LAG-3 agonist antibodies

Abstract

To provide methods of making and using agonist antibodies for use in the prevention, down-regulation and / or amelioration of autoimmune and / or immune tolerance-related disorders.SOLUTION: The present disclosure provides antibodies that bind to and agonize human LAG-3, and methods for treating autoimmune disease that utilize the antibodies to promote T-cell receptor signaling downregulation.SELECTED DRAWING: None

Description

Technical Field

[0001] The present disclosure belongs to the field of medicine. In particular, the present disclosure relates to novel antibodies that agonize human LAG-3, compositions comprising such antibodies, and methods of using such antibodies for the treatment of autoimmune diseases.

[0002] (Sequence Listing) This application has been filed together with a sequence listing in ST.26 XML format. The sequence listing is provided as a file entitled "30922_WO.xml" created on December 6, 2024, and is 108 kilobytes in size. The sequence listing information in ST.26 XML format is hereby incorporated by reference in its entirety.

Background Art

[0003] The immune checkpoint pathway regulates both autoimmune responses and anti-cancer immune responses (Isakov, N., J. Autoimmune Disorders 2016; 2(2):17). In autoimmune disease therapy, it may be desirable to enhance the effect of immune inhibitory pathways, i.e., agonize, so that the immune response is suppressed. Lymphocyte activation gene-3 (LAG-3, CD223) was first described over 30 years ago as a cell surface protein selectively expressed on activated human NK and T cells (Triebel, F., et al., J. Exp. Med. 1990; 171:1393-1405 It is well known as a cell surface inhibitory receptor that regulates T cell effector function (Hu, S et al., J. Autoimmunity 2020; 112:102504, doi.org / 10.1016 / j.jaut.2020.102504). LAG-3 is a checkpoint inhibitory receptor that disrupts the initiating phosphorylation events in T cell receptor signaling to suppress T cell activation and function.

[0004] LAG-3 agonist antibodies have been reported in International Publication No. WO2017 / 037203 and International Publication No. WO2020 / 221928, both of which disclose the LAG-3 agonist antibody IMP761 that specifically binds to a discontinuous epitope within the extracellular Ig superfamily domain D1 of human LAG-3, where the epitope is located outside the 30 amino acid extraloop sequence of domain D1 of the LAG-3 protein. More recently, International Publication No. WO2024 / 189628 teaches antibodies against glycosylation sites on specific domains of LAG-3, particularly for use in the prevention, amelioration, or treatment of influenza virus infection. Despite being a subject of high interest and decades of intensive research, including many antibody generation campaigns, there are still no commercially available LAG-3 agonist antibodies, nor are there LAG-3 agonist antibodies in late-stage clinical trials for use in the treatment of autoimmune disorders. For example, GSK2831781, a humanized IgG1 monoclonal antibody (mAb) that is specific for the LAG protein and specifically engineered to induce target depletion of LAG-3 positive T cells, was unable to reduce inflammation in the colonic mucosa, suggesting no pharmacological effect in patients with moderate to severe active ulcerative colitis (UC). Accordingly, this study (i.e., NCT03893565) was terminated early, and GlaxoSmithKline subsequently discontinued the development of GSK2831781 on May 30, 2024 (D’Haens, G., et al., Aliment Pharmacol Ther. 2023;58(3):283-296. doi:10.1111 / apt.17557. Epub 2023 Jun 16).

[0005] Accordingly, bind to human LAG-3 with desirable association and dissociation rates for optimal agonist activity, agonize the LAG-3 signaling pathway, reduce immunogenic risk, do not significantly deplete T cells, reduce T cell proliferation by promoting downregulation of T cell receptor signaling (e.g., potent gene repression of CD4 and CD8 activity), agonize human LAG-3 in immunologically relevant situations to achieve in vivo efficacy instead of T cell depletion, bind strongly to Fc gamma receptors, exhibit sufficient efficacy as a molecule for the treatment and / or prevention of autoimmune disorders, and / or provide an effective alternative for drug switching when treatment is interrupted due to at least one adverse event and / or inefficacy (especially anti-drug antibody (ADA)-mediated loss of efficacy), particularly during the treatment of autoimmune disorders with another human LAG-3 agonist antibody. There is still a need for LAG-3 agonist antibodies for treating autoimmune diseases. SUMMARY OF THE INVENTION

[0006] The present disclosure provides novel anti-human LAG-3 antibodies. The present disclosure provides compositions and methods useful for the prevention, downregulation, or amelioration of autoimmune-related disorders and / or immune tolerance-related disorders by immune checkpoint stimulation using engineered human LAG-3 agonist antibodies, thereby providing an advancement in the art. The anti-human LAG-3 agonist antibodies of the present disclosure preferably improve or restore immune homeostasis by inhibiting the T cell-mediated arm of the immune response, selectively immunosuppressing activated T cells, and thereby reducing the immunogenic risk compared to well-known LAG-3 agonist antibodies, and directly addressing the underlying disease pathology. The use of such antibodies may clinically result in long-term persistence or remission of the disease being treated.

[0007] The present disclosure provides an anti-human LAG-3 that binds to domain 2 of human LAG-3 (SEQ ID NO: 61) and binds to an epitope comprising one or more amino acid residues within SPHHHLAESF (SEQ ID NO: 64) of human LAG-3 domain 2 (SEQ ID NO: 61).

[0008] The present disclosure also provides an anti-human LAG-3 antibody that binds to human LAG-3, the antibody comprising a human LAG-3 binding domain comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises heavy chain complementarity determining regions (HCDR): HCDR1 comprising SEQ ID NO: 65, HCDR2 comprising SEQ ID NO: 2, and HCDR3 comprising SEQ ID NO: 48, and VL comprises light chain complementarity determining regions (lLCDR): LCDR1 comprising SEQ ID NO: 66, LCDR2 comprising SEQ ID NO: 67, and LCDR3 comprising SEQ ID NO: 68.

[0009] In another embodiment, the present disclosure provides an anti-human LAG-3 antibody that binds to human LAG-3, the antibody comprising an antigen binding domain comprising VH and VL, wherein VH comprises HCDR: HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 2, and HCDR3 comprising SEQ ID NO: 48, and VL comprises LCDR: LCDR1 comprising SEQ ID NO: 49, LCDR2 comprising SEQ ID NO: 5, and LCDR3 comprising SEQ ID NO: 6.

[0010] In one embodiment, VH comprises SEQ ID NO: 50 and VL comprises SEQ ID NO: 51.

[0011] In another embodiment, the anti-human LAG-3 antibody comprises a constant heavy (CH) region comprising SEQ ID NO: 9 and a constant light (CL) region comprising SEQ ID NO: 10.

[0012] The present disclosure provides an anti-human LAG-3 antibody that binds to human LAG-3, the antibody comprising an antigen binding domain comprising VH and VL, wherein VH comprises HCDR: comprising HCDR1 comprising SEQ ID NO:1, HCDR2 comprising SEQ ID NO:2, and HCDR3 comprising SEQ ID NO:3; VL is LCDR: LCDR1 comprising SEQ ID NO:4, LCDR2 comprising SEQ ID NO:5, and Contains LCDR3 comprising SEQ ID NO:6.

[0013] In one embodiment, the VH comprises SEQ ID NO:7 and the VL comprises SEQ ID NO:8.

[0014] In another embodiment, the anti-human LAG-3 antibody comprises a CH region comprising SEQ ID NO:9 and a CL region comprising SEQ ID NO:10.

[0015] In another embodiment, the anti-human LAG-3 antibody comprises a LC comprising SEQ ID NO:12 and a HC comprising SEQ ID NO:11.

[0016] In another embodiment, the anti-human LAG-3 antibodies of the invention are LAG-3 agonist antibodies.

[0017] In another embodiment, the anti-human LAG-3 antibodies of the invention are capable of detecting human LAG-3 (e.g., SEQ ID NO:57 or SEQ ID NO:58), human LAG-3 ECD (e.g., SEQ ID NO:59), and / or cynomolgus monkey LAG3 ECD (e.g., SEQ ID NO:60) at about 1×10 mAb, as determined by methods well known in the art, including but not limited to, the use of a surface plasmon resonance (SPR) biosensor at 25° C. or 37° C., and / or by methods essentially as described herein. -7 M ~ approx. 5×10 -11 K of M D In another embodiment, an anti-human LAG-3 agonist antibody of the disclosure binds to human LAG-3 (SEQ ID NO: 57 or SEQ ID NO: 58), human LAG-3 ECD (e.g., SEQ ID NO: 59), and / or cynomolgus monkey LAG3 ECD (e.g., SEQ ID NO: 60) at about 1×10 -8 M ~ approx. 1×10 -10 K of M DIt binds with. In another embodiment, the anti-human LAG-3 antibody binds to human LAG-3 (e.g., SEQ ID NO: 57 or SEQ ID NO: 58), human LAG-3 ECD (e.g., SEQ ID NO: 59), and / or cynomolgus monkey LAG3 ECD (e.g., SEQ ID NO: 60) with a K of about 5×10 -8 M to about 5×10 -10 M. In another embodiment, the anti-human antibody binds to human LAG-3 (SEQ ID NO: 57 or SEQ ID NO: 58), human LAG-3 ECD (e.g., SEQ ID NO: 59), and / or cynomolgus monkey LAG3 ECD (e.g., SEQ ID NO: 60) with a K of about 1×10 D M to about 1×10 -9 M. In another embodiment, the anti-human antibody binds to human LAG-3 (SEQ ID NO: 57 or SEQ ID NO: 58), human LAG-3 ECD (e.g., SEQ ID NO: 59), and / or cynomolgus monkey LAG3 ECD (e.g., SEQ ID NO: 60) with a K of about 1×10 -10 M. In another embodiment, the anti-human antibody binds to human LAG-3 (SEQ ID NO: 57 or SEQ ID NO: 58), human LAG-3 ECD (e.g., SEQ ID NO: 59), and / or cynomolgus monkey LAG3 ECD (e.g., SEQ ID NO: 60) with a K of about 1×10 D M.

[0018] In another embodiment, the anti-human LAG-3 agonist antibody is of the human IgG1 or IgG4 isotype. In another embodiment, the antibody is of the human IgG1 isotype. A non-limiting example of the human IgG1 isotype of the anti-human LAG-3 agonist antibody is the amino acid sequence of SEQ ID NO: 11.

[0019] In another embodiment, the antigen-binding domain of the anti-human LAG-3 is a single-chain variable fragment (scFv).

[0020] The present disclosure also provides a nucleic acid comprising a sequence encoding one or both of SEQ ID NO: 11 or SEQ ID NO: 12.

[0021] In another embodiment, the present disclosure provides a vector comprising a nucleic acid sequence encoding one or both of SEQ ID NO: 11 and SEQ ID NO: 12.

[0022] In another embodiment, the present disclosure provides a composition comprising a first vector comprising a nucleic acid sequence encoding SEQ ID NO: 11 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO: 12.

[0023] In one embodiment, the present disclosure provides cells comprising a vector. In another embodiment, the present disclosure provides cells comprising a composition. In another embodiment, the cells are mammalian cells. In another embodiment, the cells, or the mammalian cells, are isolated. In another embodiment, the present disclosure provides a process for producing an antibody, comprising culturing the cells under conditions such that the antibody is expressed and recovering the expressed antibody from the culture medium. In another embodiment, the present disclosure provides an antibody produced by culturing the cells under conditions such that the antibody is expressed and recovering the expressed antibody from the culture medium. In another embodiment, the present disclosure provides a composition comprising the anti-human LAG-3 agonist antibody of the present invention.

[0024] The present disclosure also provides a pharmaceutical composition comprising the anti-human LAG-3 agonist antibody of the present invention and a pharmaceutically acceptable excipient, diluent, or carrier. In one p embodiment, the pharmaceutical composition comprises arginine.

[0025] The present disclosure also provides a prefilled syringe of a pharmaceutical composition comprising the anti-human LAG-3 agonist antibody of the present invention and a pharmaceutically acceptable excipient, diluent, or carrier. In one embodiment, the pharmaceutical composition comprises arginine.

[0026] The present disclosure also provides a method of treating an autoimmune disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the anti-human LAG-3 agonist antibody of the present invention. In one embodiment, the autoimmune disease is rheumatoid arthritis (RA), psoriasis (PsO), ulcerative colitis (UC), type I diabetes mellitus (T1DM), lupus nephritis (LN), or systemic lupus erythematosus (SLE). In one embodiment, the autoimmune disease is an active disease. In another embodiment, the autoimmune disease is in remission.

[0027] The present disclosure also provides the anti-human LAG-3 agonist antibody of the present invention for use in therapy.

[0028] The present disclosure also provides the anti-human LAG-3 agonist antibody of the invention for use in the treatment of autoimmune diseases. In one embodiment, the autoimmune disease is RA, PsO, UC, T1DM, LN, or SLE. In another embodiment, the autoimmune disease is an active disease. In another embodiment, the autoimmune disease is in remission.

[0029] The present disclosure also provides a pharmaceutical composition comprising the anti-human LAG-3 agonist antibody of the invention for use in the treatment of RA, PsO, UC, T1DM, LN, or SLE. In one embodiment, the autoimmune disease is an active disease. In another embodiment, the autoimmune disease is in remission.

[0030] The present disclosure also provides the use of the anti-human LAG-3 agonist antibody of the invention in the manufacture of a medicament for the treatment of RA, PsO, UC, T1DM, LN, or SLE.

[0031] Mutations that promote desired qualities are well known to those skilled in the art. However, even if such mutations exist, it is not possible to predict which, or how many, of such mutations will promote the desired qualities of any particular antibody. The present invention provides an antibody comprising amino acid residue mutations that promote one or more of desired antibody expression, assembly, reduction or elimination of C1q binding, reduction or elimination of non-specific and self-interactions, reduction of the viscosity of a composition or formulation, elimination of deamidated residues, and reduction of immunogenicity.

[0032] In embodiments referring to the treatment methods described herein, such embodiments are also further embodiments for use in the manufacture of a medicament for use in, or alternatively for use in, such treatment.

[0033] In one embodiment, the anti-human LAG-3 agonist antibody of the invention is substantially pure. In another embodiment, the anti-human LAG-3 agonist antibody of the invention is sterilized.

[0034] In one embodiment, the immune disease is RA, PsO, UC, T1DM, LN, or SLE. In another embodiment, the immune disease is RA. In another embodiment, the immune disease is PsO. In another embodiment, the immune disease is UC. In another embodiment, the immune disease is T1DM. In another embodiment, the immune disease is LN. In another embodiment, the immune disease is SLE. In another embodiment, the immune disease is an active disease. In another embodiment, the immune disease is in remission.

[0035] In another embodiment, the anti-human LAG-3 agonist antibody of the invention binds to human LAG-3 but does not deplete T cells.

[0036] In another embodiment, the anti-human LAG-3 agonist antibody of the invention agonizes the LAG-3 signaling pathway.

[0037] In another embodiment, the anti-human LAG-3 agonist antibody of the invention binds to human LAG-3 with a desirable association rate and dissociation rate for optimal agonist activity.

[0038] In another embodiment, the anti-human LAG-3 agonist antibody of the invention reduces T cell proliferation by promoting downregulation of T cell receptor signaling rather than by depletion of T cells.

[0039] In another embodiment, the anti-human LAG-3 agonist antibody of the invention agonizes human LAG-3 in an immunologically relevant context to achieve in vivo efficacy.

[0040] The present disclosure also provides a method comprising: (a) contacting an anti-human LAG-3 agonist antibody of the invention with a cell expressing an extracellular domain of human LAG-3 fused to an enzyme donor subunit; (b) incubating the contacted cells under conditions suitable for the antibody to bind to the extracellular domain of human LAG-3; (c) contacting the cells with a reporter compound; (d) assaying the amount of the reporter compound; and (e) determining the IC 50 for the antibody that binds to the cells. In one embodiment, the enzyme acceptor subunit of the reporter gene is β-galactosidase and the enzyme donor subunit is the PK1 enzyme donor subunit of β-galactosidase. In another embodiment, the antibody comprises the HC of SEQ ID NO: 11 and the LC of SEQ ID NO: 12.

DETAILED DESCRIPTION OF THE INVENTION

[0041] As used herein, the terms "a", "an", "the" and similar terms used in the context of the present disclosure (especially in the context of the claims) are to be construed to cover both the singular and the plural forms unless otherwise specified herein or clearly contradicted by the context.

[0042] As used herein, "LAG-3" refers to lymphocyte activation gene 3, also known as CD223, which belongs to the immunoglobulin superfamily. The terms LAG-3, LAG3, Lag-3, and Lag3 are synonymous.

[0043] As used herein, "hLAG-3" or "human LAG-3" refers to wild-type human LAG-3, preferably wild-type human LAG-3 having the amino acid sequence set forth in SEQ ID NO: 57 (i.e., NCBI reference sequence NP_002277) or SEQ ID NO: 58.

[0044] Unless otherwise indicated herein, "LAG-3" refers to human LAG-3.

[0045] The LAG-3 polypeptide "extracellular domain" or "ECD" refers to a form of the LAG-3 polypeptide that is essentially devoid of transmembrane and cytoplasmic domains. Preferably, the LAG-3 ECD has less than 1% of transmembrane and cytoplasmic domains, and more preferably, the LAG-3 ECD has less than 0.5% of such domains. Even more preferably, the human LAG-3 ECD polypeptide is as shown in SEQ ID NO: 59. The LAG-3 polypeptide ECD may be prepared using methods well known in the art. Alternatively, the human LAG-3 polypeptide ECD may be commercially purchased from various vendors such as Bertyn Bioreagent (Rockville, MD, USA, Cat. No. 32083).

[0046] As used herein, the term "antibody" refers to an immunoglobulin molecule that binds to an antigen. Embodiments of antibodies include monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific or multispecific antibodies, or conjugated antibodies. The antibody may be of any class (e.g., IgG, IgE, IgM, IgD, IgA) and any subclass (e.g., IgG1, IgG2, IgG3, IgG4). In one embodiment, the antibody, or bispecific antibody, of the invention is IgG1.

[0047] Exemplary antibodies of the present disclosure are immunoglobulin G (IgG)-type antibodies composed of four polypeptide chains: two heavy chains (HC) and two light chains (LC) cross-linked via interchain disulfide bonds. The amino-terminal portion of each of the four polypeptide chains contains a variable region of about 100 to 125 or more amino acids that is mainly involved in antigen recognition. The carboxy-terminal portion of each of the four polypeptide chains contains a constant region that is mainly involved in effector functions. Each HC consists of VH and a heavy-chain constant region. Each LC is composed of a light-chain variable region (VL) and a light-chain constant region. The IgG isotype may be further divided into subclasses (e.g., IgG1, IgG2, IgG3, and IgG4).

[0048] The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). The CDRs are exposed on the surface of the protein and are important regions of the antibody for antigen-binding specificity. Each VH and VL is composed of three CDRs and four FRs, and they are arranged from the amino terminus to the carboxy terminus in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In this specification, the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2, and HCDR3", and the three CDRs of the LC are referred to as "LCDR1, LCDR2, and LCDR3". The CDRs contain most of the residues that form specific interactions with the antigen.The assignment of amino acid residues to CDRs may be performed according to well-known schemes, including those described in Kabat (Kabat et al., "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991)), Chothia (Chothia et al., "Canonical structures for the hypervariable regions of immunoglobulins", Journal of Molecular Biology, 196, 901-917 (1987), Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), North (North et al., "A New Clustering of Antibody CDR Loop Conformations", Journal of Molecular Biology, 406, 228-256 (2011)), or IMGT (the international ImMunoGeneTics database available at www.imgt.org; see Lefranc et al., Nucleic Acids Res. 1999;27:209-212). In the disclosure herein, the Kabat method is used to assign amino acid residues to CDRs.

[0049] Embodiments of the disclosure also include antibody fragments (i.e., antigen-binding domains or antigen-binding fragments) that include at least a portion of an antibody that retains the ability to specifically interact with an antigen or an epitope of an antigen, such as Fab, Fab’, F(ab’)2, Fv fragments, scFv antibody fragments, scFab, disulfide-bonded Fv (sdFv), Fd fragments, etc., as used herein.

[0050] The terms "antigen binding domain" and "antigen binding fragment" are used interchangeably herein and, when used herein, refer to the portion of an antibody that binds to an antigen or an epitope of an antigen. For example, the antigen binding domain in the human LAG-3 agonist antibody of the present invention binds to human LAG-3 (SEQ ID NO: 57 or SEQ ID NO: 58), or a fragment thereof, such as LAG-3 ECD (SEQ ID NO: 59). More specifically, the antigen binding domain of the human LAG-3 agonist antibody of the present invention binds to domain 2 (D2) of human LAG-3 (e.g., SEQ ID NO: 61) or a region within D2 (e.g., SEQ ID NO: 62, SEQ ID NO: 63, and / or SEQ ID NO: 64).

[0051] The term "agonize", when used herein, refers to the ability of an antibody, antibody fragment, antigen binding domain, or binding molecule to induce or increase one or more activities or functions associated with an antigen.

[0052] The present disclosure provides anti-human LAG-3 that binds to domain 2 of human LAG-3 (SEQ ID NO: 61) and binds to an epitope comprising one or more amino acid residues within SPHHHLAESF (SEQ ID NO: 64) of human LAG-3 domain 2 (SEQ ID NO: 61). In some embodiments, the present disclosure binds to domain 2 of human LAG-3 (SEQ ID NO: 61) and binds to three or more amino acid residues within PDRPASVHWFRNRGQGRVPVRESPHHHLAESF (SEQ ID NO: 62). In some embodiments, the epitope further comprises one or more amino acid residues within PQVSPMD (SEQ ID NO: 63) of human LAG-3 domain 2.

[0053] The present disclosure provides anti-human LAG-3 that binds to domain 2 of human LAG-3 (SEQ ID NO: 61) and binds to an epitope comprising one or more amino acid residues within SMTASPPGSLRASD (SEQ ID NO: 64). In some embodiments, the present disclosure provides an anti-human LAG-3 antibody that binds to domain 2 of human LAG-3 (SEQ ID NO: 61), wherein the antibody is an agonist.

[0054] As used herein, the term "Fc region" refers to a polypeptide comprising the CH2 and CH3 domains of the constant region of an immunoglobulin, such as IgG1, IgG2, IgG3, or IgG4. Optionally, the Fc region may include a part or the whole of the hinge region of an immunoglobulin, such as IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region is a human IgG Fc region, such as a human IgG1 Fc region, a human IgG2 Fc region, a human IgG3 Fc region, or a human IgG4 Fc region. In some embodiments, the Fc region is a modified IgG Fc region with reduced or eliminated effector function as compared to the corresponding wild-type IgG Fc region. The numbering of the residues of the Fc region is based on the EU index as described in Kabat (Kabat et al, Sequences of Proteins of Immunological Interest, 5th edition, Bethesda, MD: U.S. Dept. of Health and Human Services, Public Health Service, National Institutes of Health, 1991). The boundaries of the Fc region of an immunoglobulin HC may vary, and the Fc region of a human IgG heavy chain is typically defined as a stretch from the N-terminus of the CH2 domain (e.g., amino acid residue position 231 according to EU index numbering) to the C-terminus of the CH3 domain (or the C-terminus of the immunoglobulin).

[0055] Preferably, the antibodies of the present disclosure contain an Fc portion that is a subtype of human IgG1 or IgG4. It is well known that human IgG1 binds to the Fc-gamma receptor family (FcγR) and C1q. Interaction with these receptors can induce antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Certain embodiments of the human LAG-3 agonist antibodies of the present invention include an IgG1 Fc region or an Fc region derived from human IgG1, for example, a modified IgG1 Fc region having modified Fc effector function. In some embodiments of the human LAG-3 agonist antibodies of the present invention, the IgG1 Fc region or the Fc region derived from human IgG1 generally includes a well-known set of Fc region mutations L234A, L235A, and D265S (see IMGT or EU numbering), which are commonly referred to as IgG1 AAS. The term "AAS" refers to the L234A / L235A / D265S mutation. This is an example of an effector-non-expressing mutation in the Fc region (see, for example, Pejchal et al., "Profiling the Biophysical Developability Properties of Common IgG1 Fc Effector Silencing Variants," Antibodies, 12, 54 (2023)). Other embodiments of the human LAG-3 agonist antibodies of the present invention include an IgG4 Fc region or an Fc region derived from human IgG4, for example, a modified IgG4 Fc region having modified Fc effector function. For example, in some embodiments, a well-known serine-to-proline mutation at position 228 ("S228P" according to IMGT or EU numbering) is introduced into the IgG4 Fc region. This modified IgG4 Fc region is generally referred to as IgG4P, IgG4 S228P, IgG4StoP, etc. In one embodiment, the monoclonal anti-human LAG-3 agonist antibody of the present invention does not exhibit substantial ADCC activity. In another embodiment, the monoclonal anti-human LAG-3 agonist antibody of the present invention does not exhibit ADCC activity. In another embodiment, the anti-human LAG-3 agonist antibody of the present invention does not exhibit substantial CDC activity.In another embodiment, the anti-human LAG-3 agonist antibody of the present invention does not exhibit CDC activity.

[0056] In one embodiment, the monoclonal anti-human LAG-3 agonist antibody of the present invention does not substantially deplete T cells. In another embodiment, the monoclonal anti-human LAG-3 agonist antibody of the present invention does not deplete T cells.

[0057] The isolated DNA encoding the CH region can be converted into a full-length heavy chain gene by operably linking the DNA encoding VH to another DNA molecule encoding the heavy chain constant region. The sequences of the heavy chain constant region genes of humans and other mammals are well known in the art. DNA fragments encompassing these regions can be obtained, for example, by standard PCR amplification.

[0058] The isolated DNA encoding the VL region may be converted into a full-length light chain gene by operably linking the DNA encoding VL to another DNA molecule encoding the light chain constant region. The sequences of the light chain constant region genes of humans and other mammals are well known in the art. DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region. In one embodiment, the light chain constant region of the anti-LAG-3 agonist antibody of the present invention is a kappa constant region.

[0059] As used herein, the term "autoimmune disease" refers to an undesirable condition resulting from an inappropriate or unwanted immune response against one's own cells and / or tissues or transplanted cells and / or tissues. The term "autoimmune disease" is meant to include such conditions whether mediated by a humoral or a cellular immune response. Exemplary autoimmune diseases or disorders include, but are not limited to, graft-versus-host disease (GVHD), solid organ transplant rejection, vasculitis, LN, SLE, T1DM, multiple sclerosis (MS), giant cell arteritis (GCA), PsO, psoriatic arthritis (PsA), RA, inflammatory bowel disease (IBD), UC, ankylosing spondylitis (AS), Sjögren's syndrome (SjS), autoimmune hepatitis, scleroderma, celiac disease, Addison's disease, Hashimoto's disease, Graves' disease, atrophic gastritis / pernicious anemia, acquired hypogonadism / infertility, hypoparathyroidism, Coombs positive hemolytic anemia, chronic allergic diseases (such as asthma, hay fever, or allergic rhinitis), Crohn's disease, male or female infertility, Behçet's disease, Wegener's granulomatosis, myocarditis, myositis, polymyalgia rheumatica (PMR), spontaneous abortion, vitiligo, atherosclerosis, autoimmune pancreatitis, bullous pemphigoid, chronic viral infections, and myasthenia gravis. In the present disclosure, the autoimmune diseases are GVHD, solid organ transplant rejection, vasculitis, SLE, T1DM, MS, GCA, PsO, PsA, RA, IBD, UC, AS, SjS, autoimmune hepatitis, and scleroderma.

[0060] The terms "nucleic acid" or "polynucleotide" when used interchangeably herein refer to polymers of nucleotides, including single- and / or double-stranded nucleotide-containing molecules such as DNA, cDNA, and RNA molecules incorporating natural nucleotides, modified nucleotides, and / or nucleotide analogs. The polynucleotides of the present disclosure may also contain, for example, substrates incorporated therein by DNA polymerase or RNA polymerase or a synthetic reaction.

[0061] The polynucleotides of the present disclosure can be expressed in a host cell after the sequence is operably linked to an expression control sequence. Expression vectors are typically replicable in a host organism, either as episomes or as an integrated part of the host chromosomal DNA. Generally, expression vectors contain selectable markers, such as tetracycline, neomycin, and dihydrofolate reductase, to enable the detection of those cells transformed with the desired DNA sequence.

[0062] The antibodies of the present disclosure can be readily produced in mammalian cells, and this non-limiting example includes CHO, NS0, HEK293, or COS cells. Host cells are cultured using techniques well known in the art.

[0063] Vectors containing a target polynucleotide sequence (e.g., a polynucleotide encoding the polypeptide of an antibody and an expression control sequence) can be introduced into host cells by different well-known methods depending on the type of cell host.

[0064] Various methods of protein purification can be used to purify proteins including, but not limited to, antibodies, and such methods are known in the art.

[0065] In another embodiment of the present disclosure, a cell, an antibody, or a nucleic acid encoding an antibody is provided in an isolated form. As used herein, the term "isolated" refers to a cell, protein, peptide, or nucleic acid that does not contain, or substantially does not contain, any other polymeric species found in a cellular environment. "Substantially free" means, as used herein, that the protein, peptide, or nucleic acid of interest contains (on a molar basis) more than 80%, preferably more than 90%, more preferably more than 95% of the polymeric species of interest.

[0066] The antibodies of the present disclosure, or pharmaceutical compositions containing the same, may be administered by parenteral routes, and these non-limiting examples are subcutaneous administration and intravenous administration. The antibodies of the present disclosure may be administered to a patient alone, in a single dose or multiple doses, together with a pharmaceutically acceptable carrier, diluent, or excipient. The pharmaceutical compositions of the present disclosure can be prepared by methods well known in the art (e.g., Remington: The Science and Practice of Pharmacy, 22nd ed. (2012), A. Loyd et al., Pharmaceutical Press), and contain the antibodies disclosed herein, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0067] When referring to the affinity of an anti-human LAG-3 agonist antibody for human LAG-3 (e.g., SEQ ID NO: 57 or SEQ ID NO: 58), human LAG-3 ECD (e.g., SEQ ID NO: 59), and / or cynomolgus monkey LAG3 ECD (e.g., SEQ ID NO: 60), as used herein, "binds to human LAG-3" means, unless otherwise specified, determined by methods well known in the art, including but not limited to the use of a surface plasmon resonance (SPR) biosensor at 25°C or 37°C, and / or by methods essentially as described herein, about 1×10 -7 M, about 1×10 -8 M, about 1×10 -9 M, about 5×10 -10 M, about 1×10 -10 M, or about 5×10 -11 M of K D is intended to mean. In another embodiment, the anti-human LAG-3 agonist antibody of the present disclosure binds to human LAG-3 (e.g., SEQ ID NO: 57 or SEQ ID NO: 58), human LAG-3 ECD (e.g., SEQ ID NO: 59), and / or cynomolgus monkey LAG3 ECD (e.g., SEQ ID NO: 60) with a K of about 1×10 -8 M to about 5×10 -10 M determined by methods well known in the art, including but not limited to the use of an SPR biosensor at 25°C or 37°C, and / or by methods essentially as described herein. DIt binds with. In another embodiment, the anti-human LAG-3 antibody binds to human LAG-3 (e.g., SEQ ID NO: 57 or SEQ ID NO: 58), human LAG-3 ECD (e.g., SEQ ID NO: 59), and / or cynomolgus monkey LAG3 ECD (e.g., SEQ ID NO: 60) at about 5×10 -8 M to about 5×10 -10 M, as determined by methods well known in the art, including the use of an SPR biosensor at 25°C or 37°C, and / or by methods essentially as described herein. In another embodiment, the anti-human LAG-3 antibody has a Kon value and a Koff value of about 1.0x10 -4 to about 1.0x10 -3 , and about 1.3x10 5 to about 2.0×10 5 for human LAG-3 ECD (e.g., SEQ ID NO: 59), respectively (determined by SPR using BIAcore® 8K, essentially as described herein).

[0068] The terms "selectively binds" or "specifically binds" mean that the antibodies of the invention interact with human LAG-3, or cynomolgus monkey LAG-3, more frequently, more rapidly, for a longer duration, with greater affinity, or in some combination of the above, than with other substances. In one embodiment, "specifically binds" means that the antibodies of the invention bind to human LAG-3 at a K D of about 0.1 mM or less. In another embodiment, "specifically binds" means that the antibodies of the invention bind to human LAG-3 at a K D of about 0.01 mM or less. In another embodiment, "specifically binds" means that the antibodies of the invention bind to human LAG-3 at a K D of about 0.001 mM or less. In another embodiment, "specifically binds" means that the antibodies of the invention bind to human LAG-3 at a K D of about 0.0001 mM or less.

[0069] As used herein, the terms "bind" and "binds" mean, unless otherwise specified, the ability of a protein or molecule to form a chemical bond or an attractive interaction with another protein or molecule, resulting in close proximity of two proteins or molecules as determined by common methods well known in the art.

[0070] As used herein, the term "epitope" refers to the amino acid residues of an antigen that are bound by an antibody. An epitope can be a linear epitope, a conformational epitope (discontinuous epitope), or a hybrid epitope.

[0071] The term "epitope" may be used with respect to a conformational epitope. A conformational epitope may, in some embodiments, be used to describe the region of an antigen covered by an antibody (e.g., the footprint of the antibody when bound to the antigen). In some embodiments, a conformational epitope may describe the amino acid residues of an antigen that are within a particular proximity (e.g., within a particular number of angstroms) of the amino acid residues of an antibody.

[0072] The term "epitope" may also be used with respect to a functional epitope. A functional epitope may, in some embodiments, be used to describe the amino acid residues of an antigen that interact with the amino acid residues of an antibody in a manner that contributes to the binding energy between the antigen and the antibody.

[0073] Epitopes can be determined according to different experimental techniques, also referred to as "epitope mapping techniques". The determination of epitopes may vary based on the different epitope mapping techniques used, and it is understood that it may also vary depending on the different experimental conditions used, for example, due to conformational changes or cleavage of the antigen induced by specific experimental conditions. Epitope mapping techniques include, but are not limited to, X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, site-directed mutagenesis, species swap mutagenesis, alanine scanning mutagenesis, hydrogen-deuterium exchange (HDX), and cross-blocking assays, which are well-known in the art (e.g., Rockberg and Nilvebrant, Epitope Mapping Protocols: Methods in Molecular Biology, Humana Press, 3rd ed. 2018).

[0074] As used herein, the terms "competes for binding" or "competes with" refer to two antibodies that cross-compete for binding to the same antigen (i.e., compete with each other). In some embodiments, two antibodies may compete for binding to the same antigen if they bind to spatially overlapping regions of the same antigen. In some embodiments, two antibodies may compete for binding to the same antigen, where the antibodies bind to non-overlapping regions of the antigen, but the binding of one antibody blocks the binding by the other antibody, for example, due to steric hindrance or conformational changes of the antigen induced by the first antibody. As used herein, the term "substantially pure" refers to a material that contains at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% free of contaminants, for example, an antibody of the present invention.

[0075] As used herein, the term "about" means plus or minus 10 percent of the recited value or range of values. For example, "about" 12 includes values in the range of 10.8 (including 10.8) to 13.2 (including 13.2), and "about" 10 weight percent includes compositions containing from 9 (including 9) to 11 (including 11) weight percent, etc.

[0076] As used herein, the term "adaptive immunity" includes the arm of the immune response that, in contrast to the innate arm of the immune response, is antigen-specific and exhibits an enhanced secondary antigen-specific immune response upon restimulation with the same antigen.

[0077] The term "treating" (or "treat" or "treatment") refers to delaying, interfering with, suppressing, alleviating, halting, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.

[0078] "Effective amount" means the amount of the antibody of the present invention, or a pharmaceutical composition containing such an antibody, that elicits a biological or medical response in a tissue, system, animal, mammal, or human being being explored by a researcher, physician, or other clinician, or that induces a desired therapeutic effect. The effective amount of an antibody may vary depending on factors such as the medical condition, age, gender, and weight of the individual, as well as the ability of the antibody to induce the desired response in the individual. The effective amount is also an amount in which the therapeutically beneficial effects exceed any toxic or detrimental effects of the antibody. Such benefits include any one or more of an increase in immune tolerance of a transplanted organ, a stabilized autoimmune disease or disorder, or an improvement in the signs or symptoms of an autoimmune disorder. The effective amount can be readily determined by one of ordinary skill in the art using known techniques and by observing the results obtained under similar circumstances. When determining the effective amount for a patient, a number of factors are considered by the attending diagnostician, including the size, age, and general health of the patient, the specific disease or disorder involved, the degree or involvement or severity of the disease or disorder, the response of the individual patient, the specific compound being administered, the mode of administration, the bioavailability characteristics of the preparation being administered, the dosage regimen selected, the use of concomitant medications, and other relevant circumstances, but are not limited to these several factors considered by the attending physician.

[0079] The dosing schedule for administering the antibody of the present invention may be adjusted to provide the optimal desired response (e.g., therapeutic effect).

[0080] A potential advantage of the methods disclosed herein is the potential to provide significant and / or long-term remission in patients suffering from autoimmune disorders with an acceptable safety profile including acceptable tolerability, toxicity, and / or adverse events, and thus the patient benefits from the overall treatment regimen. The effectiveness of the treatments of the present disclosure can be measured by various endpoints commonly used when evaluating treatments for various autoimmune disorders, including but not limited to American College of Rheumatology (ACR) 20, ACR50, ACR70, Psoriasis Area and Severity Index (PASI) 50, PASI75, PASI90, PASI100, Systemic Lupus Erythmatosus Disease Activity Index (SLEDAI). For example, various other approaches to determining the effectiveness of any particular treatment of the present disclosure can optionally be used, including but not limited to measurement of immune cell activation markers, measures of inflammation, visualization of cell cycle-dependent biomarker measurements, and / or measurement of response through pain assessment.

[0081] As used herein, the term "modified human IgG1" means human IgG1 that has been engineered to reduce its binding to at least one human Fc gamma receptor. Typically, this is accomplished by mutating residues that lead to reduced binding of the antibody to Fc gamma receptors, such as the P329A, L234A, and L235A mutations.

[0082] The present disclosure provides antibodies that bind to human LAG-3, or antigen-binding domains thereof, where the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), where the VH comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, where HCDR1 comprises SEQ ID NO: 65, HCDR2 comprises SEQ ID NO: 2, HCDR3 comprises SEQ ID NO: 48, HCDR1 contains SEQ ID NO: 1, HCDR2 contains SEQ ID NO: 2, HCDR3 contains SEQ ID NO: 48, HCDR1 contains SEQ ID NO: 1, HCDR2 contains SEQ ID NO: 2, HCDR3 contains SEQ ID NO: 3, HCDR1 contains SEQ ID NO: 1, HCDR2 contains SEQ ID NO: 2, HCDR3 contains SEQ ID NO: 15, HCDR1 contains SEQ ID NO: 52, HCDR2 contains SEQ ID NO: 22, HCDR3 contains SEQ ID NO: 53, HCDR1 contains SEQ ID NO: 21, HCDR2 contains SEQ ID NO: 22, HCDR3 contains SEQ ID NO: 23, HCDR1 contains SEQ ID NO: 31, HCDR2 contains SEQ ID NO: 22, HCDR3 contains SEQ ID NO: 32, or HCDR1 contains SEQ ID NO: 38, HCDR2 contains SEQ ID NO: 39, HCDR3 contains SEQ ID NO: 40, and LCDR1 contains SEQ ID NO: 66, LCDR2 contains SEQ ID NO: 67, LCDR3 contains SEQ ID NO: 68, LCDR1 contains SEQ ID NO: 49, LCDR2 contains SEQ ID NO: 5, LCDR3 contains SEQ ID NO: 6, LCDR1 contains SEQ ID NO: 4, LCDR2 contains SEQ ID NO: 5, LCDR3 contains SEQ ID NO: 6, LCDR1 contains SEQ ID NO: 16, LCDR2 contains SEQ ID NO: 5, LCDR3 contains SEQ ID NO: 6, LCDR1 contains SEQ ID NO: 24, LCDR2 contains SEQ ID NO: 25, LCDR3 contains SEQ ID NO: 54, LCDR1 contains SEQ ID NO: 24, LCDR2 contains SEQ ID NO: 25, LCDR3 contains SEQ ID NO: 26, LCDR1 contains SEQ ID NO: 24, LCDR2 contains SEQ ID NO: 25, LCDR3 contains SEQ ID NO: 33, or LCDR1 contains SEQ ID NO: 41, LCDR2 contains SEQ ID NO: 42, LCDR3 contains SEQ ID NO: 43. In some embodiments, the anti-human LAG-3 antibody of the present invention is a human LAG-3 agonist antibody.

[0083] The present disclosure provides an antibody or an antigen-binding domain thereof that binds to human LAG-3, the antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), VH is comprising a VH selected from the group consisting of SEQ ID NO: 69, SEQ ID NO: 50, SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 55, SEQ ID NO: 27, SEQ ID NO: 34, and SEQ ID NO: 44, VL is comprising a VL selected from the group consisting of SEQ ID NO: 70, SEQ ID NO: 51, SEQ ID NO: 8, SEQ ID NO: 18, SEQ ID NO: 56, SEQ ID NO: 28, SEQ ID NO: 35, and SEQ ID NO: 45. In certain embodiments, VH comprises SEQ ID NO: 69, VL comprises SEQ ID NO: 70, VH comprises SEQ ID NO: 50, VL comprises SEQ ID NO: 51, VH comprises SEQ ID NO: 7, VL comprises SEQ ID NO: 8, VH comprises SEQ ID NO: 17, VL comprises SEQ ID NO: 18, VH comprises SEQ ID NO: 55, VL comprises SEQ ID NO: 56, VH comprises SEQ ID NO: 27, VL comprises SEQ ID NO: 28, VH comprises SEQ ID NO: 34, VL comprises SEQ ID NO: 35, or VH comprises SEQ ID NO: 44, VL comprises SEQ ID NO: 45. In some embodiments, the anti-human LAG-3 antibody comprises a constant heavy (CH) region comprising SEQ ID NO: 9, and a constant light (CL) region comprising SEQ ID NO: 10. In some embodiments, the anti-human LAG-3 antibody of the present invention is a human LAG-3 agonist antibody. In some embodiments, the anti-human LAG-3 antibody is of the IgG1 subtype or IgG4 subtype or variants thereof, such as IgG1AAS or IgG4P.

[0084] The present disclosure provides an antibody or an antigen-binding domain thereof that binds to human LAG-3, the antibody being a. an HC comprising SEQ ID NO: 71 and an LC comprising SEQ ID NO: 72, b. an HC comprising SEQ ID NO: 11 and an LC comprising SEQ ID NO: 12, c. an HC comprising SEQ ID NO: 19 and an LC comprising SEQ ID NO: 20, d. an HC comprising SEQ ID NO: 29 and an LC comprising SEQ ID NO: 30, e. an HC comprising SEQ ID NO: 36 and an LC comprising SEQ ID NO: 37, or f. It includes an HC containing SEQ ID NO: 46 and an LC containing SEQ ID NO: 47.

[0085] In another embodiment, the anti-human LAG-3 antibody of the present invention is a LAG-3 agonist antibody.

[0086] In some embodiments, the anti-human LAG-3 antibody of the present invention provides an antibody or an antigen-binding domain thereof that binds to human LAG-3, and the antibody a. an HC containing a sequence having at least 95% sequence identity to SEQ ID NO: 71 and an LC containing a sequence having at least 95% sequence identity to SEQ ID NO: 72, b. an HC containing a sequence having at least 95% sequence identity to SEQ ID NO: 11 and an LC containing a sequence having at least 95% sequence identity to SEQ ID NO: 12, c. an HC containing a sequence having at least 95% sequence identity to SEQ ID NO: 19 and an LC containing a sequence having at least 95% sequence identity to SEQ ID NO: 20, d. an HC containing a sequence having at least 95% sequence identity to SEQ ID NO: 29 and an LC containing a sequence having at least 95% sequence identity to SEQ ID NO: 30, e. an HC containing a sequence having at least 95% sequence identity to SEQ ID NO: 36 and an LC containing a sequence having at least 95% sequence identity to SEQ ID NO: 37, or f. an HC containing a sequence having at least 95% sequence identity to SEQ ID NO: 46 and an LC containing a sequence having at least 95% sequence identity to SEQ ID NO: 47.

[0087] In some embodiments, the anti-human LAG-3 antibody of the present invention provides an antibody or an antigen-binding domain thereof that binds to human LAG-3, and the antibody a. an HC containing a sequence having at least 90% sequence identity to SEQ ID NO: 71 and a light chain containing a sequence having at least 90% sequence identity to SEQ ID NO: 72, b. An HC comprising a sequence having at least 90% sequence identity to SEQ ID NO: 11, and an LC comprising a sequence having at least 90% sequence identity to SEQ ID NO: 12, c. An HC comprising a sequence having at least 90% sequence identity to SEQ ID NO: 19, and a light chain comprising a sequence having at least 90% sequence identity to SEQ ID NO: 20, d. An HC comprising a sequence having at least 90% sequence identity to SEQ ID NO: 29, and a light chain comprising a sequence having at least 90% sequence identity to SEQ ID NO: 30, e. An HC comprising a sequence having at least 90% sequence identity to SEQ ID NO: 36, and a light chain comprising a sequence having at least 90% sequence identity to SEQ ID NO: 37, or f. An HC comprising a sequence having at least 90% sequence identity to SEQ ID NO: 46, and a light chain comprising a sequence having at least 90% sequence identity to SEQ ID NO: 47.

[0088] In some embodiments, the antibody of the invention comprises two HCs and two light LCs, and the HC and LC polypeptides are a. the HC of SEQ ID NO: 71 and the LC of SEQ ID NO: 72; b. the HC of SEQ ID NO: 11 and the LC of SEQ ID NO: 12; c. the HC of SEQ ID NO: 19 and the LC of SEQ ID NO: 20; d. the HC of SEQ ID NO: 29 and the LC of SEQ ID NO: 30; e. the HC of SEQ ID NO: 36 and the LC of SEQ ID NO: 37; and f. the HC of SEQ ID NO: 46 and the LC of SEQ ID NO: 47, and are selected from the group consisting of.

[0089] In another embodiment, the anti-human LAG-3 antibodies of the invention are capable of detecting human LAG-3 (e.g., SEQ ID NO:57 or SEQ ID NO:58), human LAG-3 ECD (e.g., SEQ ID NO:59), and / or cynomolgus monkey LAG3 ECD (e.g., SEQ ID NO:60) at about 1×10 mAb, as determined by methods well known in the art, including but not limited to, the use of a surface plasmon resonance (SPR) biosensor at 25° C. or 37° C., and / or by methods essentially as described herein. -7 M ~ approx. 5×10 -11 K of M D In another embodiment, an anti-human LAG-3 agonist antibody of the disclosure binds to human LAG-3 (e.g., SEQ ID NO: 57 or SEQ ID NO: 58), human LAG-3 ECD (e.g., SEQ ID NO: 59), and / or cynomolgus monkey LAG3 ECD (e.g., SEQ ID NO: 60) at about 1×10 -8 M ~ approx. 1×10 -10 K of M D In another embodiment, the anti-human LAG-3 antibody binds to human LAG-3 (e.g., SEQ ID NO: 57 or SEQ ID NO: 58), human LAG-3 ECD (e.g., SEQ ID NO: 59), and / or cynomolgus monkey LAG3 ECD (e.g., SEQ ID NO: 60) at about 5×10 -8 M ~ approx. 5×10 -10 K of M D In another embodiment, the anti-human antibody has an affinity of about 1×10 to human LAG-3 (e.g., SEQ ID NO: 57 or SEQ ID NO: 58), human LAG-3 ECD (e.g., SEQ ID NO: 59), and / or cynomolgus LAG3 ECD (e.g., SEQ ID NO: 60). -9 M ~ approx. 1×10 -10 K of M D It has affinity with

[0090] In another embodiment, the anti-human LAG-3 agonist antibody is a human IgG1 or IgG4 isotype. In another embodiment, the antibody is a human IgG1 isotype. A non-limiting example of an anti-human LAG-3 agonist antibody of the human IgG1 isotype is the amino acid sequence of SEQ ID NO:11.

[0091] In another embodiment, the antigen-binding domain of the anti-human LAG-3 is a single-chain variable fragment (scFv).

[0092] The present disclosure also provides a nucleic acid comprising a sequence encoding one or both of SEQ ID NO: 11 or SEQ ID NO: 12.

[0093] In another embodiment, the present disclosure provides a vector comprising a nucleic acid sequence encoding one or both of SEQ ID NO: 11 and SEQ ID NO: 12.

[0094] In another embodiment, the present disclosure provides a composition comprising a first vector comprising a nucleic acid sequence encoding SEQ ID NO: 11 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO: 12.

[0095] In one embodiment, the present disclosure provides a cell comprising a vector. In another embodiment, the present disclosure provides a cell comprising a composition. In another embodiment, the cell is a mammalian cell. In another embodiment, the cell, or mammalian cell, is isolated. In another embodiment, the present disclosure provides a process for producing an antibody comprising culturing the cell under conditions such that the antibody is expressed and recovering the expressed antibody from the culture medium. In another embodiment, the present disclosure provides an antibody produced by culturing the cell under conditions such that the antibody is expressed and recovering the expressed antibody from the culture medium. In another embodiment, the present disclosure provides a composition comprising the anti-human LAG-3 agonist antibody of the present invention.

[0096] The present disclosure also provides a pharmaceutical composition comprising the anti-human LAG-3 agonist antibody of the present invention and a pharmaceutically acceptable excipient, diluent, or carrier. In one embodiment, the pharmaceutical composition comprises arginine.

[0097] The present disclosure also provides a prefilled syringe of a pharmaceutical composition comprising the anti-human LAG-3 agonist antibody of the present invention and a pharmaceutically acceptable excipient, diluent, or carrier. In one embodiment, the pharmaceutical composition comprises arginine.

[0098] The present disclosure also provides a method of treating an autoimmune disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the anti-human LAG-3 agonist antibody of the present invention. In one embodiment, the autoimmune disease is RA, PsO, UC, T1DM, LN, or SLE. In one embodiment, the autoimmune disease is an active disease. In another embodiment, the autoimmune disease is in remission.

[0099] The present disclosure also provides the anti-human LAG-3 agonist antibody of the present invention for use in therapy.

[0100] The present disclosure also provides the anti-human LAG-3 agonist antibody of the present invention for use in treating an autoimmune disease. In one embodiment, the autoimmune disease is RA, PsO, UC, T1DM, LN, or SLE. In another embodiment, the autoimmune disease is an active disease. In another embodiment, the autoimmune disease is in remission.

[0101] The present disclosure also provides a pharmaceutical composition comprising the anti-human LAG-3 agonist antibody of the present invention for use in treating an autoimmune disease such as RA, PsO, UC, T1DM, LN, or SLE. In one embodiment, the autoimmune disease is an active disease. In another embodiment, the immune disease is in remission.

[0102] The present disclosure also provides the use of the anti-human LAG-3 agonist antibody of the present invention in the manufacture of a medicament for the treatment of RA, PsO, UC, T1DM, LN, or SLE.

[0103] Mutations that promote the desired quality are well known to those skilled in the art. However, even if there are such mutations, it is not possible to predict which, or how many of such mutations, will promote the desired quality of any particular antibody. The present invention provides an antibody comprising an amino acid residue mutation that promotes one or more of desired antibody expression, assembly, reduction or elimination of C1q binding, reduction or elimination of non-specific and self-interactions, reduction of the viscosity of a composition or formulation, elimination of deamidated residues, and reduction of immunogenicity.

[0104] In embodiments that refer to the treatment methods described herein, such embodiments are also further embodiments for use in the manufacture of a medicament for use in, or alternatively for use in the manufacture of a medicament for use in, such treatment.

[0105] In one embodiment, the anti-human LAG-3 agonist antibody of the present invention is substantially pure. In another embodiment, the anti-human LAG-3 agonist antibody of the present invention is sterilized.

[0106] In one embodiment, the autoimmune disease is RA, PsO, UC, T1DM, LN, or SLE. In another embodiment, the autoimmune disease is rheumatoid arthritis. In another embodiment, the autoimmune disease is ulcerative colitis. In another embodiment, the autoimmune disease is type I diabetes. In another embodiment, the autoimmune disease is lupus nephritis. In another embodiment, the autoimmune disease is systemic lupus erythematosus. In another embodiment, the immune disease is an active disease. In another embodiment, the immune disease is in remission.

[0107] In another embodiment, the anti-human LAG-3 agonist antibody of the present invention binds to human LAG-3 but does not deplete T cells.

[0108] In another embodiment, the anti-human LAG-3 agonist antibody of the present invention agonizes the LAG-3 signaling pathway.

[0109] In another embodiment, the anti-human LAG-3 agonist antibody of the present invention binds to human LAG-3 with a desirable association rate and dissociation rate for optimal agonist activity.

[0110] In another embodiment, the anti-human LAG-3 agonist antibody of the present invention decreases T cell proliferation by promoting downregulation of T cell receptor signaling, rather than by T cell depletion.

[0111] In another embodiment, the anti-human LAG-3 agonist antibody of the present invention agonizes human LAG-3 in an immunologically relevant context to achieve in vivo efficacy.

[0112] The present disclosure also provides a method comprising: (a) contacting the anti-human LAG-3 agonist antibody of the present invention with a cell expressing an extracellular domain of human LAG-3 fused to an enzyme donor subunit; (b) incubating the contacted cells under conditions suitable for the antibody to bind to the extracellular domain of human LAG-3; (c) contacting the cells with a reporter compound; (d) assaying the amount of the reporter compound; and (e) determining the IC 50 for the antibody binding to the cells. In one embodiment, the enzyme acceptor subunit of the reporter gene is β-galactosidase and the enzyme donor subunit is the PK1 enzyme donor subunit of β-galactosidase. In another embodiment, the antibody comprises the HC of SEQ ID NO: 11 and the LC of SEQ ID NO: 12.

[0113] Methods for assaying LAG-3 activity in vitro are well known to those skilled in the art (see, for example, Angin M, et al., J. Immunol. 2020;204(4):810-818).

[0114] In vivo mouse models of immune activity are well known to those skilled in the art as shown and disclosed herein (e.g., Vincelette J, et al., Arthritis Res Ther. 2007;9(6):R123.doi:10.1186 / ar2331).

[0115] The DNA molecule of the present disclosure is a DNA molecule comprising a non-natural polynucleotide sequence encoding a polypeptide having the amino acid sequence of at least one of the polypeptides in the antibody of the present disclosure.

[0116] The polynucleotide of the present disclosure may be expressed in a host cell after its sequence is operably linked to an expression control sequence. Expression vectors are typically replicable in a host organism, either as an episome or as an integrated part of the host chromosomal DNA. Generally, expression vectors contain a selectable marker, such as tetracycline, neomycin, and dihydrofolate reductase, to enable the detection of those cells transformed with the desired DNA sequence.

[0117] An expression vector containing a target polynucleotide sequence (e.g., a polynucleotide encoding the polypeptide of the antibody of the present invention and an expression control sequence) can be introduced into a host cell by different well-known methods depending on the type of the host cell. Host cells (e.g., mammalian cells) include cells that are stably or transiently transfected, transformed, transduced, or infected with one or more expression vectors that express all or part of the anti-human LAG-3 agonist antibody described herein. According to some embodiments, the host cell may be stably or transiently transfected, transformed, transduced, or infected with an expression vector that expresses the HC polypeptide and an expression vector that expresses the LC polypeptide of the anti-human LAG-3 agonist antibody or its antigen-binding domain as described herein. In some embodiments, the host cell may be stably or transiently transfected, transformed, transduced, or infected with an expression vector that expresses the HC polypeptide and the LC polypeptide of the anti-human LAG-3 agonist antibody or its antigen-binding domain as described herein. The antibody of the present disclosure or its antigen-binding domain may be easily produced in mammalian host cells, non-limiting examples of which include CHO, NS0, HEK293, or COS cells. The host cells may be cultured using techniques well known in the art.

[0118] Antibody expression in mammals typically results in glycosylation. Antibody glycosylation is typically either N-linked or O-linked. N-linked glycosylation refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment of a sugar, such as N-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid. Typically, glycosylation occurs in the Fc region of the antibody at highly conserved N-glycosylation sites (e.g., position 297 in IgG1 according to IMGT or EU index numbering). The glycosylation site can be modified to alter glycosylation (e.g., to block or reduce glycosylation, or to change the amino acid sequence to generate additional or diverse glycosylation).

[0119] Expression of antibodies in mammals from IgG subclasses can result in truncation of C-terminal amino acids from one or both heavy chains. For example, in the case of IgG1 antibodies, one or two C-terminal amino acids can be removed. For IgG1 antibodies, when a C-terminal lysine is present, it may be truncated or removed from the heavy chain during expression. Thus, the second glycine from the end may also be truncated or removed from the heavy chain (see, e.g., reference to SEQ ID NOs).

[0120] Expression of antibodies in mammals can also result in modification of N-terminal amino acids. For example, when the most N-terminal amino acid of HC or LC is glutamine, it may be modified to pyro-glutamic acid.

[0121] The antibodies of the present disclosure may be purified using various methods of protein purification, such methods being well known in the art and described, for example, in Deutscher, Methods in Enzymology 182:83-89 (1990) and Scopes, Protein Purification: Principles and Practice, 3rd Edition, Springer, NY (1994).

[0122] The sequences referred to herein are numbered according to the sequence identification numbers listed in Table 1. The sequences in Table 1 are amino acid sequences unless otherwise indicated.

[0123] [Table 1] [Examples]

[0124] Antibody Expression and Purification The anti-human LAG-3 agonist antibody of the present invention can be essentially expressed and purified as follows. An expression system for secreting the antibody using an optimal predetermined heavy chain: light chain vector ratio, or a single vector system encoding both the HC and the light chain, may transiently or stably transfect an appropriate host cell such as HEK293 or CHO. The antibodies of the present disclosure may be transfected either transiently or stably in an expression system for secreting the antibody using one or more DNA molecules encoding the HC and LC of the LAG-3 agonist antibody of the present invention.

[0125] The antibody may be purified using one of many commonly used techniques. For example, the medium may be conveniently applied to a MabSelect column (GE Healthcare), or a KappaSelect column (GE Healthcare), equilibrated with a compatible buffer such as phosphate buffered saline (pH 7.4). The column may be washed to remove non-specific binding components. The bound antibody may be eluted, for example, by a pH gradient (from 20 mM Tris buffer (pH 7.0) to 10 mM sodium citrate buffer (pH 3.0), or from phosphate buffered saline (pH 7.4) to 100 mM glycine buffer (pH 3.0), etc.). The antibody fractions may be detected by, for example, ultraviolet absorbance or SDS-PAGE, and then pooled. Further purification is optional depending on the intended use. The purified antibody may be concentrated and / or sterile filtered using common techniques. Soluble aggregates, multimers, and mispairings may be effectively removed by common techniques including size exclusion, hydrophobic interaction, ion exchange, multimodal, affinity, or hydroxyapatite chromatography. The purified antibody may be immediately frozen at -70 °C or lyophilized.

[0126] Binding to human or cynomolgus monkey LAG-3 Perform surface plasmon resonance (SPR) at 37°C to determine the binding kinetics and affinity of the LAG-3 antibody for human LAG-3 and / or cynomolgus monkey LAG-3. Using a Biacore® T200 (Cytiva, Marlborough, MA), the binding kinetics and affinity of Antibody A for human LAG-3 soluble extracellular domain (ECD), SEQ ID NO: 59, and cynomolgus monkey LAG-3 ECD (ACROBiosystems, Cat# LA3-C82H3, SEQ ID NO: 59) were measured by surface plasmon resonance. Samples were diluted in HBS-EP+ (10 mM Hepes, 150 mM NaCl, 3 mM EDTA, 0.05% Tween-20, pH 7.6) (Teknova Cat# H8022) containing 5 g / L BSA (Jackson ImmunoResearch Cat# 001-000-161) running buffer. A PrismA Series S Sensor Chip (Cat# 29650263) was purchased from Cytiva.

[0127] Binding was evaluated by the antibody capture method using multi-cycle kinetics. Each cycle was performed at either 37°C or 25°C at a flow rate of 10 μL / min for antibody capture onto the PrismA chip, 100 μL / min for analyte association (120-second contact time) and dissociation (900 seconds). Each cycle consisted of the following steps: injection of the antibody at 1 μg / mL into HBS-EP+ targeting an Rmax value of 60 RU on the flow cell, injection of the analyte into HBS-EP+ (concentration range of 500 nM to 0.25 nM by 2-fold serial dilution of human LAG-3-His ECD (SEQ ID NO: 59)), followed by a 900-second dissociation phase, and regeneration using 10 μL of 10 mM glycine hydrochloride (pH 1.5) for a 60-second contact time at a flow rate of 10 μL / min. All analyte concentrations were determined using the monomer molecular weight (MW) value. The association rate (k on ) and dissociation rate (k off) was evaluated using double reference by subtracting the reference of flow cell 1 in addition to subtracting the 0 nM blank, and was fitted to the "1:1 (Langmuir) binding" model with BIAevaluation software version 4.1. The dissociation constant (K D ) was calculated from the association rate according to the relationship K D =K off / K on . Stoichiometry = [RU max / RU 捕捉 / [MW 分析物 / MW 抗体 (where MW 抗体A is 150 kDa). Values are reported as mean ± standard deviation.

[0128] In the experiment essentially carried out as described above, the results in Table 2 were obtained. The results in Table 2 show that antibodies A, C, and D bind to human and cynomolgus monkey LAG-3-ECD with high affinity.

[0129]

Table 2

[0130] Peripheral blood mononuclear cell binding assay The ability of antibody A to bind to cell surface human or cynomolgus monkey LAG-3 can be measured using a flow cytometry assay.

[0131] Briefly, human peripheral blood mononuclear cells (PBMCs) were isolated from healthy human Trima LRS (San Diego Blood Bank; San Diego, CA) using Ficoll (GE Healthcare #17144002) and activated for 3 days at 37°C in 5% CO2 in complete RPMI (RPMI, Corning #MT10041CV; 10% FBS, Corning #35-011-CV; 1X Glutamax, Gibco #35050061; 1X P / S, Corning #30002CI; 1X BME, Gibco #21985023; 1X MEM, Gibco #11140050, 1X sodium pyruvate, Corning #25-000-CI) using 4 ng / mL of staphylococcal enterotoxin B (SEB) (Toxin Technologies, BT2021MG).

[0132] Cynomolgus monkey PBMCs were isolated from whole blood (BioIVT #NHP01WBK2-0000861) using 90% Ficoll in PBS (Corning #21-031-CM) and activated in RPMI for 3 days using 4 ng / mL of staphylococcal enterotoxin B (SEB) (Toxin Technologies, BT2021MG) as described above.

[0133] Cells were incubated with serial 4-fold dilutions of antibody A or isotype hG1 starting at 100 μg / mL in PBS + 2% FBS at 4°C for 30 minutes. Samples were washed and stained with 1 μg / mL of Alexa Fluor 647 AffiniPure F(ab’)2 fragment goat anti-human IgG, Fcγ fragment specific (Jackson Immuno Research 109-606-098) in PBS + 2% FBS at 4°C for 30 minutes. After thorough washing, human samples were stained with CD3 Biolegend #300424, CD4 Biolegend #300512, CD8 Biolegend #301006, PD-1 Biolegend 329906, non-competing LAG3 conjugated with in-house BN20-2739-10 418611, Live / Dead Near IR Invitrogen L34975A, CD19 Biolegend 302218, CD33 Biolegend 366614, and CD56 Biolegend 362512 in PBS + 2% FBS at 4°C for 30 minutes. Cynomolgus monkey samples were stained with CD3 BD 557917, CD4 BD 566910, CD8 BD 557746, PD-1 Biolegend 329906, non-competing LAG3 conjugated with in-house BN20-2739-10 418611, and Live / Dead Near IR Invitrogen L34975A in PBS + 2% FBS at 4°C for 30 minutes. Cells were washed and processed on a BioRad ZE5 cytometer, and data were analyzed with FlowJo software (BD Biosciences). The percentage of cells bound by each antibody was quantified and graphed with Prism (Graphpad, San Diego, CA).

[0134] In experiments conducted essentially as described above, the data in Table 3 were obtained. Antibody A bound to cell membrane-expressed human LAG-3 and cynomolgus monkey LAG-3 (human n = 5, cynomolgus monkey n = 3).

[0135]

Table 3

[0136] In Vitro Inhibition of T Cell Proliferation The ability of the anti-human LAG-3 agonist antibody of the present invention to inhibit T cell proliferation was measured as follows.

[0137] Briefly, human peripheral blood mononuclear cells (PBMCs) were isolated from healthy human Trima LRS (San Diego Blood Bank; San Diego, CA) using Ficoll (GE Healthcare #17144002). The isolated PBMCs were labeled with a proliferation dye (Invitrogen Cat#6084290) at 37°C for 20 minutes and then washed thoroughly.

[0138] The labeled PBMCs were treated with 12-point, 4-fold serial dilutions of each antibody, starting at 100 ug / mL, in complete RPMI (RPMI, Corning #MT10041CV; 10% FCS, Corning #MT35011CV; 1X Glutamax, Gibco #35050061; 1X P / S, Corning #30002CI; 1X BME, Gibco #21985023; 1X MEM, Gibco #11140050, 1X sodium pyruvate, Corning #25-000-CI) at room temperature for 30 minutes. Next, the cells were stimulated with 5% CO2 at 37°C for 3 days with SEB (Toxin Technologies, BT2021MG) at a final concentration of 4 ng / mL in complete RPMI. Next, the cells were washed thoroughly.

[0139] The samples were stained with CD3 Biolegend #300424, CD4 Biolegend #300512, CD8 Biolegend #301006, PD-1 Biolegend 329906, unconjugated LAG3 418611 conjugated with in-house BN20-2739-10, Live / Dead Near IR Invitrogen L34975A, CD19 Biolegend 302218, CD33 Biolegend 366614, CD56 Biolegend 362512 in PBS + 2% FBS at 4°C for 30 minutes. After washing thoroughly, the cells were processed on a BioRad ZE5 cytometer and the data were analyzed with Flowjo software (BD Biosciences). The percentage of proliferated cells was quantified and graphed with Prism (Graphpad, San Diego, CA).

[0140] In the experiments conducted essentially as described above, antibody A inhibited human primary T cell proliferation as shown in Table 4 (n = 9).

[0141]

Table 4

[0142] In vivo inhibition of graft-versus-host disease (GVHD) in a humanized mouse model To demonstrate the immunomodulatory activity of the LAG-3 agonist antibody of the present invention, a humanized model of xenogeneic GvHD can be utilized. This model is created by transplanting human PBMCs into immunodeficient mice. Human immune cells recognize the mice as foreign and initiate an immune response, resulting in a significant increase in human pro-inflammatory cytokines, immune cell activation, proliferation, and extravasation into tissues, ultimately leading to weight loss and multi-organ dysfunction. Importantly, the inflammatory response is driven by human cells, and thus human-specific therapies can be investigated in the model.

[0143] Briefly described, female NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ, JAX Labs, stock number 05557) are housed three per cage under a 12-hour light:dark cycle at 72°C and allowed free access to food and water (n = 76). Human PBMCs are isolated from LRS tubes obtained from blood donors (San Diego Blood Bank) using SepMate 50 Ficoll preparation tubes according to the manufacturer's instructions (STEMCELL Technologies, Vancouver, BC). Freshly isolated PBMCs are suspended in PBS at 1.2 × 10 8 cells / mL and 100 μL of the PBMC suspension is transplanted intravenously into the mice on day 0 (1.2 × 10 7 / mouse). PBMCs were given to 72 mice, but 4 mice remained as non-engraftment controls.

[0144] On day 1 after engraftment, the mice are divided into nine weight-matched groups (n groups (n = 8 / group)) and the antibody is administered subcutaneously at 0.1, 1.0, or 3.0 mg / kg. Administration is continued weekly for the remainder of the experiment. Health checks and body weight measurements are performed regularly. Mice that have lost 20% of their starting body weight or are clearly suffering are euthanized. Clinical signs common to this model are ruffled fur, hunched body, lethargy, and respiratory or locomotor difficulty.

[0145] When most of the isotype control mice require euthanasia due to disease progression, sacrifice all the mice. For all sacrificed mice, collect blood into EDTA tubes by cardiac puncture under isoflurane anesthesia. Additionally, obtain a provisional blood sample on day 10 from the retro-orbital sinus. Clarify the blood from both collections by centrifugation for human plasma cytokine analysis. Calculate the weight change as a percentage of their baseline weight: (weight on day (x) / weight on day 0)*100. Mice that require euthanasia before the end of the study will have their last weight measurement carried through to the end. Plasma cytokines are measured using Mesoscale Discovery Human Th1 / Th2 10-Vplex (Rockville, Maryland) according to the manufacturer's instructions. Graph the data and calculate the statistics using Prism Software (GraphPad, San Diego, CA). Determine the difference in weight between groups by 2-way RM-ANOVA with Tukey's post hoc test. Determine the difference in plasma cytokine levels by 1-way ANOVA with Tukey's post hoc test. Differences between test groups are considered significant when p < 0.05.

[0146] As demonstrated by significant wasting in NSG mice, engraftment of human PBMCs from induced GvHD required study termination on day 36 post-engraftment. Treatment with antibody A can significantly attenuate disease progression, as measured by reduction of weight loss in mice, in a dose-dependent manner. Furthermore, the human LAG-3 agonist antibody can inhibit a significant increase in plasma human pro-inflammatory cytokines associated with disease progression. Thus, the anti-human LAG-3 agonist antibody of the present invention can attenuate the pathogenicity of human immune cells and reduce disease progression in a humanized GvHD model.

[0147] Epitope mapping by hydrogen-deuterium exchange mass spectrometry (HDX-MS) Hydrogen-deuterium exchange mass spectrometry (HDX-MS) combined with mass spectrometry was performed to determine where antibody A binds to the LAG-3 extracellular domain (LAG-3-ECD (e.g., SEQ ID NO: 59)).

[0148] The peptide identification of LAG-3-ECD was performed using a Waters Synapt G2Si Mass Spec. (trademark of Waters Corporation) instrument with Nepenthesin II (Nep II) used for maturation, and 5 μg of LAG-3-ECD protein was used at 0 exchange (1:10 dilution in 0.1X phosphate buffered saline in H2O). The mass spectrometer was set to HDMSe (mobility ESI+ mode) with a scan time of 0.4 seconds and a mass acquisition range of m / z 255 - 00, 1950.00. The data was processed using ProteinLynx Global SERVER (trademark) (PLGS) 2.3.02 (trademark of Waters Corporation). For the exchange experiment, complexes of LAG-3-ECD protein with individual anti-human LAG-3 binding proteins were prepared at a molar ratio of 1:1.2 in 10 mM sodium phosphate buffer (pH 7.4) containing 150 mM NaCl (1×PBS buffer). Using a custom TECAN sample preparation system, the experiment was initiated by adding 25 μL of D2O buffer containing 0.1×PBS to 2.5 μL of LAG-3-ECD (0.9 mg / mL) or LAG-3-ECD + protein complex at 15 °C for various time amounts (0 seconds, 10 seconds, 2 minutes, 10 minutes, and 60 minutes) (see Espada et al., J Am Soc Mass Spectrom. (12):2580 - 2583 (2019), which is hereby incorporated by reference in its entirety). The reaction was stopped at 4 °C for 2 minutes using equal amounts of 0.32 M TCEP, 3 M guanidine HCl, 0.1 M phosphate pH 2.5 and immediately frozen at -70 °C. The sample injection system consists of a UR3 robot, a LEAP PAL3 HDX autosampler, and a high-performance liquid chromatography (HPLC) system connected to a Waters Synapt G2Si Mass Spec. (trademark of Waters Corporation) (modified as described in Espada et al., 2019, J Am Soc Mass Spectrom. (12):2580 - 2583 (2019), which is hereby incorporated by reference in its entirety).The LC mobile phases consisted of water (A) and acetonitrile (B), each containing 0.2% formic acid. Each sample was thawed for 1 minute using 50 μL of 1.5 M guanidine HCl, 0.1 M phosphate pH 2.5, injected onto a Nep II column, and aged at 4 °C using mobile phase A at a flow rate of 250 μL / min for 2.5 minutes. The resulting peptides were captured on a Waters BEH Vanguard Pre-column at 4 °C and chromatographically separated at 4 °C using a Waters Acquity UPLC BEH C18 analytical column at a flow rate of 200 μL / min and a gradient of 3% - 85% mobile phase B over 7 minutes, and sent to a mass spectrometer for mass spectrometry. The Synapt G2Si was calibrated with Glu-fibrinopeptide (Waters Corporation (trademark)) before use. Mass spectra were acquired in HDMS mode over the m / z range of 255 - 1950, and the lock mass m / z was 556.2771 (Leucine Enkephalin, Waters Corporation (trademark)). The relative deuterium incorporation of each peptide was determined by processing the MS data of the deuterated sample and the non-deuterated control using the identified peptide list of DynamX 3.0 (Waters Corporation (trademark)). The free and bound states of LAG-3-ECD were compared for differences in deuterium incorporation to identify the protected regions indicating the binding epitopes.

[0149] Substantial decreases in deuterium incorporation were observed upon binding to LAG-3 at the PDRPASVHWFRNRGQGRVPVRESPHHHLAESF residues (SEQ ID NO: 62) and the PQVSPMD residues (SEQ ID NO: 63), which indicates the epitope region of antibody 1. A decrease in deuterium incorporation upon binding to LAG-3-ECD was also observed at the residues SPHHHLAESF (SEQ ID NO: 64). Collectively, the HDX-MS data indicate that the anti-human LAG-3 agonist antibody of the present invention binds mostly, if not completely, within domain 2 of LAG-3.

[0150] Sequence Listing of Amino Acid and Nucleotide Sequences Antibody A HCDR1: TVSGGSISSGGYYWS (SEQ ID NO: 1) HCDR2: YIYYSGITYYNPSLKS (SEQ ID NO: 2) HCDR3: ARSAVTTPFDY (SEQ ID NO: 3) LCDR1: KSSQSILYSSDNKNYLA (SEQ ID NO: 4) LCDR2: YWASTRES (SEQ ID NO: 5) LCDR3: QQYYSPPWT (SEQ ID NO: 6) VH: QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWSWIRQHPGKGLEWIGYIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARSAVTTPFDYWGQGTLVTVSS (SEQ ID NO: 7) VL: DIVMTQSPDSLAVSLGERATINCKSSQSILYSSDNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSPPWTFGQGTKVEIK (SEQ ID NO: 8) Constant region of the heavy chain: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 9) Constant region of the light chain: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 10) Heavy chain: QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWSWIRQHPGKGLEWIGYIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARSAVTTPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11) Light chain: DIVMTQSPDSLAVSLGERATINCKSSQSILYSSDNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSPPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12) DNA encoding the heavy chain: DNA encoding the light chain: GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTATATTATACAGCTCCGACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAGTACTACTCACCACCATGGACGTTCGGGCAGGGAACTAAGGTGGAAATAAAGCGAACCGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC (SEQ ID NO: 14) Antibody B HCDR1: TVSGGSISSGGYYWS (SEQ ID NO: 1) HCDR2: YIYYSGITYYNPSLKS (SEQ ID NO: 2) HCDR3: ASSTVTTPFDY (SEQ ID NO: 15) LCDR1: KSSQSVLYSSNNKNYLA (SEQ ID NO: 16) LCDR2: YWASTRES (SEQ ID NO: 5) LCDR3: QQYYSPPWT (SEQ ID NO: 6) VH: X1VQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWSWIRQHPGKGLEWIGYIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASSTVTTPFDYWGQGTLVTVSS (SEQ ID NO: 17) Here, X1 is Q, pyroglutamic acid, or absent VL: DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSPPWTFGQGTKVEIK (SEQ ID NO: 18) Heavy chain: X1VQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWSWIRQHPGKGLEWIGYIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASSTVTTPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX2 (SEQ ID NO: 19) Here, X1 is Q, pyroglutamic acid, or absent, and X2 is K, or absent Light chain: DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSPPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 20) Antibody C HCDR1: TVSGVSITSYYWS (SEQ ID NO: 21) HCDR2: RIFTSGSTNYNPSLKS (SEQ ID NO: 22) HCDR3: ARYDGME (SEQ ID NO: 23) LCDR1: RASQSVSSSYLA (SEQ ID NO: 24) LCDR2: YGASSRAT (SEQ ID NO: 25) LCDR3: QQYGSSPLT (SEQ ID NO: 26) VH: X1VQLQESGPGLVKPSETLSLTCTVSGVSITSYYWSWIRQPAGKGLEWIGRIFTSGSTNYNPSLKSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARYDGMEWGQGTLVTVSS (SEQ ID NO: 27) Here, X1 is Q, pyroglutamic acid, or absent VL: X1IVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGQGTKVEIK (SEQ ID NO: 28) Here, X1 is E, pyroglutamic acid, or absent Heavy chain: X1VQLQESGPGLVKPSETLSLTCTVSGVSITSYYWSWIRQPAGKGLEWIGRIFTSGSTNYNPSLKSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARYDGMEWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX2(SEQ ID NO: 29) Here, X1 is Q, pyroglutamic acid, or absent, and X2 is K, or absent Light chain: X1IVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 30) Here, X1 is E, pyroglutamic acid, or absent Antibody D HCDR1: TVSGGSIRSYYWS(SEQ ID NO: 31) HCDR2: RIFTSGSTNYNPSLKS(SEQ ID NO: 22) HCDR3: ARYEGFDV(SEQ ID NO: 32) LCDR1: RASQSVSSSYLA (SEQ ID NO: 24) LCDR2: YGASSRAT (SEQ ID NO: 25) LCDR3: QQYGSSPIT (SEQ ID NO: 33) VH: X1VQLQESGPGLVKPSETLSLTCTVSGGSIRSYYWSWIRQPAGKGLEWIGRIFTSGSTNYNPSLKSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARYEGFDVWGQGTLVTVSS (SEQ ID NO: 34) Here, X1 is Q, pyroglutamic acid, or absent VL: X1IVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPITFGQGTKVEIK (SEQ ID NO: 35) Here, X1 is E, pyroglutamic acid, or absent Heavy chain: X1VQLQESGPGLVKPSETLSLTCTVSGGSIRSYYWSWIRQPAGKGLEWIGRIFTSGSTNYNPSLKSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARYEGFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX2 (SEQ ID NO: 36) Here, X1 is Q, pyroglutamic acid, or absent, and X2 is K, or absent. Light chain: X1IVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPITFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE (SEQ ID NO: 37) Here, X1 is E, pyroglutamic acid, or absent. Antibody E HCDR1: AASGFTFSSFAMS (SEQ ID NO: 38) HCDR2: AISASGGRTYYADSVKG (SEQ ID NO: 39) HCDR3: AKDEPYSGSYHGLDV (SEQ ID NO: 40) LCDR1: RSSQSLLHSDGYNYLD (SEQ ID NO: 41) LCDR2: YLGSNRAS (SEQ ID NO: 42) LCDR3: MQAQQTPLT (SEQ ID NO: 43) VH: X1VQLVESGGGLVQPGGSLRLSCAASGFTFSSFAMSWVRQAPGKGLEWVSAISASGGRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDEPYSGSYHGLDVWGQGTLVTVSS (SEQ ID NO: 44) Here, X1 is Q, pyroglutamic acid, or absent. VL: DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSDGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQAQQTPLTFGQGTKVEIK (SEQ ID NO: 45) Heavy chain: X1VQLVESGGGLVQPGGSLRLSCAASGFTFSSFAMSWVRQAPGKGLEWVSAISASGGRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDEPYSGSYHGLDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX2 (SEQ ID NO: 46) Here, X1 is Q, pyroglutamic acid, or absent, and X2 is K, or absent Light chain: DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSDGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQAQQTPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 47) Consensus sequence Group I: Antibody A and Antibody B Consensus HCDR3: AX1SX2VTTPFDY (SEQ ID NO: 48) Here, X1 is R or S, and X2 is A or T Consensus LCDR1: KSSQSX1LYSSX2NKNYLA (SEQ ID NO: 49) Here, X1 is I or V, and X2 is D or N Consensus VH: X1VQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWSWIRQHPGKGLEWIGYIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAX2SX3VTTPFDYWGQGTLVTVSS (SEQ ID NO: 50) Here, X1 is Q, pyroglutamate, or absent, X2 is R or S, and X3 is A or T Consensus LCVR: DIVMTQSPDSLAVSLGERATINCKSSQSX1LYSSX2NKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSPPWTFGQGTKVEIK (SEQ ID NO: 51) Here, X1 is I or V, and X2 is D or N Group II: Antibody C and Antibody D HCDR1: TVSGX1SIX2SYYWS (SEQ ID NO: 52) Here, X1 is G or V, and X2 is R or T HCDR3: ARYX1GX2X3X4 (SEQ ID NO: 53) Here, X1 is E or D, X2 is F or M, X3 is D or E, and X4 is V or absent LCDR3: QQYGSSPX1T (SEQ ID NO: 54) X1 is I or L VH: X1VQLQESGPGLVKPSETLSLTCTVSGX2SIX3SYYWSWIRQPAGKGLEWIGRIFTSGSTNYNPSLKSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARYX4GX5X6X7WGQGTLVTVSS (SEQ ID NO: 55) X1 is Q, pyroglutamic acid, or absent, X2 is G or V, X3 is R or T, X4 is E or D, X5 is F or M, X6 is D or E, and X7 is V or absent. VL: X1IVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPX2TFGQGTKVEIK (SEQ ID NO: 56) Here, X1 is E, pyruvate, or absent, and X2 is I or L. Human LAG-3 amino acid sequence with a signal sequence: MWEAQFLGLLFLQPLWVAPVKPLQPGAEVPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCHIHLQEQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTELSSPGAQRSGRAPGALPAGHLLLFLILGVLSLLLLVTGAFGFHLWRRQWRPRRFSALEQGIHPPQAQSKIEELEQEPEPEPEPEPEPEPEPEPEQL(SEQ ID NO: 57) Human LAG-3 amino acid sequence without a signal sequence: LQPGAEVPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCHIHLQEQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTELSSPGAQRSGRAPGALPAGHLLLFLILGVLSLLLLVTGAFGFHLWRRQWRPRRFSALEQGIHPPQAQSKIEELEQEPEPEPEPEPEPEPEPEPEQL(SEQ ID NO: 58) Human LAG-3 ECD-His: LQPGAEVPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCHIHLQEQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTELSSPGAQRSGRAPGALPAGHGGGGSHHHHHH(SEQ ID NO: 59) Cynomolgus monkey LAG-3 ECD-His: PQPGAEISVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAPAPGHPPVPGHRPAAPYSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPARRADAGEYRATVHLRDRALSCRLRLRVGQASMTASPPGSLRTSDWVILNCSFSRPDRPASVHWFRSRGQGRVPVQGSPHHHLAESFLFLPHVGPMDSGLWGCILTYRDGFNVSIMYNLTVLGLEPATPLTVYAGAGSRVELPCRLPPAVGTQSFLTAKWAPPGGGPDLLVAGDNGDFTLRLEDVSQAQAGTYICHIRLQGQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPASGQEHFVWSPLNTPSQRSFSGPWLEAQEAQLLSQPWQCQLHQGERLLGAAVYFTELSSPGAQRSGRAPGALRAGHAAAHHHHHHSGS (SEQ ID NO: 60) D2 of human LAG-3 QASMTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLG (SEQ ID NO: 61) Epitope region of D2: PDRPASVHWFRNRGQGRVPVRESPHHHLAESF (SEQ ID NO: 62) PQVSPMD (SEQ ID NO: 63) SPHHHLAESF (SEQ ID NO: 64) Antibody A total consensus HCDR1: TVSGGSISSGGYX1WS (SEQ ID NO: 65) Here, X1 is F or Y HCDR2: (SEQ ID NO: 2) HCDR3: (SEQ ID NO: 48) LCDR1: KSSQSX1LYSSX2NX3NYLA (SEQ ID NO: 66) X1 is I or V, X2 is D or N, and X3 is R or K LCDR2: YWASTRX1S (SEQ ID NO: 67) Here, X1 is D or E LCDR3: X1QYYSPPWT (SEQ ID NO: 68) X1 is H or Q VH: X1VQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYX2WSWIRQHPGKGLEWIGYIYYSGITYYNPSLKSRX3TISVDTSKNQFSLX4LSSVTAADTAVYYCAX5SX6VTTPFDYWGQGTLVTVSS (SEQ ID NO: 69) X1 is Q, pyroglutamic acid, or absent, X2 is F or Y, X3 is L or V, X4 is N or K, X5 is R or S, and X6 is A or T VL: DIVMTQSPDX1LAVSLGERATINCKSSQSILYSSDNX2NYLAWX3QX4KPGQ PPKLLIYWASTRX5SGVPDRFSGSGSGTDFTLX6ISSLQAEDVAVYYCX7QYY SPPWTFGQGTKVEIK (SEQ ID NO: 70) Here, X1 is A or S, X2 is R or K, X2 is R or K, X3 is F or Y, X4 is L or Q, X5 is D or E, X6 is A or T, and X7 is H or Q HC: X1VQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYX2WSWIRQHPGKGLEWIGYIYYSGITYYNPSLKSRX3TISVDTSKNQFSLX4LSSVTAADTAVYYCARSAVTTPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEX5X6GGPSVFLFPPKPKDTLMISRTPEVTCVVVX7VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX8(SEQ ID NO: 71) X1 is Q, pyroglutamic acid, or absent, X2 is F or Y, X3 is L or V, X4 is N or K, X5 is L or A, X6 is L or A, X7 is D or S, X8 is K, or absent LC: DIVMTQSPDX1LAVSLGERATINCKSSQSX2LYSSDNX3NYLAWX4QX5KPGQPPKLLIYWASTRX6SGVPDRFSGSGSGTDFTLX7ISSLQAEDVAVYYCX8QYYSPPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 72) Here, X1 is A or S, X2 is I or V, X3 is R or K, X4 is F or Y, X5 is L or Q, X6 is D or E, X7 is A or T, X8 is H or Q VH (N-terminal modification) of antibody A: X1VQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWSWIRQHPGKGLEWIGYIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARSAVTTPFDYWGQGTLVTVSS(SEQ ID NO: 73) Here, X1 is Q, pyroglutamic acid, or absent Constant region of the heavy chain (C-terminal modification): ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX1(SEQ ID NO: 74) Here, X1 is K, or absent Heavy chain of antibody A (having N-terminal and C-terminal modifications): X1VQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWSWIRQHPGKGLEWIGYIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARSAVTTPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX2(SEQ ID NO: 75) Here, X1 is Q, pyroglutamic acid, or absent, and X2 is K, or absent

Claims

1. An antibody that binds to domain 2 (SEQ ID NO: 61) of human LAG-3, said antibody binding to an epitope comprising one or more amino acid residues within SPHHHLAESF (SEQ ID NO: 64).

2. An antibody that binds to domain 2 (SEQ ID NO: 61) of human LAG-3, wherein the antibody binds to three or more amino acid residues within PDRPASVHWFRNRGQGRVPVRESPHHHLAESF (SEQ ID NO: 62).

3. The antibody of claim 1 or 2, wherein the epitope further comprises one or more amino acid residues within PQVSPMD (sequence number 63) of human LAG-3 domain 2.

4. The antibody of claim 1, wherein the antibody also binds to one or more amino acid residues within SMTASPPGSLRASD (SEQ ID NO:64).

5. The antibody of any one of claims 1 to 4, wherein the epitope is determined by HDX-MS.

6. An antibody that binds to domain 2 (SEQ ID NO: 61) of human LAG-3, said antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), said VH comprising heavy chain complementarity determining regions (HCDRs): HCDR1 comprising SEQ ID NO: 65, HCDR2 comprising SEQ ID NO: 2, and HCDR3 comprising SEQ ID NO: 48, and said VL comprising light chain complementarity determining regions (LCDRs): LCDR1 comprising SEQ ID NO: 66, LCDR2 comprising SEQ ID NO: 67, and LCDR3 comprising SEQ ID NO:

68.

7. The antibody of claim 6, wherein the HCDR1 comprises SEQ ID NO: 1, the HCDR2 comprises SEQ ID NO: 2, and the HCDR3 comprises SEQ ID NO: 48, and the LCDR1 comprises SEQ ID NO: 49, the LCDR2 comprises SEQ ID NO: 5, and the LCDR3 comprises SEQ ID NO:

6.

8. The antibody of claim 7, wherein the HCDR1 comprises SEQ ID NO: 1, the HCDR2 comprises SEQ ID NO: 2, and the HCDR3 comprises SEQ ID NO: 3, and the LCDR1 comprises SEQ ID NO: 4, the LCDR2 comprises SEQ ID NO: 5, and the LCDR3 comprises SEQ ID NO:

6.

9. The antibody of claim 8, wherein the VH comprises SEQ ID NO: 7 and the VL comprises SEQ ID NO:

8.

10. The antibody of any one of claims 1 to 10, wherein the antibody is of the human IgG1 isotype.

11. The antibody of any one of claims 1 to 8, comprising a HC comprising SEQ ID NO:11 and a LC comprising SEQ ID NO:

11.

12. The antibody of any one of claims 1 to 7, wherein the antibody is of the human IgG4 isotype.

13. The antibody of any one of claims 1 to 12, which is an agonist of human LAG-3.

14. The antibody of any one of claims 1 to 13, wherein the antibody agonizes human LAG-3 as determined by an assay measuring inhibition of human or cynomolgus monkey CD8 cell proliferation.

15. The antibody of any one of claims 1 to 14, wherein the antibody binds to human LAG-3 ECD of SEQ ID NO: 58 or SEQ ID NO: 59 with a dissociation equilibrium constant (KD) of 100 nM or less.

16. The antibody of any one of claims 1 to 15, wherein the antibody binds to human LAG-3 ECD of SEQ ID NO: 58 or SEQ ID NO: 59 with a dissociation equilibrium constant (KD) of 5 nM or less.

17. The antibody is about 1×10 －７ M ~ approx. 1 x 10 －１１ K of M Ｄ The antibody of any one of claims 1 to 16, which binds to human LAG-3 ECD of SEQ ID NO:58 or SEQ ID NO:

59.

18. The antibody is about 1×10 －８ M ~ approx. 1 x 10 －１１ K of M Ｄ The antibody of any one of claims 1 to 17, which binds to human LAG-3 ECD of SEQ ID NO:58 or SEQ ID NO:

59.

19. The antibody is about 1×10 －９ M ~ approx. 1 x 10 －１１ K of M Ｄ The antibody of any one of claims 1 to 18, which binds to human LAG-3 ECD of SEQ ID NO:58 or SEQ ID NO:

59.

20. The antibody is about 1×10 －１０ M ~ approx. 1 x 10 －１１ K of M Ｄ The antibody of any one of claims 1 to 19, which binds to human LAG-3 ECD of SEQ ID NO:58 or SEQ ID NO:

59.

21. The antibody of any one of claims 1 to 20, wherein the KD is determined by surface plasmon resonance (SPR) in a 37°C assay.

22. A nucleic acid comprising a DNA sequence encoding one or both of SEQ ID NO:11 and SEQ ID NO:

12.

23. A vector comprising the nucleic acid of claim 22.

24. A composition comprising a first vector comprising a nucleic acid sequence encoding SEQ ID NO:11, and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:

12.

25. A cell comprising the vector of claim 23.

26. A cell comprising the composition of claim 24.

27. The cell of claim 25 or 26, wherein the cell is a mammalian cell.

28. 28. A process for producing an antibody, comprising culturing a cell according to any one of claims 25 to 27 under conditions such that the antibody is expressed, and recovering the expressed antibody from the culture medium.

29. An antibody produced by culturing a cell according to any one of claims 25 to 27 under conditions such that the antibody is expressed, and recovering the expressed antibody from the culture medium.

30. A composition comprising an antibody according to any one of claims 1 to 21.

31. A method of treating an autoimmune disease in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of an anti-human LAG-3 agonist antibody according to any one of claims 13 to 21.

32. 32. The method of claim 31, wherein the autoimmune disease is rheumatoid arthritis, psoriasis, ulcerative colitis, type I diabetes, lupus nephritis, or systemic lupus erythematosus.

33. An anti-human LAG-3 agonist antibody according to any one of claims 13 to 21 for use in therapy.

34. An anti-human LAG-3 agonist antibody according to any one of claims 13 to 21 for use in the treatment of an autoimmune disease.

35. 35. The use according to claim 34, wherein the autoimmune disease is rheumatoid arthritis, psoriasis, ulcerative colitis, type I diabetes, lupus nephritis, or systemic lupus erythematosus.

36. A pharmaceutical composition comprising an anti-human LAG-3 agonist antibody according to any one of claims 13 to 21 for use in the treatment of rheumatoid arthritis, psoriasis, ulcerative colitis, type I diabetes, lupus nephritis, or systemic lupus erythematosus.

37. Use of an anti-human LAG-3 agonist antibody according to any one of claims 13 to 21 in the manufacture of a medicament for the treatment of rheumatoid arthritis, psoriasis, ulcerative colitis, type I diabetes, lupus nephritis, or systemic lupus erythematosus.

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