM or 0.1-5 nM, inclusive.

[0023] In an embodiment, an anti-glycLAG3 antibody is combined with a V comprising the above H and V L The antibodies comprising the humanized domains and the CDRs thereof compete for specific binding to glycosylated LAG3. Preferably, these antibodies have human framework regions, i.e., are humanized versions of STC1317, and optionally contain human constant domains, such as constant domains from human IgG1, IgG2, IgG3, or IgG4. It will be understood by those skilled in the art that one or more amino acid substitutions can be made in the CDRs or framework regions of the humanized antibodies to improve binding affinity or other parameters. In embodiments, the anti-glycLAG3 antibodies bind to glycosylated LAG3 with a K d Less than the K expressed relative to unglycosylated LAG3 d In embodiments, the anti-glycLAG3 antibody binds to the K of glycosylated LAG3. d Less than the K expressed relative to unglycosylated LAG3 d In one embodiment, the anti-glycLAG3 antibody binds to the glycosylated LAG3 protein non-K d is the K of the antibody binding to unglycosylated LAG3 d In one embodiment, the anti-glycLAG3 antibody binds to the K of glycosylated LAG3 protein. d The K of the antibody binding to the unglycosylated LAG3 protein d In one embodiment, the antibody exhibits binding to cells expressing WT LAG3 in a cell flow cytometry binding assay as indicated by green counts / mm 2 The green counts / mm represent the binding to cells expressing unglycosylated LAG3. 2In one embodiment, the antibody is detectable directly or indirectly by a fluorescent marker or a marker. In one embodiment, the antibody is directly labeled with a fluorescent marker or a marker such as FITC. In one embodiment, the binding affinity of the STC1317 MAb or its binding domain or humanized or chimeric form to glycosylated STC1317 is 0.1-13 nM or 0.1 to 10 nM or 0.1 to 5 nM, including lower and upper limits.

[0024] In some aspects, the antibody is recombinant. In some aspects, the antibody is IgG, IgM, IgA, or an antigen-binding fragment thereof. In other aspects, the antibody is Fab', F(ab')2, F(ab')3, a monovalent scFv, a divalent scFv, a bispecific antibody, a bispecific scFv, or a single domain antibody. In some aspects, the antibody is a human antibody or a humanized antibody. In further aspects, the antibody is conjugated to an imaging agent, a chemotherapeutic agent, a toxin, or a radionuclide.

[0025] In another embodiment, provided herein is a composition comprising an antibody of the embodiments (eg, the antibody selectively binds to glycosylated LAG3 relative to unglycosylated LAG3) in a pharmaceutically acceptable carrier.

[0026] In yet another embodiment, an isolated polypeptide is provided that comprises a fragment of at least 7 (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) consecutive amino acids of human LAG3, comprising at least one amino acid corresponding to position N188, N250, N256, or N343 of human LAG3. In a further aspect, the isolated polypeptide of the embodiment comprises a fragment of at least 7 (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) consecutive amino acids of human LAG3, comprising at least one amino acid corresponding to position 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. In some aspects, a polypeptide of the embodiments is fused or conjugated to an immunogenic polypeptide (e.g., a keyhole limpet In some aspects, the polypeptide further comprises a Cys residue at the C- or N-terminus. For example, in some aspects, the polypeptide is conjugated to the immunogenic polypeptide via a disulfide bond at the Cys residue.

[0027] In another embodiment, a composition is provided comprising a polypeptide comprising a fragment of at least 7 (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) consecutive amino acids of human LAG3, wherein the fragment comprises at least one amino acid corresponding to position 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 composition is an immunogenic composition. In some aspects, the immunogenic composition further comprises an adjuvant, such as alum or Freund's adjuvant.

[0028] In another embodiment, provided herein is a method for treating a subject with cancer, comprising administering to the subject an effective amount of an antibody or isolated polypeptide of the 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 the embodiments (e.g., an antibody that selectively binds to glycosylated LAG3 relative to unglycosylated 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 cancer, liver cancer, bladder cancer, gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer. In certain aspects, the cancer is adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain / CNS tumors in adults, brain / CNS tumors in children, breast cancer, male breast cancer, adolescent cancer, childhood cancer, young adult cancer, cancer of unknown primary, Castleman's disease, cervical cancer, colon / rectal cancer, endometrial cancer, esophageal cancer, Ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), gestational trophoblastic disease, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, laryngeal or hypopharyngeal cancer, leukemia (e.g., adult acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia In some aspects, the antibody is in a pharmaceutically acceptable composition. In some aspects, the antibody is administered systemically. In specific aspects, the antibody is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or topically.

[0029] In some aspects, the method further comprises administering to the subject at least a second anti-cancer therapy. In certain aspects, wherein the second anti-cancer therapy is surgery, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy.

[0030] In yet another embodiment, provided herein are methods for assessing LAG3 glycosylation, N-linked glycosylation, or N-glycosylation, comprising contacting a sample containing LAG3 with an antibody of the embodiments (e.g., an antibody that selectively binds to glycosylated LAG3 relative to unglycosylated LAG3). In some aspects, the method is an in vitro method. In certain aspects, the sample is a cell sample.

[0031] In yet another embodiment, a method for preparing an antibody is provided, comprising administering a polypeptide according to the embodiments (e.g., a polypeptide comprising a fragment of at least 7 consecutive amino acids of human LAG3, the fragment comprising at least one amino acid corresponding to position N188, N250, N256, or N343 of human LAG3, wherein at least one of the amino acids corresponding to position N188, N250, N256, or N343 of human LAG3 is glycosylated) to an animal, and isolating the antibody from the animal. For example, the animal can be a mouse, rat, rabbit, or human. In certain aspects, the method further comprises identifying CDRs of the antibody and humanizing the sequences surrounding the CDRs to produce a humanized antibody. In yet further aspects, the method comprises recombinantly expressing the humanized antibody. Thus, in another embodiment, provided herein is an isolated antibody produced by the above method. Thus, in some embodiments, provided herein is an isolated antibody that selectively binds to a polypeptide of the embodiments (e.g., a polypeptide comprising a fragment of at least 7 contiguous amino acids of human LAG3, the fragment comprising at least one amino acid corresponding to position N188, N250, N256, or N343 of human LAG3, wherein at least one of the amino acids corresponding to position N188, N250, N256, or N343 of human LAG3 is glycosylated) relative to unglycosylated LAG3. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 LAG3 is heavily glycosylated. SDS-PAGE analysis of histidine-tagged LAG3 protein. Protein samples (0.5 μg) were pretreated with or without PNGase F for 1 h. Proteins were separated on 4-12% NuPAGE gels and stained with Coomassie Brilliant Blue.

[0033] Figure 2A and 2BDevelopment of monoclonal antibodies specific for glycosylated LAG3. Dot blot analysis of anti-LAG3 monoclonal antibodies was performed using LAG3-Fc or PNGase F-treated LAG3-Fc (0.5 μg per well). (A) Dot blot membrane depicts the carbohydrate-specific binding activity of several antibodies, including STC1317. LAG3-Fc and PNGase F-treated LAG3-Fc were blotted onto nitrocellulose membrane. Ag-Ab interactions were detected using anti-LAG3 mAb (0.5 μg purified) and an anti-mouse IgG secondary antibody. For controls, anti-LAG3 antibodies purified from culture supernatants of fusion hybridomas derived from LAG3B (from Balb / c) and LAG3N (from NZW), respectively, were used. (B) Sample layout of the corresponding 96-well dot blot assay plate.

[0034] Figure 3A and 3B Sensorgram of anti-LAG3 antibody analyzed by Octet. From high-throughput K D Summary of screening data. Data were fit to a 1:1 binding model to extract association and dissociation rates. KD was calculated using the ratio kd:ka. The graph shows the response versus time, indicating the progression of the interaction. Sensorgrams of anti-LAG3 antibodies STC1301-1316 (A) and STC1317-STC1322 (B).

[0035] Figure 4 KD determination of STC1317 via Biocore. The binding affinity (reduced equilibrium dissociation constant [KD] value) of STC1317 was determined using a Biacore binding assay. Six concentrations of LAG3, each diluted 2-fold, were passed through the chip. The graph depicts the response over time, showing the progression of the STC1317 interaction.

[0036] Figure 5A A and B. Increased secretion of IFN-γ and IL-2 in the presence of STC1317. The effect of STC1317 on T cell proliferation (T) in response to stimulatory cells (DCs, dendritic cells). The figures show IL-2 (A) and IFN-γ (B) cytokine levels in the presence of STC1317 and control mouse IgG. Cytokines in the supernatant were quantified by ELISA on day 5. DETAILED DESCRIPTION

[0037] N-glycosylation is a post-translational modification that begins in the endoplasmic reticulum (ER) and is subsequently processed in the Golgi apparatus (Schwarz & Aebi, Current Opinion in Structural Biology 21, 576-582 (2011)). This type of modification is first catalyzed by a membrane-associated oligosaccharyltransferase (OST) complex, which transfers preformed glycans composed of oligosaccharides to asparagine (Asn) side chain acceptors located within the NXT motif (-Asn-X-Ser / Thr) (Cheung and Reithmeier, Methods 41(4):451-59 (2007); Helenius and Aebi, Science 291(5512):2364-69 (2001)). The addition or removal of sugars from preformed glycans is mediated by a group of glycotransferases and glycosidases, respectively, which tightly regulate the N-glycosylation cascade in a cell- and location-dependent manner.

[0038] Galectin-3 (Gal-3) acts as a regulator of antigen-specific T cell activation. LAG3 expression is associated with Gal-3, and functional LAG3 is essential for Gal-3-mediated suppression of cytotoxic T lymphocyte immune responses. LAG3 can be extensively glycosylated. Glycosylation is thought to be a target for Gal-3 binding. See Long et al. (2018), page 182. Therefore, anti-glycLAG3 antibodies can exhibit enhanced Gal-3 inhibition relative to more general LAG3 antibodies.

[0039] As used herein, unless otherwise indicated, the term "lymphocyte activation gene-3" or "LAG3" refers to LAG3 from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys (cynomolgus), dogs, and rodents (e.g., mice and rats). Unless otherwise indicated, LAG3 also includes various LAG3 isoforms, related LAG3 polypeptides, including SNP variants thereof, and various modified forms of LAG3, including but not limited to phosphorylated LAG3, glycosylated LAG3, and ubiquitinated LAG3.

[0040] An exemplary amino acid sequence of human LAG3 is provided below, with the N-linked glycosylation sites indicated in bold and underlined (N188, N250, N256, and N343): MWEAQFLGLLFLQPLWVAPVKPLQPGAEVPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMTASPPGSLRASDWVIL N CSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVSPMDSGPWGCILTYRDGF N VSIMY N LTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCHIHLQEQQL N ATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTELSSPGAQRSGRAPGALPAGHLLLFLILGVLSLLLLVTGAFGFHLWRRQWRPRRFSALEQGIHPPQAQSKIEELEQEPEPEPEPEPEPEPEPEPEQL (SEQ ID NO: 1).

[0041] As shown in Table 1 below, both N-glycosylation sites are located in the extracellular domain of LAG3.

[0042] The specific glycosylation site of a particular LAG3 isoform or variant may differ from the amino acid at position 188, 250, 256, or 343 of the particular LAG3 isoform or variant, which are located in the extracellular domain of LAG3. Based on sequence alignments and other general knowledge in the art, one of ordinary skill in the art will be able to determine that any particular LAG3 isoform or variant corresponds to the glycosylation sites N188, N250, N256, and N343 of the human LAG3 exemplified above. Thus, also provided herein are antibodies that selectively bind to glycosylated forms of LAG3 isoforms or variants relative to unglycosylated LAG3 isoforms or variants. The glycosylation sites of a LAG3 isoform or variant may be the corresponding sites of N188, N250, N256, and N343 of the human LAG3 sequence provided above. Also provided herein are polypeptides comprising a fragment of at least 7 (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) contiguous amino acids of a LAG3 isoform or variant, comprising at least one amino acid corresponding to position N188, N250, N256, or N343 of the exemplary human LAG3 sequences provided above.

[0043] As used herein, and unless otherwise indicated, the articles "a," "an," and "the" refer to one or more of the grammatical objects of the article. For example, an antibody refers to one antibody or more than one antibody.

[0044] As used herein, and unless otherwise specified, the terms "or" and "and / or" are used interchangeably unless expressly indicated to refer only to alternatives or to alternatives that are mutually exclusive. As used herein, unless otherwise specified, "another" means at least a second or more.

[0045] As used herein, and unless otherwise indicated, the term "about" indicates that a value includes the inherent variation of error for the device, method being employed to determine the value, or the variation that exists among the study subjects.

[0046] As used herein, and unless otherwise indicated, the term "antibody" refers to a polypeptide product of B cells in the class of immunoglobulin (or "Ig") polypeptides that is capable of binding to a specific molecular antigen, such as IgG, IgM, IgA, IgD, IgE, and other molecules having antigen-binding fragments thereof. Antibodies are composed of two pairs of identical polypeptide chains, each pair having one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), and each amino-terminal portion of each chain comprises about 100 to about 130 or more amino acids, and each carboxyl-terminal portion of each chain comprises a constant region (see Borrebaeck (ed.) (1995) Antibody Engineering, 2nd edition, Oxford University Press.; Kuby (1997) Immunology , 2nd ed., WH Freeman and Company, New York). Herein, specific molecular antigens include 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.

[0047] As used herein, and unless otherwise indicated, the term "isolated" when used to refer to an antibody, Fab or polynucleotide, means that the molecule referred to is free of at least one component found in nature. The term includes antibodies, Fab or polynucleotides that have been removed from some or all of the other components found in their natural environment. The components of the antibody's natural environment include, for example, red blood cells, white blood cells, platelets, plasma, proteins, nucleic acids, salts and nutrients. The components of the natural environment of an Fab or polynucleotide include, for example, lipid membranes, organelles, proteins, nucleic acids, salts and nutrients. The antibodies, Fab or polynucleotides of the present invention may also be free of or substantially free of all of these components or any other component of the cell from which they were isolated or recombinantly produced.

[0048] As used herein, and unless otherwise indicated, the term "monoclonal antibody" refers to an antibody that is the product of a single cell clone or hybridoma or a cell population derived from a single cell. Monoclonal antibody also means an antibody produced by recombinant methods from immunoglobulin genes encoding heavy and light chains to produce a single molecule immunoglobulin species. The amino acid sequence of the antibody in a monoclonal antibody preparation is essentially homogeneous, and the binding activity of the antibodies in such a preparation exhibits essentially the same antigen-binding activity. In contrast, polyclonal antibodies are obtained from different B cells in a population, which are combinations of immunoglobulin molecules that bind to a specific antigen. Each immunoglobulin of a polyclonal antibody can bind to a different epitope of the same antigen. Methods for producing monoclonal and polyclonal antibodies are well known in the art (Harlow and Lane, 1996). Antibodies:ALaboratoryManual , Cold Spring Harbor Laboratory Press (1989) and Borrebaeck (editor), AntibodyEngineering: APractical Guide , WH Freeman and Co., Publishers, New York, pp. 103-120 (1991)).

[0049] As used herein, and unless otherwise indicated, the term "human antibody" refers to antibodies having human variable regions and / or human constant regions corresponding to human germline immunoglobulin sequences, or portions thereof. Kabat et al. (1991) Sequences of Pr oteinsofImmunologicalInterest These human germline immunoglobulin sequences are described in NIH Publication No. 91-3242, Fifth Edition, US Department of Health and Human Services, 5th Edition. As used herein, human antibodies may include antibodies that bind to 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.

[0050] As used herein, and unless otherwise indicated, the term "chimeric antibody" refers to antibodies in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of one or more polypeptide chains is identical or homologous to the corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

[0051] As used herein, and unless otherwise indicated, the term "humanized antibody" refers to a chimeric antibody comprising a human immunoglobulin (e.g., receptor antibody), wherein native complementary determining region ("CDR") residues are replaced with residues from corresponding CDRs of non-human species (e.g., donor antibody, such as mouse, rat, rabbit, or non-human primate) with desired specificity, affinity, and ability. In some cases, one or more FR region residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, the humanized antibody may have residues not found in the receptor antibody or the donor antibody. These modifications are made in order to further improve antibody performance. The humanized antibody heavy chain or light chain may have substantially all of at least one or more variable regions, wherein all or substantially all of the CDRs correspond to the CDRs of non-human immunoglobulins, and all or substantially all of the FRs are those of human immunoglobulin sequences. The humanized antibody may have at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a constant region of a human immunoglobulin. For more details, see Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992); Carter et al., Proc. Natl. Acd. Sci. USA 89:4285-4289 (1992); and U.S. Patent Nos. 6,800,738, 6,719,971, 6,639,055, 6,407,213 and 6,054,297.

[0052] As used herein, and unless otherwise indicated, the term "recombinant antibody" refers to an antibody prepared, expressed, produced or separated by recombinant means. Recombinant antibodies can be antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies separated from a recombinant combinatorial antibody library, antibodies separated from transgenic and / or transchromosomal animals (e.g., mice or cattle) of human immunoglobulin genes (see, e.g., Taylor, LD et al., Nucl. Acids Res. 20: 6287-6295 (1992)) or antibodies prepared, expressed, produced or separated by any other means involving splicing immunoglobulin gene sequences to other DNA sequences. Such recombinant antibodies may 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 using transgenic animals for human Ig sequences, in vivo somatic mutagenesis), so that the V H and V L The amino acid sequence of the region may be derived from human germline V H and V L sequences that are related to, but do not naturally occur in the human antibody germline repertoire in vivo.

[0053] As used herein, and unless otherwise indicated, the term "antigen-binding fragment" and similar terms refer to a portion of an antibody that includes the amino acid residues that immunospecifically bind to an antigen and confer specificity and affinity for the antibody. An antigen-binding fragment may be referred to as a functional fragment of an antibody. An antigen-binding fragment may be monovalent, divalent, or multivalent.

[0054] Molecules having antigen binding fragments include, for example, Fd, Fv, Fab, F(ab'), F(ab)2, F(ab')2, F(ab)3, F(ab')3, single-chain Fv (scFv), diabodies, triabodies, tetrabodies, minibodies, or single-domain antibodies. ScFv can be a monovalent scFv or a divalent scFv. Other molecules having antigen binding fragments can include, for example, heavy or light chain polypeptides, variable region polypeptides, or CDR polypeptides, or portions thereof, as long as such antigen binding fragments retain binding activity. Such antigen binding fragments can be found, for example, in Harlow and Lane, Antibodies:ALaboratoryManual , Cold Spring Harbor Laboratory, New York (1989); Myers (ed.), Molec. Biology andBiotechnology: A Comprehensive DeskReference , New York: VCH Publisher, Inc.; Huston et al., Cell Biophysics, 22: 189-224 (1993); Plückthun and Skerra, Meth. Enzymol., 178: 497-515 (1989); and Day, ED, Advanced Immunochemistry, 2nd ed., Wiley-Liss, Inc., New York, NY (1990). An antigen-binding fragment can be a polypeptide having an amino acid sequence of at least 5 consecutive amino acid residues, at least 10 consecutive amino acid residues, at least 15 consecutive amino acid residues, at least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino acid residues, at least 60 consecutive amino acid residues, at least 70 consecutive amino acid residues, at least 80 consecutive amino acid residues, at least 90 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least 125 consecutive amino acid residues, at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues, at least 200 consecutive amino acid residues, or at least 250 consecutive amino acid residues.

[0055] The heavy chain of an antibody refers to a polypeptide chain of approximately 50-70 kDa, wherein the amino-terminal portion includes a variable region of approximately 120 to 130 or more amino acids, and the carboxyl-terminal portion includes a constant region. Based on the amino acid sequence of the heavy chain constant region, the constant region can be one of five different types, called alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ). Different heavy chains have different sizes: α, δ, and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When combined with a light chain, these different types of heavy chains give rise to the five well-known classes of antibodies: IgA, IgD, IgE, IgG, and IgM, which include the four subclasses of IgG, namely IgG1, IgG2, IgG3, and IgG4. The heavy chain can be a human heavy chain.

[0056] The light chain of an antibody refers to a polypeptide chain of about 25 kDa, wherein the amino terminal portion includes a variable region of about 100 to about 110 or more amino acids, and the carboxyl terminal portion includes a constant region. The approximate length of a light chain is 211 to 217 amino acids. Based on the amino acid sequence of the constant domain, there are two different types, called lambda (λ) and kappa (κ). Light chain amino acid sequences are well known in the art. The light chain can be a human light chain.

[0057] The variable domain or variable region of an antibody refers to a part of the light chain or heavy chain of an antibody, which is generally located at the amino terminus of the light chain or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and is used for the binding and specificity of each specific antibody to its specific antigen. The sequence of the variable domains between different antibodies varies greatly. The variability of the sequence is concentrated in the CDRs, while the less variable parts of the variable domains are called framework regions (FRs). The CDRs of the light and heavy chains are primarily responsible for the interaction between the antibody and the antigen. The amino acid position numbering used herein is according to the EU index, as in Kabat et al. (1991). Sequences of proteins of immunological interest (USDepartment of Health and Human Services, Washington, DC) 5th Edition. The variable region can be a human variable region.

[0058] CDR refers to the V H One of the three hypervariable regions (H1, H2 or H3) within the non-framework region of the β-sheet framework, or an antibody V L One of the three hypervariable regions (L1, L2 or L3) within the non-framework region of the β-sheet framework. Thus, CDRs are variable region sequences interspersed within the framework region sequences. CDR regions are well known to those skilled in the art and have been defined by, for example, Kabat as regions with the highest variability within the antibody variable domain (Kabat et al., J. Biol. Chem. 252: 6609-6616 (1977); Kabat, Adv. Prot. Chem. 32: 1-75 (1978)). Chothia also structurally defined CDR region sequences as those residues that are not part of the conserved β-sheet framework and are therefore able to adapt to different conformations (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)). Both terms are recognized in the art. The positions of CDRs within canonical antibody variable domains have been determined by comparing various 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 in the hypervariable region varies among different antibodies, additional residues associated with canonical positions are often numbered with a, b, c, etc., next to the residue numbers in the canonical variable domain numbering scheme (Al-Lazikani et al., supra (1997)). This nomenclature is also well known to those skilled in the art.

[0059] A universal numbering system, ImMunoGeneTics (IMGT) Information (Lafranc et al., 2003, Dev. Comp. Immunol., 27(1):55-77). IMGT is a comprehensive information system specifically for immunoglobulins (Ig), T cell receptors (TRs), and major histocompatibility complexes (MHCs) of humans and other vertebrates. In this article, CDRs are indicated based on amino acid sequence and position in the light or heavy chain. Since the "position" of CDRs within the immunoglobulin V domain structure is conserved between species and is present in structures called loops, a numbering system is used that aligns variable domain sequences based on structural features, CDRs, and framework residues, and is easily identified. This information can be used to transplant and replace CDR residues from an immunoglobulin from one species into a receptor framework, typically from a human antibody. Another numbering system (AHon) has been developed by Honegger et al., 2001, J. Mol. Biol., 309:657-670. The correspondence between numbering systems, including, for example, Kabat numbering and the IMGT unique numbering system, is well known to those skilled in the art (see, for example, Kabat, supra; Chothia et al., supra; Martin, 2010, Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag; and Lefranc et al., 1999, Nuc. Acids Res., 27:209-212).

[0060] AbM and Contact methods also define CDR region sequences.AbM hypervariable region represents a compromise between Kabat CDR and Chothia structural loops, and is used by the AbM antibody modeling software of Oxford Molecular (see, for example, Martin, 2010, Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag). " contact " hypervariable region is based on the analysis of available complex crystal structure.The residue from each in these hypervariable regions or CDR is as described below.

[0061] An exemplary description of the CDR region sequences is shown in Table 2 below. The positions of the CDRs within canonical antibody variable regions have been determined by comparing various 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 the hypervariable regions varies among different antibodies, additional residues relative to canonical positions are typically numbered with a, b, c, etc., next to the residue number in the canonical variable region numbering scheme (Al-Lazikani et al., supra). This nomenclature is also well known to those skilled in the art.

[0062] Table 2. Exemplary Description of CDR Region Sequences

[0063]

[0064]

[0065] One or more CDRs can also be covalently or non-covalently incorporated into a molecule to make it an immunoadhesin. An immunoadhesin can incorporate one or more CDRs as part of a larger polypeptide chain, can covalently link one or more CDRs to another polypeptide chain, or can non-covalently incorporate one or more CDRs. The CDRs allow the immunoadhesin to bind to a specific target antigen.

[0066] As used herein and unless otherwise indicated, the term "binding" or "binding to..." refers to an interaction between molecules. The interaction can be, for example, a non-covalent interaction, including hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions. The strength of the total non-covalent interactions between an antibody and a single epitope of a target molecule (e.g., glycosylated human LAG3) is the affinity of the antibody for that epitope. "Binding affinity" generally refers to the strength of the sum of the non-covalent interactions between a single binding site of a molecule (e.g., a binding protein such as an antibody) and its binding partner (e.g., an antigen).

[0067] The affinity of a binding molecule X (e.g., an antibody) for its binding partner Y (e.g., the antibody's cognate antigen) can often be expressed in terms of the dissociation constant (K d ) or equilibrium dissociation constant (K D ) represents. Low-affinity antibodies generally bind antigen slowly and dissociate easily, while high-affinity antibodies generally bind antigen faster and remain bound longer. Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present disclosure. D ” or “K D The "value" can be measured by assays known in the art, such as by binding assays.D It can be measured in a radiolabeled antigen binding assay (RIA), for example, using a Fab form of the antibody of interest and its antigen (Chen et al., (1999) J. Mol. Biol. 293:865-881). D or K D Values ​​can also be measured by surface plasmon resonance using Biacore, using, for example, a BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ), or by biolayer interferometry, using, for example, an Octet QK384 system (ForteBio, Menlo Park, CA). As used herein, and unless otherwise indicated, an antibody is considered to "selectively bind" a first antigen relative to a second antigen if it binds the first antigen with a higher affinity than the second antigen. An antibody generally does not bind to a completely unrelated antigen.

[0068] As used herein, and unless otherwise indicated, the term "polypeptide" as used herein includes oligopeptides having 2 to 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, such as more than 30 amino acids, more than 50 amino acids, more than 100 amino acids, more than 150 amino acids, more than 200 amino acids, more than 300 amino acids, more than 400 amino acids, more than 500 amino acids, or more than 600 amino acids. Polypeptides can be produced, for example, by recombinant expression or by chemical synthesis. The polypeptides of the present disclosure can be post-translationally modified or chemically modified (e.g., glycosylation, carbamylation, phosphorylation, biotinylation, attachment of fluorescent dyes, etc.). Polypeptides can be glycosylated at specific sites. Polypeptides can include non-natural amino acids that are not encoded by the natural genetic code. For example, the polypeptide may include a methylated backbone structure, a peptidomimetic backbone structure (poly-N-substituted glycine), L-amino acids, R-amino acids, etc. The polypeptide may have a wild-type sequence, a naturally occurring variant sequence, a mutant sequence (e.g., a point mutant, a deletion mutant), etc.

[0069] Anti-glycLAG3 antibodies

[0070] Provided herein are isolated antibodies that selectively bind to glycosylated LAG3 relative to unglycosylated 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 antibodies provided herein are antigen-binding fragments that selectively bind to glycosylated LAG3 relative to unglycosylated LAG3.

[0071] 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 glycosylated only at N188. In some embodiments, the isolated antibodies selectively bind to human LAG3 glycosylated only at N250. In some embodiments, the isolated antibodies selectively bind to human LAG3 glycosylated only at N256. In some embodiments, the isolated antibodies selectively bind to human LAG3 glycosylated only at N343. In some embodiments, the isolated antibodies selectively bind to human LAG3 glycosylated only at N188 and N250. In some embodiments, the isolated antibodies selectively bind to human LAG3 glycosylated only at N188 and N256. In some embodiments, the isolated antibodies selectively bind to human LAG3 glycosylated only at N188 and N343. In some embodiments, the isolated antibody selectively binds to human LAG3 that is glycosylated only at N250 and N256. In some embodiments, the isolated antibody selectively binds to human LAG3 that is glycosylated only at N250 and N343. In some embodiments, the isolated antibody selectively binds to human LAG3 that is glycosylated only at N256 and N343. In some embodiments, the isolated antibody selectively binds to human LAG3 that is glycosylated only at N188, N250, and N256. In some embodiments, the isolated antibody selectively binds to human LAG3 that is glycosylated only at N188, N250, and N343. In some embodiments, the isolated antibody selectively binds to human LAG3 that is glycosylated only at N188, N256, and N343. In some embodiments, the isolated antibody selectively binds to human LAG3 that is glycosylated only at N250, N256, and N343. In some embodiments, the isolated antibody selectively binds human LAG3 having N188, N250, N256, and N343 glycosylation.

[0072] In certain aspects, the anti-glycLAG3 antibody binds to LAG3 and masks or screens one or more glycosylation motifs to block binding or other interactions of the molecule with the motif, and can block glycosylation of LAG3 at the glycosylation site. In specific embodiments, the anti-glycLAG3 antibody masks glycosylation sites at one or more of N188, N250, N256, and N343.

[0073] In some embodiments, the antibodies provided herein selectively bind to one or more glycosylation motifs of LAG3. In some embodiments, the antibodies selectively bind to a glycopeptide having a glycosylation motif and an adjacent peptide. In some embodiments, the antibodies selectively bind to glycosylated LAG3, wherein the d Less than the K expressed relative to unglycosylated LAG3 d In certain embodiments, the antigen-binding fragment binds to glycosylated LAG3 with a K d Less than 50% of the Kd exhibited relative to unglycosylated LAG3. In some embodiments, the antibody binds to glycosylated LAG3 with a Kd of d Less than the K expressed relative to unglycosylated LAG3 d In a further aspect, the antibody binds to glycosylated LAG3 with a K d is relative to the K of unglycosylated LAG3 d at most one tenth.

[0074] Monoclonal antibodies that preferentially bind to glycosylated LAG3 are provided, particularly STC1317 as described herein. Humanized and chimeric forms of STC1317 and antibodies that compete for binding to STC1317 are also provided. The heavy and light chain variable domains of STC1317 are provided in Table 3 below.

[0075] In a specific aspect, an anti-glycLAG3 monoclonal antibody STC1317 is provided, which has the amino acid sequences of SEQ ID NOs: 3 and 5, respectively (mature V H and V L Provided herein are heavy and light chain variable domains having the amino acid sequence of the SEQ ID NO: 1 region (SEQ ID NO: 24), and antigen-binding portions thereof, as well as humanized and chimeric forms thereof. Provided herein are anti-glycLAG3 antibodies that compete with STC1317 MAb for binding to LAG3 and / or bind to the same epitope as STC1317.

[0076] The monoclonal nucleic acid (DNA) and corresponding amino acid sequences of the heavy and light chain variable (V) domains of STC1317 mAb are provided as shown in Table 3 below. SEQ ID NOs: 2 and 3 are STC1317 V H The nucleotide and amino acid sequences of the domain, and SEQ ID NO: 4 and 5 are STC1317κV LNucleotide and amino acid sequences of the mature forms of the domains. Table 4 provides the Chothia, AbM, Kabat, and Contact heavy and light chain V domain CDRs of STC1317.

[0077] In one embodiment, an anti-glycLAG3 antibody that specifically and preferentially binds to glycosylated LAG3 comprises a V having an amino acid sequence of SEQ ID NO: 3. H domain and / or a V domain having an amino acid sequence of SEQ ID NO: 5 L In one embodiment, the anti-glycLAG3 antibody is combined with a V domain comprising SEQ ID NO: 3 H domain and V of SEQ ID NO: 5 L In other embodiments, the anti-glycLAG3 antibody comprises a V domain that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 3. H domain and / or 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: 5 L These anti-glycLAG3 antibodies can be chimeric antibodies and comprise human constant domains, such as constant domains from human IgG1, IgG2, IgG3, or IgG4.

[0078] In one embodiment, an anti-glycLAG3 antibody that specifically and preferentially binds to glycosylated LAG3 comprises V H domain, the V H The domain comprises Chothia CDRs 1-3 having the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively; AbM CDRs 1-3 comprising the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 8, respectively; Kabat CDRs 1-3 comprising the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 8, respectively; or Contact CDRs 1-3 comprising the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively, or a combination thereof. In one embodiment, the anti-glycLAG3 antibody is conjugated to a V domain comprising ... H The V domain of the antibody competes for specific binding to glycosylated LAG3. HThe domain comprises Chothia CDRs 1-3 having the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively; AbM CDRs 1-3 comprising the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 8, respectively; Kabat CDRs 1-3 comprising the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 8, respectively; or Contact CDRs 1-3 comprising the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively, or a combination thereof. In one embodiment, an anti-glycLAG3 antibody that specifically and preferentially binds to glycosylated LAG3 comprises V L domain, the V L The domain comprises Chothia, AbM or Kabat CDRs 1-3 having the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively; or comprises Contact CDRs 1-3 having the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively, or a combination thereof. In one embodiment, the anti-glycLAG3 antibody is conjugated to a V domain comprising L The V domain of the antibody competes for specific binding to glycosylated LAG3. L The domain comprises Chothia, AbM or Kabat CDRs 1-3 having the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively; or comprises Contact CDRs 1-3 having the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively, or a combination thereof. In one embodiment, the anti-glycLAG3 antibody comprises or competes for binding to a specific antibody comprising V H domain and contains V L domain, the V HThe domain comprises Chothia CDRs 1-3 having the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively; AbM CDRs 1-3 having the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 8, respectively; Kabat CDRs 1-3 having the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 8, respectively; or Contact CDRs 1-3 having the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively; the V L The domain comprises Chothia, AbM or Kabat CDRs 1-3 having the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively; or comprises Contact CDRs 1-3 having the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively. Preferably, V H and V L The domains have CDRs of the same class, ie both have Chothia, AbM, Kabat or Contact CDRs.

[0079] In other embodiments, the anti-glycLAG3 antibody has a V region comprising CDR H1, CDR H2, and CDR H3. H The anti-glycLAG3 antibody may have a V domain, wherein the CDR H1, CDR H2, and CDR H3 have an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions in one, two, or three of the following CDRs: a CDR having an amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively, or a CDR having an amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 8, respectively, or a CDR having an amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 8, respectively, or a CDR having an amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively. L domain, the V LThe domain comprises CDR L1, CDR L2, and CDR L3 having an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions in 1, 2, or 3 CDRs: CDRs having the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively, or CDRs having the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively. The anti-glycLAG3 antibody may have a V H and V L In some embodiments, the amino acid substitutions are conservative substitutions.

[0080] Preferably, the aforementioned antibody has human framework regions, ie, is a humanized form of STC1317, and optionally comprises a human constant domain, such as a constant domain from human IgG1, IgG2, IgG3 or IgG4.

[0081] Those skilled in the art will appreciate that one or more amino acid substitutions may be made in the CDR and / or framework regions of a humanized antibody to improve binding affinity or other parameters. H and V L In one embodiment, the anti-glycLAG3 antibody binds to glycosylated LAG3, and its K d Less than the K expressed relative to unglycosylated LAG3 d In embodiments, the anti-glycLAG3 antibody binds to glycosylated LAG3, whose K d Less than the K expressed relative to unglycosylated LAG3 d In one embodiment, the anti-glycLAG3 antibody binds to glycosylated LAG3 protein with a K d is the K of the antibody binding to unglycosylated LAG3 d In one embodiment, the anti-glycLAG3 antibody binds to glycosylated LAG3 protein with a K d The K of the antibody binding to the unglycosylated LAG3 protein d In one embodiment, the antibody exhibits binding to cells expressing WT LAG3 in a cell flow cytometry binding assay as indicated by green counts / mm 2 The green counts / mm represent the binding to cells expressing unglycosylated LAG3. 2In one embodiment, the antibody can be detected directly or indirectly by a fluorescent marker or label. In one embodiment, the antibody is directly labeled with a fluorescent marker or label, such as FITC. In one embodiment, the binding affinity of the STC1317 MAb or its binding domain or humanized or chimeric form for glycosylated LAG3 is 0.1-13 nM or 0.1-5 nM, inclusive.

[0082] Another embodiment provides isolated nucleic acid molecules encoding anti-glycLAG3 V H domain and / or anti-glycLAG3 antibody V L domain, encoding V H The nucleic acid of the domain comprises a nucleotide sequence at least 90-98% identical to SEQ ID NO: 2, encoding V L The nucleic acid encoding the V domain comprises a nucleotide sequence that is at least 90-98% identical to SEQ ID NO: 4. H and / or V L The nucleotide sequence of the domain is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 2 or SEQ ID NO: 4, respectively.

[0083] Table 3 below provides the nucleotide and amino acid sequences of the heavy and light chain variable domains of STC1317.

[0084] Table 3. Nucleotide and amino acid sequences of the heavy and light chain variable domains of STC1317

[0085]

[0086] The CDR sequences of the STC1317 antibody according to Chothia, AbM, Kabat, and Contact CDRs are provided in Table 4 below. Thus, a humanized form of STC1317 is provided that preferentially binds to glycosylated LAG3 compared to unglycosylated LAG3 comprising the CDRs of Table 4 below grafted into human framework regions.

[0087] Table 4. CDR sequences of STC1317

[0088]

[0089]

[0090] In some embodiments, the anti-glycLAG3 antibodies provided herein can be IgG, IgM, IgA, IgD, or IgE. The anti-glycLAG3 antibodies can also be chimeric antibodies, affinity matured antibodies, humanized antibodies, or human antibodies. The anti-glycLAG3 antibodies can also be camelized antibodies, intrabodies, or anti-idiotypic (anti-Id) antibodies. In some embodiments, the anti-glycLAG3 antibodies can be polyclonal antibodies or monoclonal antibodies.

[0091] In some embodiments, the antibodies provided herein are antigen-binding fragments that selectively bind to glycosylated LAG3 relative to unglycosylated LAG3. The antigen-binding fragment can 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 can be a monovalent scFv or a bivalent scFv.

[0092] Polyclonal or monoclonal antibodies, antigen-binding fragments and binding domains and CDRs (including engineered forms of any of the foregoing) specific for glycosylated LAG3, one or more corresponding epitopes thereof, or conjugates of any of the foregoing, whether such antigens or epitopes are isolated from natural sources or are synthetic derivatives or variants of natural compounds, can be generated by known means and as described herein.

[0093] Antibodies can be produced from any animal source (including birds and mammals). In some embodiments, the antibody is a sheep, mouse (e.g., mouse and rat), rabbit, goat, guinea pig, camel, horse or chicken antibody. In addition, newer technology allows the development and screening of human antibodies from human combinatorial antibody libraries. For example, phage antibody expression technology allows the production of specific antibodies in the absence of animal immunity, as described in U.S. Patent number 6,946,546, which is incorporated herein by reference in its entirety. These techniques are further described in Marks et al., Bio / Technol., 10:779-783 (1992); Stemmer, Nature, 370:389-391 (1994); Gram et al., Proc. Natl. Acad. Sci. USA, 89:3576-3580 (1992); Barbas et al., Proc. Natl. Acad. Sci. USA, 91:3809-3813 (1994) and Schier et al., Gene, 169(2):147-155 (1996); the entire contents of which are incorporated herein by reference.

[0094] Methods for producing polyclonal antibodies in various animal species and for producing various types of monoclonal antibodies (including humanized, chimeric, and fully human monoclonal antibodies) are well known in the art. For example, the following U.S. patents provide useful descriptions of such methods and are incorporated herein by reference: U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,196,265; 4,275,149; 4,277,437; 4,366,241; 4,469,797; 4,472,50 9; 4,606,855; 4,703,003; 4,742,159; 4,767,720; 4,816,567; 4,867,973; 4,938,948; 4,946,778; 5,021,236; 5,164,296; 5,196,066; 5,223,409; 5,403,484; 5,420,2 53; 5,565,332; 5,571,698; 5,627,052; 5,656,434; 5,770,376; 5,789,208; 5,821,337; 5,844,091; 5,858,657; 5,861,155; 5,871,907; 5,969,108; 6,054,297; 6,165, 464; 6,365,157; ​​6,406,867; 6,709,659; 6,709,873; 6,753,407; 6,814,965; 6,849,259; 6,861,572; 6,875,434; 6,891,024; 7,407,659; and 8,178,098; the entire contents of which are incorporated herein by reference.

[0095] In some embodiments, the anti-glycLAG3 antibody may be a monoclonal antibody. In some embodiments, the anti-glycLAG3 antibody may be a polyclonal antibody. An animal may be inoculated with an antigen, such as a glycosylated LAG3 polypeptide, to produce antibodies specific for the glycosylated LAG3 polypeptide. Antigens are often bound or conjugated to another molecule to enhance the immune response. A conjugate can be any peptide, polypeptide, protein, or non-proteinaceous substance that is bound to an antigen for eliciting an immune response in an animal. The antibodies produced by an animal in response to antigen inoculation comprise a variety of different molecules (polyclonal antibodies) produced by a variety of individual antibody-producing B lymphocytes. Under the correct conditions for the production of polyclonal antibodies in an animal, the majority of the antibodies in the animal's serum recognize collective epitopes on the antigenic compound of the immunized animal.

[0096] In some embodiments, monoclonal antibodies are prepared by affinity purification. This specificity can be further enhanced to select only those antibodies that recognize target antigens or epitopes. The method for generating monoclonal antibodies (MAb) can be identical with the method for preparing polyclonal antibodies. In some embodiments, rodents such as mice and rats are used to produce monoclonal antibodies. In some embodiments, rabbits, sheep or frog cells are used to produce monoclonal antibodies. The application of rats is well-known and can provide some advantages. Mice (for example, BALB / c mice) are conventionally used, and high-percentage stable fusions are usually provided.

[0097] Hybridoma technology involves fusing a single B lymphocyte from a mouse previously immunized with a glycosylated LAG3 polypeptide with an immortalized myeloma cell (usually a mouse myeloma). This technology provides a means to propagate a single antibody-producing cell for an unlimited number of generations, thereby producing unlimited quantities of structurally identical antibodies (monoclonal antibodies) with the same antigen or epitope specificity.

[0098] Anti-glycLAG3 antibodies can be produced by any method known in the art for producing polypeptides, such as in vitro synthesis, recombinant DNA production, and the like. Humanized antibodies can be produced by recombinant DNA technology. The antibodies described herein can also be produced using recombinant immunoglobulin expression technology. The recombinant production of immunoglobulin molecules, including humanized antibodies, is described in U.S. Patent No. 4,816,397 (Boss et al.), U.S. Patent Nos. 6,331,415 and 4,816,567 (both to Cabilly et al.), British Patent GB 2,188,638 (Winter et al.), and British Patent GB 2,209,757; the entire contents of which are incorporated herein by reference. Techniques for recombinant expression of immunoglobulins, including humanized immunoglobulins, can also be found in Goeddel et al., GeneExpressionTechnology Methods in Enzymology Vol.185Academic Press(1991)andBorreback, Antibody Engineering , WH Freeman (1992); the entire contents of which are incorporated herein by reference. Additional information on the production, design and expression of recombinant antibodies can be found in Mayforth, Designing Antibodies ,Academic Press,San Diego (1993).

[0099] Developed a method for replacing the light chain and heavy chain constant domains of monoclonal antibodies with similar domains of human origin and keeping the variable region of foreign antibodies intact. Alternatively, fully human monoclonal antibodies are produced in transgenic mice or rats of human immunoglobulin genes. Also developed is a method for converting the variable domains of monoclonal antibodies into a more human form by recombinantly constructing antibody variable domains with 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, llama) monoclonal antibodies, and the framework regions are derived from human amino acid sequences. It is believed that replacing the amino acid sequence unique to rodents in antibodies with the amino acid sequence found in the corresponding position of human antibodies will reduce the possibility of adverse immune reactions occurring during treatment use. Gene mutations or other changes may also occur in the hybridomas or other cells that produce antibodies, which may or may not change the binding specificity of the antibodies produced by the hybridomas.

[0100] Engineered antibodies can be produced by using monoclonal antibodies and other antibodies and recombinant DNA technology to produce other antibodies or chimeric molecules that retain the antigen or epitope specificity of the original antibody, i.e., the molecule has a binding domain. Such technology can involve introducing DNA encoding the immunoglobulin variable region or CDR of an antibody into the genetic material of the framework region, constant region, or constant region plus framework region of a different antibody. See, for example, U.S. Patent Nos. 5,091,513 and 6,881,557, which are incorporated herein by reference.

[0101] In certain embodiments, the anti-glycLAG3 antibody is a human antibody. Human antibodies can be prepared by various methods known in the art, including the above-mentioned phage display method, using antibody libraries 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). Transgenic mice that cannot express functional endogenous immunoglobulins but can express human immunoglobulin genes can be used to produce human antibodies. For example, human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, in addition to human heavy and light chain genes, human variable regions, constant regions, and diversity regions can be introduced into mouse embryonic stem cells. Mouse heavy and light chain immunoglobulin genes can be rendered functional individually or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. Modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. Chimeric mice are then bred to produce homozygous progeny expressing human antibodies. Transgenic mice are immunized using conventional methods using a selected antigen, such as all or part of a glycosylated LAG3 polypeptide. Monoclonal antibodies against the antigen can be obtained from immunized transgenic mice using conventional hybridoma technology (see, e.g., U.S. Patent No. 5,916,771). The human immunoglobulin transgenes carried by the transgenic mice rearrange during B cell differentiation, followed by class switching and somatic mutation. Therefore, using this technology, therapeutically useful IgG, IgA, IgM, and IgE antibodies can be produced. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93, which is incorporated herein by reference in its entirety). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, for example, International Publication Nos. WO 98 / 24893, WO 96 / 34096, and WO 96 / 33735; and U.S. Patent Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598, the entire contents of which are incorporated herein by reference. Additionally, companies such as Abgenix, Inc. (Freemont, Calif.) and Medarex (Princeton, NJ) may be working to provide human antibodies against selected antigens using similar technology as described above.

[0102] In one embodiment, the antibody is a chimeric antibody, for example, comprising an antigen-binding sequence from a non-human donor transplanted to an antibody of a heterologous non-human, human or humanized sequence (for example, a framework and / or constant domain sequence). In one embodiment, the non-human donor is a rat. In one embodiment, the antigen-binding sequence is synthesized, for example, by mutagenesis (for example, phage display screening of a human phage library, etc.). In one embodiment, the chimeric antibody provided herein has a mouse V region and a human C region. In one embodiment, the mouse light chain V region is fused to a human kappa light chain. In one embodiment, the mouse heavy chain V region is fused to a human IgG1 C region.

[0103] Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., J. Immunol. Methods 125:191-202 (1989); and U.S. Patent Nos. 6,311,415, 5,807,715, 4,816,567, and 4,816,397; all of which are incorporated herein by reference in their entirety. Chimeric antibodies comprising one or more CDRs from a non-human species and framework regions from a human immunoglobulin molecule can be produced using a variety of techniques known in the art, including, for example, CDR grafting (EP 239,400; International Publication No. WO 91 / 09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneer 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 entirety.

[0104] An exemplary method for producing a recombinant chimeric anti-glycLAG3 antibody may comprise the following: a) constructing an expression vector by conventional molecular biology methods, the expression vector encoding and expressing an antibody heavy chain in which the CDRs and variable regions of a murine anti-glycLAG3 monoclonal antibody are fused to an Fc region derived from a human immunoglobulin, thereby producing a vector for expressing the chimeric antibody heavy chain; b) constructing an expression vector by conventional molecular biology methods, the expression vector encoding and expressing the antibody light chain of a murine anti-glycLAG3 monoclonal antibody, thereby producing a vector for expressing the chimeric antibody light chain; c) transferring the expression vector into a host cell by conventional molecular biology methods to produce a transfected host cell for expressing the chimeric antibody; and d) culturing the transfected cells using conventional cell culture techniques, thereby producing the chimeric antibody.

[0105] An exemplary method for producing a recombinant humanized anti-glycLAG3 antibody may comprise the following: a) constructing an expression vector by conventional molecular biology methods, which encodes and expresses an antibody heavy chain, wherein the minimal portion of the CDR and variable region framework required to maintain the binding specificity of the donor antibody is derived from a non-human immunoglobulin, such as a murine anti-glycLAG3 monoclonal antibody, and the remainder of the antibody is derived from a human immunoglobulin, thereby producing a vector for expressing the humanized antibody heavy chain; b) constructing an expression vector by conventional molecular biology methods, which encodes and expresses an antibody light chain, wherein the minimal portion of the CDR and variable region framework required to maintain the binding specificity of the donor antibody is derived from a non-human immunoglobulin, such as a murine anti-glycLAG3 monoclonal antibody, and the remainder of the antibody is derived from a human immunoglobulin, thereby producing a vector for expressing the humanized antibody light chain; c) transferring the expression vector into a host cell by conventional molecular biology methods to produce a transfected host cell for expressing the humanized antibody; and d) culturing the transfected cells using conventional cell culture techniques, thereby producing the humanized antibody.

[0106] For any exemplary method, host cells can be co-transfected with such expression vectors, which may include different selection markers, but are preferably identical except for the heavy and light chain coding sequences. This program provides 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 of the heavy and light chains may include cDNA or genomic DNA or both. The host cell used to express the recombinant antibody may be a bacterial cell such as Escherichia coli, or more preferably a eukaryotic cell such as Chinese hamster ovary (CHO) cell or HEK-293 cell. The selection of expression vectors depends on the selection of the host cell, and may be selected to have desired expression and regulatory characteristics in the selected host cell. Other cell lines that may be used include, but are not limited to, CHO-K1, NSO, and PER.C6 (Crucell, Leiden, the Netherlands). In addition, when selecting a host cell to consider species-specific codon usage preferences and enhancing protein expression, codon usage may be optimized. For example, for expression in CHO cells, DNA encoding the antibody can incorporate codons preferentially used by Cricetulus griseus (from which Chinese hamster ovary cells are derived). Codon optimization methods can be used to promote improved expression in 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)).

[0107] In one embodiment, the antibody is an immunoglobulin single variable domain derived from a camelid antibody, preferably a heavy chain camelid antibody, without the light chain, which is referred to as V H H domain sequences or Nanobodies TM Nanobody TM (Nb) is the smallest functional fragment or single variable domain (V) of a naturally occurring single-chain antibody. HH), and are known to those skilled in the art. They are derived from heavy chain-only antibodies seen in camelids (Hamers-Casterman et al., Nature 363: 446-448 (1993); Desmyter et al., Nat. Struct. Biol., 803-811 (1996)). In the family "Camelids", immunoglobulins lacking polypeptide light chains are found. "Camelids" include 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 herein designated Nanobodies TM or V H Nb antibodies. The small size and unique biophysical properties of Nb antibodies outperform conventional antibody fragments in recognizing unusual or hidden epitopes and binding to cavities or active sites of protein targets. Furthermore, Nb antibodies can be designed as multispecific and multivalent antibodies, attached to reporter molecules, or humanized. Nb antibodies are stable, survive the gastrointestinal system, and can be easily manufactured.

[0108] By unifying two antigen-binding sites of different specificities into a single construct, bispecific antibodies have the ability to bind two discrete antigens with extremely high specificity and therefore have great potential as therapeutic agents. Bispecific antibodies can initially be prepared by fusing two hybridomas, each of which can produce a different immunoglobulin. Bispecific antibodies can also be produced by connecting two scFv antibody fragments while omitting the Fc portion present in complete immunoglobulins. Each scFv unit in such a construct can be composed of an antibody heavy chain (V H ) and light chain (V L ) are connected to each other by a synthetic polypeptide linker, which is usually genetically engineered to have minimal immunogenicity while maintaining maximum resistance to proteolysis. The individual scFv units can be linked by a variety of techniques, including the incorporation of a short (usually less than 10 amino acids) polypeptide spacer that bridges the two scFv units to produce bispecific single-chain antibodies. The resulting bispecific single-chain antibody is thus a single polypeptide chain containing two V domains with different specificities. H / V L The substance of each scFv unit wherein V H and V LThe domains are separated by a polypeptide linker long enough to allow intramolecular association between the two domains, and wherein the scFv units thus formed are serially connected to one another by a polypeptide spacer that is kept short enough to prevent, for example, the V of one scFv unit from being too large. H domain and another scFv unit V L Unwanted association between.

[0109] Examples of antigen-binding fragments include, but are not limited to: (i) L 、V H 、C L and C H1 Fab fragments composed of V H and C H1 (iii) an "Fv" fragment consisting of the VL and VH domains of a single antibody; (iv) a "dAb" fragment consisting of the VH domain; (v) isolated CDR regions; (vi) a F(ab')2 fragment, a bivalent fragment comprising two linked Fab fragments; (vii) a single-chain Fv molecule ("scFv"), in which the V H domain and V L The domains are connected by a peptide linker that allows the two domains to associate to form a binding domain; (viii) bispecific single chain F v Dimers (U.S. Patent No. 5,091,513); and (ix) diabodies, multivalent or multispecific fragments constructed by gene fusion (U.S. Patent Application Publication No. 20050214860). Fv, scFv or diabody molecules can be stabilized by incorporating disulfide bonds connecting the VH and VL domains. Minibodies with scFvs linked to the CH3 domain can also be prepared (Hu et al., Cancer Res., 56:3055-3061 (1996)).

[0110] Antibody-like binding peptide mimetics are also contemplated in embodiments. Liu et al., Cell Mol. Biol., 49:209-216 (2003) describe "antibody-like binding peptide mimetics" (ABiPs), which are peptides that act as pared-down antibodies and have some of the advantages of longer serum half-lives and less cumbersome synthetic methods.

[0111] Glycosylated LAG3 peptide

[0112] In yet another embodiment, a composition is provided comprising a polypeptide comprising a fragment of at least 7 (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) consecutive amino acids of human LAG3, wherein the fragment comprises at least one amino acid corresponding to position 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.

[0113] In some embodiments, polypeptides comprising at least seven contiguous amino acids of human LAG3 are also provided herein, comprising at least one amino acid corresponding to position 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 comprises at least seven contiguous amino acids of human LAG3, comprising the amino acid corresponding to position N188 being glycosylated. In some embodiments, the polypeptide comprises at least seven contiguous amino acids of human LAG3, comprising the amino acid corresponding to position N250 being glycosylated. In some embodiments, the polypeptide comprises at least seven contiguous amino acids of human LAG3, comprising the amino acid corresponding to position N256 being glycosylated. In some embodiments, the polypeptide comprises at least seven contiguous amino acids of human LAG3, comprising the amino acid corresponding to position N343 being glycosylated.

[0114] For example, the polypeptide can be a fragment of amino acids 182-190 or 187-194 of human LAG3, wherein N188 is glycosylated. For another example, the polypeptide can be a fragment of amino acids 247-254 or 248-256 of human LAG3, wherein N250 is glycosylated, or a fragment of amino acids 252-259 or 255-265 of human LAG3, wherein N256 is glycosylated. For another example, the polypeptide can be a fragment of amino acids 250-260 or 249-257 of human LAG3, wherein N250 and N256 are glycosylated. For another example, the polypeptide can be a fragment of amino acids 340-347 or 342-349 of human LAG3, wherein 343 is glycosylated. One of ordinary skill in the art will understand that polypeptides contemplated herein include any and all polypeptides having at least seven contiguous amino acids of human LAG3, including at least one amino acid corresponding to position N188, N250, N256, or N343 of human LAG3, wherein at least one of the amino acids corresponding to position N188, N250, N256, or N343 of human LAG3 is glycosylated.

[0115] 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. In some embodiments, provided herein are compositions comprising at least two polypeptides provided herein. The at least two polypeptides can be separate molecules or linked into 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.

[0116] In some embodiments, the polypeptides provided herein include non-natural amino acids. In some embodiments, non-natural amino acids are methylated at the α-amino group to produce peptides with a methylated backbone. In some embodiments, non-natural amino acids are R-amino acids. In some embodiments, non-natural amino acids can include dyes (e.g., fluorescent dyes) or affinity tags. In some embodiments, the polypeptides provided herein include chemical modifications. Chemical modifications include, for example, chemical modifications with biotin or fluorescent dyes. Those skilled in the art will recognize that methods for introducing non-natural amino acids into polypeptides and chemically modifying polypeptides are well known in the art.

[0117] In some embodiments, a polypeptide of the embodiments is fused or conjugated to an immunogenic polypeptide (e.g., a keyhole limpet In some aspects, the polypeptide further comprises a Cys residue at the C- or N-terminus. For example, in some aspects, the polypeptide is conjugated to the immunogenic polypeptide via a disulfide bond at the Cys residue.

[0118] In another embodiment, provided herein are immunogenic compositions comprising a polypeptide comprising a fragment of at least 7 consecutive amino acids of human LAG3, the fragment comprising at least one amino acid corresponding to position N188, N250, N256, or N343 of human LAG3, wherein at least one of the amino acids corresponding to position 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.

[0119] In some embodiments, methods for producing antibodies are provided, comprising administering a polypeptide to an animal and isolating the antibody from the animal, wherein the polypeptide comprises a fragment of at least seven contiguous amino acids of human LAG3, the fragment comprising 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 can 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 produce a humanized antibody. In further aspects, the method comprises recombinantly expressing the humanized antibody. Thus, in another 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 the embodiments (e.g., a polypeptide comprising a fragment of at least 7 contiguous amino acids of human LAG3, said fragment comprising at least one amino acid corresponding to position N188, N250, N256, or N343 of human LAG3, wherein at least one of said amino acids corresponding to position N188, N250, N256, or N343 of human LAG3 is glycosylated relative to unglycosylated LAG3).

[0120] The polypeptides provided herein can be prepared by any method known in the art. For example, polypeptides can be prepared by chemical synthesis or recombinant production. Exemplary methods for expressing and purifying recombinant polypeptides can be found in, for example, Scopes 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 accomplished using methods well known in the art (see Kelley and Winkler, 1990, see: Genetic Engineering Principles and Methods , Setlow JK, ed., Plenum Press, NY, Vol. 12, pp 1-19; Stewart et al., 1984, JM Young, JD, Solid Phase Peptide Synthesis , Pierce Chemical Co., Rockford, Ill; Marglin and Merrifield, Ann. Rev. Biochem, 39: 841-866, at 862 (1970). Merrifield, RB, 1963, J. Am. Chern. Soc. 85: 2149-2154; Chemical Approaches to the Synthesis of Peptides and Proteins ,Williams et al., eds., 1997, CRC Press, Boca Raton Fla.; Solid Phase Peptide Synthesis:A Practical Approach , Atherton & Sheppard, eds., 1989, IRL Press, Oxford, England; see also USPNs. 4,105,603; 3,972,859; 3,842,067; and 3,862,925).

[0121] Modifications and derivatives

[0122] Antibodies against glycosylated LAG3 may have the ability to neutralize or counteract the effects of glycosylated LAG3, regardless of the animal species, monoclonal cell line, or other source of the antibody. Certain animal species may be less preferred for generating therapeutic antibodies because they may be more likely to cause an allergic response due to activation of the complement system by the Fc portion of the antibody. However, intact antibodies can be enzymatically digested into Fc (complement binding) fragments, as well as antibody fragments having binding domains or CDRs. Removal of the Fc portion reduces the likelihood that the antibody fragment will elicit an undesirable immune response, and thus, Fc-free antibodies may be used for prophylactic or therapeutic treatments. 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 antibodies to animals that have been generated in other species or have sequences from other species.

[0123] The binding properties of anti-glycLAG3 antibodies can be further improved by screening for variants that exhibit the desired properties. For example, such improvements can be achieved using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequences encoding them. In a specific embodiment, such phage can be used to display antigen-binding fragments, such as Fabs and Fvs or disulfide-stabilized Fvs, expressed from a library or combinatorial antibody library (e.g., human or murine). Phage expressing antigen-binding fragments that bind to the target antigen can be selected or identified using antigen, for example, using labeled antigen or antigen bound or captured to a solid surface or beads. The phage used in these methods are typically filamentous phage, including fd and M13. The antigen-binding fragments are expressed as recombinant fusion proteins to the phage gene III or gene VIII protein. Examples of phage display methods that can be used to prepare antibodies or polypeptides as described herein include those disclosed in Brinkman et al., J Immunol Methods, 182:41-50 (1995); Ames et al., J. Immunol. Methods, 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol., 24:952-958 (1994); Persic et al., Gene, 187:9-18 (1997); Burton et al., Adv. Immunol. 57:191-280 (1994); PCT Publications WO 92 / 001047; WO 90 / 02809; WO 91 / 10737; WO 92 / 01047; WO 92 / 18619; WO 93 / 11236; WO 95 / 15982; WO 95 / 20401; and U.S. Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; all of which are incorporated herein by reference in their entirety.

[0124] As described in the above references, after phage selection, the antibody coding regions can be isolated from the phage and used to produce whole antibodies, including humanized antibodies, or any other desired fragments, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast and bacteria, for example, as described in detail below. For example, techniques for recombinantly producing Fab, Fab' and F(ab')2 fragments can also be used using methods known in the art, such as those disclosed in PCT Publication WO 92 / 22324; Mullinax, RL et al., BioTechniques, 12(6):864-869 (1992); and Sawai et al., Am. J. Reprod. Immunol, 34:26-34 (1995); and Better, M. et al. Science 240:1041-1043 (1988); all of which are incorporated herein by reference in their entirety. Examples of techniques that can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston, JS et al., Methods in Enzymology 203:46-88 (1991); Shu, L et al., Proc. Natl. Acad. Sci. (USA) 90:7995-7999; and Skerra. A. et al., Science 240:1038-1040 (1988); all of which are incorporated herein by reference in their entirety.

[0125] Phage display technology can be used to increase the affinity of the anti-glycLAG3 antibodies described herein. This technology can be used to obtain high-affinity antibodies that can be used in the combination methods described herein. This technology is called affinity maturation and uses mutagenesis or CDR walking and reselection to identify antibodies that bind to the antigen with higher affinity than the original or parent antibody using such receptors or ligands (or their extracellular domains) or antigenic fragments (see, for example, Glaser, SM et al., J. Immunol. 149: 3903-3913 (1992)). Mutagenesis of entire codons rather than individual nucleotides produces a semi-random library of amino acid mutations. Libraries can be constructed from a set of variant clones, each of which differs in a single amino acid change in a single CDR and contains variants representing every possible amino acid substitution for each CDR residue. Mutants with increased binding affinity for the antigen can be screened by contacting immobilized mutants with labeled antigen. Any screening method known in the art can be used to identify mutant antibodies with increased avidity for the antigen (e.g., ELISA) (see, e.g., Wu, H. et al., Proc. Natl. Acad. Sci. (USA) 95(11):6037-6042 (1998); Yelton, DE et al., J. Immunol. 155:1994-2004 (1995). CDR walking that randomizes the light chain can also be used. (See Schier et al., J. Mol. Biol. 263:551-567 (1996)).

[0126] Random mutagenesis can be used together with phage display methods to identify improved CDR and / or variable regions. Phage display technology can alternatively be used to increase (or reduce) CDR affinity by directed mutagenesis (e.g., affinity maturation or "CDR-walking"). This technology uses target antigen or its antigenic fragment to identify antibodies with CDRs that bind antigens with higher (or lower) affinity than initial or parental antibodies (see, e.g., Glaser, SM et al., J. Immunol. 149: 3903-3913 (1992)).

[0127] 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 entirety.

[0128] 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 relative to a "parent" (or wild-type) molecule. Such amino acid substitutions or additions can introduce naturally occurring (i.e., DNA-encoded) or non-naturally occurring amino acid residues. Such amino acids can be glycosylated (e.g., having altered levels of mannose, 2-N-acetylglucosamine, galactose, fucose, glucose, sialic acid, 5-N-acetylneuraminic acid, 5-hydroxyacetylneuraminic acid, etc.), acetylated, pegylated, phosphorylated, amidated, derivatized with known protecting / blocking groups, proteolytically cleaved, linked to cellular ligands or other proteins, etc. In some embodiments, the altered carbohydrate modifications modulate one or more of the following: antibody solubility, promotion of antibody subcellular transport and secretion, promotion of antibody assembly, conformational integrity, and antibody-mediated effector function. In some embodiments, the altered carbohydrate modifications enhance antibody-mediated effector function relative to an antibody lacking the carbohydrate modifications. Carbohydrate modifications that result in altered antibody-mediated effector functions are well known in the art (e.g., see Shields, RL et al., J. Biol. Chem. 277(30):26733-26740 (2002); Davies J. et al. Biotechnology & Bioengineering 74(4):288-294 (2001); all of which are incorporated herein by reference in their entirety). Methods for altering carbohydrate content are known to those skilled in the art, see, for example, Wallick, SC et al., J. Exp. Med. 168(3):1099-1109 (1988); Tao, MH et al., J. Immunol. 143(8):2595-2601 (1989); Routledge, EG et al., Transplantation 60(8):847-53 (1995); Elliott, S. et al., Nature Biotechnol. 21:414-21 (2003); Shields, RL et al., J. Biol. Chem. 277(30):26733-26740 (2002); all of which are incorporated herein by reference in their entirety.

[0129] Substitution variants can include an exchange of one amino acid with another amino acid at one or more sites within the antibodies or polypeptides provided herein, and can be designed to modulate one or more properties of the antibodies or polypeptides, with or without loss of other functions or performances. The substitution can be conservative, i.e., an amino acid is replaced by an amino acid with a similar shape and charge. Conservative substitutions are well known in the art and include, for example, the following changes: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamic acid to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine, or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Alternatively, the substitution may be non-conservative, thereby affecting the function or activity of the polypeptide.Non-conservative changes typically involve replacing one residue with a chemically different residue, such as replacing a non-polar or uncharged amino acid with a polar or charged amino acid, or vice versa.

[0130] In some embodiments, the antibody may comprise a first set of CDRs as listed in Table 4, but with substitutions (e.g., conservative substitutions) at residues that are not conserved in another or all other CDR sets. For example, the antibody may comprise a Chothia, Abm, or Contact type CDR set, but with one or more substitutions at residues within the heavy chain CDR1 sequence (SEQ ID No: 6, 9, or 13) that do not correspond to those in the Kabat type CDR1 sequence (SEQ ID NO: 11). Similarly, residues 1-4 on SEQ ID NO: 20 may be substituted, for example, with conservative substitutions. Similarly, the trailing residues on SEQ ID NO: 18 and / or SEQ ID NO: 8 may be substituted, for example, with conservative substitutions. These are just a few examples, but one of ordinary skill will understand which substitutions can be made based on what is shown in Table 4.

[0131] In some embodiments, an antibody having a first set of CDRs (e.g., Chothia, AbM, Kabat, and Contact) can comprise a framework region having one or more amino acid substitutions such that the one or more substituted residues are identical at the corresponding position (assessed by sequence alignment and / or according to Kabat numbering) to a second set of CDRs having a leading (i.e., N-terminal) or trailing (i.e., C-terminal) residue that is not present in the CDRs in the first set. For example, the framework region adjacent to the C-terminus of the light chain Contact-type CDR2 has a substituted residue corresponding to the trailing 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 the light chain AbM, Kabat, or Chothia-type CDR2 has a substituted residue corresponding to one or more of the 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 replacement residue corresponding to one or more of the 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 replacement residue corresponding to one or more of the trailing 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 replacement residue corresponding to one or more of the 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 replacement residue corresponding to one or more of the 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 ordinary skill will understand what framework permutations can be made based on what is shown in Table 4.

[0132] In some embodiments, humanized antibodies are derivative antibodies. Such humanized antibodies include amino acid residue replacement, deletion or addition in one or more non-human CDRs. Compared with non-derivative humanized antibodies, humanized antibody derivatives can have substantially the same combination, better combination or worse combination. In some embodiments, one, two, three, four or five amino acid residues in a CDR are mutated, for example, replaced, deleted or added.

[0133] In some embodiments, the polypeptide is a derivatized polypeptide. Such polypeptides include amino acid residue substitutions, deletions, or additions compared to wild-type human LAG3. A derivatized polypeptide can have substantially the same binding, better binding, or worse binding than an anti-glycLAG3 antibody compared to a non-derivatized polypeptide. In some embodiments, one, two, three, four, or five amino acid residues of human LAG3 have been mutated, e.g., substituted, deleted, or added.

[0134] Antibodies or polypeptides described herein can be modified by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, and the like. In one embodiment, the derived polypeptide or derived antibody has a function similar or identical to that of the parent polypeptide or antibody. In another embodiment, the derived polypeptide or derived antibody exhibits altered activity relative to the parent polypeptide or parent antibody. For example, the derived antibody (or fragment thereof) may bind more tightly to its epitope than the parent antibody or have greater tolerance to proteolysis.

[0135] The substitutions, additions or deletions in the derivatized antibodies can be in the Fc region of the antibody and can therefore be used to change the binding affinity of the antibody to one or more FcγRs. Methods for modifying antibodies with altered binding to one or more FcγRs are known in the art, see, for example, PCT Publication Nos. WO 04 / 029207, WO 04 / 029092, WO 04 / 028564, WO99 / 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 entirety. In some embodiments, antibodies or other molecules may have altered affinity for activating FcγRs (e.g., FcγRIIIA). Preferably, such modifications also have altered Fc-mediated effector functions. Modifications that affect Fc-mediated effector functions are well known in the art (see U.S. Patent No. 6,194,551 and WO 00 / 42072). In some embodiments, modifications of the Fc region result in antibodies with altered antibody-mediated effector functions, altered binding to other Fc receptors (e.g., Fc activating receptors), altered antibody-dependent cell-mediated cytotoxicity (ADCC) activity, altered C1q binding activity, altered complement-dependent cytotoxicity activity (CDC), phagocytic activity, or any combination thereof.

[0136] Derivative antibodies or polypeptides can also have a modified half-life (e.g., serum half-life) of the parent molecule or antibody in mammals, preferably humans. In some embodiments, this change results in a half-life greater than 15 days, preferably greater than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months or greater than 5 months. The increased half-life of the humanized antibody or polypeptide in mammals, preferably humans, results in a higher serum titer of the antibody or polypeptide in mammals, and thus reduces the frequency of administration of the antibody or polypeptide and / or reduces the concentration of the antibody or polypeptide to be administered. Antibodies or polypeptides with increased in vivo half-life can be produced by techniques known to those skilled in the art. For example, antibodies or polypeptides with increased in vivo half-life can be produced by modifying (e.g., replacing, deleting or adding) amino acid residues identified as participating in the interaction between the Fc domain and the FcRn receptor. The humanized antibodies described herein can be engineered to increase biological half-life (see, e.g., U.S. Patent No. 6,277,375). For example, the humanized antibodies described herein can be engineered in the Fc-hinge domain to have increased in vivo half-life or serum half-life.

[0137] As described herein, antibodies or polypeptides with increased in vivo half-life can be produced by attaching polymer molecules such as high molecular weight polyethylene glycol (PEG) to the antibody or polypeptide. PEG can be connected to the antibody or polypeptide with or without a multifunctional linker, by site-specific conjugation of PEG to the N or C-terminal end of the molecule or antibody or by the ε-amino group present on the lysine residues. Straight or branched polymer derivatizations that cause minimal loss of biological activity can be used. The degree of conjugation can be closely monitored by SDS-PAGE and mass spectrometry to ensure that the PEG molecule is correctly conjugated to the antibody. Unreacted PEG can be separated from the antibody-PEG conjugate by, for example, size exclusion or ion exchange chromatography.

[0138] The antibodies or polypeptides described herein can also be modified by the methods and coupling agents described by Davis et al. (see U.S. Patent No. 4,179,337) to provide compositions that can be injected into the circulatory system of mammals without substantially any immunogenic response. The removal of the Fc portion can reduce the likelihood that the antibody fragment will elicit an undesirable immune response, and therefore, antibodies without Fc can be used for prophylactic or therapeutic treatments. As described above, antibodies can also be constructed to be chimeric, partially or fully human to reduce or eliminate the adverse immunological consequences caused by administering antibodies to animals that have been produced in other species or have sequences from other species.

[0139] Fusions and conjugates

[0140] The anti-glycLAG3 antibodies or glycosylated LAG3 polypeptides provided herein can also be expressed as fusion proteins with other proteins or chemically conjugated to another moiety.

[0141] In some embodiments, provided herein are antibodies or polypeptides having an Fc portion, wherein the Fc portion can differ by isotype or subclass, can be chimeric or hybrid, and / or can be modified, for example, to improve effector function, half-life control, tissue accessibility, enhance biophysical properties (such as stability), and improve production efficiency (and at a lower cost). Many modifications that can be used to construct the disclosed fusion proteins and methods for preparing them are known in the art, see, for example, Mueller, JP et al., Mol. Immun. 34(6): 441-452 (1997), Swann, PG, Curr. Opin. Immun. 20: 493-499 (2008), and Presta, LG, Curr. Opin. Immun. 20: 460-470 (2008). In some embodiments, the Fc region is a native IgG1, IgG2, or IgG4 Fc region. In some embodiments, the Fc region is a hybrid, for example, a chimera having an IgG2 / IgG4 Fc constant region. Modifications to the Fc region include, but are not limited to, modifications to IgG4 to prevent binding to Fcγ receptors and complement, modifications to IgG1 to improve binding to one or more Fcγ receptors, modifications to IgG1 to minimize effector function (amino acid changes), IgG1 with altered / no glycans (usually by changing the expression host), and IgG1 with altered pH dependency for binding to FcRn. The Fc region may include the entire hinge region, or less than the entire hinge region.

[0142] Another embodiment includes IgG2-4 hybrids and IgG4 mutants that have reduced binding to FcRs, which increases their half-life. Representative IgG2-4 hybrids and IgG4 mutants are described in Angal et al., Molec. Immunol. 30(1):105-108 (1993); Mueller et al., Mol. Immun. 34(6):441-452 (1997); and U.S. Patent No. 6,982,323; all of which are incorporated herein by reference in their entirety. In some embodiments, the IgG1 and / or IgG2 domains are deleted, for example, Angal et al. describe IgG1 and IgG2 in which serine 241 is replaced by proline.

[0143] In some embodiments, provided herein are fusion proteins or polypeptides having at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acids.

[0144] In some embodiments, provided herein are anti-glycLAG3 antibodies or glycosylated LAG3 polypeptides that are linked to, covalently bound to, or complexed with at least one moiety. Such moieties can include, but are not limited to, moieties that enhance the efficacy of the molecule as a diagnostic or therapeutic agent. In some embodiments, the moiety can include an imaging agent, a toxin, a therapeutic enzyme, an antibiotic, a radiolabeled nucleotide, or the like.

[0145] In some embodiments, a moiety can be 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), a protein (e.g., tumor necrosis factor, an interferon (e.g., α-interferon, β-interferon), a nerve growth factor, a platelet-derived growth factor, a tissue plasminogen activator, or an apoptotic agent (e.g., tumor necrosis factor-α, tumor necrosis factor-β)), a biological response modifier (e.g., a lymphokine (e.g., interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), leukin-3 ("IL-4"), or a combination thereof), a cytokine (e.g., IL-6), a cytokine (e.g., IL-7), a cytokine (e.g., IL-8), a cytokine (e.g., IL-9), a cytokine (e.g., IL-10), a cytokine (e.g., IL-11), a cytokine (e.g., IL-12), a cytokine (e.g., IL-13), a cytokine (e.g., IL-14), a cytokine (e.g., IL-15), a cytokine (e.g., IL-16), a cytokine (e.g., IL-17), a cytokine (e.g., IL-18), a cytokine (e.g., IL-29), a cytokine (e.g., interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or macrophage colony stimulating factor ("M-CSF"), or growth factors (e.g., growth hormone ("GH")), cytotoxins (e.g., cytostatic or cytocidal agents such as paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione (dihydroxy anthracin dione), mitoxantrone, plicamycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE; e.g., vedotin), and puromycin and its analogs or homologs), antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil dacarbazine), alkylating agents (e.g., dichloromethane, thiotepa chlorambucil, melphalan, (carmustine; BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, plicamycin, and anthramycin (AMC)), or antimitotic agents (e.g., vincristine and vinblastine).

[0146] The techniques for conjugating these therapeutic moieties to antibodies are well known; see, for example, 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 MONOCLONALANTIBODIES FOR CANCER DETECTION AND THERAPY, Baldwin et al. (eds.), 1985, pp. 303-16, Academic Press; Thorpe et al., Immunol. Rev. 62: 119-158 (1982); Carter et al., Cancer J. 14(3): 154-169 (2008); Alley et al., Curr. Opin. Chem. Biol. 14(4): 529-537 (2010); Carter et al., Amer. Assoc. Cancer Res. Educ. Book. 2005(1):147-154(2005); Carter et al., Cancer J. 14(3):154-169(2008); Chari, Acc. Chem Res. 41(1):98-107(2008); Doronina et al., Nat. Biotechnol.21(7):778-784 (2003); Ducry et al., Bioconjug Chem. 21(1):5-13 (2010); Senter, Curr. Opin. Chem. Biol. 13(3):235-244 (2009); and Teicher, Curr Cancer Drug Targets. 9(8):982-1004 (2009). Auristatin E) (MMAE), such as vedotin; or a combination thereof.

[0147] In a preferred embodiment, the antibody is conjugated to maytansine, which is a benzobridge macrolide first isolated from the bark of the Ethiopian shrub Maytenus ovatus. This cytotoxic agent and its derivatives (such as maytansine) bind to the tubulin near the vinca alkaloid binding site. They are considered to have high affinity for the tubulin at the end of the microtubules, and have lower affinity for the sites distributed throughout the microtubules. The inhibition of microtubule dynamics causes cells to stagnate in the G2 / M phase of the cell cycle, ultimately leading to cell death due to apoptosis. (Oroudjev et al., Mol. Cancer Ther., 10L2700-2713 (2010)). Two maytansine derivatives (containing thiol maytansine) include DM1 and DM4 (ImmunoGen, Inc., Waltham, MA) and are widely used in combination with irreversible and reversible linkers. In particular, DM1 attached to an antibody with a thioether linker is referred to as "emtansine"; DM1 attached to an antibody with an SPP linker is referred to as "mertansine." DM4 attached with an SPDB linker is referred to as "ravtansine"; and DM4 attached with an sSPDB linker is referred to as "soravtansine." (ImmunoGen, Inc., Waltham, MA). In one embodiment, the anti-glycLAG3 antibody-ADC comprises a maytansinoid payload, DM1, that acts on tubulin. In one embodiment, the anti-glycLAG3 antibody-ADC comprises a maytansinoid payload, DM4, that acts on tubulin. In one embodiment, the anti-glycLAG3 antibody-ADC comprises a payload that acts on DNA, such as DGN462 (ImmunoGen, Inc., Waltham, MA). 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. 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.

[0148] In a specific embodiment, the cytotoxic agent conjugated to the anti-glycLAG3 antibody is MMAE (monomethyl auristatin E (or demethyl auristatin E)), a highly toxic anti-tumor agent whose antimitotic activity involves inhibiting cell division by blocking tubulin polymerization. Vedotin is an international nonproprietary name that refers to the MMAE in the MMAE-antibody conjugate plus its linker to the antibody. In more specific embodiments, the ADC is STC1317 (chimeric or humanized form)-MMAE or STC1317 (chimeric or humanized form)-MMAE.

[0149] Many chemical linkers are known and used to conjugate cytotoxic or DNA-acting drug payloads to antibodies to generate ADCs. Certain linkers used alone or in combination to generate ADCs containing anti-glycLAG3 antibodies, particularly those that are internalized upon binding to their targets as described herein, include SMCC (4-(N-maleimidomethyl)cyclohexanecarboxylic acid N-hydroxysuccinimide ester); SPDB (N-succinimidyl 3-(2-pyridyldithio)butyrate); SPP (N-succinimidyl 4-(2-pyridyldithio)pentanoate); sulfo-SPDB or sSPDB (N-succinimidyl-4-(2-pyridyldithio)-2-sulfobutyrate); the thioether linker succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (MCC); and vc (valine-citrulline dipeptide linker). For example, engineered linkers (e.g., SMCC, SPDB, S-SPDB) (Immunogen, Inc.) have been designed to stabilize the ADC before it binds to the tumor and then optimize payload efficacy after the ADC is internalized within the cancer cell. Other linkers, such as the dipeptide vc linker, which is a cathepsin-cleavable linker, can be used to conjugate antibodies to cytotoxic agents, such as auristatin, a mitotic inhibitor derived from dolastatin 10, such as monomethyl auristatin E (MMAE), e.g., vitin. Cytotoxins can be conjugated to antibodies such that more than one toxin molecule is attached to each antibody molecule, e.g., an average of 2, 3, 4, 5, 6, 7, or 8 toxin molecules per antibody.

[0150] In one embodiment, MMAE is indirectly linked to the antibody cysteine ​​via a maleimidocaproyl (MC) linker, which is coupled to valine-citrulline-p-aminobenzyloxycarbonyl-MMAE (MC-vc-PAB-MMAE). In the linear structure of "MC-vc-PAB-MMAE", "MC" consists of maleimide and caproic acid and is the portion that connects the antibody, typically connected via a cysteine ​​group on the H chain. Furthermore, "MC" is attached to a "vc" linker consisting of valine (Val) and citrulline (Cit), and the linker is a cathepsin-cleavable linker that can be cleaved by cathepsins within tumors or cancer cells. "vc" is attached to a spacer "PAB", i.e., p-aminobenzoic acid, to which the MMAE cytotoxin is attached. MC-vc-PAB-MMAE ADC releases free, membrane-permeable MMAE when cleaved by a protease such as cathepsin B. In one embodiment, the antibody linker is stable in extracellular fluid but is cleaved by cathepsins once the ADC enters the tumor or cancer cell, thereby activating the anti-mitotic mechanism of MMAE or other toxin drugs. In another embodiment, monomethyl auristatin F (MMAF) is linked to the antibody cysteine ​​via a maleimidocaproyl group (MC-MMAF). Compared to the MC-vc-PAB-MMAE ADC, the MC-MMAF ADC, like the MCC-DM1 ADC, is not cleavable and must be internalized and degraded intracellularly, releasing the cysteine-MC-MMAF as the active drug within the cell.

[0151] In one embodiment, the cytotoxic payload is released in the lysosome after the ADC is internalized into the cell. In the lysosome, the lysosomal enzyme digests the antibody component of the ADC. After lysosomal degradation, the drug (and drug linker) payload is released into the cytoplasm, where the drug binds to the intracellular target, ultimately leading to cell death. Ideally, the released payload is fully active while the linker is still connected. In other embodiments where the target bound to the ADC results in poor transport to the lysosome, a linker that is stable outside the target cell but cuts the payload from the antibody component once it enters the cell provides an alternative mode of releasing the payload inside the cell but outside the lysosome. In other embodiments, the linker is stable in the extracellular fluid, but is cut by cathepsin once the ADC enters the tumor or cancer cell, thereby activating the anti-mitotic or other cytotoxic mechanisms of the toxin drug. In other embodiments, the payload released by the action of the cleavable linker can enter adjacent cancer cells and kill them by the bystander effect, thereby enhancing the targeting and tumor killing activity of the ADC.

[0152] In some embodiments, the antibodies and polypeptides described herein can be conjugated to a marker (e.g., a peptide) to facilitate purification. In some embodiments, the marker is a hexa-histidine peptide, a hemagglutinin "HA" tag (SEQ ID NO: 22: YPYDVPDYA), which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, IA et al., Cell, 37: 767-778 (1984)), or a "Flag" tag (Knappik, A. et al., Biotechniques 17 (4): 754-761 (1994)).

[0153] In some embodiments, the moiety can be an imaging agent that can be detected 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.

[0154] 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; 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), phosphorus ( 32P), praseodymium ( 142 Pr), promethium ( 149 Pm), rhenium ( 186 Re、 188 Re), rhodium ( 105 Rh), ruthenium ( 97 Ru), Samarium ( 153 Sm), Scandium ( 47 Sc), selenium ( 75 Se), strontium ( 85 Sr), sulfur ( 35 S), technetium ( 99 Tc), thallium ( 201 Ti), tin ( 113 Sn, 117 Sn), tritium ( 3 H), xenon ( 133 Xe), Ytterbium ( 169 Yb, 175 Yb), yttrium ( 90 Y), zinc ( 65 Zn); positron-emitting metals using various positron emission tomography techniques, and non-radioactive paramagnetic metal ions.

[0155] The imaging agent can be conjugated to an antibody or polypeptide provided herein directly or through an intermediate (e.g., a linker known in the art) using techniques known in the art. Regarding metal ions that can be conjugated to antibodies and other molecules described herein for use as diagnostic agents, see, for example, U.S. Patent No. 4,741,900. Some conjugation methods involve the use of metal chelate complexes, for example, organic chelating agents such as diethylenetriaminepentaacetic anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-toluenesulfonamide; and / or tetrachloro-3-6α-diphenyl glycoluril-3 connected to the antibody. Monoclonal antibodies can also react with enzymes in the presence of coupling agents such as glutaraldehyde or periodate. Conjugates with fluorescein markers can be prepared in the presence of these coupling agents or by reacting with isothiocyanates.

[0156] In some embodiments, an antibody or polypeptide as described herein can be conjugated to a second antibody to form an antibody heteroconjugate, as described by Segal in U.S. Patent No. 4,676,980. Such heteroconjugate antibodies can also 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, LIGHT, ICOS, B7-H3, B7-H7, B7-H7CR, CD70, CD47) or a cytokine (e.g., IL-7, IL-15, IL-12, IL-4 TGF-β, IL-10, IL-17, IFNγ, Flt3, BLys) or a chemokine (e.g., CCL21).

[0157] In some embodiments, the anti-glycLAG3 antibodies or glycosylated LAG3 polypeptides described herein can also be attached to a solid support, which can be used for immunoassays or purification of a target antigen or other molecule capable of binding to a target antigen that has been immobilized on the support by binding to an antibody or antigen-binding fragment described herein. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0158] Protein purification

[0159] Protein purification techniques are well known to those skilled in the art. These techniques relate, on one level, to homogenizing and crudely fractionating cells, tissues, or organs into polypeptide and non-polypeptide fractions. Unless otherwise indicated, chromatography and electrophoresis techniques can be used to further purify the target protein or polypeptide to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suitable for preparing pure peptides are ion exchange chromatography, size exclusion chromatography, reversed-phase chromatography, hydroxyapatite chromatography, polyacrylamide gel electrophoresis, affinity chromatography, immunoaffinity chromatography, and isoelectric focusing. A particularly effective method for purifying peptides is fast liquid chromatography (FPLC) or even high performance liquid chromatography (HPLC). As is well known in the art, it is believed that the order in which the various purification steps are performed can be changed, or certain steps can be omitted, and still produce a suitable method for preparing a substantially purified polypeptide.

[0160] Purified polypeptide means a composition that is separable from other components, wherein the polypeptide is purified to any degree relative to its naturally available state. Thus, an isolated or purified polypeptide also refers to a polypeptide that is removed from the environment in which it may naturally occur. Generally speaking, "purified" refers to a polypeptide composition that has been fractionated to remove various other components and in which the composition substantially retains the biological activity expressed by it. When the term "substantially purified" is used, the designation is intended to refer to a composition in which the polypeptide forms the major component of the composition, e.g., constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the protein in the composition.

[0161] According to the present disclosure, various methods for determining the degree of purification of a quantitative polypeptide are known to those skilled in the art. These include, for example, determining the specific activity of the active fraction, or analyzing and assessing the amount of polypeptide in the fraction by SDS / PAGE. A preferred method for assessing fraction purity is to calculate the specific activity of the fraction, compare it with the specific activity of the initial extract, and thereby calculate the purity thereof, assessed by a "purification factor." Of course, the actual unit used to represent the amount of activity will depend on the selected specific assay technique performed after purification, and whether the expressed polypeptide exhibits detectable activity.

[0162] It is not generally required that a polypeptide is always provided in its most purified state. In fact, it is contemplated that in certain embodiments, products with a lower degree of basic purification may be useful. Partial purification can be accomplished by combining fewer purification steps or by using different forms of the same general purification scheme. For example, it is understood that cation exchange column chromatography performed using HPLC equipment will typically produce a greater "fold" of purification than the same technique using a low-pressure chromatography system. Methods with a relatively low degree of purification may have advantages in terms of overall recovery of the protein product or maintaining the activity of the expressed protein.

[0163] Affinity chromatography is a chromatographic procedure that relies on the specific affinity between the substance to be separated and the molecule to which it can specifically bind. This is a receptor-ligand type of interaction. Column materials are synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb substances from the solution. Elution occurs by changing the conditions to conditions where binding does not occur (e.g., changing pH, ionic strength, temperature, etc.). The matrix should be a substance that does not adsorb molecules to any significant extent and has a wide range of chemical, physical and thermal stability. The ligand should be coupled in a manner that does not affect its binding properties. The ligand should also provide relatively tight binding. The substance should be eluted without destroying the sample or the ligand.

[0164] Size exclusion chromatography (SEC) is a chromatographic method in which molecules in a solution are separated based on their size, or more technically, their hydrodynamic volume. It is commonly applied to macromolecules or macromolecular complexes, such as proteins and industrial polymers. Typically, when an aqueous solution is used to transport the sample through the column, the technique is called gel filtration chromatography, while when an organic solvent is used as the mobile phase, it is called gel permeation chromatography. The basic principle of SEC is that particles of different sizes will elute (filter) through the stationary phase at different rates. This results in a separation of a solution of particles based on size. If all particles are loaded simultaneously or nearly simultaneously, particles of the same size should elute together.

[0165] High-performance liquid chromatography (or high-pressure liquid chromatography, HPLC) is a form of column chromatography commonly used in biochemistry and analytical chemistry to separate, identify, and quantify compounds. HPLC uses a column filled with a chromatographic filler (stationary phase), a pump to move one or more mobile phases through the column, and a detector to display the retention time of molecules. Retention times vary depending on the interaction between the stationary phase, the analyte, and the solvent or solvents used.

[0166] Also provided herein is a method for assessing LAG3 glycosylation, N-linked glycosylation, or N-glycosylation, comprising contacting a sample containing LAG3 with an antibody of the embodiments (e.g., an antibody that selectively binds to glycosylated LAG3 relative to unglycosylated LAG3). In some aspects, the method is an in vitro method. In certain aspects, the sample is a cell sample.

[0167] Nucleic Acids

[0168] The present disclosure also contemplates nucleic acid molecules (DNA or RNA) encoding any of the anti-glycLAG3 antibodies or glycosylated LAG3 polypeptides described herein. Also provided herein are vector molecules (e.g., plasmids) configured to transport or replicate such nucleic acid molecules. Nucleic acids can be single-stranded, double-stranded, and can contain both single-stranded and double-stranded portions.

[0169] pharmaceutical preparations

[0170] When pursuing clinical applications of pharmaceutical compositions containing antibodies, it is often beneficial to prepare pharmaceutical or therapeutic compositions that are suitable for the intended application. Generally, pharmaceutical compositions can comprise an effective amount of an anti-glycLAG3 antibody or glycosylated LAG3 polypeptide as described herein, or another agent dissolved or dispersed in a pharmaceutically acceptable carrier.

[0171] Also provided herein are compositions comprising an anti-glycLAG3 antibody or glycosylated LAG3 polypeptide as described herein. In some embodiments, the composition can comprise at least 0.1% by weight of the antibody or polypeptide. In some embodiments, the composition can 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 can comprise from about 2% to about 75%, from about 25% to about 60%, from about 30% to about 50%, or any range therein, of the weight of the composition. The amount of active compound in each therapeutically useful composition can be prepared so that a suitable dosage will be obtained in any given unit dose of the compound. Those skilled in the art of preparing such pharmaceutical formulations will consider a variety of factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life and other pharmacological considerations, and therefore, a variety of dosages and treatment regimens may be desirable.

[0172] The composition can be a pharmaceutical composition comprising an anti-glycLAG3 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 regulatory agency of the U.S. federal or state government, or a carrier listed in the U.S. Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias for use in animals, more particularly humans.

[0173] As used herein, and unless otherwise indicated, the term "carrier" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), excipient, stabilizer, or vehicle administered with a therapeutic agent. A "pharmaceutically acceptable carrier" is a carrier that is nontoxic to cells or mammals exposed thereto at the dosage and concentration employed, and can be a sterile liquid, such as water and oil, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. A pharmaceutically acceptable molecular entity or composition does not produce adverse, allergic, or other adverse reactions when properly administered to an animal, such as a human. In view of the present disclosure, the preparation of pharmaceutical compositions with antibodies or additional active ingredients is known to those skilled in the art, as exemplified by Remington's Pharmaceutical Sciences, 18th edition, 1990, incorporated herein by reference. In addition, for animal (e.g., human) administration, it should be understood that the formulation should meet the sterility, pyrogenicity, general safety, and purity standards required by the FDA Office of Biological Standards.

[0174] Compositions are contemplated to include between about 0.001 mg and about 10 mg of total antibody or polypeptide per milliliter (ml). Thus, the concentration of the antibody or polypeptide in the composition can be about, at least about, or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml or more (or any range derivable therein). 47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% can be an anti-glycLAG3 antibody or a glycosylated LAG3 polypeptide.

[0175] In light of the present disclosure, the preparation of pharmaceutical compositions having antibodies or other polypeptides as described herein as active ingredients is known to those skilled in the art, as exemplified in Remington's Pharmaceutical Sciences, 18th edition, 1990, incorporated herein by reference. In addition, for animal (e.g., human) administration, it will be understood that the preparations should meet the sterility, pyrogenicity, general safety and purity standards required by the FDA Office of Biological Standards.

[0176] Pharmaceutically acceptable carriers include liquids, semisolids (i.e., pastes) or solid carriers. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, adhesives, fillers, etc., or combinations thereof. Pharmaceutically acceptable carriers include aqueous solvents (e.g., water, alcohol / water solutions, ethanol, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils and injectable organic esters such as ethyl oleate), dispersion media, coating agents (e.g., lecithin), surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, inert gases, parabens (e.g., methylparaben, propylparaben), chlorobutane, In some embodiments, the present invention provides a pharmaceutical composition comprising ...

[0177] In some embodiments, the pharmaceutically acceptable carrier can be an aqueous pH buffered solution. Examples include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (e.g., less than about 10 amino acid residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN®. TM , polyethylene glycol (PEG) and PLURONICS TM .

[0178] In some embodiments, pharmaceutically acceptable carriers can be sterile liquids, such as water and oil, including those oils from petroleum, animal, plant or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water can be a carrier, particularly when the intravenous pharmaceutical composition is administered. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, ethylene glycol, water, ethanol, polysorbate-80, etc. Compositions can also contain a small amount of wetting agent or emulsifier, or pH buffer. These compositions can take the form of solution, suspension, emulsion, tablet, pill, capsule, powder, sustained-release formulation, etc.

[0179] Some embodiments of the present disclosure can have different types of carriers, depending on whether it is used in solid, liquid or aerosol form, and for route of administration (such as injection), whether it needs to be sterile. Compositions can be formulated for intravenous, intradermal, transdermal, intrathecal, intraarterial, intraperitoneal, intranasal, intravaginal, intrarectal, intramuscular, subcutaneous, transmucosal, oral, surface, local, by suction (such as, aerosol inhalation), by injection, by infusion, by continuous infusion, by local perfusion direct bathing target cells, via catheter, through lavage, in lipid composition (such as, liposome) or by other methods known to those of ordinary skill in the art or aforementioned any combination (see, for example, Remington's Pharmaceutical Sciences, the 18th edition, 1990, incorporated herein by reference). Typically, such compositions can be prepared into liquid solutions or suspensions; It is also possible to prepare a solid form suitable for preparing a solution or suspension after adding a liquid before injection; And, it is also possible to emulsify preparations.

[0180] Anti-glycLAG3 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 those formed with free amino groups of the protein composition, or those formed with inorganic acids (such as hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, or mandelic acid). Salts formed with free carboxyl groups can also be derived from inorganic bases, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide; or organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, or procaine.

[0181] In a further embodiment, provided herein is a pharmaceutical composition with lipid. Lipid can broadly include a class of substances that are characteristically insoluble in water and can be extracted with organic solvents. Examples include compounds containing long-chain aliphatic hydrocarbons and derivatives thereof. Lipid can be naturally occurring or synthetic (i.e., designed or produced by people). Lipid can be a biological substance. Biological lipids are well known in the art and include, for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, sphingolipids, glycolipids, sulfolipids, lipids with ether- and ester-connected fatty acids, polymerizable lipids and combinations thereof. Compounds other than those specifically described herein that are understood to be lipids by those skilled in the art can also be used.

[0182] Those of ordinary skill in the art will be familiar with a range of techniques that can be used to disperse compositions in lipid vehicles. For example, the antibody or polypeptide can be dispersed in a solution containing lipids, dissolved in lipids, emulsified with lipids, mixed with lipids, combined with lipids, covalently bound to lipids, contained in lipids as a suspension, contained in or complexed with micelles or liposomes, or otherwise associated with lipids or lipid structures by any means known to those of ordinary skill in the art. The dispersion may or may not result in the formation of liposomes.

[0183] Typically, the ingredients of the composition are provided separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or anhydrous concentrate in a hermetically sealed container, such as an ampoule or sachet indicating the amount of active agent. When the composition is administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical grade water or saline. In the case of administration of the composition by injection, sterile water for injection or saline can be provided in an ampoule so that the ingredients can be mixed before administration.

[0184] The amount of active ingredient in each therapeutically useful composition can be prepared so that a suitable dosage will be obtained in any given unit dose of the compound. Those skilled in the art of preparing such pharmaceutical formulations can consider a variety of factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life and other pharmacological considerations, and therefore, a variety of dosages and treatment regimens may be desirable.

[0185] Unit dose or dosage form refers to a physically discrete unit suitable for a subject, each unit containing a predetermined amount of the pharmaceutical composition, the amount being calculated to produce the desired response associated with its administration (i.e., appropriate route and treatment regimen) discussed above. The amount to be administered depends on the desired effect, depending on 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 degree of disease penetration, previous or concurrent therapeutic interventions, the patient's idiopathic disease, the route of administration, and the efficacy, stability, and toxicity of the specific therapeutic substance. In other non-limiting examples, the dosage can have a range of from about 1 μg / kg / body weight, about 5 μg / kg / body weight, about 10 μg / kg / body weight, about 50 μg / kg / body weight, about 100 μg / kg / body weight, about 200 μg / kg / body weight, about 350 μg / kg / body weight, about 500 μg / kg / body weight, about 1 mg / kg / body weight, about 5 mg / kg / body weight, about 10 mg / kg / body weight, about 50 mg / kg / body weight, about 100 mg / kg / body weight, about 200 mg / kg / body weight, about 350 mg / kg / body weight, about 500 mg / kg / body weight, to about 1000 mg / kg / body weight or more per administration, and any range derivable therein. In non-limiting examples of ranges derivable from the numbers listed herein, a range of about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 μg / kg / body weight to about 500 mg / kg / body weight, etc. can be administered according to the above numbers. In any event, the practitioner responsible for administration will determine the concentration of the active ingredient(s) in the composition and the appropriate dosage(s) for an individual subject.

[0186] As will be understood by those skilled in the art, the compositions described herein are not limited by the specific properties of therapeutic preparations. For example, such compositions can be provided in a preparation together with a physiologically tolerable liquid, gel or solid carrier, diluent and excipient. These therapeutic preparations can be applied to mammals for veterinary use, such as for domestic animals, and for clinical use in humans, in a manner similar to other therapeutic agents. In general, the dosage required for the therapeutic effect will vary according to the specific requirements of the type of use and mode of administration and individual subjects. The actual dosage of the composition administered to an animal patient (including a human patient) can be determined by physical and physiological factors, such as body weight, severity of the disease, the type of disease being treated, previous or simultaneous therapeutic interventions, the patient's idiopathic disease, and route of administration. Depending on dosage and route of administration, the number of administrations of the preferred dosage and / or effective dose can vary according to the reaction of the subject. In any case, the practitioner responsible for administration will determine the concentration of one or more active ingredients in the composition and one or more suitable dosages for individual subjects.

[0187] Treatment of diseases

[0188] As used herein, unless otherwise indicated, the term "subject" refers to an animal that is the subject of treatment, observation, and / or experiment. "Animal" includes vertebrates and invertebrates, such as fish, shellfish, reptiles, birds, and especially mammals. "Mammals" include, but are not limited to, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cattle, horses, primates, such as monkeys, chimpanzees, apes, and humans. In some embodiments, the subject is a human.

[0189] As used herein, and unless otherwise indicated, the terms "cancer" or "cancerous" refer to the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, hematological cancers and solid tumors.

[0190] As used herein, and unless otherwise indicated, the term "treatment" refers to the process and mode of administering or applying a therapeutic agent to a subject or performing an operation on a subject for the purpose of obtaining a therapeutic benefit for a disease or health-related condition. For example, treatment can include administering a therapeutically effective amount of an anti-glycLAG3 antibody to a subject. When used in reference to a cancer patient, the term "treatment" refers to an action that may reduce the severity of cancer or delay or slow the progression of cancer, including (a) inhibiting cancer growth, reducing cancer growth rate, arresting progression, reducing cancer invasiveness, or preventing cancer metastasis, and (b) causing cancer regression, delaying or minimizing one or more symptoms associated with the presence of cancer, or prolonging the survival of a cancer patient.

[0191] As used herein, and unless otherwise indicated, the term "therapeutically effective amount" refers to an amount of an agent (e.g., an antibody or polypeptide described herein or other agent described herein) sufficient to reduce and / or ameliorate the severity and / or duration of a given disease, disorder, or condition and / or symptoms associated therewith. A therapeutically effective amount of an agent (including a therapeutic agent) can be an amount necessary to: (i) reduce or alleviate the development or progression of a given disease, disorder, or condition, (ii) reduce or alleviate the recurrence, development, or onset of a given disease, disorder, or condition, and / or (iii) improve or enhance the prophylactic or therapeutic effect of another therapy (e.g., a therapy other than the 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) can vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the substance / molecule / agent to elicit a desired response in the individual. A therapeutically effective amount includes an amount in which any toxic or deleterious effects of the substance / molecule / agent are outweighed by the therapeutically beneficial effects.

[0192] As used herein, and unless otherwise indicated, the term "administering" refers to the act of injecting or otherwise physically delivering a substance present outside the body into a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, 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 typically occurs after the onset of the disease, disorder, or condition, or a symptom thereof. When preventing a disease, disorder, or condition, or a symptom thereof, administration of the substance typically occurs before the onset of the disease, disorder, or condition, or a symptom thereof.

[0193] Also provided herein are therapeutic uses of anti-glycLAG3 antibodies and glycosylated LAG3 polypeptides. These antibodies or polypeptides can be used to modulate the activity of LAG3 / MHCII signaling. These antibodies or polypeptides can also be used to treat diseases by inhibiting the inhibitory activity of LAG3 in T cell activation or proliferation and cytokine secretion. Thus, provided herein are uses of such antibodies or polypeptides for upregulating a subject's immune system by inhibiting or blocking LAG3 signaling. In some embodiments, provided herein are uses of antibodies or polypeptides for blocking LAG3 binding to Gal-3, MHCII, liver sinusoidal endothelial cell lectin (LSECtin), and / or CD3.

[0194] In some embodiments, provided herein are therapeutic uses of anti-glycLAG3 antibodies and glycosylated LAG3 polypeptides for the treatment of cancer. Upregulation of the immune system is particularly desirable in the treatment of cancer, and thus methods of treating cancer are also provided herein. Cancer refers to a neoplasm or tumor caused by abnormal, uncontrolled cell growth. Cancer can be primary or metastatic. In specific embodiments, the cancer cells are positive for MCHII.

[0195] In certain aspects, the polypeptides or antibodies of the embodiments (e.g., glycosylated LAG3 polypeptides or antibodies that bind to glycosylated LAG3) can be administered to treat cancer. In specific embodiments, the anti-glycLAG3 antibody is a chimeric or humanized form of STC1317. Cancers for which this treatment method is applicable include any malignant cell type, such as those found in solid tumors or hematological tumors. Exemplary solid tumors may include, but are not limited to, tumors of organs selected from the group consisting of pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. Exemplary hematological tumors include myeloid tumors, T or B cell malignancies, leukemias, lymphomas, blastomas, myelomas, and the like. Other examples of cancers that can be treated using the methods provided herein include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia, squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, stomach cancer or gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, Kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, various head and neck cancers, melanoma, superficial spreading melanoma, lentigo melanoma, acral lentigo melanoma, nodular melanoma, and B-cell lymphomas (including low-grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, multiple myeloma, acute myeloid leukemia (AML), and chronic myeloblastic leukemia.

[0196] Cancer may specifically be of the following histological types, but is not limited to: malignant neoplasms; carcinoma; undifferentiated carcinoma; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatricoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; bile duct carcinoma; hepatocellular carcinoma; combined hepatocellular and bile duct carcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma, familial polyposis coli; solid carcinoma; malignant carcinoid tumor; bronchiolar-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe cell carcinoma; oncocytic carcinoma; oncocytic adenocarcinoma; basophilic cell carcinoma; clear cell adenocarcinoma; granulosa cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-cystic sclerosing carcinoma; adrenocortical carcinoma; endometrioid carcinoma carcinoma); skin appendage carcinoma; apocrine gland carcinoma; sebaceous gland carcinoma; cervical gland carcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous transformation; malignant thymoma; malignant ovarian stromal tumor; malignant thecoma cell tumor; malignant granulosa cell tumor; malignant testicular blastoma (androblastoma) ma, malignant); Sertoli cell carcinoma; malignant Leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extramammary paraganglioma; pheochromocytoma; glomus sarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma within giant nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; mesenchymal Sarcoma; malignant mixed tumor; mixed Müllerian tumor; Wilms' tumor; hepatoblastoma; carcinosarcoma; malignant mesenchymal tumor; malignant Brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma; malignant goiter; choriocarcinoma; malignant mesonephroblastoma; angiosarcoma; malignant hemangioendothelioma; Kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; malignant odontoma ameloblastoma; ameloblastic odontosarcoma; malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pinealoma; chordoma; malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectodermal tumor; cerebellar sarcoma; ganglioblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant neurilemoma; malignant granular cell tumor;Malignant lymphoma; Hodgkin's disease; Hodgkin's; paragranuloma; small lymphocytic malignant lymphoma; diffuse large cell malignant lymphoma; follicular malignant lymphoma; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small bowel disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0197] In some embodiments, the antibodies or polypeptides provided herein can be used to treat 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.

[0198] Polypeptides or antibodies can be used herein as anti-tumor agents in a variety of ways. Provided herein are methods of using polypeptides or antibodies as anti-tumor agents, thus comprising contacting a tumor cell population with a therapeutically effective amount of the polypeptide or antibody for a period of time sufficient to inhibit tumor cell growth.

[0199] 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 encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibody or fusion protein, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), constructing the nucleic acid as part of a retroviral or other vector, etc.

[0200] The methods of administration provided herein include, but are not limited to, injection, such as by parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural, and mucosal (e.g., intranasal and oral routes). In some embodiments, the antibodies, other molecules, or pharmaceutical compositions provided herein are administered intramuscularly, intravenously, subcutaneously, intravenously, intraperitoneally, orally, intramuscularly, subcutaneously, intracavitary, transdermally, or transdermally. The composition can be administered by any convenient route, such as by infusion or rapid push, by absorption of epithelial or mucocutaneous linings (e.g., oral mucosa, rectal, and intestinal mucosa), and can be administered together with other bioactive agents. Administration can be systemic or local. In addition, pulmonary administration can also be employed, for example, by using an inhaler or nebulizer, and formulated together with an atomizer. 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; 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 entirety. In some embodiments, the antibodies, other molecules, or pharmaceutical compositions provided herein are administered topically to the area in need of treatment, which can be achieved, for example, by local infusion, by injection, or by implantation of a porous, non-porous, or gel-like material, including membranes, e.g., sialastic membranes, or fibers. In some embodiments, when administering an antibody or other molecule described herein, care is taken to use a material that the antibody or other molecule is not absorbed by.

[0201] In some embodiments, the antibodies or polypeptides provided herein are formulated in liposomes for targeted delivery. Liposomes are vesicles composed of concentric, ordered phospholipid bilayers that encapsulate an aqueous phase. Liposomes typically have various types of lipids, phospholipids, and / or surfactants. The components of the liposomes are arranged in a bilayer configuration, similar to the lipid arrangement of biological membranes. Liposomes can be useful delivery vehicles, in part due to their biocompatibility, low immunogenicity, and low toxicity. Methods for preparing liposomes are known in the art and are provided herein, see, for example, Epstein et al., 1985, Proc. Natl. Acad. Sci. USA, 82: 3688; Hwang et al., 1980 Proc. Natl. Acad. Sci. USA, 77: 4030-4; U.S. Patent Nos. 4,485,045 and 4,544,545; all of which are incorporated herein by reference in their entirety.

[0202] Also provided herein is a method for preparing liposomes with extended serum half-life (i.e., enhanced circulation time), such as those disclosed in U.S. Patent number 5,013,556. In some embodiments, the liposomes used in the method provided herein will not be quickly cleared from the circulation, i.e., will not be absorbed into the mononuclear phagocyte system (MPS). Also provided herein is a sterically stabilized liposome, which is prepared using conventional methods known to those skilled in the art. The sterically stabilized liposome can include a lipid component with a large and highly flexible hydrophilic portion, which reduces the adverse reaction of the liposome with serum proteins, reduces the opsonization (oposonization) by serum components, and reduces the recognition by MPS. Polyethylene glycol can be used to prepare sterically stabilized liposomes. For the preparation of liposomes and sterically stabilized liposomes, see, e.g., Bendas et al., 2001 BioDrugs, 15(4):215-224; Allen et al., 1987 FEBS Lett. 223:42-6; Klibanov et al., 1990 FEBS Lett., 268:235-7; Blum et al., 1990, Biochim. Biophys. Acta., 1029:91-7; Torchilin et al., 1996, J. Liposome Res. 6:99-116; Litzinger et al., 1994, Biochim. Biophys. Acta, 1190:99-107; Maruyama et al., 1991, Chem. Pharm. Bull., 39:1620-2; Klibanov et al., 1991, Biochim Biophys Acta, 1062; 142-8; Allen et al., 1994, Adv. Drug Deliv. Rev, 13: 285-309, the entire contents of which are incorporated herein by reference.

[0203] Also provided herein are liposomes suitable for specific organ targeting, see, for example, U.S. Patent number 4,544,545, or liposomes suitable for specific cell targeting, see, for example, U.S. Patent Application Publication No. 2005 / 0074403, the entire contents of which are incorporated herein by reference. Particularly useful liposomes for compositions and methods provided herein can be produced by reverse phase evaporation with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derived phosphatidylethanolamine (PEG-PE). Liposomes can be extruded through a filter with a limited pore size to produce liposomes with a desired diameter. In some embodiments, molecules with antigen binding fragments, for example, F (ab'), can be conjugated to liposomes using previously described methods, see, for example, Martin et al., 1982, J. Biol. Chem. 257: 286-288, which are incorporated herein by reference in their entirety.

[0204] Humanized or chimeric antibodies as described herein can also be formulated into immunoliposomes. Immunoliposomes refer to liposome compositions in which antibodies or fragments thereof are covalently or non-covalently attached to the surface of the liposomes. Chemical methods for attaching antibodies to the surface of liposomes are known in the art, see, for example, U.S. Patent No. 6,787,153; Allen et al., 1995, Stealth Liposomes, Boca Rotan: CRC Press, 233-44; Hansen et al., 1995, Biochim.Biophys.Acta, 1239: 133-144, which are incorporated herein by reference in their entirety. In some embodiments, the immunoliposomes for the methods and compositions provided herein are further spatially stabilized. In some embodiments, humanized antibodies as described herein are covalently or non-covalently attached to a hydrophobic anchor that is stably rooted in the lipid bilayer of the liposome. Examples of hydrophobic anchors include, but are not limited to, phospholipids, such as phosphatidylethanolamine (PE), phosphatidylinositol (PI). To achieve covalent linkage between the antibody and the hydrophobic anchor, any biochemical strategy known in the art can be used, see, for example, J. Thomas August, ed., 1997, GeneTherapy:Advances in Pharmacology, Volume 40, Academic Press, San Diego, Calif., p. 399-435, which is incorporated herein by reference in its entirety. For example, functional groups on the antibody molecule can react with reactive groups on a hydrophobic anchor associated with the liposome, for example, amino groups of lysine side chains on the antibody can be coupled to liposome-associated N-glutaryl-phosphatidylethanolamine activated with a water-soluble carbodiimide; or thiol groups of reduced antibodies can be coupled to the liposome via a thiol-reactive anchor (e.g., 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, all of which are incorporated herein by reference. Immunoliposome formulations with anti-glycosylated LAG3 antibodies can be particularly effective as therapeutic agents because they deliver the active ingredient to the cytoplasm of target cells (i.e., cells containing the receptor to which the antibody binds). In some embodiments, the immunoliposomes can have an increased half-life in the blood, particularly in target cells, and can be internalized into the cytoplasm of target cells, thereby avoiding loss of the therapeutic agent or degradation through the endolysosomal pathway.

[0205] The immunoliposome compositions provided herein can have one or more vesicle-forming lipids, antibodies of the present invention or other molecules or fragments or derivatives thereof, and optional hydrophilic polymers. The vesicle-forming lipids can be lipids with two hydrocarbon chains (e.g., acyl chains and polar head groups). The example of vesicle-forming lipids includes phospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol, sphingomyelin and glycolipids, such as cerebrosides, gangliosides. Other lipids that can be used for the preparations provided herein are well known to those skilled in the art and are included in this specification. In some embodiments, the immunoliposome compositions further include hydrophilic polymers, such as polyethylene glycol and ganglioside GM1, which increase the serum half-life of the liposome. The method of conjugating hydrophilic polymers to liposomes is well known in the art and is included in this specification. Other exemplary immunoliposomes and methods for their preparation can be found in, for example, U.S. Patent Application Publication No. 2003 / 0044407; PCT International Publication No. WO 97 / 38731, Vingerhoeads et al., 1994, Immunomethods, 4:259-72; Maruyama, 2000, Biol. Pharm. Bull. 23(7):791-799; Abra et al., 2002, Journal of Liposome Research, 12(1&2):1-3; Park, 2002, Bioscience Reports, 22(2):267-281; ​​Bendas et al., 2001 BioDrugs, 14(4):215-224, J. Thomas August ed., 1997, Gene Therapy: Advances inPharmacology , Volume 40, Academic Press, San Diego, Calif., pp. 399-435; all of which are incorporated herein by reference in their entirety.

[0206] Also provided herein are methods for treating cancer patients by administering to the patient a unit dose of an anti-glycLAG3 antibody. Also provided herein are methods for treating cancer patients by administering to the patient a unit dose of a glycosylated LAG3 polypeptide. A unit dose refers to a physically discrete unit suitable as a unit dosage form for a subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in combination with the required diluent (i.e., carrier or vehicle).

[0207] The antibody, polypeptide or composition is administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The amount to be administered depends on the subject to be treated, the ability of the subject's system to utilize the active ingredient, and the desired degree of therapeutic effect. The precise amount of the active ingredient to be administered depends on the practitioner's judgment and is unique to each individual subject. However, suitable dosage ranges for systemic application are disclosed herein and depend on the route of administration. Suitable regimens for initial administration and booster administration are also contemplated, and typically include an initial administration followed by repeated administration at one or more hour intervals through subsequent injection or other administration. Exemplary multiple administrations are described herein and can be used to maintain sustained high serum and tissue levels of the polypeptide or antibody. Alternatively, continuous intravenous infusion is contemplated that is sufficient to maintain the concentration in the blood within the range specified for in vivo therapy.

[0208] A therapeutically effective amount is a predetermined amount calculated to achieve the desired effect. Generally, the dosage will vary with the patient's age, condition, sex, and degree of disease, and can be determined by a person skilled in the art. If any complications arise, the dosage can be adjusted by the individual physician.

[0209] In some embodiments, provided herein are antibodies, polypeptides or pharmaceutical compositions packaged in airtight sealed containers, such as ampoules or sachets. In one embodiment, provided herein are antibodies, polypeptides or pharmaceutical compositions in the form of dry sterile lyophilized powder or anhydrous concentrates provided in airtight sealed containers, and can be reconstituted to an appropriate concentration for administration to a subject, for example, with water or saline. In some embodiments, provided herein are antibodies, polypeptides or pharmaceutical compositions with a unit dose 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 provided in airtight sealed containers with dry sterile lyophilized powder. Provided herein are freeze-dried antibodies, polypeptides or pharmaceutical compositions should be stored at 2 to 8 ° C in their original containers and should be administered within 12 hours, preferably within 6 hours, within 5 hours, within 3 hours or within 1 hour after reconstruction. In an alternative embodiment, provided herein are antibodies, polypeptides or pharmaceutical compositions provided in liquid form in airtight sealed containers indicating the quantity and concentration of the antibody, polypeptide or pharmaceutical composition. In some embodiments, the liquid form of an antibody, polypeptide, or pharmaceutical composition provided herein is provided in a hermetically sealed container at at least 1 mg / ml, more preferably at least 2.5 mg / ml, at least 5 mg / ml, at least 8 mg / ml, at least 10 mg / ml, at least 15 mg / ml, at least 25 mg / ml, at least 50 mg / ml, at least 100 mg / ml, at least 150 mg / ml, at least 200 mg / ml.

[0210] The precise dose used in the formulation will also depend on the route of administration and the severity of the condition, and should be determined according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems. For anti-glycLAG3 antibodies or glycosylated LAG3 polypeptides, the dose administered to a patient will generally be between 0.01 mg / kg and 100 mg / kg of the patient's body weight. In some embodiments, the dosage administered to the patient is 0.01mg / kg to 20mg / kg, 0.01mg / kg to 10mg / kg, 0.01mg / kg to 5mg / kg, 0.01 to 2mg / kg, 0.01 to 1mg / kg, 0.01mg / kg to 0.75mg / kg, 0.01mg / kg to 0.5mg / kg, 0.01mg / kg to 0.25mg / kg, 0.01 to 0.15mg / kg, 0.01 to 0.10mg / kg, 0.01 to 0.05mg / kg or 0.01 to 0.025mg / kg patient body weight. The dosage administered to the patient can be 0.2mg / kg, 0.3mg / kg, 1mg / kg, 3mg / kg, 6mg / kg or 10mg / kg. It is estimated that the dosage as low as 0.01mg / kg shows obvious pharmacodynamic effect. It is estimated that the dosage level of 0.10-1mg / kg is the most suitable. It is also expected that higher dosage (for example, 1-30mg / kg) is active. Generally, due to the immune response to exogenous polypeptides, the half-life of human antibodies in the human body is longer than that of antibodies from other species. Therefore, it is possible to implement lower dosages of human antibodies and lower frequency administration. In addition, by modification (for example lipidation), by enhancing the uptake and tissue penetration of antibodies, it is possible to reduce the dosage and frequency of the antibody or polypeptide provided herein.

[0211] In another embodiment, the composition can be delivered in a controlled release or sustained release system. Any technology known to those skilled in the art can be used to produce a sustained release formulation with one or more antibodies, molecules or pharmaceutical compositions provided herein. See, for example, U.S. Patent No. 4,526,938; PCT Publication WO 91 / 05548; PCT Publication WO 96 / 20698; Ning et al., Radiotherapy & Oncology 39: 179-189 (1996), Song et al., PDA Journal of Pharmaceutical Science & Technology 50: 372-397 (1995); Cleek et al., Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24: 853-854 (1997); and Lam et al., Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24: 759-760 (1997); all of which are incorporated herein by reference in their entirety. In one embodiment, a pump can be used in a controlled release system (see Langer, supra; Sefton, 1987, CRC Crit. Ref Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery 88:507; and Saudek et al., 1989, N. Engl. J. Med. 321:574).In another embodiment, polymeric materials can be used to achieve controlled release of antibodies or polypeptides (see, e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Press., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J., Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 7 1:105); U.S. Patent No. 5,679,377; U.S. Patent No. 5,916,597; U.S. Patent No. 5,912,015; U.S. Patent No. 5,989,463; U.S. Patent No. 5,128,326; PCT Publication No. WO 99 / 15154; and PCT Publication No. WO 99 / 20253); all of which are incorporated herein by reference in their entirety.

[0212] Examples of polymers that can be used in sustained-release formulations include, but are not limited to, poly(hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate)), poly(methacrylic acid), polyglycolide (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. In yet another embodiment, a controlled-release system can be placed near the therapeutic target (e.g., the lungs), thereby requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). In another embodiment, a polymer composition that can be used as a controlled-release implant is used according to Dunn et al. (see U.S. Pat. No. 5,945,155, which is incorporated herein by reference in its entirety). Based on the therapeutic effect of in situ controlled release of the bioactive material from the polymer system, implantation can generally occur anywhere in the patient's body where treatment is required.

[0213] In another embodiment, non-polymer sustained delivery system is used, thus the non-polymer implant in the subject's body is used as a drug delivery system. After implantation in vivo, the organic solvent of the implant will dissipate, disperse or leach into the tissue fluid around from the composition, and the non-polymer material will gradually condense or precipitate to form a solid microporous matrix (referring to U.S. Patent number 5,888,533). Controlled release system has also been discussed in the commentary of Langer (1990, Science 249:1527-1533). Any technology well known to those skilled in the art can be used for producing the sustained release formulation comprising one or more therapeutic agents provided herein. See, e.g., U.S. Patent No. 4,526,938; International Publication Nos. WO 91 / 05548 and WO 96 / 20698; Ning et al., 1996, Radiotherapy & Oncology 39:179-189; Song et al., 1995, PDA Journal of Pharmaceutical Science & Technology 50:372-397; Cleek et al., 1997, Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854; and Lam et al., 1997, Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760; all of which are incorporated herein by reference in their entirety.

[0214] Also provided herein are embodiments in which the composition has a nucleic acid encoding an antibody or polypeptide as provided herein, wherein the nucleic acid can be administered in vivo to promote expression of the antibody or polypeptide it encodes by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, for example, by using a retroviral vector (see U.S. Patent No. 4,980,286), or by direct injection, or by using microparticle bombardment (e.g., a gene gun; Biolistics, Dupont), or by coating it with lipids or cell surface receptors or transfection agents, or by administering it in conjunction with a homeobox-like peptide that is 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 the host cell DNA by homologous recombination for expression.

[0215] Treatment of a subject with the antibody, polypeptide or pharmaceutical composition provided herein of a therapeutically effective amount can include a single treatment or a series of treatments. It is contemplated that the antibody, polypeptide or pharmaceutical composition provided herein can be systemically or locally administered to treat a disease, such as, in a cancer patient suffering from locally advanced or metastatic cancer, suppressing tumor cell growth or killing cancer cells. They can be administered intravenously, intrathecally and / or intraperitoneally. They can be administered alone or in combination with antiproliferative drugs. In one embodiment, they are administered before surgery or other procedures to reduce the patient's cancer load. Alternatively, they can be administered postoperatively to ensure that any remaining cancer (for example, cancer that surgery fails to eliminate) will not survive. In some embodiments, they can be administered after the primary cancer subsides to prevent metastasis.

[0216] Combination therapy

[0217] In certain embodiments, the compositions and methods of the present invention involve administering a glycosylated LAG3 polypeptide or an antibody that selectively binds to glycosylated LAG3 in combination with a second or additional therapy. Such therapies can be used to treat any disease associated with LAG3 or glycosylated LAG3. For example, the disease can be cancer, and the second therapy can be an anti-cancer or anti-hyperproliferative therapy.

[0218] Methods and compositions, including combined therapies, enhance the therapeutic or protective effect, and / or increase the therapeutic effect of another anti-cancer or anti-hyperproliferative therapy. Treatment and prevention methods and compositions can be provided in a combined amount that effectively achieves the desired effect, such as killing cancer cells and / or inhibiting cell hyperproliferation. The method 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 raises the immune system without a direct cytotoxic effect. Tissues, tumors or cells can be exposed to one or more compositions or pharmacological preparations comprising one or more agents (such as antibodies or anticancer agents), or by exposing tissues, tumors and / or cells to two or more different compositions or preparations, wherein one composition provides 1) polypeptides or antibodies, 2) anticancer agents, or 3) both polypeptides or antibodies and anticancer agents. In addition, it is contemplated that such combined therapies can be used in combination with chemotherapy, radiotherapy, surgical therapy or immunotherapy.

[0219] As applied to cells, the terms "contacting" and "exposing" are used herein to describe the process of delivering a therapeutic polypeptide or antibody and a chemotherapeutic or radiotherapeutic agent to or placing them in direct juxtaposition with a target cell. For example, to achieve cell killing, the two agents are delivered to the cell in a combined amount effective to kill the cell or prevent it from dividing.

[0220] The anti-glycLAG3 antibody or glycosylated LAG3 polypeptide can be administered before, during, after, or in various combinations of the second or additional anticancer treatment. The interval between administrations can vary from simultaneous to a few minutes to several days to several weeks. In embodiments where the antibody or polypeptide is provided to the patient separately from the anticancer agent, it is generally ensured that a long period of time does not elapse between each delivery time so that the two compounds can still exert a beneficial combined effect on the patient. In such cases, it is expected that the anti-glycLAG3 antibody or glycosylated LAG3 polypeptide and the second therapy can be provided to the patient within about 12 to 24 or 72 hours of each other, and more specifically within about 6-12 hours of each other. In some cases, the treatment period can be significantly extended, with a few days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks) between each administration.

[0221] In specific embodiments, anti-glycLAG3 antibodies are administered in combination with one or more other anti-LAG3 antibodies, including relatlimab (BMS-986016), LAG525, REGN3767, MGD013, FS118, TSR-033, or IMP321, to patients for the treatment of cancer. In other embodiments, anti-glycLAG3 antibodies are administered in combination with one or more anti-PD-1 antibodies, and in a specific embodiment, the anti-PD-1 antibody is durvalumab, nivolumab, pebrolizumab, avelumab, atezolizumab, or cemiplimab, administered to patients for the treatment of cancer. In other embodiments, the anti-glycLAG3 antibody is administered with an agent that inhibits LAG3, CTLA-4, PD-L1, or PD-1 activity, such as an immunoadhesin having 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.

[0222] In certain embodiments, the anti-glycLAG3 antibody is administered in combination with an antibody that preferentially binds to glycosylated PD-1 over unglycosylated PD-1. In particular, the anti-glycLAG3 antibody can be administered in combination with a chimeric or humanized form of the anti-PD-1 antibody STM418 or STM432, which preferentially binds to glycosylated PD-1 over unglycosylated PD-1, and the amino acid sequences (and encoding nucleotide sequences) of the heavy and light chain variable domains are disclosed in PCT publication WO 2017 / 096026, entitled “Antibodies Specific To Glycosylated PD-1 And Methods Of Use It Of,” published on June 8, 2017, which is incorporated herein by reference.

[0223] In certain embodiments, the anti-glycLAG3 antibody is administered in combination with an antibody that preferentially binds to glycosylated PD-L1 over unglycosylated PD-L1. In particular, the anti-glycLAG3 antibody can be administered in combination with a chimeric or humanized form of the anti-PD-L1 antibody STM004 or STM115, which preferentially binds to glycosylated PD-L1 over unglycosylated PD-L1, and the amino acid sequences (and encoding nucleotide sequences) of the heavy and light chain variable domains are disclosed in PCT publication WO 2016 / 160792, entitled “Antibodies Specific To Glycosylated PD-L1 And Methods Of Use It Of,” published on October 6, 2016, which is incorporated herein by reference. The anti-glyc-LAG3 antibodies are also administered in combination with chimeric or humanized forms of the anti-PD-L1 antibodies STM073 and SMT108, which preferentially bind to glycosylated PD-L1 compared to unglycosylated PD-L1, and the amino acid sequences (and encoding nucleotide sequences) of the heavy and light chain variable domains are disclosed in U.S. Provisional Application No. 62 / 314,652, filed on March 29, 2016, entitled “Dual Function Antibodies Specific To Glycosylated PD-L1 And Methods Of Use It Of,” which is incorporated herein by reference.

[0224] In certain embodiments, a course of treatment may last from 1 to 90 days or longer (this range includes the days in between). It is contemplated that one agent may be administered on any day from day 1 to day 90 (this range includes the days in between) or any combination thereof, and another agent may be administered on any day from day 1 to day 90 (this range includes the days in between) or any combination thereof. An agent may be administered to a patient once or multiple times within a single day (24 hours). In addition, after a course of treatment, it is contemplated that there is a period of time during which anticancer therapy is not administered. This period may last from 1 to 7 days, and / or from 1 to 5 weeks, and / or from 1 to 12 months or longer (this range includes the days in between), depending on the patient's condition, such as their prognosis, strength, health, etc. Treatment cycles may be repeated as needed.

[0225] Various combinations can be used. Some examples of treatments with an anti-glycLAG3 antibody or glycosylated LAG3 polypeptide as "A" and a second anticancer therapy as "B" are listed below: A / B / AB / A / BB / B / AA / B / BB / A / AA / B / B / BB / A / B / BB / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / AB / A / B / AB / A / A / B / AB / A / A / BA / A / A / BB / A / A / A / AA / B / A / AA / A / A / B / A.

[0226] Any antibody, polypeptide or pharmaceutical composition provided herein is administered in combination with a second therapy to the patient and the general protocol for administering such a second therapy will be followed, taking into account the toxicity, if any, of the second therapy. Therefore, in some embodiments, there is a step of monitoring the toxicity attributable to the combination therapy.

[0227] chemotherapy

[0228] According to an embodiment of the present invention, a variety of chemotherapeutics can be used as a second therapy. Chemotherapeutics can be compounds or compositions used in the treatment of cancer. These agents or medicines can be classified according to their intracellular activity patterns, for example, whether and at what stage they affect the cell cycle. Alternatively, agents can be characterized based on their ability to directly cross-link DNA, be embedded in DNA, or by affecting nucleic acid synthesis to induce chromosomes and mitotic aberrations.

[0229] Examples of chemotherapeutic agents include: alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquinone, meturedopa, and uredopa; ethyleneimines and methylmelamines including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; annonaceous lactones (particularly bratacin and bratacinone); camptothecins (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogues adolesin, kazelesin and biselesin); Nostoc cyclic peptides (particularly Nostoc cyclic peptide 1 and Nostoc cyclic peptide 8); dolastatin; duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1); sclerocortin; sphingomyelin; sarcodictyin; spongestatin; nitrogen mustards such as chlorambucil, naphthiazolin, clofosamide, estramustine, ifosfamide, mechlorethamine, chlorambucil, nitrogen mustard hydrochloride, melphalan, nitroceramide, phenylephrine, prednimustine, trofosfamide, and uracil mustard; nitrosureas such as carmustine, chlorozolin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as enediyne antibiotics (e.g., kacilmustine, chlorambucil, chlorambucil, estramustine, ifosfamide, mechlorethamine, nitrogen mustard hydrochloride, melphalan, nitroceramide, phenylephrine, prednimustine, trofosfamide, and uracil mustard; nitrosureas such as carmustine, chlorozolin, fotemustine, lomustine, nimustine, and ranimustine; dynemycins, including dynemycin A; bisphosphonates such as clodronate; esperamicins; and the neocarcin chromophores and related chromoprotein enediyne antibiotic chromophores, aclacinomycins, actinomycins, authrarnycins, azaserine, bleomycins, actinomycin C, carabicin, carminomycins, chromomycins, dactinomycins, daunorubicin, detoxibacin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyano-doxorubicin), daunorubicin, ... morpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, mexilomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycin, peplomycin, potfiromycin, puromycin, triferon-doxorubicin, rhodorubicin, streptozocin, streptozocin, tuberculin, ubenimex, zinstatin and daurubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as leucovorin, pteropterin and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiabendine and thioguanine;Pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as captestosterone, drotosterol propionate, cyclothiocarb, melastane, and testolactone; anti-adrenal drugs such as mitotane and trilostane; folic acid supplements such as frolinic acid; aceglucuronolide; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; colcemid; diazocine; elformithine; elliptonium acetate; epothilones; etoglucosamine; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansines such as maytansine and Ansamitocin; mitoxantrone; mitoxantrone; mopidanmol; nitraerine; pentostatin; methambucil; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; sizolan; spirogermanium; tinuzzoic acid; triazoline; 2,2',2"-trichlorotriethylamine; trichothecenes (particularly T-2 toxin, verracurin A, and bacilli) oxaliplatin (e.g., paclitaxel and gemcitabine); 6-thioguanine; mercaptopurine; platinum coordination complexes such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; cyclophosphamide; taxanes, such as paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone Anthraquinone; vincristine; vinorelbine; noxolin; teniposide; edatrexate; daunorubicin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, plicamycin, gemcitabine, navelbine, farnesyl-protein transferase inhibitors, trans-platinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

[0230] Radiation therapy

[0231] Another conventional anticancer therapy that can be used in combination with the methods and compositions described herein is radiotherapy or radiation therapy. Radiotherapy includes the use of gamma rays, X-rays and / or directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors, such as microwaves, proton beam irradiation (U.S. Patent Nos. 5,760,395 and 4,870,287; all of which are incorporated herein by reference in their entirety) and ultraviolet (UV) irradiation are also contemplated. Most likely, all of these factors will cause extensive damage to DNA, DNA precursors, replication and repair of DNA, and assembly and maintenance of chromosomes.

[0232] The tumor microenvironment is inherently suppressive due to the presence of myeloid-derived suppressor cells and regulatory T cells, which infiltrate the tumor and suppress the immune response. Furthermore, the expression of certain inhibitory molecules on T cells and antigen-presenting cells (APCs) can limit an effective immune response. Radiation mediates its antitumor effects by inducing tumor cell apoptosis, senescence, and autophagy, and in some cases, can stimulate a more effective immune response.

[0233] The abscopal effect is a physiological process whereby targeted radiation of a primary tumor induces an anti-tumor response at distant sites that are not within the radiation field. The mechanisms responsible for the abscopal effect are thought to be immune-mediated, involving 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 therapy, agents that can trigger the abscopal effect are particularly advantageous in treating metastatic tumors, which are often more difficult to treat once they have spread to secondary sites in the body.

[0234] The anti-glycLAG3 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, simultaneously with, or after radiation therapy to achieve a synergistic abscopal effect.

[0235] In some embodiments, a therapeutically effective amount of an antibody, polypeptide, or pharmaceutical composition described herein is administered that effectively sensitizes a tumor in a host to radiation. The radiation can be ionizing radiation, particularly gamma radiation. In some embodiments, gamma radiation is emitted by a linear accelerator or a radionuclide. The radiation of the tumor with a radionuclide can be external or internal.

[0236] In some embodiments, administration of an antibody, polypeptide, or pharmaceutical composition described herein begins up to one month, particularly up to 10 days or one week, before irradiation of the tumor. In addition, irradiation of the tumor is segmented, with administration of an antibody, polypeptide, or pharmaceutical composition described herein maintained in the interval between the first and last radiation sessions.

[0237] Radiation can also be X-ray radiation. X-ray dosage ranges range from daily doses of 50 to 200 roentgens for extended periods of time (3 to 4 weeks) to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, depending on the half-life of the isotope, the strength and type of radiation emitted, and uptake by the neoplastic cells.

[0238] Immunotherapy

[0239] Those skilled in the art will appreciate that immunotherapy can be combined or used in conjunction with the methods of the embodiments. In the context of cancer treatment, immunotherapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab This is one such example. Checkpoint inhibitors such as ipilimumab, pembrolizumab, nivolumab and atezolizumab are other examples. Immune effectors can be, for example, antibodies specific for some markers on the surface of tumor cells. A single antibody can serve as an effector of therapy, or it can recruit other cells to actually affect cell killing. Antibodies can also be conjugated to agents or toxins (e.g., chemotherapeutic agents, radionuclides, ricin A chain, cholera toxin, pertussis toxin) and used only as targeting agents. Alternatively, the effector can be a lymphocyte carrying a surface molecule that interacts directly or indirectly with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0240] In one aspect of immunotherapy, tumor cells carry some markers that are easy to target, that is, they are not present on most other cells. There are many tumor markers and any of these can be suitable for targeting in the context of this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B and p155. An alternative aspect of immunotherapy is to combine anticancer effects with immunostimulatory effects. There are also immunostimulatory molecules, including: cytokines, such as IL-2, IL-4, IL-12, GM-CSF, γ-IFN, chemokines, such as MIP-1, MCP-1, IL-8 and growth factors, such as FLT3 ligand.

[0241] Examples of immunotherapies currently under investigation or use are immune adjuvants, 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 therapies, 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. Patent 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. Patent No. 5,824,311); all of which are incorporated herein by reference in their entirety. It is contemplated that one or more anti-cancer therapies can be used with the therapies described herein that include the use of anti-glycLAG3 antibodies or glycosylated LAG3 polypeptides.

[0242] Operation

[0243] Approximately 60% of cancer patients will undergo some type of surgery, including preventive, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection, in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed, and may be used in combination with other therapies, such as the treatment of the present embodiment, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to the physical removal of at least part of the tumor. In addition to tumor resection, surgical treatments also include laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs surgery).

[0244] After removal of some or all of the cancer cells, tissue, or tumor, a cavity may form in the body. Treatment can be completed by perfusing, directly injecting, or topically applying additional anticancer 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. These treatments can also be given at different dosages.

[0245] Other medicines

[0246] It is contemplated that other agents may be used in combination with certain aspects of this embodiment to improve the therapeutic efficacy of the treatment. These additional agents include agents that affect the upregulation of cell surface receptors and the connection of GAPs, cell growth inhibitors and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of hyperproliferative cells to apoptosis inducers, or other biological agents. Increasing intercellular signaling by increasing the number of GAP connections can increase the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cell growth inhibitors or differentiation agents may be used in combination with certain aspects of this embodiment to improve the anti-hyperproliferative efficacy of the treatment. It is contemplated that cell adhesion inhibitors may be used to improve the efficacy of the present embodiment. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. Further contemplation of other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as antibody c225, may be used in combination with certain aspects of the embodiments of the present invention to improve therapeutic efficacy.

[0247] Kits and diagnostic agents

[0248] In various aspects, provided herein are kits comprising therapeutic agents and / or other therapeutic agents and delivery agents. In some embodiments, kits for preparing and / or administering the therapies provided herein are contemplated. The kits can include one or more sealed vials containing any of the pharmaceutical compositions provided herein. The kits can include, for example, at least one anti-glycLAG3 antibody or glycosylated LAG3 polypeptide, and reagents for preparing, formulating, and / or administering the components provided herein or performing one or more steps of the methods provided herein.

[0249] In some embodiments, the kit can include an anti-glycLAG3 antibody and at least one auxiliary reagent. In some embodiments, the kit can include a glycosylated LAG3 polypeptide and at least one auxiliary reagent.

[0250] In some embodiments, the kit further comprises a second anticancer agent. The second anticancer agent can be a chemotherapeutic agent, an immunotherapeutic agent, a hormonal therapeutic agent, or a cytokine.

[0251] In some embodiments, the kit may further comprise suitable container means, which is a container that does not react with the components of the kit, such as an eppendorf tube, an assay plate, a syringe, a bottle or a test tube. The container may be made of a sterilizable material (such as plastic or glass).

[0252] The kit can also include an instruction chart outlining the procedural steps of the methods described herein, and will follow substantially the same procedures as described herein or known to those of ordinary skill. The instruction information can be in a computer-readable medium comprising machine-readable instructions that, when executed using a computer, results in a real or virtual program showing the delivery of a pharmaceutically effective amount of an antibody or polypeptide provided herein. The kit can also include a public notice in the form of a governmental agency regulation governing the manufacture, use, or sale of a drug or biological product, reflecting the approval of the agency for manufacture, use, or sale with respect to human administration.

[0253] Example

[0254] It should be understood that modifications that do not significantly change the nature and spirit of the various embodiments described herein are also contemplated.Accordingly, the following examples are intended to be illustrative and not limiting in any way.

[0255] Materials and methods

[0256] K by Octet D Determine and bin. For high-throughput K D For screening, the antibody ligand was loaded onto the sensor at 20 nM solution. A baseline was established in PBS containing 1 mg / ml bovine serum albumin (assay buffer), and the association step was performed by immersing the sensor in a single concentration of analyte in assay buffer. Dissociation was performed and monitored in fresh assay buffer. All experiments were performed with sensor shaking at 1,000 rpm. Data were fitted to a 1:1 binding model using ForteBio's data analysis software to extract association and dissociation rates. The ratio k was used. d / k a Calculate K D. In a typical epitope binning assay, the antigen LAG3-His (10 nM) was preincubated with the secondary antibody (10 nM) for 1 hour at room temperature. A control antibody (20 nM) was loaded onto the AMC sensor (ForteBio) and the remaining Fc binding sites on the sensor were blocked with a whole mouse IgG antibody (Jackson ImmunoResearch). The sensor was exposed to the preincubated antigen-secondary antibody mixture. The raw data was processed using ForteBio's Data Analysis Software 7.0, and the competitive binding of the antibody pairs was evaluated. Additional binding of the secondary antibody indicates an unoccupied epitope (non-competitor), while no binding indicates epitope blocking (competitor).

[0257] Glycosylation analysis of LAG3. To confirm the glycosylation of LAG3 protein, cell lysates were treated with the enzymes PNGase F, Endo H, and β-glycosidase (New England BioLabs, Ipswich, MA, USA) as described by the manufacturer.

[0258] Example 1: LAG3 is heavily glycosylated.

[0259] Based on the UniProt database (http: / / www.uniprot.org), human LAG3 is glycosylated at N188, N250, N256, and N343. To confirm the glycosylation of the LAG3 protein, we treated the LAG3 protein with PNGase F (New England BioLabs) as described by the manufacturer. When the LAG3 protein is expressed in mammalian cells, the size of the protein (57.5 kDa) is larger than the calculated molecular weight (46.41 kDa). Treatment with PNGase F removes the oligosaccharide moiety from the protein, resulting in a reduction in the protein size to the expected 46.41 kDa, as shown in Figure 2. Figure 1 This result confirmed the glycosylation of LAG3 protein ( Figure 1 ).

[0260] Example 2: Production of anti-LAG3 antibodies.

[0261] We obtained LAG3-His protein purified from 293F cells overexpressing heavily glycosylated LAG3 from Novoprotein. Hybridomas producing monoclonal antibodies against glycosylated human LAG3 were generated according to standard protocols by fusing SP2 / 0 murine myeloma cells with splenocytes isolated from Balb / C (n=4) and NZW mice immunized with human LAG3 (n=4; Antibody Solutions, Inc., Sunnyvale, CA, USA). Prior to fusion, sera from immunized mice were validated for binding to the LAG3 immunogen using FACS analysis. Over 3,000 hybridomas producing monoclonal antibodies (mAbs) were generated. The antibody-producing hybridomas were further tested for specificity. Of these, 22 candidate mAb-producing hybridomas were selected by FACS using 293T cells expressing LAG3 WT or 4NQ (deglycosylated form), grown in DCGF medium (Antibody Solutions), and the mAb-containing supernatant was concentrated and purified. Purified mAbs were tested for their ability to bind to glycosylated LAG3 but not to deglycosylated LAG3 using dot blot analysis. The results of this assay showed that of the 22 mAbs tested, 3 mAbs specifically bound to glycosylated LAG3 (Figure 2).

[0262] To facilitate purification of each antibody from hybridoma supernatants, the isotype of each antibody was determined by ELISA. Antibodies against each antibody isotype were used according to the manufacturer's instructions (Sigma-Aldrich, Cat. # ISO2). The isotype 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 and protein A was used for IgG2a and IgG2b (Table 5).

[0263] Table 5. Isotypes of anti-LAG3 antibodies.

[0264] mAbs Isotype mAbs Isotype STC1301 G1 STC1312 G2b STC1302 G2a / G3 STC1313 G3 / G2a STC1303 G2a / G2b STC1314 G2b STC1304 G2a STC1315 G1 STC1305 G1 / A STC1316 G2a STC1306 G2a STC1317 G1 / G2a STC1307 G2a / A STC1318 G2a STC1308 A STC1319 G1 STC1309 M STC1320 G2b / G1 STC1310 G2b STC1321 G2b STC1311 G2a STC1322 M

[0265] For high-throughput K DFor screening, the antibody ligand was loaded onto the Octet sensor (Anti-Mouse IgG Fc Capture (AMC) Biosensors) using a 20 nM solution. A baseline was established in PBS containing 1 mg / mL bovine serum albumin (assay buffer), and the association step was performed by immersing the sensor in a single concentration of analyte in assay buffer. Dissociation was performed and monitored in fresh assay buffer. All experiments were performed with the sensor vibrating at 1,000 revolutions per minute. ForteBio (Menlo Park, CA, USA) data analysis software was used to fit the data to a 1:1 binding model to extract association and dissociation rates. K was calculated using the ratio kd:ka. D The data are summarized in Figure 3 and Table 6. Among them, STC1317 showed the strongest binding affinity with a KD of 2.85x 10 9 M.

[0266] Table 6. Kinetic parameters of anti-LAG3 antibodies determined by Octet.

[0267] Sample ID answer KD(M) kon(1 / Ms) kdis(1 / s) STC1301 -0.0048 3.42E+13 1.86E-03 6.37E+10 STC1302 -0.0024 1.82E+04 6.10E+01 1.11E+06 STC1303 -0.0001 2.73E+13 7.45E-03 2.04E+11 STC1304 -0.0043 <1.0E-12 9.31E-04 <1.0E-07 STC1305 -0.0094 <1.0E-12 9.31E-04 <1.0E-07 STC1306 0.0025 <1.0E-12 4.59E+04 <1.0E-07 STC1307 -0.0032 <1.0E-12 4.66E-04 <1.0E-07 STC1308 0.0226 <1.0E-12 9.35E+05 <1.0E-07 STC1309 0.0012 8.99E-06 6.43E+03 5.78E-02 STC1310 -0.0025 1.31E+09 6.90E+04 9.01E+13 STC1311 -0.0025 2.63E+11 1.22E+02 3.21E+13 STC1312 -0.0046 1.58E+20 1.04E+03 1.64E+23 STC1314 -0.004 5.16E+07 4.48E+03 2.31E+11 STC1315 0.0051 <1.0E-12 1.72E+100 1.67E-02 STC1316 0.0149 2.36E-11 6.15E+08 1.45E-02 STC1317 0.3111 2.85E-09 2.61E+05 7.42E-04 STC1318 0.0012 2.16E-07 1.60E+08 3.44E+01 STC1319 0.0129 1.74E-09 5.59E+05 9.72E-04 STC1320 0.0017 1.44E-06 4.02E+04 5.77E-02 STC1321 -0.0033 1.83E-12 7.45E-03 <1.0E-07 STC1322 -0.0036 <1.0E-12 1.86E-03 <1.0E-07

[0268] Example 3. Antibody Sequencing

[0269] Hybridoma cell pellets were frozen at -80 ° C and total RNA was isolated using RNeasy Plus Mini Kit (Qiagen, Hilden, DE) and quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). According to the manufacturer's instructions (Clontech, Mountain View, CA, USA), cDNA synthesis and rapid amplification of cDNA ends (RACE) were performed using the SMARTer RACE 5' / 3' kit. PCR primers were purchased from Novagen Mouse Ig-Primer set (Merck KGaA, Darmstadt, DE, Germany) for Ig-specific PCR amplification. 0.5 μg of heavy chain primer set (AF), light (kappa) chain primer set (AG) and template RNA were used per 25 μL reaction volume. After gel separation, the PCR product was connected to the pCR 2.1 vector, transformed into competent cells, and selected on 100 μg / mL ampicillin and 50 μL X-gal / IPTG agar plates. After 24 hours, white colonies were picked. After cultivation, plasmid DNA was isolated and sequenced using M13 forward and reverse primers. The sequences and CDRs of the STC1317 heavy chain and kappa light chain variable regions are listed in Tables 3 and 4, respectively.

[0270] Example 4. Determination of KD by Biocore analysis

[0271] 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 expressed in HBS-EP + Buffer was flowed through the chip at 2 μg / mL. Next, six concentrations of LAG3, each diluted 2-fold, were passed through the chip. The sensing data were analyzed using Biacore X100 Evaluation Software version 2.0.1, with a 1:1 binding kinetics. The KD values ​​of STC1317 for Fc-tagged and His-tagged LAG3 proteins were found to be 0.021 nM and 0.406 nM, respectively. This indicates that STC1317 has a very strong binding affinity for LAG3 ( Figure 4 and Table 7 ).

[0272] Table 7. BIACORE assay of anti-glyc-LAG3 antibody STC1317 binding to LAG3-His.

[0273] antigen answer KD(M) kon(1 / Ms) kdis(1 / s) Measured by the following LAG3-Fc fusion 0.3796 2.10E-11 3.45E+05 7.25E-06 Octet LGG3-His tagging 0.3111 2.85E-09 2.61E+05 7.42E-04 Octet LAG3-His tag - 4.06E-10 9.43E+05 3.83E-04 Biacore

[0274] Example 5. Effect of anti-GlycLAG3 antibodies on T cell proliferation.

[0275] To evaluate the efficacy of STC1317 in vitro, mixed lymphocyte reactions (MLRs) were performed using dendritic cells (DCs, induced by PBMCs from 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 pan-DC enrichment kit (Miltenyi Biotech #130-100-777) according to the manufacturer's recommendations and used to stimulate allogeneic memory or naive CD4 + CD4 T cells were also enriched from PBMCs of another allogeneic donor (Immunospot #CTL-CP1) using CD4 microbeads (Miltenyi Biotech #130-045-101). + T cells. DC (1×10 4 DC / well) and 1×10 5T cells / well were co-cultured in 96-well flat-bottom plates (Nunc) in the presence of STC1317. After 5 days of culture, the concentrations of IFN-γ and IL-2 in the culture supernatant were determined by cytokine ELISA kits (BioLegend) according to the manufacturer's instructions. As shown in Figure 5, the secretion of T cell proliferation markers IFN-γ and IL-2 was significantly increased in the presence of STC1317.

[0276] Example 6: Antibody Humanization—Framework Regions

[0277] As mentioned above, for some purposes, including, for example, for the in vivo treatment of human diseases, it is preferred to use humanized derivatives of mouse monoclonal antibodies. In order to form this humanized antibody, first the framework sequence (" parent " sequence) of mouse monoclonal antibodies is compared with the framework sequence of one group of " acceptor " human antibodies, to identify the difference in the framework sequence. Humanization is achieved by replacing the mismatched framework residues between the parent and the acceptor. The displacement at potential important positions can be, for example, at the Vernier district, VH / VL interchain interface or CDR standard category determination position displacement analysis expected back mutation (see Foote, J. et al., J.Molec.Biol.224:487-499 (1992)).

[0278] The Conserved Domain Database (COD) (Marchler-Bauer et al. (2011) Nucleic Acids Res. 39: D225-D229) can be used to determine the domain content of each amino acid chain and the approximate boundaries of each domain. Variable domain boundaries and CDR boundaries can be accurately determined according to several commonly used definitions (Kabat, E.A. 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. Molec. Biol. 309(3): 657-670 (2001)).

[0279] Use MAFFT (Katoh, K. et al., Nucleic Acids Res.30:3059-3066 (2002)) to generate the multiple alignment of parental sequence and mouse and people's germline sequence, and the entries in each alignment are sorted according to the sequence identity with the parental sequence.By clustering with 100% sequence identity and excluding redundant entries, the reference set is simplified to one group of unique sequence.

[0280] The optimal receptor framework selection is based on the entire parent antibody sequence identity with the receptor in the framework of the two chains; however, the positions constituting the VH / VL interchain interface are of particular interest. In addition, the CDR loop lengths and CDR positions responsible for the discrete set of canonical structures defined for the 5 CDRs (Chothia, C. et al., J. Mol. Biol. 196:901-917 (1987); Martin, AC et al., J. Molec. Biol 263:800-815 (1996); Al-Laziniki, B. et al., J. Molec. Biol. 273:927-948 (1997)) are compared with the germline to determine which germline frameworks have the same interface residues and are known to support similar CDR loop conformations.

[0281] Based on the sequence alignment of the parent antibody with the human germline, the closest matching entry was determined. The preferred human germline was selected based on the following ranking criteria: (1) sequence identity throughout the framework; (2) identical or compatible interchain interface residues; (3) support for loops with the canonical conformation of the parent CDRs; (4) the combination of heavy and light germlines found in the expressed antibody; and (5) the presence of N-glycosylation sites that must be removed.

[0282] Structural models of the Fv region of the humanized antibody were generated. Candidate structural template fragments of the FR and CDR, as well as the complete Fv, were selected based on their sequence identity to the target and qualitative crystallographic measurements of the template structure (e.g., resolution in angstroms). denoted) scoring, ranking, and selection from the antibody database.

[0283] In order to structurally compare CDR and FR templates, 5 residues on either side of CDR are included in the CDR template. The alignment of the fragments is generated based on the overlapping segments and the generated structural sequence alignment. Template fragments are processed by MODELLER (Sall, A. et al.; J.Molec.Biol.234:779-815 (1993)) and compared together. This scheme creates conformational constraints derived from a set of aligned structural templates. By conjugate gradient and simulated annealing optimization procedures, a set of structures that meet the constraints is created. The model structure is selected from this set based on energy scoring, and the energy scoring is derived from the scoring of the protein structure and the satisfaction of the conformational constraints. The model is checked and the side chains in different positions between the target and the template are optimized and energy is minimized using a side chain optimization algorithm. A set of visualization and computational tools is used to evaluate CDR conformational variability, local stacking, and surface analysis to select one or more preferred models.

[0284] Build the structural model of parental antibody and check defects, such as stress, bond angle or dihedral angle in bad atomic stacking, bond length.These defects may indicate potential problems of antibody structure stability.Modeling scheme is intended to minimize this type of defect.The initial structural model of humanized Fv comprises all safe displacements (i.e., displacement that should not affect binding affinity or stability) and cautious displacement (i.e., carrying out position displacement, but the position may be very important to binding affinity).Do not change the displacement at the position related to the risk that is considered to reduce binding affinity or stability reduction.Template search and selection are performed separately with parental template search, so as to create good independent model, rather than the variant model of the close match of parent.Along with the assessment of carrying out potential displacement, model can be updated to reflect the influence of preferred displacement and back mutation.

[0285] Example 7: Antibody humanization—constant region:

[0286] The variable region (VH) of the STC1317 heavy chain and the variable region (VL) of its kappa light chain 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 chain 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 on HPLC by protein A affinity column.

[0287] Throughout this application, various publications are cited. The disclosures of these publications are hereby incorporated by reference in their entirety into this application for the purpose of more fully describing the prior art to which this disclosure relates. Although examples of certain specific embodiments are provided herein, it will be apparent to those skilled in the art that various changes and modifications may be made. Such modifications are also intended to fall within the scope of the appended claims. Sequence Listing <110> STCUBE & CO. Yoo, Stephen S. <120> Antibodies specific for glycosylated LAG3 and methods of use thereof <130> 24258.0014P1 <150> 62 / 912,867 <151> 2019-10-09 <160> twenty one <170> PatentIn version 3.5 <210> 1 <211> 525 <212> PRT <213> Homo sapiens <400> 1 Met Trp Glu Ala Gln Phe Leu Gly Leu Leu Phe Leu Gln Pro Leu Trp 1 5 10 15 Val Ala Pro Val Lys Pro Leu Gln Pro Gly Ala Glu Val Pro Val Val 20 25 30 Trp Ala Gln Glu Gly Ala Pro Ala Gln Leu Pro Cys Ser Pro Thr Ile 35 40 45 Pro Leu Gln Asp Leu Ser Leu Leu Arg Arg Ala Gly Val Thr Trp Gln 50 55 60 His Gln Pro Asp Ser Gly Pro Pro Ala Ala Ala Pro Gly His Pro Leu 65 70 75 80 Ala Pro Gly Pro His Pro Ala Ala Pro Ser Ser Trp Gly Pro Arg Pro 85 90 95 Arg Arg Tyr Thr Val Leu Ser Val Gly Pro Gly Gly Leu Arg Ser Gly 100 105 110 Arg Leu Pro Leu Gln Pro Arg Val Gln Leu Asp Glu Arg Gly Arg Gln 115 120 125 Arg Gly Asp Phe Ser Leu Trp Leu Arg Pro Ala Arg Arg Ala Asp Ala 130 135 140 Gly Glu Tyr Arg Ala Ala Val His Leu Arg Asp Arg Ala Leu Ser Cys 145 150 155 160 Arg Leu Arg Leu Arg Leu Gly Gln Ala Ser Met Thr Ala Ser Pro Pro 165 170 175 Gly Ser Leu Arg Ala Ser Asp Trp Val Ile Leu Asn Cys Ser Phe Ser 180 185 190 Arg Pro Asp Arg Pro Ala Ser Val His Trp Phe Arg Asn Arg Gly Gln 195 200 205 Gly Arg Val Pro Val Arg Glu Ser Pro His His His Leu Ala Glu Ser 210 215 220 Phe Leu Phe Leu Pro Gln Val Ser Pro Met Asp Ser Gly Pro Trp Gly 225 230 235 240 Cys Ile Leu Thr Tyr Arg Asp Gly Phe Asn Val Ser Ile Met Tyr Asn 245 250 255 Leu Thr Val Leu Gly Leu Glu Pro Pro Thr Pro Leu Thr Val Tyr Ala 260 265 270 Gly Ala Gly Ser Arg Val Gly Leu Pro Cys Arg Leu Pro Ala Gly Val 275 280 285 Gly Thr Arg Ser Phe Leu Thr Ala Lys Trp Thr Pro Pro Gly Gly Gly 290 295 300 Pro Asp Leu Leu Val Thr Gly Asp Asn Gly Asp Phe Thr Leu Arg Leu 305 310 315 320 Glu Asp Val Ser Gln Ala Gln Ala Gly Thr Tyr Thr Cys His Ile His 325 330 335 Leu Gln Glu Gln Gln Leu Asn Ala Thr Val Thr Leu Ala Ile Ile Thr 340 345 350 Val Thr Pro Lys Ser Phe Gly Ser Pro Gly Ser Leu Gly Lys Leu Leu 355 360 365 Cys Glu Val Thr Pro Val Ser Gly Gln Glu Arg Phe Val Trp Ser Ser 370 375 380 Leu Asp Thr Pro Ser Gln Arg Ser Phe Ser Gly Pro Trp Leu Glu Ala 385 390 395 400 Gln Glu Ala Gln Leu Leu Ser Gln Pro Trp Gln Cys Gln Leu Tyr Gln 405 410 415 Gly Glu Arg Leu Leu Gly Ala Ala Val Tyr Phe Thr Glu Leu Ser Ser 420 425 430 Pro Gly Ala Gln Arg Ser Gly Arg Ala Pro Gly Ala Leu Pro Ala Gly 435 440 445 His Leu Leu Leu Phe Leu Ile Leu Gly Val Leu Ser Leu Leu Leu Leu 450 455 460 Val Thr Gly Ala Phe Gly Phe His Leu Trp Arg Arg Gln Trp Arg Pro 465 470 475 480 Arg Arg Phe Ser Ala Leu Glu Gln Gly Ile His Pro Pro Gln Ala Gln 485 490 495 Ser Lys Ile Glu Glu Leu Glu Gln Glu Pro Glu Pro Glu Pro Glu Pro 500 505 510 Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro Glu Gln Leu 515 520 525 <210> 2 <211> 363 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct; MAb STC1317 mature heavy chain V domain <400> 2 gaagtgcagg tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60 tcctgtgcag cctctggatt cactttcagt agctatgcca tgtcttgggt tcgccagact 120 ccggcgaaga ggctggagtg ggtcgcaact attagtggtg gtggtagtta cacctactat 180 ccagacagtg taaagggccg attcaccatc tccagagaca atgccaagaa caccctgtac 240 ctgcaaatga gcagtctgag gtctgaggac acagccatgt attactgtgc aagggggaag 300 tatggtaact acgactatga tatggactac tggggtcaag gaacctcagt caccgtctcc 360 tca 363 <210> 3 <211> 121 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct; MAb STC1317 mature heavy chain V domain <400> 3 Glu Val Gln Val Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Thr Pro Ala Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gly Lys Tyr Gly Asn Tyr Asp Tyr Asp Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 4 <211> 330 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct; MAb STC1317 light chain V domain <400> 4 gacattgtgc tgacacagtc tcctgcttcc ttagctgtat ctctggggca gagggccacc 60 atctcataca gggccagcaa aagtgtcagt acatctggct atagttatat gcactggaac 120 caacagaaac cagtacagcc acccagactc ctcatctatc ttgtatccaa cctagaatct 180 ggggtccctg ccaggttcag tggcagtggg tctggggacag acttcaccct caacatccat 240 cctgtggagg aggaggatgc tgcaacctat tactgtcagc acattaggga gctttacacg 300 ttcggagggg ggaccaagct ggaaataaaa 330 <210> 5 <211> 110 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct; MAb STC1317 light chain V domain <400> 5 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro Val Gln Pro Pro 35 40 45 Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg 85 90 95 Glu Leu Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 6 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct; STC1807 heavy chain CDR1 <400> 6 Gly Phe Thr Phe Ser Ser Tyr 1 5 <210> 7 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct; STC1807 heavy chain CDR2 <400> 7 Ser Gly Gly Gly Ser Tyr 1 5 <210> 8 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct; STC1807 heavy chain CDR3 <400> 8 Gly Lys Tyr Gly Asn Tyr Asp Tyr Asp Met Asp Tyr 1 5 10 <210> 9 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct; STC1807 heavy chain CDR1 <400> 9 Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser 1 5 10 <210> 10 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct; STC1807 heavy chain CDR2 <400> 10 Thr Ile Ser Gly Gly Gly Ser Tyr Thr Tyr 1 5 10 <210> 11 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct; STC1807 heavy chain CDR1 <400> 11 Ser Tyr Ala Met Ser 1 5 <210> 12 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct; STC1807 heavy chain CDR2 <400> 12 Thr Ile Ser Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val Lys 1 5 10 15 Gly <210> 13 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct; STC1807 heavy chain CDR1 <400> 13 Ser Ser Tyr Ala Met Ser 1 5 <210> 14 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct; STC1807 heavy chain CDR2 <400> 14 Trp Val Ala Thr Ile Ser Gly Gly Gly Ser Tyr Thr Tyr 1 5 10 <210> 15 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct; STC1807 heavy chain CDR3 <400> 15 Ala Arg Gly Lys Tyr Gly Asn Tyr Asp Tyr Asp Met Asp 1 5 10 <210> 16 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct; STC1807 light chain CDR1 <400> 16 Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr Met His 1 5 10 15 <210> 17 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct; STC1807 light chain CDR2 <400> 17 Leu Val Ser Asn Leu Glu Ser 1 5 <210> 18 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct; STC1807 light chain CDR3 <400> 18 Gln His Ile Arg Glu Leu Tyr Thr 1 5 <210> 19 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct; STC1807 light chain CDR1 <400> 19 Ser Thr Ser Gly Tyr Ser Tyr Met His Trp Asn 1 5 10 <210> 20 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct; STC1807 light chain CDR2 <400> 20 Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu 1 5 10 <210> twenty one <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct; STC1807 light chain CDR3 <400> twenty one Gln His Ile Arg Glu Leu Tyr 1 5

Claims

1. An isolated monoclonal antibody that selectively binds to glycosylated LAG3 relative to the unglycosylated form of LAG3, wherein the antibody comprises a V H domain and V L domain, where: i) V H The domain comprises, according to the Chothia numbering system, a CDRH1 consisting of the amino acid sequence of SEQ ID NO: 6, a CDR H2 consisting of the amino acid sequence of SEQ ID NO: 7, and a CDR H3 consisting of the amino acid sequence of SEQ ID NO: 8, and The V L A domain comprising, according to the Chothia numbering system, a CDRL1 consisting of the amino acid sequence of SEQ ID NO: 16, a CDR L2 consisting of the amino acid sequence of SEQ ID NO: 17, and a CDR L3 consisting of the amino acid sequence of SEQ ID NO: 18; or ii) V H The domain comprises, according to the AbM numbering system, a CDRH1 consisting of the amino acid sequence of SEQ ID NO: 9, a CDR H2 consisting of the amino acid sequence of SEQ ID NO: 10, and a CDR H3 consisting of the amino acid sequence of SEQ ID NO: 8, and The V L a domain comprising, according to the AbM numbering system, a CDR L1 consisting of the amino acid sequence of SEQ ID NO: 16, a CDR L2 consisting of the amino acid sequence of SEQ ID NO: 17, and a CDR L3 consisting of the amino acid sequence of SEQ ID NO: 18; or iii) V H The domain comprises, according to the Kabat numbering system, a CDR H1 consisting of the amino acid sequence of SEQ ID NO: 11, a CDR H2 consisting of the amino acid sequence of SEQ ID NO: 12, and a CDR H3 consisting of the amino acid sequence of SEQ ID NO: 8, and The V L A domain comprising, according to the Kabat numbering system, a CDRL1 consisting of the amino acid sequence of SEQ ID NO: 16, a CDR L2 consisting of the amino acid sequence of SEQ ID NO: 17, and a CDR L3 consisting of the amino acid sequence of SEQ ID NO: 18; or iv) V H The domain comprises: according to the Contact numbering system, a CDR H1 consisting of the amino acid sequence of SEQ ID NO: 13, a CDR H2 consisting of the amino acid sequence of SEQ ID NO: 14, and a CDR H3 consisting of the amino acid sequence of SEQ ID NO: 15, and The V L The domain comprises: according to the Contact numbering system, CDRL1 consisting of the amino acid sequence of SEQ ID NO: 19, CDR L2 consisting of the amino acid sequence of SEQ ID NO: 20, and CDR L3 consisting of the amino acid sequence of SEQ ID NO:

21.

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

3. The isolated monoclonal antibody of claim 1, wherein the antibody blocks the binding of LAG3 to one or more of Gal-3, MHCII, and LSECtin.

4. The isolated monoclonal antibody of claim 1, wherein the antibody selectively binds LAG3 glycosylated at position N188, N250, N256, N343, or any combination thereof, relative to aglycosylated LAG3.

5. The isolated monoclonal antibody of claim 1, wherein the antibody has a binding affinity for glycosylated LAG3 of 0.1-10 nM, inclusive.

6. The isolated monoclonal antibody of claim 1, wherein the antibody masks glycosylation of LAG3 at one or more of N188, N250, N256, or N145.

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

5.

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

3.

9. The isolated monoclonal antibody of claim 1, wherein V L The domain consists of 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 monoclonal antibody of claim 1, which has human framework regions.

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

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

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

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

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

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

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

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

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

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

21. A method for assessing LAG3 glycosylation for non-diagnostic and non-therapeutic purposes, the method comprising contacting a sample containing LAG3 with the isolated monoclonal antibody of any one of claims 1 to 15 or the conjugate of claim 16.

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

23. Use of the isolated monoclonal antibody of any one of claims 1 to 15 or the conjugate of claim 16 in the preparation of a kit for assessing LAG3 glycosylation in a sample.

24. The use according to claim 23, wherein the sample is a cell sample.

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