Engineered antibodies that bind lag3, production methods, pharmaceutical compositions and uses thereof

Abstract

This invention provides a fully human anti-LAG3 IgG antibody that is engineered to have amino acid sequences in its heavy chain variable region and / or light chain variable region to improve antigen binding, cell binding, T cell activation and cytokine release capabilities.

Description

Technical Field

[0001] This invention provides a fully human anti-LAG3 IgG antibody, which is engineered to have amino acid sequences in its heavy chain variable region and / or light chain variable region to improve antigen binding, cell binding, T cell activation and cytokine release capabilities. Prior Technology

[0002] Lymphocyte activation gene-3 (or LAG3) (also known as CD223) is a member of the immunoglobulin superfamily and is structurally and genetically related to CD4. LAG3 is not expressed in resting peripheral blood lymphocytes but is expressed in activated T cells and NK cells. LAG3 is a membrane protein encoded by a gene located on the distal part of the short arm of chromosome 12 near the CD4 gene, indicating that the LAG3 gene can evolve through gene replication (Triebel et al. (1990) J. Exp. Med. 171:1393-1405). []

[0003] Similar to CD4, LAG3 has been shown to interact with class II MHC molecules, but unlike CD4, LAG3 does not interact with the human immunodeficiency virus gp120 protein (Baixeras et al. (1992) J. Exp. Med. 176:327-337). Studies using soluble LAG3 immunoglobulin fusion protein (sLAG3Ig) have confirmed that LAG3 binds directly and specifically to class II MHC on the cell surface (Huard et al. (1996) Eur. J. Immunol. 26:1180-1186).

[0004] In in vitro studies of antigen-specific T cell responses, the addition of anti-LAG3 antibody led to increased T cell proliferation, higher expression of activated antigens (such as CD25), and higher concentrations of cytokines (such as interferon-γ and interleukin-4), supporting the role of LAG3 / class II MHC interaction in downregulating antigen-independent stimulation of CD4+ T lymphocytes (Huard et al. (1994) Eur. J. Immunol. 24:3216-3221). The intracytoplasmic region of LAG3 has been shown to interact with a protein called LAP, which is considered a signal transduction molecule involved in the downregulation of the CD3 / TCR activation pathway (Iouzalen et al. (2001) Eur. J. Immunol. 31:2885-2891). Furthermore, it has been shown that CD4+CD25+ regulatory T cells (Tregs) exhibit LAG3 upon activation, and antibodies against LAG3 inhibit it both in vitro and in vivo through the inhibition of induced Treg cells, indicating that LAG3 contributes to the inhibitory activity of Treg cells (Huang, C. et al. (2004) Immunity 21:503-513). Moreover, it has been shown that LAG3 negatively regulates T cell homeostasis in both T cell-dependent and independent mechanisms by regulating T cells (Workman and Vignali (2005) J. Immunol. 174:688-695).

[0005] In some cases, LAG3 has also been shown to have immunostimulatory effects. For example, compared with untransfected tumor cells, LAG3-transfected tumor cells transplanted into syngeneic mice showed reduced growth or complete regression, indicating that LAG3 expression on tumor cells stimulates antitumor responses by triggering antigen-presenting cells via class II MHC molecules (Prigent et al. (1999) Eur. J. Immunol. 29:3867-3876). Furthermore, soluble LAG3 Ig fusion proteins have been shown to simultaneously stimulate humoral and cellular immune responses when administered to mice along with antigens, indicating that soluble LAG3 Ig can be used as a vaccine adjuvant (El Mir and Triebel (2000) J. Immunol. 164:5583-5589). In addition, soluble human LAG3 Ig has been shown to amplify type I tumor-specific immunity in vitro (Casati et al. (2006) Cancer Res. 66:4450-4460). For further information on the functional activity of LAG3, see Triebel (2003) Trends Immunol. 24:619-622. In light of the above, other reagents for modulating the activity of LAG3 are of interest. Summary of the Invention

[0006] This technology still requires effective treatment based on LAG3, particularly anti-LAG3 antibodies. This invention provides antibodies engineered to exhibit higher affinity for their target antigens and improved cell-killing ability compared to parental antibodies. This invention includes the following examples. Example 1 is a fully human anti-LAG3 antibody comprising a heavy chain variable region having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4, 6, 8, or 10; and a light chain variable region having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 5, 7, 9, or 11.

[0007] Example 2 is a LAG3-binding protein comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:4, 6, 8 or 10; and the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:5, 7, 9 or 11.

[0008] Example 3 is the LAG3 binding protein of Example 2, wherein the heavy chain variable region contains the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:4 and the light chain variable region contains the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:5; or the heavy chain variable region contains the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:6 and the light chain variable region contains the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:7; or the heavy chain variable region contains the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:8 and the light chain variable region contains the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:9; or the heavy chain variable region contains the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:10 and the light chain variable region contains the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO:11.

[0009] Example 4 is the LAG3 binding protein of Example 2 or 3, which is an antibody or antibody fragment.

[0010] Example 5 is the fully human anti-LAG3 antibody of Example 1 or the LAG3 binding protein of any one of Examples 2 to 4, wherein the heavy chain variable region sequence and the light chain variable region sequence comprise SEQ ID NO:4 and 5 (hereinafter referred to as 1C5-3A6), SEQ ID NO:6 and 7 (hereinafter referred to as 2D12-2E3-N102D), SEQ ID NO:8 and 9 (hereinafter referred to as 5C8) or SEQ ID NO:10 and 11 (hereinafter referred to as 8D9).

[0011] Example 6 is a Fab fully human anti-LAG3 antibody, comprising a variable domain region from the heavy chain and a variable domain region from the light chain, wherein the variable domain region from the heavy chain comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4, 6, 8 or 10, and wherein the variable domain region from the light chain comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 5, 7, 9 or 11.

[0012] Example 7 is the Fab fully human anti-LAG3 antibody of Example 6, wherein the variable domain region from the heavy chain and the variable domain region from the light chain include SEQ ID NO:4 and 5 (hereinafter referred to as 1C5-3A6), SEQ ID NO:6 and 7 (SEQ ID NO:4 and 5 (hereinafter referred to as 1C5-3A6), SEQ ID NO:6 and 7 (hereinafter referred to as 2D12-2E3-N102D), SEQ ID NO:8 and 9 (hereinafter referred to as 5C8), or SEQ ID NO:10 and 11 (hereinafter referred to as 8D9).

[0013] Example 8 is a single-chain human anti-LAG3 antibody, comprising a variable domain region from the heavy chain and a variable domain region from the light chain, wherein the variable domain region from the heavy chain comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4, 6, 8 or 10, and wherein the variable domain region from the light chain comprises a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 5, 7, 9 or 11.

[0014] Example 9 is the LAG3 binding protein of any one of Examples 2 to 5 or the single-chain human anti-LAG3 antibody of Example 8, wherein the variable domain region from the heavy chain and the variable domain region from the light chain are bound together with the peptide linker.

[0015] Example 10 is the single-chain human anti-LAG3 antibody of Example 8, wherein the variable domain region from the heavy chain and the variable domain region from the light chain include SEQ ID NO:4 and 5 (referred to herein as 1C5-3A6), SEQ ID NO:6 and 7 (referred to herein as 2D12-2E3-N102D), SEQ ID NO:8 and 9 (referred to herein as 5C8), or SEQ ID NO:10 and 11 (referred to herein as 8D9).

[0016] Example 11 is any of the LAG3-binding proteins or fully human anti-LAG3 antibodies in the preceding examples, wherein the antibody binds to human LAG3 at a concentration of 10⁻⁸ M or less (KD).

[0017] Example 12 is any one of the LAG3 binding proteins or fully human anti-LAG3 antibodies in Examples 1 to 7, wherein the antibody binds to cynomolgus monkey LAG3 at a concentration of 10⁻⁶ M or less.

[0018] Example 13 refers to any of the LAG3 binding proteins or fully human anti-LAG3 antibodies in the preceding examples, which contain IgG4 class antibodies.

[0019] Example 14 is a pharmaceutical composition comprising a pharmaceutically acceptable excipient and any of the LAG3-binding proteins or fully human anti-LAG3 antibodies from the preceding examples.

[0020] Example 15 is a kit comprising any one of Examples 1 to 13, the LAG3 binding protein, the Fab fully human anti-LAG3 antibody, the single-chain human anti-LAG3 antibody, or the fully human anti-LAG3 antibody.

[0021] Example 16 is a nucleic acid that encodes the heavy chain variable region of any of the LAG3 binding proteins or fully human anti-LAG3 antibodies in Examples 1 to 13.

[0022] Example 17 is a nucleic acid that encodes the light chain variable region of any of the LAG3 binding proteins or fully human anti-LAG3 antibodies in Examples 1 to 13.

[0023] Example 18 is a nucleic acid that encodes [(a)(i)] The heavy chain variable region of any one of the fully human anti-LAG3 antibodies in Examples 1 to 13, and [(ii)] The light chain variable region of any one of the fully human anti-LAG3 antibodies in Examples 1 to 13; or [(b)] LAG3 binding protein of any one of Examples 2 to 5, 9, or 11 to 13.

[0024] Example 19 is a vector containing the nucleic acid of Example 16.

[0025] Example 20 is a vector containing the nucleic acid of Example 17.

[0026] Example 21 is a vector containing the nucleic acid of Example 18.

[0027] Example 22 is a host cell that carries the vector of Example 19.

[0028] Example 23 is the host cell of Example 22, wherein the vector includes an expression vector, and wherein the host cell expresses a heavy chain variable region.

[0029] Example 24 is a host cell that carries the vector of Example 20.

[0030] Example 25 is the host cell of Example 24, wherein the vector includes an expression vector, and wherein the host cell expresses a light chain variable region.

[0031] Example 26 is a host cell that carries a first vector containing the vector of Example 19 and a second vector containing the vector of Example 20.

[0032] Example 27 is the host cell of Example 26, wherein the first vector includes a first expression vector, the second vector includes a second expression vector, and the host cell expresses a heavy chain variable region and a light chain variable region.

[0033] Example 28 is a host cell that carries the vector of Example 21.

[0034] Example 29 is the host cell of Example 28, wherein the vector includes an expression vector, and wherein the host cell expresses a heavy chain variable region and a light chain variable region.

[0035] Example 30 is a method for preparing the heavy chain variable region of LAG3 binding protein, or fully human anti-LAG3 antibody, or Fab fully human anti-LAG3 antibody, or single-chain human anti-LAG3 antibody. The method includes culturing the host cell population of Example 23 under conditions suitable for expressing the heavy chain variable region of fully human anti-LAG3 antibody, or Fab fully human anti-LAG3 antibody, or single-chain human anti-LAG3 antibody.

[0036] Example 31 is the method of Example 30, which further includes: recovering the expressed LAG3 binding protein, or a fully human anti-LAG3 antibody, or a Fab fully human anti-LAG3 antibody, or the heavy chain variable region of a single-chain human anti-LAG3 antibody from a host cell.

[0037] Example 32 is a method for preparing the light chain variable region of a fully human anti-LAG3 antibody, or a Fab fully human anti-LAG3 antibody, or a single-chain human anti-LAG3 antibody. The method includes culturing the host cell population of Example 25 under conditions suitable for expressing the light chain variable region of the fully human anti-LAG3 antibody, or the Fab fully human anti-LAG3 antibody, or the single-chain human anti-LAG3 antibody.

[0038] Example 33 is the method of Example 32, which further includes: recovering the expressed light chain variable region of a fully human anti-LAG3 antibody, or a Fab fully human anti-LAG3 antibody, or a single-chain human anti-LAG3 antibody from a host cell.

[0039] Example 34 is a method for preparing (i) a fully human anti-LAG3 antibody, or a Fab fully human anti-LAG3 antibody, or the heavy chain variable region of a single-chain human anti-LAG3 antibody, and (ii) a fully human anti-LAG3 antibody, or a Fab fully human anti-LAG3 antibody, or the light chain variable region of a single-chain human anti-LAG3 antibody, the method comprising: culturing the host cell population of Example 27 under conditions suitable for expressing (i) the heavy chain variable region of the fully human anti-LAG3 antibody, or a Fab fully human anti-LAG3 antibody, or the single-chain human anti-LAG3 antibody, and (ii) the light chain variable region of the fully human anti-LAG3 antibody, or a Fab fully human anti-LAG3 antibody, or the single-chain human anti-LAG3 antibody.

[0040] Example 35 is the method of Example 34, which further includes: recovering from host cells (i) the expressed heavy chain variable region of a fully human anti-LAG3 antibody, or a Fab fully human anti-LAG3 antibody, or a single-chain human anti-LAG3 antibody, and (ii) the expressed light chain variable region of a fully human anti-LAG3 antibody, or a Fab fully human anti-LAG3 antibody, or a single-chain human anti-LAG3 antibody.

[0041] Example 36 is a method for preparing (i) a fully human anti-LAG3 antibody, or a Fab fully human anti-LAG3 antibody, or a heavy chain variable region of a single-chain human anti-LAG3 antibody, and (ii) a fully human anti-LAG3 antibody, or a Fab fully human anti-LAG3 antibody, or a single-chain human anti-LAG3 antibody, the method comprising: culturing the host cells of Example 29 under conditions suitable for expressing (i) the heavy chain variable region of the fully human anti-LAG3 antibody, or a Fab fully human anti-LAG3 antibody, or a single-chain human anti-LAG3 antibody, and (ii) the light chain variable region of the fully human anti-LAG3 antibody, or a Fab fully human anti-LAG3 antibody, or a single-chain human anti-LAG3 antibody.

[0042] Example 37 is the method of Example 36, which further includes: recovering from host cells (i) the expressed heavy chain variable region of a fully human anti-LAG3 antibody, or a Fab fully human anti-LAG3 antibody, or a single-chain human anti-LAG3 antibody, and (ii) the expressed light chain variable region of a fully human anti-LAG3 antibody, or a Fab fully human anti-LAG3 antibody, or a single-chain human anti-LAG3 antibody.

[0043] Example 38 is a method for treating an individual with a disease associated with LAG3 overexpression, the method comprising administering to the individual an effective amount of a therapeutic composition comprising any one of Examples 1 to 13 of the LAG3 binding protein or a human anti-LAG3 antibody.

[0044] Example 39 refers to any one of Examples 1 to 13, which is a LAG3-binding protein or a human anti-LAG3 antibody, used to treat diseases associated with LAG3 overexpression.

[0045] Example 40 is the method of Example 38, or the LAG3 binding protein or human anti-LAG3 antibody used as in Example 39, wherein the disease is bone cancer, pancreatic cancer, skin cancer, head or neck cancer, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, cancer of the anus, stomach cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small bowel cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue cancer. Tumors, urethral cancer, penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal cord axis tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, or cancers induced by asbestos.

[0046] Example 41 refers to the LAG3 binding protein or human anti-LAG3 antibody of any one of Examples 1 to 13, used in the method of any one of Examples 38. Simple Explanation of the Diagram

[0047] [picture] [1] Shows the amino acid sequences of LAG3 target antigens from humans (SEQ ID NO:1), cynomolgus monkeys (SEQ ID NO:2) and mice (SEQ ID NO:3). []

[0048] [picture] [2] The amino acid sequences of the heavy chain variable region and light chain variable region of the fully human anti-LAG3 antibodies 1C5-3A6 (SEQ ID NO:4 and 5), 2D12-2E3-N102D (SEQ ID NO:6 and 7), 5C8 (SEQ ID NO:8 and 9), and 8D9 (SEQ ID NO:10 and 11) are shown.

[0049] [picture] [3] Results of a dose-dependent ELISA analysis of the binding of soluble human LAG3 antigen are shown. The experiment is described in Example 1.

[0050] [picture] [4] A series of SPR sensing plots showing the binding kinetics of the anti-LAG3 antibody and the measured KD values ​​are shown. The Biacore experiment is described in Example 2.

[0051] [picture] [5] Bar graph showing the results of ELISA analysis of the cross-reactivity of various anti-LAG3 antibodies with human, mouse, or cynomolgus monkey LAG3 proteins. The experiment is described in Example 3. []

[0052] [picture] [6] A graph comparing the binding ability of various anti-LAG3 antibodies to the reporter Jurkat cell line expressing human LAG3 / NFAT-Luc. The experiment is described in Example 4.

[0053] [picture] [7A] This graph compares the binding affinity of various anti-LAG3 antibodies to PBMCs of cynomolgus monkeys activated by Staphylococcal Enterotoxin (SEB). Values ​​along the Y-axis are ∆MFI. The experiment is described in Example 5.

[0054] [picture] [7B] This graph compares the binding ability of various anti-LAG3 antibodies to PBMCs of cynomolgus monkeys activated with Staphylococcus aureus enterotoxin (SEB) using the same data generated from the self-described experiment in Figure 7A. Values ​​along the Y-axis represent ∆LAG3+ cells. The experiment is described in Example 5.

[0055] [picture] [8A] Bar graph comparing the functional activities of various anti-LAG3 antibodies in the presence of Jurkat cell lines expressing human LAG3 / NFAT-Luc and Raji cells activated by Staphylococcus aureus enterotoxin (SEB). Experiment #1 is described in Example 6.

[0056] [picture] [8B] Bar graph comparing the functional activities of various anti-LAG3 antibodies in the presence of Jurkat cell lines expressing human LAG3 / NFAT-Luc and Raji cells activated by Staphylococcus aureus enterotoxin (SEB). Experiment #2 is described in Example 6.

[0057] [picture] [9] This is a bar graph showing the results of epitope mapping analysis comparing the binding of the anti-LAG3 antibody described herein with that of a competitor anti-LAG3 antibody. The experiment is described in Example 7. []

[0058] [picture] [10A] Bar graphs comparing interferon-γ release induced by various anti-LAG3 antibodies were used from PBMCs from donor #1. The experiment is described in Example 8.

[0059] [picture]

[10] Bar graphs of interferon-γ release induced by various anti-LAG3 antibodies were compared using PBMCs from donor #2 in the B lineage. The experiment is described in Example 8. Implementation

[0060] [Cross-reference to related applications] []

[0061] This application claims priority to U.S. Provisional Patent Application No. 63 / 004,798, filed April 3, 2020, which is incorporated herein by reference in its entirety for all purposes.

[0062] Throughout this application, various publications, patents, and / or patent applications are cited. The disclosures of these publications, patents, and / or patent applications are incorporated herein by reference in their entirety to provide a more comprehensive description of the state-of-the-art technology to which these disclosures pertain. In the event of any conflict between any material incorporated by reference and the express content of this application, the express content shall prevail. [definition] []

[0063] Unless otherwise defined, the technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art. Generally, the terms used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, transgenic cell production, protein chemistry, nucleic acid chemistry, and hybridization as described herein are well-known and commonly used in this art. Unless otherwise indicated, the methods and techniques provided herein are generally performed according to procedures well known in this art and described in the various general and more specific references cited and discussed herein. See, for example, Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), and Ausubel et al., *Current Protocols in Molecular Biology*, Greene Publishing Associates (1992).Many basic texts describe standard antibody production processes, including Borrebaeck (ed.) *Antibody Engineering*, 2nd ed., Freeman and Company, NY, 1995; McCafferty et al., *Antibody Engineering, A Practical Approach IRL* at Oxford Press, Oxford, England, 1996; and Paul (1995) *Antibody Engineering Protocols*, Humana Press, Towata, NJ, 1995; Paul (ed.), *Fundamental Immunology*, Raven Press, NY, 1993; Coligan (1991) *Current Protocols in Immunology*, Wiley / Greene, NY; Harlow and Lane (1989) *Antibodies: A Laboratory Manual*, Cold Spring Harbor Press, NY; Stites et al. (eds.) *Basic and Clinical Immunology* (4th ed.), Lange Medical Publications, Los Altos, Calif., and the references cited therein; and *Coding Monoclonal Antibodies: Principles and Practice* (2nd ed.). Press, New York, NY, 1986, and Kohler and Milstein, Nature 256: 495-497, 1975. All references cited herein are incorporated herein by reference in their entirety. Enzymatic reactions and enrichment / purification techniques are well-known and commonly achieved in this technique or performed as described herein according to the manufacturer's instructions. The terminology described herein and used in conjunction with analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry and their laboratory procedures and techniques is well-known and commonly used in this technique. Standard techniques can be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and for the treatment of patients.

[0064] The headings provided herein are not intended to limit the various forms of the invention, which can be understood by referring to this specification in its entirety.

[0065] Unless the context otherwise requires, singular terms shall include plural terms and plural terms shall include singular terms. Unless explicitly and clearly limited to a single indicator, the singular forms of “a,” “an,” and “the,” and the singular use of any word, shall include plural indicators.

[0066] It should be understood that the use of substitution (e.g., "or") in this document means any one or both of the substitutions or any combination thereof.

[0067] As used herein, the term "and / or" means each of a specific characteristic or component, together or apart from the other. For example, the term "and / or" as used in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A only" and "B only". Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to cover each of the following states: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; "A only"; "B only"; and "C only".

[0068] As used herein, the terms “comprising,” “including,” “having,” and “containing,” and their grammatical variations, as used herein, are intended to be non-restrictive, meaning that one or more items in the list do not preclude other items that may be substituted for or added to the listed items. It should be understood that whenever the language “comprising” is used to describe a state, other similar states described as “consisting of” and / or “substantially consisting of” are also provided.

[0069] As used herein, the term "about" refers to a value or composition within an acceptable margin of error for a particular value or composition, as determined by a person of ordinary skill, the margin of error depending in part on the method of determination or determination of that value or composition, i.e., the limitations of the measurement system. For example, "about" or "approximately" may mean that each technical practice is within one or more standard deviations. Alternatively, depending on the limitations of the measurement system, "about" or "approximately" may mean a range of up to 10% (i.e., ±10%) or greater. For example, about 5 mg may include any number between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, these terms may mean up to an order of magnitude or up to 5 times a certain value. When a particular value or composition is provided in this invention, unless otherwise stated, the meaning of "about" or "approximately" should be assumed to be within an acceptable margin of error for that particular value or composition.

[0070] The terms “peptide,” “polypeptide,” and “protein,” and other related terms used herein, are used interchangeably and refer to polymers of amino acids and are not limited to any particular length. Polypeptides may contain both natural and non-natural amino acids. Polypeptides include recombinant or chemically synthesized forms. Polypeptides also include precursor molecules that have not yet undergone cleavage (e.g., by cleavage of secretory messaging peptides or by non-enzymatic cleavage at certain amino acid residues). Polypeptides include mature molecules that have undergone cleavage. These terms encompass natural and artificial proteins, protein fragments and polypeptide analogs (such as mutant proteins, variants, chimeric proteins, and fusion proteins), and proteins that are translated or otherwise covalently or non-covalently modified. This document describes polypeptides comprising the amino acid sequence of a LAG3-binding protein (e.g., an anti-LAG3 antibody or its antigen-binding portion) prepared using a recombinant procedure. []

[0071] The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide,” and other related terms used herein, are used interchangeably and refer to polymers of nucleotides and are not limited to any particular length. Nucleic acids include recombinant and chemically synthesized forms. Nucleic acids include DNA molecules (cDNA or genomic DNA), RNA molecules (e.g., mRNA), DNA or RNA analogs produced using nucleotide analogs (e.g., peptide nucleic acids and non-naturally generated nucleotide analogs), and hybrids thereof. Nucleic acid molecules may be single-stranded or double-stranded. In one embodiment, the nucleic acid molecule of the present invention comprises a continuous open reading frame encoding an antibody or a fragment thereof or scFv, derivative, mutant protein, or variant thereof. In one embodiment, the nucleic acid comprises one type of polynucleotide or a mixture of two or more different types of polynucleotides. Nucleic acids encoding antibody light chains, antibody heavy chains, anti-LAG3 antibodies, or antigen-binding portions thereof are described herein. In one embodiment, the nucleic acid encodes a heavy chain variable region comprising at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4, 6, 8, or 10. In one embodiment, the nucleic acid encoding includes a light chain variable region that is at least 95% sequence identical to the amino acid sequence of SEQ ID NO: 5, 7, 9 or 11. []

[0072] The terms "recovery," "recovering," and other related terms refer to the acquisition of a protein (e.g., an antibody or its antigen-binding portion) from host cell culture medium, host cell lysate, or host cell membrane. In one embodiment, the protein is presented as a recombinant protein in the host cell, which is fused to a secretion signaling peptide sequence mediating the secretion of the presented protein. The secreted protein can be recovered from the host cell culture medium. In one embodiment, the protein is presented as a recombinant protein in the host cell, which lacks the secretion signaling peptide sequence recoverable from host cell lysate. In one embodiment, the protein is presented as a membrane-bound protein in the host cell, which can be recovered using a detergent to release the presented protein from the host cell membrane. In one embodiment, regardless of the method used to recover the protein, a procedure for removing cellular debris from the recovered protein can be performed. For example, the recovered protein can be subjected to chromatography, gel electrophoresis, and / or dialysis. In one embodiment, the chromatography includes any one or any combination of two or more procedures, including affinity chromatography, hydroxyapatite chromatography, ion exchange chromatography, reversed-phase chromatography, and / or chromatography on silica. In one embodiment, affinity chromatography includes protein A or G (a cell wall component from Staphylococcus aureus). []

[0073] The term "isolated" refers to a protein (e.g., an antibody or its antigen-binding portion) or polynucleotide that is substantially free of other cellular material. Isolation, using protein purification techniques well known in the art, renders a protein substantially free of its native components (or components associated with cell expression systems or chemical synthesis methods used to produce antibodies). In some embodiments, the term "isolated" also refers to a protein or polynucleotide that is substantially free of other molecules of the same kind (e.g., other proteins or polynucleotides with different amino acid or nucleotide sequences). Techniques well known in the art, including low-resolution methods (such as gel electrophoresis) and high-resolution methods (such as HPLC or mass spectrometry), can be used to analyze the homogeneity purity of the desired molecule. In one embodiment, any of the antibody light chain, antibody heavy chain, anti-LAG3 antibody, or antigen-binding protein described herein can be isolated. []

[0074] As used herein, "antigen-binding protein" and related terms (e.g., LAG3-binding protein) refer to proteins comprising a portion that binds to an antigen (e.g., LAG3) and, where appropriate, a scaffold or framework portion that allows the antigen-binding portion to adopt a conformation that promotes the binding of the antigen-binding protein to the antigen. Examples of antigen-binding proteins include antibodies, antibody fragments (e.g., the antigen-binding portion of an antibody), antibody derivatives, and antibody analogs. Antigen-binding proteins may include, for example, alternative protein scaffolds or artificial scaffolds having grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds comprising mutations introduced to, for example, stabilize the three-dimensional structure of the antigen-binding protein, and fully synthetic scaffolds comprising, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, Vol. 53, Issue 1:121-129; Roque et al., 2004, Biotechnol. Prog. 20:639-654. Additionally, peptide antibody mimics ("PAMs") and antibody mimic-based scaffolds utilizing fibrone junction components as the scaffold can be used. This article describes antigen-binding proteins that bind to LAG3.

[0075] Antigen-binding proteins may have structures such as immunoglobulins. In one embodiment, "immunoglobulin" refers to a tetrameric molecule composed of two pairs of identical polypeptide chains, each pair having a "light chain" (approximately 25 kDa) and a "heavy chain" (approximately 50 to 70 kDa). The amino-terminal portion of each chain includes a variable region of approximately 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxyl-terminal portion of each chain defines a constant region primarily responsible for effector functions. Human light chains are classified as κ or λ light chains. Heavy chains are classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by "J" regions of approximately 12 or more amino acids, wherein the heavy chain also includes "D" regions of approximately 10 amino acids. See Fundamental Immunology, Chapter 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)) (which is incorporated herein by reference in its entirety for all purposes). The variable regions of each light / heavy chain pair form antibody-binding sites, giving the intact immunoglobulin two antigen-binding sites. In one embodiment, the antigen-binding protein may be a synthetic molecule having a structure different from that of the tetrameric immunoglobulin molecule but still binding to a target antigen or binding to two or more target antigens. For example, synthetic antigen-binding proteins may comprise antibody fragments, one to six or more polypeptide chains, asymmetric assembly of polypeptides, or other synthetic molecules. This article describes an antigen-binding protein with immunoglobulin-like properties that specifically bind to LAG3. [ , ]

[0076] The variable regions of the immunoglobulin chain exhibit the same general structure as the relatively conserved framework regions (FRs) linked by three hypervariable regions (also known as complementarity-determining regions or CDRs). From the N-terminus to the C-terminus, both the light and heavy chains contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0077] One or more CDRs can be covalently or nonvalently incorporated into a molecule to make it an antigen-binding protein. Antigen-binding proteins can incorporate CDRs as part of a larger polypeptide chain, covalently link CDRs to another polypeptide chain, or nonvalently incorporate CDRs. CDRs allow antigen-binding proteins to specifically bind to the specific antigen of interest.

[0078] The allocation of amino acids to their respective domains is based on the definitions provided by Kabat et al. in Sequences of Proteins of Immunological Interest, 5th Edition, US Dept. of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242, 1991. Other numbering systems for amino acids in the immunoglobulin chain include IMGT.RTM. (international ImMunoGeneTics information system; Lefranc et al., Dev. Comp. Immunol. 29:185-203;2005) and AHo (Honegger and Pluckthun, J. Mol. Biol. 309(3):657-670;2001); Chothia (Al-Lazikani et al., 1997 Journal of Molecular Biology 273:927-948); Contact (Maccallum et al., 1996 Journal of Molecular Biology 262:732-745), and Aho (Honegger and Pluckthun 2001 Journal of Molecular Biology 309:657-670).

[0079] As used herein, "antibody" and "antibodies" and related terms refer to a complete immunoglobulin or its antigen-binding portion that specifically binds to an antigen. Antigen-binding portions can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of a complete antibody. Antigen-binding portions include, in particular, Fab, Fab', F(ab')2, Fv, domain antibodies (dAb), and complementarity-determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, bifunctional antibodies (diabodies), trifunctional antibodies (triabodies), tetrafunctional antibodies (tetrabodies), and polypeptides containing at least a portion of an immunoglobulin sufficient to confer specific antigen binding to that polypeptide.

[0080] Antibodies include recombinant antibodies and antigen-binding moieties. Antibodies include non-human, chimeric, humanized, and fully human antibodies. Antibodies include monospecific and multispecific antibodies (e.g., bispecific, trispecific, and higher-order specificities). Antibodies include tetrameric antibodies, light chain monomers, heavy chain monomers, light chain dimers, and heavy chain dimers. Antibodies include F(ab')2 fragments, Fab' fragments, and Fab fragments. Antibodies include single-domain antibodies, monovalent antibodies, single-chain antibodies, single-chain variable fragments (scFv), camelified antibodies, affibodies, disulfide-linked Fvs (sdFv), anti-idiotype antibodies (anti-Id), and minibodies. Antibodies include single-strain and multi-strain populations. This article describes anti-LAG3 antibodies containing light and / or heavy chains.

[0081] As used herein, the terms "antigen-binding domain," "antigen-binding region," or "antigen-binding site," and other related terms, refer to a portion of an antigen-binding protein containing amino acid residues (or other portions) that interact with the antigen and contribute to the antigen-binding protein's specificity and affinity for the antigen. For an antibody that specifically binds to its antigen, the antibody will contain at least a portion of at least one of its CDR domains. This article describes the antigen-binding domain from an anti-LAG3 antibody.

[0082] As used herein in the context of antibodies, antigen-binding proteins, or antibody fragments, the terms "specific binding" and other related terms refer to the non-covalent or covalent preferential binding of an antigen relative to other molecules or portions (e.g., the antibody specifically binds to a particular antigen relative to other available antigens). In one embodiment, an antibody specifically binds to a target antigen if it binds to the antigen with a dissociation constant KD of 10⁻⁵ M or less, or 10⁻⁶ M or less, or 10⁻⁷ M or less, or 10⁻⁸ M or less, or 10⁻⁹ M or less, or 10⁻¹⁰ M or less. This document describes anti-LAG3 antibodies that specifically bind to LAG3.

[0083] In one embodiment, the dissociation constant (KD) can be measured using BIACORE surface plasma resonance (SPR) analysis. Surface plasma resonance refers to an optical phenomenon that allows for the analysis of real-time interactions by detecting changes in protein concentration within a biosensor matrix, for example, using a BIACORE system (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ).

[0084] As used herein, "antigen determinant" and related terms refer to a portion of an antigen bound by an antigen-binding protein (e.g., an antibody or its antigen-binding portion). An antigen determinant may comprise portions of two or more antigens bound by an antigen-binding protein. An antigen determinant may comprise discontinuous portions of an antigen or two or more antigens (e.g., amino acid residues that are discontinuous in the primary sequence of the antigen but sufficiently close to each other in the tertiary and quaternary structures for binding by an antigen-binding protein). Generally, the variable region (particularly the CDR) of an antibody interacts with the antigen determinant. This article describes an anti-LAG3 antibody and its antigen-binding protein that bind to the antigen determinant of the LAG3 polypeptide (antigen).

[0085] As used herein, the terms "antibody fragment," "antibody moiety," "antigen-binding fragment of an antibody," or "antigen-binding portion of an antibody," and other related terms, refer to molecules other than intact antibodies that contain a portion of an intact antibody that binds to the antigen bound by the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; Fd; and Fv fragments, as well as dAb; bifunctional antibodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and polypeptides that contain at least a portion of an antibody sufficient to confer specific antigen binding to the polypeptide. Antigen-binding portions of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding portions particularly include Fab, Fab', F(ab')2, Fv, domain antibodies (dAb), and complementarity-determining region (CDR) fragments, chimeric antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, and polypeptides that contain at least a portion of an immunoglobulin sufficient to confer antigen-binding properties to the antibody fragment. This document describes an antigen-binding fragment of an anti-LAG3 antibody.

[0086] The terms "Fab," "Fab fragment," and other related terms refer to a monovalent fragment comprising a variable light chain region (VL), a constant light chain region (CL), a variable heavy chain region (VH), and a first constant region (CH1). Fab fragments can bind antigens. The F(ab')2 fragment is a bivalent fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region. F(Ab')2 has antigen-binding capability. The Fd fragment comprises the VH and CH1 regions. The Fv fragment comprises the VL and VH regions. Fv fragments can bind antigens. dAb fragments are antigen-binding fragments having a VH domain, a VL domain, or either a VH or VL domain (US Patents 6,846,634 and 6,696,245; US Publication Nos. 2002 / 02512, 2004 / 0202995, 2004 / 0038291, 2004 / 0009507, and 2003 / 0039958; and Ward et al., Nature 341:544-546, 1989). This document describes Fab fragments comprising the antigen-binding portion of an anti-LAG3 antibody.

[0087] Single-chain antibodies (scFvs) are antibodies in which the VL and VH regions are linked via linkers (e.g., synthetic sequences of amino acid residues) to form a continuous protein chain. Preferably, the linkers are long enough to allow the protein chain to fold itself and form a monovalent antigen-binding site (see, for example, Bird et al., 1988, Science 242:423-26 and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83). This article describes single-chain antibodies comprising the antigen-binding portion of an anti-LAG3 antibody.

[0088] Bifunctional antibodies are bivalent antibodies comprising two polypeptide chains, each containing VH and VL domains linked by a linker. This linker is too short to allow pairing between the two domains on the same chain, thus allowing each domain to pair with a complementary domain on the other polypeptide chain (see, for example, Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-48, and Poljak et al., 1994, Structure 2:1121-23). ​​If the two polypeptide chains of a bifunctional antibody are identical, the resulting bifunctional antibody will have two identical antigen-binding sites due to their pairing. Polypeptide chains with different sequences can be used to prepare bifunctional antibodies with two different antigen-binding sites. Similarly, trifunctional and tetrafunctional antibodies are antibodies comprising three and four polypeptide chains, respectively, forming three and four antigen-binding sites, which may be identical or different. Bifunctional, trifunctional, and tetrafunctional antibody constructs can be prepared using the antigen-binding portion of any anti-LAG3 antibody described herein.

[0089] The term "human antibody" refers to an antibody having one or more variable and constant regions derived from human immunoglobulin sequences. In one embodiment, all variable and constant domains are derived from human immunoglobulin sequences (e.g., a fully human antibody). Such antibodies can be prepared in various ways, including, as described below, by recombinant methods or by immunizing mice with the antigen of interest, the mice being genetically modified to express antibodies derived from human heavy and / or light chain encoding genes. This document describes a fully human anti-LAG3 antibody and its antigen-binding protein.

[0090] "Humanized" antibodies refer to antibodies that have sequences differing from those of antibodies derived from non-human species by one or more amino acid substitutions, deletions, and / or additions, such that when the humanized antibody is administered to a human individual, it is unlikely to induce an immune response, and / or induces a milder immune response compared to antibodies from non-human species. In one embodiment, certain amino acids in the framework and constant domains of the heavy and / or light chains of the non-human species antibody are mutated to produce a humanized antibody. In another embodiment, a constant domain from a human antibody is fused to a variable domain of a non-human species. In yet another embodiment, one or more amino acid residues in one or more CDR sequences of a non-human antibody are altered to reduce the potential immunogenicity of the non-human antibody when administered to a human individual, wherein the altered amino acid residues are not essential for the antibody's immunospecific binding to its antigen, or the alteration to the amino acid sequence is a conserved alteration such that the binding of the humanized antibody to the antigen is not significantly worse than the binding of the non-human antibody to the antigen. Examples of how to prepare humanized antibodies can be found in U.S. Patents 6,054,297, 5,886,152 and 5,877,293.

[0091] As used herein, the term "chimeric antibody" and related terms refer to an antibody comprising one or more regions of a first antibody and one or more regions of one or more other antibodies. In one embodiment, one or more of the CDRs are derived from a human antibody. In another embodiment, all CDRs are derived from a human antibody. In yet another embodiment, CDRs from more than one human antibody are mixed and matched in the chimeric antibody. For example, the chimeric antibody may comprise CDR1 from the light chain of a first human antibody, CDR2 and CDR3 from the light chain of a second human antibody, and CDRs from the heavy chain of a third antibody. In another instance, the CDRs are derived from different species, such as humans and mice, or humans and rabbits, or humans and goats. Those skilled in the art will appreciate that other combinations are possible.

[0092] Furthermore, the framework region may be derived from one of the same antibodies, from one or more different antibodies (such as human antibodies), or from humanized antibodies. In one example of a chimeric antibody, a portion of the heavy chain and / or light chain is identical, homologous, or derived from an antibody of a particular species or belonging to a particular antibody class or subclass, while the remaining portion of that chain is identical, homologous, or derived from an antibody of another species or belonging to another antibody class or subclass. This also includes fragments of such antibodies that exhibit the desired biological activity (i.e., the ability to specifically bind to a target antigen). Chimeric antibodies may be prepared from a portion of any of the anti-LAG3 antibodies described herein.

[0093] As used herein, the term "variant" polypeptide and "variant" of polypeptide refers to a polypeptide comprising one or more amino acid residues inserted, deleted, and / or substituted relative to a reference polypeptide sequence. Polypeptide variants include fusion proteins. In the same manner, variant polynucleotides comprise polypeptides comprising one or more nucleotide sequences inserted, deleted, and / or substituted relative to another polynucleotide sequence. Polynucleotide variants include fusion polynucleotides.

[0094] As used herein, the term "derivative" of polypeptide refers to a polypeptide (e.g., an antibody) that has been chemically modified, for example, by binding to another chemical moiety, such as polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation. Unless otherwise indicated, the term "antibody" includes, in addition to antibodies comprising two full-length heavy chains and two full-length light chains, its derivatives, variants, fragments, and mutant proteins, as exemplified below.

[0095] As used herein, the term "Fc" or "Fc region" refers to the portion of the constant region of an antibody heavy chain that begins within or after the hinge region and terminates at the C-terminus of the heavy chain. The Fc region includes at least a portion of the CH and CH3 regions and may or may not include a portion of the hinge region. Two polypeptide chains, each carrying half of the Fc region, can dimerize to form the complete Fc domain. The Fc domain can bind to Fc cell surface receptors and some proteins of the immune complement system. The Fc domain exhibits effector functions, including any one or any combination of two or more of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADP), phagocytosis, and / or cell binding. The Fc domain can bind Fc receptors, including FcγRI (e.g., CD64), FcγRII (e.g., CD32), and / or FcγRIII (e.g., CD16a).

[0096] As used herein, "labeled antibody" or related terms refer to antibodies and their antigen-binding portions that are unlabeled or conjugated to a detectable label or portion for detection, wherein the detectable label or portion is radioactive, colorimetric, antigenic, enzymatic, detectable beads (such as magnetic or electron-dense (e.g., gold) beads), biotin, streptavidin, or protein A. A variety of labels may be used, including but not limited to radionuclides, fluorescent agents, enzymes, enzyme receptors, enzyme cofactors, enzyme inhibitors, and ligands (e.g., biotin, haptens). Any anti-LAG3 antibody described herein may be unlabeled or conjugated to a detectable label or portion.

[0097] As used in this article, "percentage of similarity" or "percentage of homology" and related terms refer to a quantitative measurement of the similarity between two peptides or two polynucleotide sequences. The percentage of similarity between two peptide sequences is a function of the number of identical amino acids at the aligned positions shared by the two peptide sequences. Considering the number and length of nicks, it may be necessary to introduce nicks to optimize the alignment of the two peptide sequences. Similarly, the percentage of similarity between two polynucleotide sequences is a function of the number of identical nucleotides at the aligned positions shared by the two polynucleotide sequences. Considering the number and length of nicks, it may be necessary to introduce nicks to optimize the alignment of the two polynucleotide sequences. Sequence comparisons and determination of the percentage of similarity between two peptide sequences or two polynucleotide sequences can be achieved using mathematical algorithms. For example, the "percentage of similarity" or "percentage of homology" between two peptides or two polynucleotide sequences can be determined by comparing these sequences using the GAP computer program (GCG Wisconsin Package (version 10.3) (Accelrys, San Diego, Calif.)) with its default parameters. Statements such as "containing a sequence that is at least X% identical to Y" relative to a test sequence mean that when compared with the above sequence Y, the test sequence contains residues that are at least X% identical to the residues of Y. []

[0098] In one embodiment, the amino acid sequence of the test antibody may be similar to, but not identical to, the amino acid sequence of any of the light chain and / or heavy chain peptides constituting any of the anti-LAG3 antibody or its antigen-binding protein described herein. The similarity between the test antibody and the peptide may be at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% similarity to any of the light chain and / or heavy chain peptides constituting any of the anti-LAG3 antibody or its antigen-binding moiety described herein. In one embodiment, the similar peptide may contain amino acid substitutions within the heavy chain and / or light chain. In one embodiment, amino acid substitutions include one or more conserved amino acid substitutions. A "conserved amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue with a side chain (R group) having similar chemical properties (e.g., charge or hydrophobicity). Generally, conserved amino acid substitutions do not substantially alter the functional properties of the protein. Skilled technicians can introduce up to 5% of conserved and / or non-conserved amino acid substitutions into the variable regions of the heavy chain and / or light chain without negatively affecting the physical structure, binding capacity, or cell-killing ability of the antibody. Well-known methods for identifying and performing conserved amino acid substitutions in the variable regions designed to preserve or modify antibody binding characteristics are described in: Brummel et al., 1993 Biochemistry 32:1180-1187; Kobayashi et al., 1999 Protein Engineering 12(10):879-884; and Burks et al., 1997 Proc. Natl. Acad. Sci. USA 94:412-417. Methods for identifying and performing non-conserved amino acid substitutions in the variable regions of the heavy chain and / or light chain to preserve or modify antigen binding are also known (Near et al., 1993 Molecular Immunology 30(4):369-377). Therefore, skilled practitioners can predict and modify up to 5% of the amino acids in the heavy chain variable region and / or light chain variable region without significantly reducing the antigen-binding ability of the antibody. Where two or more amino acid sequences differ from each other through conserved substitution, the percentage of sequence identity or similarity can be adjusted upwards to correct for the conservatism of the substitution. Those skilled in this technique are familiar with the methods for making such adjustments. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331, which is incorporated herein by reference in its entirety.Examples of amino acid groups with side chains having similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxy side chains: serine and threonine; (3) acetylamine side chains: aspartic acid and glutamic acid; (4) aromatic side chains: phenylalanine, tyrosine and tryptophan; (5) basic side chains: lysine, arginine and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine ​​and methionine. []

[0099] Antibodies can be obtained from sources containing immunoglobulins with varying antigen specificity, such as serum or plasma. If such antibodies are affinity purified, they can be enriched for specific antigen specificity. Such antibody-rich formulations typically consist of less than about 10% antibodies with specific binding activity against a particular antigen. Repeating affinity purification on these formulations can increase the proportion of antibodies with specific binding activity against the antigen. Antibodies prepared in this manner are commonly referred to as "monospecific." Monospecific antibody formulations can consist of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 99.9% antibodies with specific binding activity against a particular antigen. Antibodies can be produced using recombinant nucleic acid technology as described below.

[0100] As used herein, "vector" and related terms refer to nucleic acid molecules (e.g., DNA or RNA) that are operatively linked to foreign genetic material (e.g., nucleic acid transgenes). Vectors can be used as mediators for introducing foreign genetic material into cells (e.g., host cells). Vectors may include at least one restriction endonuclease recognition sequence for inserting transgenes into the vector. Vectors may include at least one gene sequence conferred with antibiotic resistance or selectable features to aid in the selection of host cells carrying vector-transgene constructs. Vectors may be single-stranded or double-stranded nucleic acid molecules. Vectors may be linear or circular nucleic acid molecules. Donor nucleic acids used in gene editing methods employing zinc finger nucleases, TALEN, or CRISPR / Cas may be a type of vector. One type of vector is a "plast," which refers to a linear or circular double-stranded extrachromosomal DNA molecule that can be linked to a transgene and is capable of replicating, transcribing, and / or translating the transgene in a host cell. Viral vectors typically contain a viral RNA or DNA backbone sequence that can be linked to a transgene. The viral backbone sequence may be modified to disable infection but retain the viral backbone and co-linked transgenes for insertion into the host cell genome. Examples of viral vectors include retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, baculoviruses, papovaviral viruses, vaccinia virus, herpes simplex virus, and Epstein-Barr viral vectors. Some vectors can replicate autonomously in the host cells to which they are introduced (e.g., bacterial vectors containing bacterial origins of replication and free-living mammalian vectors). Upon introduction into a host cell, other vectors (e.g., non-free-living mammalian vectors) are integrated into the host cell's genome and thereby replicate along with the host genome. []

[0101] "Expression vector" is a vector that may contain one or more regulatory sequences (such as inducible and / or constitutive promoters and enhancers). An expression vector may include a ribosome-binding site and / or adenosine monophosphate (AMP) site. The regulatory sequence directs the transcription, or transcription and translation, of a transgene linked to the expression vector transduced into a host cell. The regulatory sequence controls the degree, timing, and / or location of transgene expression. The regulatory sequence may exert its effect, for example, directly on the transgene or through the action of one or more other molecules (e.g., polypeptides that bind to the regulatory sequence and / or nucleic acid). The regulatory sequence may be part of the vector. Other examples of regulatory sequences are described, for example, in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif., and Baron et al., 1995, Nucleic Acids Res. 23:3605-3606. An expression vector may contain a nucleic acid encoding at least a portion of any of the light chain, heavy chain, or anti-LAG3 antibody described herein. []

[0102] This document describes a vector (e.g., an expression vector) operatively linked to a nucleic acid encoding an antibody light chain, antibody heavy chain, anti-LAG3 antibody, or an antigen-binding portion thereof. In one embodiment, the vector is operatively linked to a nucleic acid encoding a heavy chain variable region having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4, 6, 8, or 10. In one embodiment, the nucleic acid encodes a light chain variable region having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 5, 7, 9, or 11. []

[0103] The transgenic line is "operably linked" to the vector when a link exists between the transgene and the vector to allow the functionalization or expression of the transgene sequence contained in the vector. In one embodiment, the transgenic line is "operably linked" to the regulatory sequence when the regulatory sequence affects the expression of the transgene (e.g., the degree, timing, or location of expression). []

[0104] As used herein, the terms "transfected," "transformed," "transduced," or other related terms refer to the process of transferring or introducing a foreign nucleic acid (e.g., a transgenic gene) into a host cell. A "transfected," "transformed," or "transduced" host cell is one that has been transfected, transformed, or transduced with a foreign nucleic acid (transgenic gene). Host cells include primary individual cells and their progeny. A foreign nucleic acid encoding at least a portion of any of the light chain, heavy chain, or anti-LAG3 antibody described herein may be introduced into a host cell. An expression vector containing at least a portion of any of the light chain, heavy chain, or anti-LAG3 antibody described herein may be introduced into a host cell, and the host cell may express a polypeptide containing at least a portion of the light chain, heavy chain, or anti-LAG3 antibody. []

[0105] As used herein, the terms "host cell" or "host cell population" or related terms refer to cells (or populations thereof) in which foreign (exogenous or transgenic) nucleic acids have been introduced. The foreign nucleic acid may include an expression vector operatively linked to a transgene, and the host cell may be used to express nucleic acids and / or polypeptides encoded by the foreign nucleic acid (transgene). Host cells (or populations thereof) may be cultured cells or may be derived from an individual. Host cells (or populations thereof) include primary individual cells and their progeny without regard to the number of subcultures. Progeny cells may or may not carry the same genetic material as the parent cells. Host cells include progeny cells. In one embodiment, host cell describes any cell (including its progeny) that has been modified, transfected, transduced, transformed, and / or manipulated in any way to express an antibody, as disclosed herein. In one instance, host cells (or populations thereof) may be introduced using an expression vector operatively linked to a nucleic acid encoding a desired antibody or its antigen-binding portion as described herein. Host cells and populations thereof may carry an expression vector stably integrated into the host's genome or may carry an extrachromosomal expression vector. In one embodiment, the host cell and its population may carry an extrachromosomal vector that is present after several cell divisions, or is present momentarily and then present, or is lost after several cell divisions. []

[0106] Transgenic host cells can be prepared using non-viral methods, including well-known engineered nucleases (including zinc finger nucleases), TALENS, or CRISPR / Cas. Genome editing technologies (such as zinc finger nucleases) can be used to introduce transgenes into the host cell's genome. Zinc finger nucleases comprise a pair of chimeric proteins, each containing a non-specific endonuclease domain fused to a restriction endonuclease (e.g., FokI) derived from the DNA-binding domain of an engineered zinc finger motif. The DNA-binding domain can be engineered to bind to a specific sequence in the host genome, and the endonuclease domain performs double-stranded cleavage. The donor DNA carries the transgene (e.g., encoding either the CAR or DAR construct described herein) and a side-joint sequence homologous to a region on either side of the desired insertion site in the host cell's genome. The host cell's DNA repair mechanisms can precisely insert the transgene via homologous DNA repair. Zinc finger nucleases have been used to prepare transgenic mammalian host cells (US Patents 9,597,357, 9,616,090, 9,816,074, and 8,945,868). Transgenic host cells can be prepared using TALENs (transcription activator-like effector nucleases), which are similar to zinc finger nucleases in that they contain a non-specific endonuclease domain fused to a DNA-binding domain that delivers precise transgenic insertion. Similar to zinc finger nucleases, TALENs also introduce double-stranded cleavage into the host DNA. Transgenic host cells can also be prepared using CRISPR (regularly spaced clustered short palindromic repeats). CRISPR employs a Cas endonuclease coupled to a guide RNA for target-specific donor DNA integration. The guide RNA comprises a conserved polynucleotide containing a prototypical spacer adjacent motif (PAM) sequence upstream of the gRNA-binding region in the target DNA and hybridizes to the host cell target site, where the Cas endonuclease cleaves the double-stranded target DNA. The guide RNA can be designed to hybridize to a specific target site. Similar to zinc finger nucleases and TALEN, CRISPR / Cas systems can be used to introduce site-specific insertions of donor DNA having side-joint sequences homologous to the insertion site. Examples of CRISPR / Cas systems for genome modification are described, for example, in U.S. Patent Nos. 8,697,359, 10,000,772, 9,790,490, and U.S. Patent Application Publication No. US 2018 / 0346927. In one embodiment, transgenic host cells can be prepared using zinc finger nucleases, TALEN, or CRISPR / Cas systems, and the host target site can be the TRAC gene (T cell receptor α constant region). The donor DNA can contain, for example, any of the nucleic acids encoding the CAR or DAR constructs described herein. The donor DNA can be co-delivered into host cells using zinc finger nucleases, TALEN, or CRISPR / Cas systems via electroporation, nuclear staining, or lipid transfection.

[0107] The host cell may be a prokaryote, such as *Escherichia coli*, or a eukaryote, such as a single-celled eukaryote (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cells), a mammalian cell (e.g., human cells, monkey cells, hamster cells, rat cells, mouse cells, or insect cells), or a fusion tumor. In one embodiment, the host cell may be introduced with an expression vector operatively linked to a nucleic acid encoding a desired antibody, thereby generating a transfected / transformed host cell, which is then cultured under conditions suitable for expressing the antibody via the transfected / transformed host cell, and the antibody is recovered from the transfected / transformed host cell (e.g., from host cell lysate) or from the culture medium as needed. In one embodiment, the host cell comprises non-human cells, including CHO, BHK, NSO, SP2 / 0, and YB2 / 0. In one embodiment, the host cell comprises human cells, including HEK293, HT-1080, Huh-7, and PER.C6. Examples of host cells include the COS-7 monkey kidney cell line (ATCC CRL 1651) (see Gluzman et al., 1981, Cell 23: 175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells or their derivatives such as Veggie CHO and related cell lines grown in serum-free medium (see Rasmussen et al., 1998, Cytotechnology 28:31) or CHO strain DX-B 11, which is deficient in DHFR (see Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216-20), HeLa cells, the BHK (ATCC CRL 10) cell line, and the CV1 / EBNA cell line derived from the African green monkey kidney cell line CV1 (ATCC CCL 70) (see McMahan et al., 1991, EMBO J). 10:2821), human embryonic kidney cells, such as 293, 293 EBNA, or MSR 293, human epidermal A431 cells, human Colo 205 cells, other transformed primate cell lines, normal diploid cells, in vitro cultured cell lines derived from primary tissues, primary explants, HL-60, U937, HaK, or Jurkat cells. In one embodiment, the host cell includes lymphoid cells such as Y0, NSO, or Sp20. In one embodiment, the host cell is a mammalian host cell, but not a human host cell. Typically, the host cell is a cultured cell that can be transformed or transfected with a nucleic acid encoding a polypeptide, which can then be expressed in the host cell. The phrases "transgenic host cell" or "recombinant host cell" can be used to refer to a host cell that has been transformed or transfected with a nucleic acid to be expressed.Host cells can also be cells that contain nucleic acids but do not express them to the desired extent, unless regulatory sequences are introduced into the host cell to make it operatively linked to nucleic acids. It should be understood that the term host cell refers not only to a specific individual cell but also to its offspring or potential offspring. Because certain modifications can occur in offspring due to, for example, mutations or environmental influences, such offspring may indeed differ from the parent cell, but are still included within the scope of the terminology used herein. []

[0108] This document describes a host cell or population of host cells carrying a vector (e.g., an expression vector) operatively linked to a nucleic acid encoding an antibody light chain, antibody heavy chain, anti-LAG3 antibody, or an antigen-binding portion thereof. In one embodiment, the host cell carries a nucleic acid operatively linked to a heavy chain variable region comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4, 6, 8, or 10. In one embodiment, the nucleic acid encodes a light chain variable region comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 5, 7, 9, or 11. []

[0109] The polypeptides (e.g., antibodies and antigen-binding proteins) of this invention can be produced using any method known in this art. In one example, the polypeptide is produced by a recombinant nucleic acid method, by inserting a nucleic acid sequence (e.g., DNA) encoding the polypeptide into a recombinant expression vector, introducing the recombinant expression vector into a host cell, and allowing the host cell to express the polypeptide under conditions that promote expression.

[0110] General techniques for manipulating recombinant nucleic acids are described, for example, in Sambrook et al., *Molecular Cloning: A Laboratory Manual*, Volumes 1–3, Cold Spring Harbor Laboratory Press, 2nd ed., 1989, or F. Ausubel et al., *Current Protocols in Molecular Biology* (Green Publishing and Wiley-Interscience: New York, 1987), and are regularly updated, and are incorporated herein by reference in their entirety. Nucleic acids encoding polypeptides (e.g., DNA) are operatively linked to an expression vector carrying one or more suitable transcriptional or translational regulatory elements derived from mammalian, viral, or insect genes. Such regulatory elements include transcription promoters, optional operator sequences controlling transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences controlling the termination of transcription and translation. Expression vectors may include origins or replication vectors that confer the ability to replicate in host cells. Expression vectors may include genes that confer selection to promote recognition of transgenic host cells (e.g., transformants).

[0111] Recombinant DNA can also encode any type of protein tag sequence that can be used to purify proteins. Examples of protein tags include, but are not limited to, histidine tags, FLAG tags, myc tags, HA tags, or GST tags. Suitable selection and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts can be found in Cloning Vectors: A Laboratory Manual (Elsevier, NY, 1985).

[0112] The expression vector construct can be introduced into host cells using methods suitable for the host cell. Various methods are known in this technology for introducing nucleic acids into host cells, including but not limited to electroporation; transfection with calcium chloride, rubidium chloride, calcium phosphate, DEAE-glucan, or other substances; viral transfection; non-viral transfection; microprojectile bombardment; lipid transfection; and infection (e.g., where the vector is an infectious agent). Suitable host cells include prokaryotes, yeast, mammalian cells, or bacterial cells.

[0113] Suitable bacteria include Gram-negative or Gram-positive organisms such as *Escherichia coli* or *Bacillus* spp. Yeast (preferably from *Saccharomyces* species, such as *Saccharomyces cerevisiae* cells) can also be used to produce peptides. Various mammalian or insect cell culture systems can also be used to express recombinant proteins. Luckow and Summers (Bio / Technology, 6:47, 1988) commented on baculovirus systems used to produce heterologous proteins in insect cells. Examples of suitable mammalian host cell lines include endothelial cells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3, Chinese hamster ovary (CHO), human embryonic kidney cells, HeLa, 293, 293T, and BHK cell lines. Purified peptide lines are prepared by culturing suitable host / vector systems to express recombinant proteins. For many applications, the small size of many peptides disclosed in this paper presents a preferred expression method in *E. coli*. Proteins are then purified from culture medium or cell extracts. Any of the light chain, heavy chain, or anti-LAG3 antibody or its antigen-binding protein can be expressed via transgenic host cells.

[0114] The antibodies and antigen-binding proteins revealed in this article can also be produced using cell translation systems. For this purpose, the nucleic acids encoding the polypeptides must be modified to allow in vitro transcription to produce mRNA and to allow cell-free translation of the mRNA in specific cell-free systems (eukaryotic systems such as mammalian or yeast cell-free translation systems or prokaryotic systems such as bacterial cell-free translation systems)).

[0115] The nucleic acids encoding any of the various polypeptides disclosed herein are chemically synthesizable. Codons can be selectively used to modify expression in cells. This codon usage will depend on the cell type selected. Specific codon usage patterns have been developed for Escherichia coli and other bacteria, as well as mammalian cells, plant cells, yeast cells, and insect cells. See, for example: Mayfield et al., Proc. Natl. Acad. Sci. USA. 2003 100(2):438-42; Sinclair et al. Protein Expr. Purif. 2002 (1):96-105; Connell N D. Curr. Opin. Biotechnol. 2001 12(5):446-9; Makrides et al. Microbiol. Rev. 1996 60(3):512-38; and Sharp et al. Yeast. 1991 7(7):657-78.

[0116] The antibodies and antigen-binding proteins described in this article can also be produced by chemical synthesis (e.g., by the method described in Solid Phase Peptide Synthesis, 2nd Edition, 1984, The Pierce Chemical Co., Rockford, Ill.). Protein modifications can also be produced by chemical synthesis.

[0117] The antibodies and antigen-binding proteins described herein can be purified using protein isolation / purification methods generally known in the field of protein chemistry. Non-limiting examples include extraction, recrystallization, salting out (e.g., with ammonium sulfate or sodium sulfate), centrifugation, dialysis, ultrafiltration, adsorption chromatography, ion exchange chromatography, hydrophobic chromatography, normal-phase chromatography, reverse-phase chromatography, gel filtration, gel permeation chromatography, affinity chromatography, electrophoresis, countercurrent distribution, or any combination thereof. After purification, the peptides can be exchanged into different buffers and / or concentrated using any of the various methods known in this art (including, but not limited to, filtration and dialysis).

[0118] The purified antibodies and antigen-binding proteins described herein are preferably at least 65% pure, at least 75% pure, at least 85% pure, more preferably at least 95% pure, and most preferably at least 98% pure. Regardless of the exact value of purity, the peptides are pure enough for use in pharmaceutical products. Any of the light chain, heavy chain, or anti-LAG3 antibodies or their antigen-binding proteins described herein can be expressed in transgenic host cells and then purified to approximately 65 to 98% purity or higher using any method known in this art.

[0119] In some embodiments, the antibodies and antigen-binding proteins described herein may further include post-translational modifications. Exemplary post-translational protein modifications include phosphorylation, acetylation, methylation, ADP-ribosylation, ubiquitination, glycosylation, carbonylation, ubiquitin-like glycosylation, biotinylation, or the addition of peptide side chains or hydrophobic groups. Thus, the modified peptides may contain non-amino acid elements, such as lipids, polysaccharides, or monosaccharides and phosphates. A preferred form of glycosylation is sialylation, which binds one or more sialic acid moieties to the peptide. The sialic acid moieties improve solubility and serum half-life while also reducing the potential immunogenicity of the protein. See Raju et al. Biochemistry. 2001 31;40(30):8868-76.

[0120] In one embodiment, the antibodies and antigen-binding proteins described herein may be modified into soluble peptides, comprising linking the antibodies and antigen-binding proteins to a non-protein polymer. In one embodiment, the non-protein polymer includes polyethylene glycol (“PEG”), polypropylene glycol, or polyoxyalkylene oxide, in the manner described in Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192, or 4,179,337.

[0121] PEG is a water-soluble polymer that is commercially available or can be prepared by ring-opening polymerization of ethylene glycol using methods well known in this art (Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, pp. 138-161). The term "PEG" is used broadly to cover any polyethylene glycol molecule, regardless of size or modification at the PEG terminus, and can be represented by the following formula: X—O(CH2 CH2 O)n—CH2 CH2 OH (1), where n is 20 to 2300 and X is H or a terminal modification, such as a C1-4 alkyl group. In one embodiment, PEG is terminated at one end with a hydroxyl or methoxy group, i.e., X is H or CH3 ("methoxyPEG"). PEG may contain other chemical groups required for the binding reaction; this is due to the chemical synthesis of the molecule; or it may be a spacer group at the optimal distance from the part of the molecule. In addition, such PEG may consist of one or more PEG side chains linked together. PEG having more than one PEG chain is called multi-arm or branched PEG. Branched PEGs can be prepared, for example, by adding polyethylene oxide to various polyols, including glycerol, neopentyl tertrol, and sorbitol. For example, tetra-armed branched PEGs can be prepared from neopentyl tertrol and ethylene oxide. Branched PEGs are described, for example, in EP-A 0 473 084 and U.S. Patent No. 5,932,462. One form of PEG comprises two PEG side chains (PEG2) linked via a primary amino group of a lysine (Monfardini et al., Bioconjugate Chem. 6 (1995) 62-69).

[0122] The serum clearance rate of PEG-modified peptides relative to the clearance rate of unmodified antibodies and antigen-binding proteins binding to the peptides can be adjusted (e.g., increased or decreased) by approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or even 90%. PEG-modified antibodies and antigen-binding proteins may have a prolonged half-life (t1 / 2) relative to the half-life of unmodified peptides. The half-life of PEG-modified peptides can be prolonged by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, or 500%, or even up to 1000%, relative to the half-life of unmodified antibodies and antigen-binding proteins. In some embodiments, the protein half-life is determined in vitro, such as in buffered saline solution or in serum. In other embodiments, the protein half-life is the in vivo half-life, such as the half-life of the protein in the serum or other body fluids of an animal.

[0123] This invention provides therapeutic compositions comprising any of the light chains, heavy chains, or anti-LAG3 antibodies or their antigen-binding proteins described herein, and pharmaceutically acceptable excipients. Excipients encompass carriers, stabilizers, and excipients. Examples of pharmaceutically acceptable excipients include, for example, inert diluents or fillers (e.g., sucrose and sorbitol), lubricants, slip agents, and anti-adhesion agents (e.g., magnesium stearate, zinc stearate, stearic acid, silicon dioxide, hydrogenated vegetable oil, or talc). Other examples include buffers, stabilizers, preservatives, nonionic detergents, antioxidants, and isotonic agents.

[0124] Therapeutic compositions and methods of their preparation are well known in the art and can be found, for example, in "Remington: The Science and Practice of Pharmacy" (20th edition, edited by AR Gennaro A R., 2000, Lippincott Williams & Wilkins, Philadelphia, Pa.). Therapeutic compositions formulated for non-enteral administration may contain, and may contain, excipients, sterile water, saline, polyalkylene glycols (such as polyethylene glycol), plant-derived oils, or hydrogenated naphthalene. Biocompatible, biodegradable lactide polymers, lactide / glycolic acid copolymers, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the antibodies (or their antigen-binding portions) described herein. Nanoparticle formulations (e.g., biodegradable nanoparticles, solid lipid nanoparticles, liposomes) may be used to control the biodistribution of the antibodies (or their antigen-binding portions). Other potentially suitable non-enteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. The concentration of antibodies (or their antigen-binding proteins) in a formulation varies depending on many factors, including the dosage and route of administration of the drug to be administered.

[0125] Any of the anti-LAG3 antibodies (or their antigen-binding portions) may be administered as a pharmaceutically acceptable salt (such as non-toxic acid addition salts or metal complexes commonly used in the pharmaceutical industry). Examples of acid addition salts include organic acids such as acetic acid, lactic acid, pyric acid, maleic acid, citric acid, malic acid, ascorbic acid, succinic acid, benzoic acid, palmitic acid, succinic acid, salicylic acid, tartaric acid, methanesulfonic acid, toluenesulfonic acid, or trifluoroacetic acid or the like; polymeric acids such as tannic acid, carboxymethyl cellulose or the like; and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, or the like. Metal complexes include zinc, iron, and the like. In one example, the antibody (or its antigen-binding portion) is formulated in the presence of sodium acetate to increase thermal stability.

[0126] Any of the anti-LAG3 antibodies (or their antigen-binding portions) that can be formulated for oral use include tablets containing a mixture of the active ingredient and a non-toxic, pharmaceutically acceptable excipient. Formulations for oral use may also be provided as chewable tablets, hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, or soft gelatin capsules in which the active ingredient is mixed with an aqueous or oil-based medium.

[0127] As used herein, the term "individual" refers to humans and non-human animals, including vertebrates, mammals, and non-mammals. In one embodiment, an individual may be a human, a non-human primate, ape, monkey, rodent (e.g., mouse and rat), cow, pig, horse, canine, feline, goat, wolf (lupine), frog, or fish.

[0128] The terms "administering" and their grammatical variations refer to the physical introduction of a pharmaceutical agent into an individual using any of the various methods and delivery systems known to those skilled in the art. Exemplary administration routes of the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other non-enteric administration routes, such as by injection or infusion. As used herein, the phrase "non-enteric administration" means administration modes other than enteric and local administration (usually by injection), and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrasheath, intralymphatic, intralesional, intracystic, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, intra-articular, subcystic, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, as well as intracorporeal electroporation. In some embodiments, the formulation is administered via a non-enteric route, such as orally. Other non-enteral routes include local, epidermal, or mucosal administration, such as intranasal, vaginal, rectal, sublingual, or local administration. Administration may also be performed, for example, once, multiple times, and / or over one or more extended periods. Any of the anti-LAG3 antibodies (or their antigen-binding proteins) described herein may be administered to individuals using methods and routes of delivery known to this technology.

[0129] The terms "effective amount," "therapeutic effective amount," or "effective dose," or related terms, are used interchangeably and refer to the amount of an antibody or antigen-binding protein (such as any of the anti-LAG3 antibody or its antigen-binding moiety described herein) sufficient, when administered to an individual, to achieve a measurable improvement or prevention of a disease or ailment associated with tumor or cancer antigen expression. When used alone or in combination, the therapeutically effective amount of the antibodies described herein will depend on the relative activity of the antibody and combination (e.g., in inhibiting cell growth) and will vary depending on the individual being treated and the nature of the disease, the individual's weight, age, and sex, the severity of the individual's disease, the method of administration, and similar factors, which can be readily determined by someone skilled in the art. []

[0130] In one embodiment, the effective therapeutic dose will depend on certain characteristics of the individual being treated and the disease being treated, and can be determined by a person skilled in the art using known techniques. Generally, the peptide is administered at a dose of about 0.01 g / kg / day to about 50 mg / kg / day, preferably 0.01 mg / kg / day to about 30 mg / kg / day, and most preferably 0.1 mg / kg / day to about 20 mg / kg / day. The peptide can be administered daily (e.g., once daily, twice daily, three times daily, or four times daily) or preferably less frequently (e.g., weekly, bi-weekly, tri-weekly, monthly, or quarterly). Additionally, as is known in the art, adjustments may be necessary based on age and weight, general health, sex, diet, administration time, drug interactions, and disease severity. []

[0131] This invention provides a method for treating an individual suffering from a disease associated with the expression of LAG3. The disease includes cancer or tumor cells expressing tumor-associated antigens. In one embodiment, the cancer or tumor includes prostate cancer, breast cancer, ovarian cancer, head and neck cancer, bladder cancer, skin cancer, colorectal cancer, anal cancer, rectal cancer, pancreatic cancer, lung cancer (including non-small cell lung cancer and small cell lung cancer), leiomyomas, brain cancer, gliomas, glioblastomas, esophageal cancer, liver cancer, kidney cancer, stomach cancer, colon cancer, cervical cancer, uterine cancer, endometrial cancer, vulvar cancer, laryngeal cancer, vaginal cancer, bone cancer, nasal cavity cancer, paranasal sinus cancer, nasopharyngeal cancer, oral cavity cancer, oropharyngeal cancer, laryngeal cancer, lower larynx cancer, salivary gland cancer, ureteral cancer, urethral cancer, penile cancer, and testicular cancer.

[0132] In one embodiment, cancer includes hematologic cancers, including leukemia, lymphoma, myeloma, and B-cell lymphoma. Hematologic cancers include multiple myeloma (MM), non-Hodgkin's lymphoma (NHL) (including Burkitt's lymphoma (BL)), B-cell chronic lymphocytic leukemia (B-CLL), systemic lupus erythematosus (SLE), B- and T-cell acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma, chronic myeloid leukemia (CML), hairy cell leukemia (HCL), follicular lymphoma, Waldenstrom's macroglobulinemia, mantle cell lymphoma, and Hodgkin's lymphoma. (HL), plasma cell myeloma, precursor B-cell lymphoblastic leukemia / lymphoma, plasmacytoma, giant cell myeloma, plasma cell myeloma, heavy chain myeloma, light chain or Bence-Jones myeloma, lymphomatoid granulomatosis, post-transplant lymphoproliferative disorders, immune dysregulation, rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, antiphospholipid syndrome, Chagas' disease, Graves' disease, Wegener's granulomatosis, polyarteritis nodosa, Sjogren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, antiphospholipid syndrome, ANCA-associated vasculitis, Goodpasture's disease, Kawasaki disease Diseases, autoimmune hemolytic anemia and rapidly progressive glomerulonephritis, heavy chain disease, primary or immune cell-associated amyloidosis, and monoclonal immunoglobulin elevation of unknown significance.

[0133] This invention provides engineered LAG3-binding proteins that specifically bind to LAG3, particularly anti-LAG3 antibodies or their antigen-binding portions, and their uses. The anti-LAG3 antibodies exhibit modified characteristics, including modified binding to the LAG3 antigen, modified binding to LAG3-expressing cells, and / or a higher degree of cytokine-releasing activity. The anti-LAG3 antibodies can cross-react (bind) with LAG3 antigens in cynomolgus monkeys and / or mice. []

[0134] This invention provides a fully human anti-LAG3 antibody and its fragments, which can act as an immune checkpoint inhibitor and can be used in immunotherapy for the treatment of diseases such as cancer. []

[0135] In some embodiments, the present invention provides a fully human antibody of an antigen-binding protein, such as an IgG class, which binds to an antigenic determinant of a LAG3 polypeptide (e.g., a target antigen) or a fragment of the LAG3 polypeptide. In one embodiment, the LAG3 target antigen comprises a polypeptide having a wild-type or polymorphic or mutant amino acid sequence. In one embodiment, the LAG3 target antigen comprises a human LAG3 polypeptide (UniProtKB P18627; SEQ ID NO:1), a cynomolgus monkey LAG3 polypeptide (NCBI accession number XP 005570011.1; SEQ ID NO:2), or a mouse LAG3 polypeptide (UniProtKB Q61790; SEQ ID NO:3), or a fragment of any of these LAG3 polypeptides shown in Figure 1. The LAG3 target antigen may be prepared by recombinant methods or chemically synthesized. The LAG3 target antigen may be in a soluble form or a membrane-bound form (e.g., expressed by cells or phages). In one embodiment, the LAG3 target antigen comprises an extracellular portion of a cell surface LAG3 antigen. In one embodiment, the LAG3 target antigen is expressed by cells (e.g., cancer cell lines or non-cancer cell lines that naturally express LAG3) or by cell lines engineered to express LAG3 (such as CHO, HeLa, HEK293, Jurkat, or Panoply™ (from Creative Biogene, Shirley, New York)). Cell lines that do not naturally express LAG3 are not expected to bind to anti-LAG3 antibodies. The LAG3 target antigen may be a fusion protein or, for example, bound to a detectable moiety (such as a fluorophore). The LAG3 target antigen may be a recombinant polypeptide with or without an affinity tag (such as a histidine tag). The LAG3 target antigen may be a biotinylated LAG3 protein from humans, mice, or cynomolgus monkeys (e.g., catalog number LA3-H82Fb, biotinylated human LAG3 protein from AcroBiosystems). []

[0136] In one embodiment, wild-type and / or mutant human LAG3 antigens can be used in analyses comparing the binding ability of any of the anti-LAG3 antibodies described herein and / or in antigenic determinant mapping analyses. []

[0137] This invention provides antigen-binding proteins, such as fully human antibodies of the IgG class, wherein the antibody binds to the antigenic determinant of a LAG3 polypeptide (target antigen), wherein the antibody comprises a heavy chain variable region having at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity with the amino acid sequences or combinations thereof of SEQ ID NO: 4, 6, 8, or 10, and the anti-LAG3 antibody comprises a light chain variable region having at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity with the amino acid sequences or combinations thereof of SEQ ID NO: 5, 7, 9, or 11, respectively. The amino acid sequences of the heavy chain variable regions and light chain variable regions of antibodies 1C5-3A6, 2D12-2E3-N102D, 5C8, and 8D9 are shown in Figure 2. []

[0138] The present invention provides an antigen-binding protein, such as a fully human antibody of the IgG class, wherein the antibody binds an antigenic determinant of a LAG3 polypeptide (target antigen), wherein the antibody comprises a weight-variable region having at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity with an amino acid sequence or combination thereof of SEQ ID NO: 4, 6, 8 or 10. []

[0139] The present invention provides an antigen-binding portion, such as a fully human antibody of the IgG class, wherein the antibody binds an antigenic determinant of a LAG3 polypeptide (target antigen), wherein the antibody comprises a light chain variable region having 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity with an amino acid sequence or combination thereof of SEQ ID NO: 5, 7, 9 or 11. []

[0140] In one embodiment, the anti-LAG3 antibody is an isolated antibody. In another embodiment, the anti-LAG3 antibody is a recombinant antibody. []

[0141] In one embodiment, the anti-LAG3 antibody comprises an IgG1, IgG2, IgG3, or IgG4 class antibody. In one embodiment, the anti-LAG3 antibody comprises an IgG1 or IgG4 class antibody. In one embodiment, the hinge region of the anti-LAG3 antibody may be mutated to change the number of potential disulfide bonds formed. In one embodiment, the anti-LAG3 antibody comprises a hinge region having an amino acid sequence CPPC, CPSC, SPPC, or SPSC. In one embodiment, the anti-LAG3 antibody comprises a heavy chain constant region having a hinge region in which the amino acid sequence CPSC, SPPC, or SPSC is substituted for the CPPC sequence. In one embodiment, the heavy chain of the anti-LAG3 antibody may be mutated to eliminate one or more known isomerizations of NG motifs (e.g., as part of an NGR motif). In one embodiment, the isomerization site may bind an integrin. In one embodiment, the anti-LAG3 antibody comprises a heavy chain including an SGR motif replacing an NGR motif. In one embodiment, the anti-LAG3 antibody comprises a heavy chain variable region in which an SGR motif replaces an NGR motif. In one embodiment, the heavy and / or light chains of the anti-LAG3 antibody may be mutated, and the mutated antibody exhibits the same or similar binding ability to the LAG3 antigen and / or LAG3-expressing cells. []

[0142] In one embodiment, the anti-LAG3 antibody or a fragment thereof comprises an antigen-binding moiety that binds to the antigenic determinant of the LAG3 polypeptide (target antigen) with a binding affinity (KD) of 10⁻⁶ M or less, 10⁻⁷ M or less, 10⁻⁸ M or less, 10⁻⁹ M or less, or 10⁻¹⁰ M or less (see Figure 4). In one embodiment, the binding between anti-LAG3 antibodies or fragments thereof can be detected and measured using surface plasma resonance, flow cytometry, and / or ELISA. []

[0143] This invention provides an anti-LAG3 antibody that binds to an antigenic determinant of a human LAG3 polypeptide and is capable of binding (e.g., cross-reacting) to an antigenic determinant of a LAG3 polypeptide (e.g., a homologous antigen) from at least one of non-human animals (such as mice, rats, goats, rabbits, hamsters, and / or monkeys (e.g., cynomolgus monkeys)). In one embodiment, the anti-LAG3 antibody binds to mouse LAG3 with a binding affinity KD of 10⁻⁵ M or less, or 10⁻⁶ M or less, or 10⁻⁷ M or less, or 10⁻⁸ M or less, or 10⁻⁹ M or less, or 10⁻¹⁰ M or less. In another embodiment, the anti-LAG3 antibody binds to cynomolgus monkey LAG3 with a binding affinity KD of 10⁻⁵ M or less, or 10⁻⁶ M or less, or 10⁻⁷ M or less, or 10⁻⁸ M or less, or 10⁻⁹ M or less, or 10⁻¹⁰ M or less.

[0144] This invention provides a fully human antibody that binds to a LAG3 peptide, wherein the antibody comprises both a heavy chain and a light chain, and wherein the amino acid sequence of the heavy / light chain variable region has at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with any of the following amino acid sequences: SEQ ID NO: 4 and 5 (hereinafter referred to as 1C5-3A6), SEQ ID NO: 6 and 7 (hereinafter referred to as 2D12-2E3-N102D), SEQ ID NO: 8 and 9 (hereinafter referred to as 5C8), or SEQ ID NO: 10 and 11 (hereinafter referred to as 8D9). The amino acid sequences of the heavy chain variable region and light chain variable region of antibodies 1C5-3A6, 2D12-2E3-N102D, 5C8, and 8D9 are shown in Figure 2. []

[0145] This invention provides a Fab fully human antibody fragment comprising a variable region from the antibody heavy chain and a variable region from the antibody light chain. In some embodiments, the sequence of the variable region from the heavy chain has at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identity with the amino acid sequences or combinations thereof of SEQ ID NO: 4, 6, 8, or 10, and the sequence of the variable region from the light chain has at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identity with the amino acid sequences or combinations thereof of SEQ ID NO: 5, 7, 9, or 11. In some embodiments, the sequence from the variable region of the heavy chain has at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% similarity to the amino acid sequences or combinations thereof of SEQ ID NO: 4, 6, 8, or 10, and the sequence from the variable region of the light chain has at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% similarity to the amino acid sequences or combinations thereof of SEQ ID NO: 5, 7, 9, or 11. The amino acid sequences of the variable regions of the heavy and light chains of antibodies 1C5-3A6, 2D12-2E3-N102D, 5C8, and 8D9 are shown in Figure 2. []

[0146] This invention provides a Fab fully human antibody fragment comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of the heavy chain / light chain variable regions have at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% similarity to any of the following amino acid sequences: SEQ ID NO: 4 and 5 (hereinafter referred to as 1C5-3A6), SEQ ID NO: 6 and 7 (hereinafter referred to as 2D12-2E3-N102D), SEQ ID NO: 8 and 9 (hereinafter referred to as 5C8), or SEQ ID NO: 10 and 11 (hereinafter referred to as 8D9). []

[0147] This invention provides a single-chain fully human antibody comprising a polypeptide chain having a variable region from a fully human heavy chain and a variable region from a fully human light chain, and, if desired, a linker (e.g., a peptide linker) connecting the heavy chain variable region and the light chain variable region. In some embodiments, the heavy chain variable region comprises at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with the amino acid sequence or combination thereof of SEQ ID NO: 4, 6, 8, or 10, and the light chain variable region comprises at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with the amino acid sequence or combination thereof of SEQ ID NO: 5, 7, 9, or 11. In some embodiments, the heavy chain variable region contains at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with the amino acid sequence or combination thereof of SEQ ID NO:4, 6, 8, or 10, and the light chain variable region contains at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with the amino acid sequence or combination thereof of SEQ ID NO:5, 7, 9, or 11. []

[0148] This invention provides a single-chain fully human antibody comprising a polypeptide having a heavy chain variable region and a light chain variable region, wherein the amino acid sequence set of the heavy chain / light chain variable region has at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identity with any of the following amino acid sequence sets: SEQ ID NO: SEQ ID NO: 4 and 5 (hereinafter referred to as 1C5-3A6), SEQ ID NO: 6 and 7 (hereinafter referred to as 2D12-2E3-N102D), SEQ ID NO: 8 and 9 (hereinafter referred to as 5C8), or SEQ ID NO: 10 and 11 (hereinafter referred to as 8D9). In one embodiment, the single-chain fully human antibody comprises an optional linker (e.g., a peptide linker) for conjugating the heavy chain variable region and the light chain variable region. []

[0149] This invention provides therapeutic compositions comprising any of the anti-LAG3 antibodies or antigen-binding proteins thereof described herein and pharmaceutically acceptable excipients. Excipients encompass carriers and stabilizers. In one embodiment, the therapeutic composition comprises an anti-LAG3 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of the heavy / light chain variable regions have at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identity with any of the following groups of amino acid sequences: SEQ ID NO: SEQ ID NO: 4 and 5 (hereinafter referred to as 1C5-3A6), SEQ ID NO: 6 and 7 (hereinafter referred to as 2D12-2E3-N102D), SEQ ID NO: 8 and 9 (hereinafter referred to as 5C8), or SEQ ID NO: 10 and 11 (hereinafter referred to as 8D9). []

[0150] The present invention provides a kit comprising any of the disclosed fully human anti-LAG3 antibodies or any of their antigen-binding fragments or any of the Fab fragments or single-chain antibodies described herein. []

[0151] This invention provides a nucleic acid encoding an antibody heavy chain variable region having at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity, of the amino acid sequence or combination thereof with SEQ ID NO:4, 6, 8, or 10. This invention also provides a nucleic acid encoding an antibody light chain variable region having at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity, of the amino acid sequence or combination thereof with SEQ ID NO:5, 7, 9, or 11. []

[0152] This invention provides a first nucleic acid encoding an antibody heavy chain variable region having at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity, of the amino acid sequence of SEQ ID NO:4, 6, 8, or 10, or a combination thereof. This invention provides a second nucleic acid encoding an antibody light chain variable region having at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity, of the amino acid sequence of SEQ ID NO:5, 7, 9, or 11, or a combination thereof. []

[0153] The present invention provides a nucleic acid encoding an antibody heavy chain variable region having at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity with the amino acid sequence or combination thereof of SEQ ID NO:4, 6, 8 or 10, and the nucleic acid encoding an antibody light chain variable region having at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity with the amino acid sequence or combination thereof of SEQ ID NO:5, 7, 9 or 11. []

[0154] This invention provides a nucleic acid encoding an antibody comprising both a heavy chain and a light chain, wherein the amino acid sequence of the heavy / light chain variable region has at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with any of the following amino acid sequences: SEQ ID NO: SEQ ID NO: 4 and 5 (hereinafter referred to as 1C5-3A6), SEQ ID NO: 6 and 7 (hereinafter referred to as 2D12-2E3-N102D), SEQ ID NO: 8 and 9 (hereinafter referred to as 5C8), or SEQ ID NO: 10 and 11 (hereinafter referred to as 8D9). []

[0155] This invention provides a nucleic acid encoding a Fab complete human antibody fragment, the Fab complete human antibody fragment comprising a variable region from the heavy chain and a variable region from the light chain, wherein the amino acid sequence from the variable region of the heavy chain has at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% similarity to the amino acid sequences of SEQ ID NO: 4, 6, 8, or 10, or combinations thereof. This invention also provides a nucleic acid encoding a Fab complete human antibody fragment, the Fab complete human antibody fragment comprising a variable region from the heavy chain and a variable region from the light chain, wherein the amino acid sequence from the variable region of the light chain has at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% similarity to the amino acid sequences of SEQ ID NO: 5, 7, 9, or 11, or combinations thereof. []

[0156] This invention provides a first nucleic acid encoding a Fab complete human antibody fragment, the Fab complete human antibody fragment comprising a variable region from the heavy chain and a variable region from the light chain, wherein the amino acid sequence of the variable region from the heavy chain has at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% similarity to the amino acid sequences of SEQ ID NO: 4, 6, 8, or 10, or combinations thereof. This invention also provides a second nucleic acid encoding a Fab complete human antibody fragment, the Fab complete human antibody fragment comprising a variable region from the heavy chain and a variable region from the light chain, wherein the amino acid sequence of the variable region from the light chain has at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% similarity to the amino acid sequences of SEQ ID NO: 5, 7, 9, or 11, or combinations thereof. []

[0157] This invention provides a nucleic acid encoding a Fab fully human antibody fragment comprising a variable region from the heavy chain and a variable region from the light chain, wherein the amino acid sequence of the variable region from the heavy chain has at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% similarity to the amino acid sequences of SEQ ID NO:4, 6, 8, or 10, or combinations thereof, and the nucleic acid encodes a variable region from the light chain, wherein the amino acid sequence of the variable region from the light chain has at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% similarity to the amino acid sequences of SEQ ID NO:5, 7, 9, or 11, or combinations thereof. []

[0158] This invention provides a nucleic acid encoding a Fab complete human antibody fragment comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of the heavy chain / light chain variable regions have at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identity with any of the following amino acid sequences: SEQ ID NO: SEQ ID NO: 4 and 5 (hereinafter referred to as 1C5-3A6), SEQ ID NO: 6 and 7 (hereinafter referred to as 2D12-2E3-N102D), SEQ ID NO: 8 and 9 (hereinafter referred to as 5C8), or SEQ ID NO: 10 and 11 (hereinafter referred to as 8D9). []

[0159] This invention provides a nucleic acid encoding a single-chain fully human antibody, the single-chain fully human antibody comprising a polypeptide chain having a variable region from a fully human heavy chain and a variable region from a fully human light chain, and, if desired, a linker (e.g., a peptide linker) connecting the heavy chain variable region and the light chain variable region. In some embodiments, the nucleic acid encodes a heavy chain variable region comprising at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with the amino acid sequence or combination thereof of SEQ ID NO: 4, 6, 8, or 10, and the nucleic acid encodes a light chain variable region comprising at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with the amino acid sequence or combination thereof of SEQ ID NO: 5, 7, 9, or 11. In one embodiment, the peptide linker comprises an amino acid sequence (GGGGS)N (SEQ ID NO: 12), wherein N is 1 to 6. In another embodiment, the peptide linker comprises an amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 13). []

[0160] This invention provides a nucleic acid encoding a single-chain fully human antibody, the single-chain fully human antibody comprising a variable region from a fully human heavy chain and a variable region from a fully human light chain, and, if desired, a linker (e.g., a peptide linker) connecting the heavy chain variable region and the light chain variable region. In some embodiments, the heavy chain variable region comprises at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with the amino acid sequence or combination thereof of SEQ ID NO: 4, 6, 8, or 10, and the light chain variable region comprises at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with the amino acid sequence or combination thereof of SEQ ID NO: 5, 7, 9, or 11. In one embodiment, the peptide linker comprises the amino acid sequence (GGGGS)N (SEQ ID NO: 12), where N is 1 to 6. In one embodiment, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 13). []

[0161] This invention provides a nucleic acid encoding a single-chain fully human antibody, wherein the single-chain fully human antibody is a single-chain antibody comprising a polypeptide chain having a heavy chain variable region and a light chain variable region, wherein the amino acid sequence set of the heavy chain / light chain variable region has at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identity with any of the following amino acid sequences: SEQ ID NO: SEQ ID NO: 4 and 5 (hereinafter referred to as 1C5-3A6), SEQ ID NO: 6 and 7 (hereinafter referred to as 2D12-2E3-N102D), SEQ ID NO: 8 and 9 (hereinafter referred to as 5C8), or SEQ ID NO: 10 and 11 (hereinafter referred to as 8D9). In one embodiment, the nucleic acid encodes a single-chain fully human antibody comprising an optional linker (e.g., a peptide linker) that joins the variable heavy chain region and the variable light chain region. In one embodiment, the peptide linker comprises the amino acid sequence (GGGGS)N (SEQ ID NO: 12), wherein N is 1 to 6. In one embodiment, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 13). []

[0162] The present invention provides a first vector (e.g., a first expression vector) operably linked to a first nucleic acid encoding a heavy chain variable region, the heavy chain variable region comprising at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID No: 4, 6, 8, or 10, and a second vector (e.g., a second expression vector) operably linked to a second nucleic acid encoding a light chain variable region, the light chain variable region comprising at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 5, 7, 9, or 11. []

[0163] The present invention provides a first vector (e.g., a first expression vector) operably linked to a first nucleic acid encoding a heavy chain variable region, the heavy chain variable region comprising at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 4, 6, 8, or 10. In one embodiment, the first vector is also operably linked to a second nucleic acid encoding a light chain variable region, the light chain variable region comprising at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 5, 7, 9, or 11. []

[0164] The present invention provides a vector (e.g., an expression vector) operatively linked to a nucleic acid encoding a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region having at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with any of the following amino acid sequences: SEQ ID NO: SEQ ID NO: 4 and 5 (hereinafter referred to as 1C5-3A6), SEQ ID NO: 6 and 7 (hereinafter referred to as 2D12-2E3-N102D), SEQ ID NO: 8 and 9 (hereinafter referred to as 5C8), or SEQ ID NO: 10 and 11 (hereinafter referred to as 8D9). []

[0165] The present invention provides a first vector (e.g., a first expression vector) operably linked to a first nucleic acid encoding a Fab complete human antibody fragment, the Fab complete human antibody fragment comprising a heavy chain variable region having at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 4, 6, 8, or 10. The present invention also provides a second vector (e.g., a second expression vector) operably linked to a second nucleic acid encoding a Fab complete human antibody fragment, the Fab complete human antibody fragment comprising a light chain variable region having at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 5, 7, 9, or 11. []

[0166] The present invention provides a first vector (e.g., a first expression vector) operably linked to a first nucleic acid encoding a Fab fully human antibody fragment comprising a heavy chain variable region, the heavy chain variable region comprising at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 4, 6, 8, or 10. In one embodiment, the first vector is also operably linked to a second nucleic acid encoding a light chain variable region comprising at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 5, 7, 9, or 11. []

[0167] The present invention provides a vector (e.g., an expression vector) operatively liganded to a nucleic acid encoding a Fab fully human antibody fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region have at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with any of the following amino acid sequences: SEQ ID NO: SEQ ID NO: 4 and 5 (hereinafter referred to as 1C5-3A6), SEQ ID NO: 6 and 7 (hereinafter referred to as 2D12-2E3-N102D), SEQ ID NO: 8 and 9 (hereinafter referred to as 5C8), or SEQ ID NO: 10 and 11 (hereinafter referred to as 8D9). []

[0168] The present invention provides a first vector (e.g., a first expression vector) operably linked to a nucleic acid encoding a single-chain complete human antibody, the single-chain complete human antibody comprising a polypeptide chain having a variable region from a complete human heavy chain and a variable region from a complete human light chain, and, if desired, a linker (e.g., a peptide linker) for conjugating the heavy chain variable region and the light chain variable region, wherein the heavy chain variable region comprises at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with the amino acid sequence or combination thereof of SEQ ID NO: 4, 6, 8, or 10, and wherein the light chain variable region comprises at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with the amino acid sequence or combination thereof of SEQ ID NO: 5, 7, 9, or 11, and at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with the amino acid sequence or combination thereof of SEQ ID NO: 5, 7, 9, or 11. In one embodiment, the peptide linker comprises the amino acid sequence (GGGGS)N (SEQ ID NO: 12), wherein N is 1 to 6. In one embodiment, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 13). []

[0169] The present invention provides a first vector (e.g., a first expression vector) operably linked to a nucleic acid encoding a single-chain complete human antibody, the single-chain complete human antibody comprising a polypeptide chain having a variable region from a complete human heavy chain and a variable region from a complete human light chain, and, if desired, a linker (e.g., a peptide linker) for conjugating the variable light chain and the variable heavy chain regions, wherein the variable region of the heavy chain comprises at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with any of the following amino acid sequences: SEQ ID NO: SEQ ID NO: 4 and 5 (hereinafter referred to as 1C5-3A6), SEQ ID NO: 6 and 7 (hereinafter referred to as 2D12-2E3-N102D), SEQ ID NO: 8 and 9 (hereinafter referred to as 5C8), or SEQ ID NO: 10 and 11 (hereinafter referred to as 8D9). In one embodiment, the peptide linker comprises the amino acid sequence (GGGGS)N (SEQ ID NO: 12), where N is 1 to 6. In one embodiment, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 13). []

[0170] The present invention provides a host cell or a population of host cells, wherein the host cell or a single host cell from the host cell population carries a first vector (e.g., a first expression vector) operatively linked to a first nucleic acid encoding a first nucleic acid of a heavy chain variable region, the heavy chain variable region comprising at least 95% sequence identity with an amino acid sequence of SEQ ID NO: 4, 6, 8, or 10, and the host cell or a single host cell from the host cell population carries a second vector (e.g., a second expression vector) operatively linked to a second nucleic acid encoding a second nucleic acid of a light chain variable region, the light chain variable region comprising at least 95% sequence identity with an amino acid sequence of SEQ ID NO: 5, 7, 9, or 11. In one embodiment, the first expression vector directs the expression of the heavy chain variable region in the host cell or the host cell population, and the second expression vector directs the expression of the light chain variable region in the host cell or the host cell population. []

[0171] The present invention provides a first host cell or a first host cell population, wherein the first host cell or a single host cell from the first host cell population carries a first vector (e.g., a first expression vector) operatively linked to a first nucleic acid encoding a heavy chain variable region, the heavy chain variable region comprising at least 95% sequence identity with an amino acid sequence of SEQ ID NO: 4, 6, 8, or 10; and a second host cell or a second host cell population, wherein the second host cell or a single host cell from the second host cell population carries a second vector (e.g., a second expression vector) operatively linked to a second nucleic acid encoding a light chain variable region, the light chain variable region comprising at least 95% sequence identity with an amino acid sequence of SEQ ID NO: 5, 7, 9, or 11. In one embodiment, the first expression vector directs the expression of the heavy chain variable region in the first host cell, and the second expression vector directs the expression of the light chain variable region in the second host cell. []

[0172] The present invention provides a host cell or a population of host cells, wherein the host cell or a single host cell from the host cell population carries a vector (e.g., an expression vector) operatively linked to a first nucleic acid encoding a heavy chain variable region comprising at least 95% sequence identity with an amino acid sequence of SEQ ID NO: 4, 6, 8, or 10, and the vector in the host cell is also operatively linked to a second nucleic acid encoding a light chain variable region comprising at least 95% sequence identity with an amino acid sequence of SEQ ID NO: 5, 7, 9, or 11. In one embodiment, the expression vector directs the expression of the heavy chain variable region and the light chain variable region in the host cell. []

[0173] This invention provides a host cell or a population of host cells, wherein the host cell or a single host cell from the host cell population carries a first vector (e.g., a first expression vector) operatively linked to a first nucleic acid encoding a Fab complete human antibody fragment, the Fab complete human antibody fragment comprising a variable region from the heavy chain, wherein the amino acid sequence of the variable region from the heavy chain is at least 95% identical to the amino acid sequences of SEQ ID NO: 4, 6, 8, or 10, or combinations thereof. In one embodiment, the host cell or a single host cell from the host cell population also carries a second vector (e.g., a second expression vector) operatively linked to a second nucleic acid encoding a variable region from the light chain, wherein the amino acid sequence of the variable region from the light chain is at least 95% identical to the amino acid sequences of SEQ ID NO: 5, 7, 9, or 11, or combinations thereof. In one embodiment, the first expression vector directs the expression of the heavy chain variable region in the host cell and the second expression vector directs the expression of the light chain variable region in the host cell. []

[0174] The present invention provides a first host cell or a first host cell population, wherein the first host cell or a single host cell from the first host cell population carries a first vector (e.g., a first expression vector) operatively linked to a first nucleic acid encoding a Fab complete human antibody fragment, the Fab complete human antibody fragment comprising a variable region from the heavy chain, wherein the amino acid sequence of the variable region from the heavy chain is at least 95% identical to the amino acid sequences of SEQ ID NO: 4, 6, 8, or 10, or combinations thereof. The present invention also provides a second host cell or a second host cell population, wherein the second host cell or a single host cell from the second host cell population carries a second vector (e.g., a second expression vector) operatively linked to a second nucleic acid encoding a variable region from the light chain, wherein the amino acid sequence of the variable region from the light chain is at least 95% identical to the amino acid sequences of SEQ ID NO: 5, 7, 9, or 11, or combinations thereof. In one embodiment, the first expression vector directs the expression of the heavy chain variable region in the first host cell, and the second expression vector directs the expression of the light chain variable region in the second host cell. []

[0175] This invention provides a host cell or a population of host cells, wherein the host cell or a single host cell from the host cell population carries a vector (e.g., an expression vector) operatively linked to a first nucleic acid encoding a Fab complete human antibody fragment, the Fab complete human antibody fragment comprising a variable region from the heavy chain, wherein the amino acid sequence of the variable region from the heavy chain is at least 95% identical to the amino acid sequences of SEQ ID NO: 4, 6, 8, or 10, or combinations thereof, and the vector in the host cell is also operatively linked to a second nucleic acid encoding a variable region from the light chain, wherein the amino acid sequence of the variable region from the light chain is at least 95% identical to the amino acid sequences of SEQ ID NO: 5, 7, 9, or 11, or combinations thereof. In one embodiment, the expression vector directs the expression of the heavy chain variable region and the light chain variable region in the host cell. []

[0176] The present invention provides a host cell or a population of host cells, wherein the host cell or a single host cell from the host cell population carries a first vector (e.g., a first expression vector) operatively linked to a nucleic acid encoding a single-chain fully human antibody, the single-chain fully human antibody comprising a polypeptide chain having a variable region from a fully human heavy chain and a variable region from a fully human light chain, and, if desired, a linker (e.g., a peptide linker) for conjugating the heavy chain variable region and the light chain variable region, wherein the heavy chain variable region contains at least 95% sequence identity with an amino acid sequence or combination thereof of SEQ ID NO: 4, 6, 8, or 10, and wherein the light chain variable region contains at least 95% sequence identity with an amino acid sequence or combination thereof of SEQ ID NO: 5, 7, 9, or 11. In one embodiment, the first vector comprises a nucleic acid sequence encoding a peptide linker comprising an amino acid sequence (GGGGS)N (SEQ ID NO: 12), wherein N is 1 to 6. In one embodiment, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 13). In one embodiment, the expression vector guides the expression of the single-chain antibody in the host cell. []

[0177] This invention provides a method for preparing a first polypeptide having an antibody heavy chain variable region, the method comprising: culturing a first host cell population (e.g., a plurality of cells) carrying a first expression vector under conditions suitable for expressing the first polypeptide having an antibody heavy chain variable region having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4, 6, 8, or 10. In one embodiment, the method further comprises: recovering from the first host cell population the expressed first polypeptide having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4, 6, 8, or 10.

[0178] This invention provides a method for preparing a second polypeptide having an antibody light chain variable region, the method comprising: culturing a second host cell population (e.g., a plurality of cells) carrying a second expression vector under conditions suitable for expressing a second polypeptide having an antibody light chain variable region having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 5, 7, 9, or 11. In one embodiment, the method further comprises: recovering from the second host cell population the expressed second polypeptide having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 5, 7, 9, or 11.

[0179] This invention provides a method for preparing a first polypeptide having an antibody heavy chain variable region and a second polypeptide having an antibody light chain variable region. The method comprises: culturing a population (e.g., a plurality of) of host cells carrying a first expression vector (operably linked to a first nucleic acid encoding the first polypeptide) and a second expression vector (operably linked to a second nucleic acid encoding the second polypeptide) under conditions suitable for expressing the first polypeptide having an antibody heavy chain variable region having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4, 6, 8, or 10, and suitable for expressing the second polypeptide having an antibody light chain variable region having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 5, 7, 9, or 11. In one embodiment, the method further comprises: recovering from the host cell population the expressed first polypeptide having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4, 6, 8, or 10, and recovering from the host cell population the expressed second polypeptide having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 5, 7, 9, or 11.

[0180] This invention provides a method for preparing a first polypeptide having an antibody heavy chain variable region, the method comprising: culturing a first host cell population (e.g., a plurality of cells) carrying a first expression vector under conditions suitable for expressing the first polypeptide having an antibody heavy chain variable region having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4, 6, 8, or 10. In one embodiment, the method further comprises: recovering from the first host cell population the expressed first polypeptide having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4, 6, 8, or 10.

[0181] This invention provides a method for preparing a second polypeptide having an antibody light chain variable region, the method comprising: culturing a second host cell population (e.g., a plurality of cells) carrying a second expression vector under conditions suitable for expressing a second polypeptide having an antibody light chain variable region having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 5, 7, 9, or 11. In one embodiment, the method further comprises: recovering from the second host cell population the expressed second polypeptide having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 5, 7, 9, or 11.

[0182] This invention provides a method for preparing a first polypeptide having an antibody heavy chain variable region and a second polypeptide having an antibody light chain variable region. The method comprises: culturing a population (e.g., a plurality of) of host cells carrying an expression vector (operably linked to a first nucleic acid encoding the first polypeptide and operably linked to a second nucleic acid encoding the second polypeptide) under conditions suitable for expressing the first polypeptide having an antibody heavy chain variable region having at least 95% sequence identity with the amino acid sequences of SEQ ID NO: 4, 6, 8, or 10, and suitable for expressing the second polypeptide having an antibody light chain variable region having at least 95% sequence identity with the amino acid sequences of SEQ ID NO: 5, 7, 9, or 11. In one embodiment, the method further comprises: recovering from the host cell population the expressed first polypeptide having at least 95% sequence identity with the amino acid sequences of SEQ ID NO: 4, 6, 8, or 10, and recovering from the host cell population the expressed second polypeptide having at least 95% sequence identity with the amino acid sequences of SEQ ID NO: 5, 7, 9, or 11.

[0183] This invention provides a method for treating an individual suffering from a disease associated with LAG3 overexpression or LAG3 mal-expression, the method comprising administering to the individual an effective amount of a therapeutic composition comprising any one or any combination of two or three of the fully human anti-LAG3 antibodies described herein. In one embodiment, the disease is a cancer, tumor, autoimmune disease, or infectious disease caused by a virus or pathogenic virus or pathogenic bacteria or pathogenic fungus or pathogenic parasite.

[0184] In one embodiment, the anti-LAG3 antibody may be combined with other forms of immunotherapy, such as interferon therapy (e.g., interferon, GM-CSF, G-CSF, IL-2) or bispecific antibody therapy, which provides enhanced tumor antigen presentation (see, for example, Holliger (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure 2:1121-1123).

[0185] In one embodiment, the disease is a cancer selected from the group consisting of: bone cancer, pancreatic cancer, skin cancer, head or neck cancer, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, cancer of the anus, stomach cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small bowel cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or Acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, and chronic lymphocytic leukemia), childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal cord axis tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, and asbestos-induced cancers.

[0186] In one embodiment, the disease is a cancer or tumor selected from the group consisting of: non-Hodgkin's lymphoma (NHL), Burke's lymphoma (BL), multiple myeloma (MM), B-cell chronic lymphocytic leukemia (B-CLL), B and T-cell acute lymphoblastic leukemia (ALL), T-cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin's lymphoma (HL), chronic myeloid leukemia (CML), non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), acute osteomyelitis... Myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma (MM), breast cancer, ovarian cancer, head and neck cancer, bladder cancer, melanoma, colorectal cancer, pancreatic cancer, lung cancer, leiomyosarcoma, leiomyosarcoma, glioma, glioblastoma, and solid tumors, of which solid tumors are selected from the following groups: breast tumors, ovarian tumors, lung tumors, pancreatic tumors, prostate tumors, melanoma, colorectal tumors, lung tumors, head and neck tumors, bladder tumors, esophageal tumors, liver tumors, and kidney tumors.

[0187] In one embodiment, the disease is an autoimmune disease, including Alzheimer's disease, allergies, asthma, celiac disease, Crohn's disease, Graves' disease, inflammatory bowel disease (IBD), lupus, multiple sclerosis, myasthenia gravis, polymyalgia rheumatica, rheumatoid arthritis, type I diabetes, or vasculitis.

[0188] In one embodiment, the disease is selected from the group of infectious diseases comprising: HIV, hepatitis (types A, B, and C), influenza, herpes, Giardia, malaria, Leishmania, Staphylococcus aureus, Pseudomonas aeruginosa, flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, and arbovirus encephalitis virus.

[0189] In one embodiment, the disease is caused by pathogenic viruses, including HIV, hepatitis (type A, B, or C), herpesviruses (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein-Barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, Coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, human papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, and arbovirus encephalitis virus.

[0190] In one embodiment, the disease is caused by pathogenic bacteria, including Chlamydia, Rickettsial bacteria, Mycobacterium, Staphylococcus, Streptococcus, Pneumococcus, Neisseria meningitidis, Neisseria gonorrhoeae, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Corynebacterium diphtheriae, Salmonella, Bacillus, Vibrio cholerae, Clostridium tetani, Clostridium botulinum, Bacillus anthracis, Yersinia pestis, Leptospirosis, and Lyme disease bacteria.

[0191] In one embodiment, the disease is caused by pathogenic fungi, including Candida species (such as Candida albicans, Candida krusei, Candida glabrata, and Candida tropicalis), Cryptococcus neoformans, Aspergillus species (such as Fumigatus and Niger), Mucorales species (such as Mucor, Absidia, and Rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, or Histoplasma capsulatum.

[0192] In one embodiment, the disease is caused by pathogenic parasites, including Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, or Nippostrongylus brasiliensis. [Example] []

[0193] The following examples are illustrative and can be used to further understand embodiments of the invention, and should not be construed in any way as limiting the scope of this teaching. []

[0194] [Example] [1] [Regarding soluble] [LAG3] [Dose-dependent binding of proteins] [Of] [ELISA] [analyze] [:] []

[0195] The Novartis 525 antibody system was internally selected and generated. Anti-LAG3 antibodies (1C5-3A6, 2D12-2E3-N102D, 5C8, and 8D9) and competing antibodies (Novartis 525) were plated on enzyme-linked immunosorbent assay (ELISA) plates overnight at 4°C in 1X phosphate-buffered saline (PBS1X) at 5 µg / mL (20 µL / well). The plates were washed three times with 250 µL / well of PBS-T (supplemented with 0.05% Tween 20 in PBS1X). Subsequently, the plates were blocked for 1 hour at room temperature (RT) with 100 µL / well of casein (Thermo Fisher, catalog number 37528) in PBS. After washing three times with 250 µL / well PBS-T, purified human LAG3 His-tagged protein (Sino Biological; catalog number 16498-H08H) was serially diluted into the wells (maximum concentration 10 µg / mL; dilution 1:3) and incubated at room temperature for 1 hour. The plate was washed three times with 250 µL / well PBS-T and binding was visualized by applying 50 µL / well SureBlue™ TMB-1 component microporous peroxidase receptor (SeraCare; catalog number 5120-0075). Readings were terminated using 25 µL / well 0.16 M sulfuric acid (H₂SO₄) stop solution and read at 450 nM on a plate reader. The results are shown in Figure 3.

[0196] [Example] [2] [Using surface plasma resonance] (Biacore) [Determine binding affinity.] []

[0197] Binding kinetics of anti-LAG3 antibodies (1C5-3A6, 2D12-2E3-N102D, 5C8, and 8D9) and competing antibodies (Novartis 525) were determined using surface plasma resonance (SPR). Anti-human crystallizable fragment region (Fc region) antibodies were immobilized on a CM5 sensor chip to approximately 8,000 RU using a standard N-hydroxysuccinimide / N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (NHS / EDC) conjugation method. Antibodies (2 µg / mL) were captured at a flow rate of 10 µL / min for 60 seconds. Recombinant human LAG3-His (Sino Biological; catalog number 16498-H08H) was serially diluted in a running buffer of 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, and 0.05% v / v surfactant P20 (HBS-EP+). All measurements were performed in HBS-EP+ buffer at a flow rate of 30 μL / min. A 1:1 (Langmuir) binding model was used to fit the data. All BIACORE analyses were performed at room temperature. The results are shown in Figure 4. The measured KD values ​​are listed in Table 1 below. []

[0198] [surface] [1] [Measured] [K, D , ] [value] [] [1C5-3A6] [5C8] [2D12-2E3-N102D] [8D9] [Novartis 525] 6.43x10⁻¹¹ 6.15x10-12 8.42x10-10 Not measured 6.16 x 10⁻¹²

[0199] [Example] [3] [With crab-eating macaques and mice] [LAG3] Cross-reactivity of antigens []

[0200] Human, mouse, or cynomolgus monkey LAG3-His tagged recombinant protein was plated overnight at 4°C in 50 µL / well of 1X phosphate-buffered saline (PBS1X) at a concentration of 2 µg / mL. The plates were washed three times with 250 µL / well of PBS-T (supplemented with 0.05% Tween 20 in PBS1X). Subsequently, the plates were blocked for 1 hour at room temperature with 100 µL / well of casein (Thermo Fisher, PBS37528) in PBS. After washing three times with 250 µL / well of PBS-T, the purified antibody was incubated for 1.5 hours at room temperature in 50 µL / well of PBS-T at a concentration of 2 µg / mL. The plate was washed three times with 250 µL / well of PBS-T, and HRP-bound goat anti-human Fc secondary antibody (Sera Care; catalog number 5220-0279) was diluted 1:5,000 and incubated at room temperature at 50 µL / well for 1 hour. Subsequently, the plate was washed three times with 250 µL / well of PBS-T, and binding was visualized by applying 50 µL / well of SureBlue™ TMB-1 component microwell peroxidase receptor (SeraCare; catalog number 5120-0075). Readings were terminated using 25 µL / well of 0.16 M sulfuric acid (H₂SO₄) stop solution and read at 450 nM on a plate reader. The results are shown in Figure 5. []

[0201] [Example] [4] [:right] [LAG3] [The binding of cells] []

[0202] Human LAG3 / NFAT-Luc receptor Jurkat cell line (BPS Biosciences; catalog number 71278) was cultured in complete RPMI (RPMI 1640 + 10% FCS + Pen / Strep) supplemented with 1 mg / mL G418 and 200 µg / mL hygromycin B. Wild-type (WT) Jurkat cells were also cultured in complete RPMI. []

[0203] Cells were seeded at 80,000 cells / well in 96-well V-bottom plates and washed twice with 170 µL / well of FACS buffer (PBS 1X + 2% FCS / FBS + 0.1% sodium azide). Anti-LAG3 and isotype control antibodies were diluted in FACS buffer at various concentrations (from 10 to 0.003 µg / mL; dilution 1:5) and cultured with human LAG3 or WT Jurkat cells at 100 µL / well for 20 min at 4°C. After washing twice with 170 µL / well of FACS buffer, cells were cultured at 4°C with 100 µL / well of AF647-binding goat anti-human IgG secondary antibody (Southern Biotech; catalog number 2040-31, batch K471X873C; dilution 1:2,000 in FACS buffer) for 20 min. Cells were washed twice, resuspended in 140 µL / well FACS buffer, and collected on an Attune NxT via flow cytometry. Data were analyzed using FlowJo v10. ΔMFI was calculated as [(geometric mean of LAG3 Jurkat expression) – (geometric mean of WT Jurkat expression)]. The results are shown in Figure 6. []

[0204] [Example] [5] [:right] [SEB] [Animated Crab-Eating Macaque] [PBMC] [Specific binding.] []

[0205] [SEB] [Activation]: Fresh peripheral blood mononuclear cells (PBMCs) from cynomolgus monkeys were isolated and seeded at 2.0E+05 cells / well in 200 µL of complete RPMI (RPMI 1640 + 10% FCS + Pen / Strep) containing 200 U / mL of recombinant human IL-2 (Miltenyi Biotec; catalog number 130-097-748) or with or without 300 ng / mL of Staphylococcus aureus enterotoxin B (SEB) (catalog number 122, batch 1224171) from Toxin Technology, Inc. The PBMCs were cultured in a tissue culture incubator at 37°C and 5% CO2 for 4 days. []

[0206] [Combined Analysis]: On day 4, untreated and activated cynomolgus monkey PBMCs were washed twice with 170 µL / well FACS buffer (PBS 1X + 2% FCS / FBS + 0.1% sodium azide) and transferred to 96-well V-bottom plates at a density of 80,000 cells. Anti-LAG3 and isotype control antibodies were diluted in FACS buffer at various concentrations (from 10 to 0.016 µg / mL; dilution 1:5) and incubated at 4°C with untreated and activated cynomolgus monkey PBMCs at a density of 100 µL / well for 20 minutes. After washing twice in 170 µL / well FACS buffer, cells were incubated for 20 minutes at 4°C with 100 µL / well of AF647-bound goat anti-human IgG secondary antibody (Southern Biotech; catalog number 2040-31, batch K471X873C; dilution 1:2,000 in FACS buffer). Cells were washed twice, resuspended in 150 µL / well FACS buffer, and collected on an Attune NxT flow cytometer. Data were analyzed using FlowJo v10. []

[0207] ΔMFI was calculated as [(geometric mean of PBMCs of SEB-activated cynomolgus monkeys) – (geometric mean of PBMCs of untreated cynomolgus monkeys)]. See Figure 7A. []

[0208] ΔLAG3+ cells were calculated as [(LAG3+ cells in SEB-activated cynomolgus monkey PBMCs) – (LAG3+ cells in untreated cynomolgus monkey PBMCs)]. See Figure 7B. []

[0209] [Example] [6] [Detection resistance] [LAG3] [Functional activity of antibodies in reporter assays.] []

[0210] [analyze] [principle] When Raji and LAG3 / NFAT reporter Jurkat cells are co-cultured in the presence of superantigens (such as Staphylococcus aureus enterotoxin E), the superantigen cross-links the TCR complex on the effector cells (reporter Jurkat) and class II MHC on the target cells (Raji), leading to TCR activation and NFAT luciferase signaling. []

[0211] The interaction between LAG3 and class II MHC prevents this cross-linking and inhibits TCR activation and NFAT luciferase signaling. []

[0212] In the presence of anti-LAG-3 antibody: the interaction between LAG3 and MHC-II is blocked, and T cell activation is promoted by superantigen and NFAT-luciferase signals on effector cells. []

[0213] Human LAG3 / NFAT-Luc reporter Jurkat cell line (BPS Biosciences; catalog number 71278) was cultured in complete RPMI (RPMI 1640 + 10% FCS + Pen / Strep) supplemented with 1 mg / mL G418 and 200 µg / mL hygromycin B. RAJI cells (ATCC; catalog number CCL-86) were cultured in complete RPMI. []

[0214] [LAG3] [Preparation of Spectroradioactive Cells:] Human LAG3 / NFAT-Luc reporter Jurkat cells (40,000 cells / well) were seeded in complete RPMI in 96-well white clear-bottomed analytical plates (Costar; catalog number 3610) and pre-cultured with 10, 1, or 0.1 µg / mL (final concentration) of anti-LAG3 or isotype control antibody (final volume 60 µL / well). During pre-culture, the cells were maintained in a tissue culture incubator at 37°C and 5% CO2 for 30 minutes. []

[0215] [Antigen-presenting cells] [-RAJI] [Cell Preparation:] RAJI cells were resuspended at 0.75E+06 cells / mL in complete RPMI containing 0.1 ng / mL Staphylococcus aureus enterotoxin E (SEE) (catalog number ET404) from Toxin Technology, Inc. During pre-culture, cells were maintained in a tissue culture incubator at 37°C and 5% CO2 for 30 minutes. []

[0216] [Analysis Initiation and Reading:] 40 µL of SEE-activated RAJI cells were directly transferred to wells containing pre-cultured human LAG3 / NFAT-Luc reporter Jurkat cells (total volume 100 µL / well). The 96-well white clear plate was incubated at 37°C and 5% CO2 in a tissue culture incubator for 5–6 hours. Luciferase information was displayed using a ONE-step luciferase assay system (BPS Biosciences; catalog number 60690-1) with a 100 µL / well coating. The plate was then incubated at room temperature for 15 minutes with gentle agitation. Information was measured using a Spark® TECAN reader. Data were analyzed by subtracting the average background luminescence (cell-free control wells) from the luminescence readings of other wells. The fold induction rate of NFAT luciferase reporter expression was calculated as [(luminescence of antibody-treated wells minus background luminescence) / (average luminescence of untreated control wells minus background luminescence)]. The report sub-analysis was performed twice, and the results are shown in Figures 8A and 8B. []

[0217] [Example] [7] [Antigenic determinant map] []

[0218] Purified anti-LAG3 monoclonal antibody was plated onto ELISA Maxisorp™ plates overnight at 4°C in 50 µL / well of 1X phosphate-buffered saline (PBS1X). The plates were washed three times with 250 µL / well of PBS-T (supplemented with 0.05% Tween 20 in PBS1X). Then, the plates were blocked for 1 hour at room temperature with 100 µL / well of casein (Thermo Fisher, catalog number 37528) in PBS. After washing three times with 250 µL / well of PBS-T, recombinant human LAG3-Fc tag protein (Sino Biological; catalog number 16498-H05H) was incubated for 1.5 hours at room temperature in 50 µL / well of PBS-T at 2 µg / mL. The plate was washed three times with 250 µL / well PBS-T, and a biotinylated anti-LAG3 Novartis 525 antibody line (NVS525-biotinylated line internally selected, produced, and biotinylated) was incubated at 5 µg / mL in PBS-T (50 µL / well) for 1 hour at room temperature. After washing three times in PBS-T, streptavidin-HRP molecules (Thermo Fisher; catalog number N100) were diluted 1:5,000 in PBS-T and incubated at 50 µL / well for 30 minutes at room temperature for detection of bound NVS525-biotin. After washing the plate three times with 250 µL / well PBS-T, binding was visualized by applying 50 µL / well SureBlue™ TMB-1 component microwell peroxidase receptor (SeraCare; catalog number 5120-0075). The message was terminated using a 25 µL / well solution of 0.16 M sulfuric acid (H₂SO₄) and read at 450 nM on a plate reader. The results are shown in Figure 9. []

[0219] [Example] [8] [Cytoin Release Analysis] []

[0220] Fresh PBMCs (from two different donors) were isolated and diluted 2.0E+06 cells / mL in complete RPMI (RPMI 1640 + 10% FCS + Pen / Strep) supplemented with recombinant hIL-2 (1,000 U / mL). Cells were seeded at 2.0E+05 cells / well in U-shaped plates (100 µL / well). Next, anti-LAG-3 or isotype control antibody lines were diluted 10, 1, and 0.1 µg / mL (final concentration) in complete RPMI containing SEB (Staphylococcal enterotoxin B, List Biological laboratories; catalog number 122) (a mixture of 50 µL of antibody preparation and 50 µL of SEB, both at 2X concentration), to a final concentration of 100 ng / mL. The plates were incubated at 37°C for four days. On day 4, the cells and supernatant were transferred to V-shaped plates and centrifuged at 1,400 rpm for 5 minutes. The supernatant was transferred to a V-bottom 96-well plate and frozen at -80°C until analysis of intercytokine contents (IFNγ or other pro-inflammatory intercytokines) was performed. []

[0221] [Cytocin Detection]: The pro-inflammatory group 1 (human) kit (catalog number K15049D) from Meso Scale Discovery (MSD) was used according to the manufacturer's recommendations. In short, the analytical sample was thawed at room temperature. Meanwhile, a cytocin standard was prepared by serial dilution of calibrator 1 in Eppendorf™ tubes using diluent 2 (catalog number R51BB-3; lot M0020102). The analytical sample was then diluted with diluent 2 in 96-well ultra-low adhesion plates (Corning; catalog number 7007; lot 18219008) (a final dilution typically in the range of 1:200 to 1:2,000 is required to obtain a high number of samples within the standard curve range). []

[0222] Next, deseal the MSD 96-well plate and wash three times with 300 µl / well KPL buffer (Sera Care, catalog 5150-0008; lot 10430811). Following the previously prepared plate layout, transfer each diluted sample and calibrator to the MSD plate at 50 µl / well (avoiding air bubbles). Seal the plate using an adhesive plate sealer (VWR; catalog 60941-120) and incubate at room temperature with gentle agitation for 2 hours. Wash the plate three times with 300 µl / well KPL buffer and apply 25 µl / well of the SULFO-tagged detection antibody (1:50 dilution) prepared in diluent 3. Seal the plate and incubate in the dark at room temperature with gentle agitation for 2 hours. Subsequently, wash the plate three times with 300 µl / well KPL buffer. The information was displayed by adding 150 µl of 2X read buffer and detecting it on an MSD instrument (Meso Sector S600; Model 1201). The results are shown in Figures 10A and 10B.

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[0224]

[0225]

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[0227]

[0228]

[0229]

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Claims

1. A LAG3-binding protein comprising a heavy chain variable region having an amino acid sequence of SEQ ID No: 8 and a light chain variable region having an amino acid sequence of SEQ ID NO: 9, wherein the heavy chain variable region comprises the CDR1, CDR2 and CDR3 sequences present in SEQ ID NO: 8; and the light chain variable region comprises the CDR1, CDR2 and CDR3 sequences present in SEQ ID NO:

9.

2. The LAG3 binding protein in request item 1 is an antibody or antibody fragment.

3. The LAG3 binding protein in claim 1 is a fully human anti-LAG3 antibody.

4. The LAG3 binding protein in Request 1 is a Fab-based fully human anti-LAG3 antibody.

5. The LAG3-binding protein in claim 1 is a single-chain human anti-LAG3 antibody.

6. The LAG3-binding protein as claimed in claims 1, 2, or 5, wherein the variable domain region from the heavy chain and the variable domain region from the light chain are bound together with a peptide linker.

7. The LAG3-binding protein as claimed in claim 1 or 3, wherein the antibody binds to human LAG3 at a concentration of 10⁻⁸ M or less per kDa.

8. The LAG3-binding protein as claimed in claim 1 or 3, wherein the antibody binds to cynomolgus monkey LAG3 at a concentration of 10⁻⁶ M or less KD.

9. The LAG3 binding protein, as requested in item 1 or 3, contains an IgG4 antibody.

10. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and a LAG3-binding protein as claimed in any one of claims 1 to 9.

11. A kit comprising the LAG3-binding protein as claimed in any one of claims 1 to 9.

12. A nucleic acid encoding (a) (i) the heavy chain variable region of a LAG3-binding protein as claimed in any of claims 1 to 9, and (ii) the light chain variable region of a LAG3-binding protein as claimed in any of claims 1 to 9; or (b) a LAG3-binding protein as claimed in claim 1 or 2.

13. A vector comprising the nucleic acid as claimed in claim 12.

14. A host cell carrying the vector as claimed in claim 13.

15. The host cell of claim 14, wherein the vector comprises an expression vector, and wherein the host cell expresses the heavy chain variable region and the light chain variable region.

16. A method for preparing (i) a heavy chain variable region of a fully human anti-LAG3 antibody, or a Fab fully human anti-LAG3 antibody, or a single-chain human anti-LAG3 antibody, and (ii) a light chain variable region of a fully human anti-LAG3 antibody, or a Fab fully human anti-LAG3 antibody, or a single-chain human anti-LAG3 antibody, the method comprising: The host cell population as claimed in claim 15 shall be cultured under conditions suitable for expressing (i) the heavy chain variable region of the fully human anti-LAG3 antibody, or the Fab fully human anti-LAG3 antibody, or the single-chain human anti-LAG3 antibody, and (ii) the light chain variable region of the fully human anti-LAG3 antibody, or the Fab fully human anti-LAG3 antibody, or the single-chain human anti-LAG3 antibody.

17. The method of claim 16, further comprising: The following are recovered from the host cells: (i) the expressed heavy chain variable region of the fully human anti-LAG3 antibody, or the Fab fully human anti-LAG3 antibody, or the single-chain human anti-LAG3 antibody; and (ii) the expressed light chain variable region of the fully human anti-LAG3 antibody, or the Fab fully human anti-LAG3 antibody, or the single-chain human anti-LAG3 antibody.

18. Use of a therapeutic composition comprising any one of claims 1 to 9 of the present invention for the preparation of a medicament for treating an individual suffering from a disease associated with LAG3 overexpression.

19. As claimed in claim 18, wherein the disease is bone cancer, pancreatic cancer, skin cancer, head or neck cancer, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, cancer of the anus, stomach cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, or non-Hodgkin's lymphoma. Lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal cord axis tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer or cancer induced by asbestos.

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