KR20250001490A - Anti-LAG-3 Antibody, Antigen-Binding Fragments and Uses thereof - Google Patents

Abstract

The present invention provides antibodies or antigen-binding fragments that specifically bind to human LAG-3 with high affinity. In certain embodiments, the antibodies or antigen-binding fragments can bind to monkey LAG-3, mouse LAG-3, or both. The antibodies or antigen-binding fragments according to the present invention can inhibit binding of LAG-3 to MHC Class II molecules, inhibit binding of LAG-3 to B7-H3, and/or stimulate an antigen-specific T cell response.

Description

{Anti-LAG-3 Antibody, Antigen-Binding Fragments and Uses thereof}

The present invention relates to an antibody or antigen-binding fragment that specifically binds to Lymphocyte Activation Gene-3 (LAG-3). The present invention also relates to a polynucleotide encoding the antibody or antigen-binding fragment, a pharmaceutical composition comprising the antibody or antigen-binding fragment, and a use of the antibody or antigen-binding fragment for treating and/or preventing diseases associated with pathogenic T cells or a suppressed immune system, such as cancer.

Recently, as it has become known that some tumor cells can escape the immune system by causing an immunosuppressive state, interest in so-called immune checkpoint inhibitors that achieve anticancer effects through the immune system by preventing this immunosuppressive state or restoring it to a normal state has greatly increased.

T cells play a crucial role in the immune system, especially adaptive immunity. Due to their ability to be appropriately activated to achieve clonal expansion and effector function, their ability to selectively and directly recognize and kill antigen-expressing cells (cytotoxic T lymphocytes or CD8+ effector T cells), and their ability to orchestrate various immune responses (CD4+ helper T cells), they have been a major research subject for the purpose of modulating antitumor immunity from a therapeutic perspective.

Long-term antigen exposure causes T cell dysfunction, where they lose their ability to proliferate in the presence of antigens and gradually become unable to produce cytokines, thus failing to kill target cells. T cells with this dysfunction are called exhausted T cells, and they cannot exhibit effector functions such as cytotoxicity and cytokine secretion in response to antigen stimulation.

Lymphocyte activation gene 3 (LAG-3 or CD223) is a unique transmembrane protein with structural homology to CD4, which has four extracellular immunoglobulin superfamily-like domains (D1-D4). Like CD4, the LAG-3 protein binds to MHC class II molecules, but with higher affinity at a site distinct from that of CD4 (Huard et al. Proc. Natl. Acad. Sci. USA 94 (1997), 5744-5749). LAG-3 is a cell surface molecule expressed on activated T cells (Huard et al. Immunogenetics 39: 213-217, 1994), NK cells (Triebel et al, J Exp Med 171:1393-1405, 1990), B cells (Kisielow et al, Eur J Immnol 182: 1885-1891, 2009), and plasma dendritic cells (Workman et al, J Immunol 182: 1885-1891, 2009), and is known to play a role in T cell homeostasis and regulation of T cell function. In addition, LAG-3 is expressed on quiescent or dysfunctional T cell populations, for example, LAG-3-expressing T cells are enriched in tumor sites or infected organs (Blackburn et al, Nature Immunol 10: 29-37, 2009). Therefore, antibodies that can induce and restore immune responses by inhibiting the activity of LAG-3 are required.

For example, human antibodies that bind to LAG-3 and uses thereof are disclosed in WO 2010/019570. Other LAG-3 antibodies are described in WO 2014/140180, WO 2015/138920, WO 2016/028672, WO 2018/069500, and WO 2019/256602.

The present invention provides antibodies or antigen-binding fragments thereof, particularly human monoclonal antibodies and antigen-binding fragments thereof, which specifically bind to LAG-3 and have desirable functional properties. Such functional properties include high affinity binding to human LAG-3, binding to one or more LAG-3 selected from monkey LAG-3 (e.g., cynomolgus and/or rhesus LAG-3), and mouse LAG-3, the ability to inhibit binding of LAG-3 to a B7-H3 molecule, and/or the ability to stimulate an antigen-specific T cell response. The antibodies of the present invention can be used, for example, to detect LAG-3 protein, or to stimulate an antigen-specific T cell response, such as in a tumor-bearing subject.

The present invention provides an anti-LAG-3 antibody or an antigen-binding fragment thereof that specifically binds to LAG-3.

The anti-LAG-3 antibody or antigen-binding fragment thereof of the present invention binds to human LAG-3 and has at least one or more of the following properties:

(a) Binding to monkey LAG-3 and/or mouse LAG-3;

(b) inhibiting the binding of LAG-3 to B7-H3 molecules;

(c) inhibiting the binding of LAG-3 to MHC II molecules;

(d) blocking LAG-3-induced T cell downregulation and rescuing T cell signaling; and

(e) Inhibiting tumor growth and increasing the survival rate of cancer patients.

Preferably, the anti-LAG-3 antibody or antigen-binding fragment thereof of the present invention exhibits two of the properties (a), (b), (c), (d) and (e), more preferably, it exhibits three of the properties (a), (b), (c), (d) and (e), and most preferably, it exhibits all of (a), (b), (c), (d) and (e).

In a preferred embodiment, the antibody or antigen-binding fragment thereof of the invention stimulates a T cell response, such as interferon gamma (IFNγ) production in a mixed lymphocyte reaction assay. In another embodiment, the antibody or antigen-binding fragment thereof of the invention stimulates an immune response, such as an anti-tumor response (e.g., inhibiting tumor growth in an in vivo tumor transplantation model).

The anti-LAG-3 antibodies or antigen-binding fragments of the present invention bind to monomeric LAG-3 protein with high affinity. In certain embodiments, the anti-LAG-3 antibodies or antigen-binding fragments of the present invention bind to monomeric LAG-3 protein with a KD of less than about 10 nM, less than about 3 nM, preferably less than about 2 nM, more preferably less than about 1 nM, and most preferably less than about 0.1 nM as measured by surface plasmon resonance.

In one embodiment, the present invention comprises:

(a) a heavy chain variable domain comprising a CDR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 13, 25, 37, 49, 61 and 73, a CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 15, 27, 39, 51, 63 and 75, and a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 17, 29, 41, 53, 65 and 77; and/or

(b) provides an antibody or an antigen-binding fragment thereof that specifically binds to LAG-3, comprising a light chain variable domain comprising a CDR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 19, 31, 43, 55, 67 and 79, a CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 21, 33, 45, 57, 69 and 81, and a CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 23, 35, 47, 59, 71 and 83.

The desirable combination is

(a) a heavy chain variable domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 3; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 5; and/or

(b) a light chain variable domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9; and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

Another desirable combination is

(a) a heavy chain variable domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 15; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17; and/or

(b) a light chain variable domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 19; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21; and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 23.

Another desirable combination is

(a) a heavy chain variable domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 27; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 29; and/or

(b) a light chain variable domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 31; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 33; and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 35.

Another desirable combination is

(a) a heavy chain variable domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 37; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 39; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 41; and/or

(b) a light chain variable domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 43; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 45; and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 47.

Another desirable combination is

(a) a heavy chain variable domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 49; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 51; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 53; and/or

(b) a light chain variable domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 55; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 57; and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 59.

Another desirable combination is

(a) a heavy chain variable domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 61; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 63; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 65; and/or

(b) a light chain variable domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 67; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 69; and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 71.

Another desirable combination is

(a) a heavy chain variable domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 73; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 75; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 77; and/or

(b) a light chain variable domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 79; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 81; and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 83.

In another embodiment, the present invention comprises:

(a) a heavy chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 85, 89, 93, 97, 101, 105 and 109, and/or

(b) provides an antibody or antigen-binding fragment thereof that specifically binds to LAG-3, comprising a light chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 87, 91, 95, 99, 103, 107 and 111.

The desirable combination is

(a) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 85, and/or

(b) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 87.

Another desirable combination is

(a) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 89, and/or

(b) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 91.

Another desirable combination is

(a) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 93, and/or

(b) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 95.

Another desirable combination is

(a) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 97, and/or

(b) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 99.

Another desirable combination is

(a) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 101, and/or

(b) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 103.

Another desirable combination is

(a) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 105, and/or

(b) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 107.

Another desirable combination is

(a) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 109, and/or

(b) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 111.

In another embodiment, an antibody or antigen-binding fragment thereof according to the present invention

(a) a heavy chain variable domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 3; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 5, and having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 85, and/or

(b) a light chain variable domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9; and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 11, and having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 87.

In another embodiment, an antibody or antigen-binding fragment thereof according to the present invention

(a) a heavy chain variable domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 15; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, and having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 89, and/or

(b) a light chain variable domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 19; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21; and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 23, and having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 91.

In another embodiment, an antibody or antigen-binding fragment thereof according to the present invention

(a) a heavy chain variable domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 27; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 29, and having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 93, and/or

(b) a light chain variable domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 31; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 33; and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 35, and having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 95.

In another embodiment, an antibody or antigen-binding fragment thereof according to the present invention

(a) a heavy chain variable domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 37; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 39; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 41, and having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 97, and/or

(b) a light chain variable domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 43; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 45; and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 47, and having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 99.

In another embodiment, an antibody or antigen-binding fragment thereof according to the present invention

(a) a heavy chain variable domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 49; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 51; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 53, and having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 101, and

(b) a light chain variable domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 55; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 57; and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 59, and having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 103.

In another embodiment, an antibody or antigen-binding fragment thereof according to the present invention

(a) a heavy chain variable domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 61; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 63; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 65, and having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 105, and/or

(b) a light chain variable domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 67; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 69; and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 71, and having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 107.

In another embodiment, an antibody or antigen-binding fragment thereof according to the present invention

(a) a heavy chain variable domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 73; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 75; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 77, and having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 109, and/or

(b) a light chain variable domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 79; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 81; and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 83, and having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 111.

The antibody of the present invention can be, for example, a full-length antibody, for example, of the IgG1, IgG2 or IgG4 isotype. In a preferred embodiment, the antibody is of a modified IgG2 isotype.

Additionally, the antibody of the present invention may be an antibody fragment such as a Fab, F(ab'), (Fab')2, Fv, diabody or scFv fragment, or a single chain antibody.

The present invention also provides immunoconjugates comprising an antibody of the invention or an antigen-binding portion thereof linked to a therapeutic agent, such as a cytotoxin or a radioactive isotope.

The present invention also provides bispecific and multispecific molecules comprising an antibody of the invention or an antigen-binding portion thereof linked to another functional moiety having a binding specificity different from that of the antibody or antigen-binding portion thereof.

Also provided are compositions comprising the antibody or antigen-binding fragment thereof, or immunoconjugate or bispecific molecule of the present invention and a pharmaceutically acceptable carrier.

The present disclosure also encompasses nucleic acid molecules encoding antibodies or antigen-binding fragments thereof of the present invention, as well as expression vectors comprising such nucleic acids and host cells comprising such expression vectors.

Also provided are methods of producing anti-LAG-3 antibodies using host cells comprising such expression vectors, the methods comprising the steps of (i) expressing the antibody in the host cell and (ii) isolating the antibody from the host cell.

In another aspect, the invention relates to methods of stimulating an immune response using an anti-LAG-3 antibody or antigen-binding fragment thereof of the invention. For example, an antibody of the invention is characterized by certain functional characteristics or properties of the antibody. The ability of the antibody to stimulate an immune response, such as an antigen T cell response, can be indicated, for example, by the ability of the antibody to stimulate interferon-gamma (IFN-r) production in an antigen-specific T cell response. In certain embodiments, an antibody of the invention exhibits the ability to bind to human LAG-3 and to stimulate an antigen-specific T cell response. In certain embodiments, an antibody molecule of the invention increases the expression of IFN-r from a cell activated by a bidirectional mixed lymphocyte reaction (MLR) activated by alloreactivity of lymphocytes, as compared to the expression of IFN-r when an isotype control (e.g., IgG1 or IgG4) is used, as measured, for example, in a human whole blood ex vivo assay.

In another embodiment, the invention provides a method of stimulating an immune response in a subject, comprising administering to the subject an antibody or antigen-binding fragment of the invention such that an immune response (e.g., a mixed lymphocyte reaction) is stimulated within the subject.

In a preferred embodiment, the subject is a tumor-bearing subject and an immune response against the tumor is stimulated. In another preferred embodiment, the subject is a virus-bearing subject and an immune response against the virus is stimulated.

In another aspect, the invention provides a method of inhibiting the growth of tumor cells in a subject, comprising administering to the subject an antibody of the invention such that the growth of the tumor is inhibited in the subject. In another aspect, the invention provides a method of treating a viral infection in a subject, comprising administering to the subject an antibody of the invention such that the viral infection is treated.

In another aspect, the invention provides a method for stimulating an immune response in a subject, comprising administering to the subject an anti-LAG-3 antibody and at least one additional immunostimulatory antibody, such as an anti-PD-1 antibody, an anti-PD-L1 antibody and/or an anti-CTLA-4 antibody, to stimulate an immune response in the subject, for example, to inhibit tumor growth or stimulate an antiviral response. In one embodiment, the subject is administered an anti-LAG-3 antibody and an anti-PD-1 antibody. In another embodiment, the subject is administered an anti-LAG-3 antibody and an anti-PD-L1 antibody. In another embodiment, the subject is administered an anti-LAG-3 antibody and an anti-CTLA-4 antibody. In one embodiment, the anti-LAG-3 antibody is a human antibody, such as an antibody according to the invention. Alternatively, the anti-LAG-3 antibody can be, for example, a chimeric or humanized antibody. In another embodiment, the at least one additional immunostimulatory antibody (e.g., an anti-PD-1, anti-PD-L1, and/or anti-CTLA-4 antibody) is a human antibody. Alternatively, the at least one additional immunostimulatory antibody can be, for example, a chimeric or humanized antibody.

In another aspect, the present invention relates to a method for preparing an anti-LAG-3 antibody, the method comprising the following steps:

(a) (i) a heavy chain variable domain sequence comprising a CDR-H1 sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2, 14, 26, 38, 50 and 74, a CDR-H2 sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4, 16, 28, 40, 52, 64 and 76, and a CDR-H3 sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6, 18, 30, 42, 54, 66 and 78; and (ii) providing a light chain variable domain sequence comprising a CDR-L1 sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8, 20, 32, 44, 56, 68 and 80, a CDR-L2 sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 10, 22, 34, 46, 58, 70 and 82, and a CDR-L3 sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 12, 24, 36, 48, 60, 72 and 84; and optionally,

(b) generating at least one altered antibody sequence by altering at least one amino acid residue within the heavy chain variable region antibody sequence and/or the light chain variable region antibody sequence.

The present invention also relates to embodiments of a LAG-3 binding antibody comprising an Fc region, wherein the Fc region can be a variant Fc region comprising one or more amino acid modifications that decrease the affinity of the variant Fc region for an FcγR and/or improve the serum half-life.

The present invention encompasses anti-LAG-3 antibodies having altered glycosylation patterns. In some embodiments, modifications may be useful to remove undesirable glycosylation sites, or antibodies lacking a fucose moiety present in the oligosaccharide chain may be useful, for example, to enhance antibody-dependent cellular cytotoxicity (ADCC) function (Shield et al. (2002) JBC 277:26733). In other embodiments, modifications in galactosylation may be made to alter complement-dependent cytotoxicity (CDC).

The antibody or antigen-binding fragment thereof according to the present invention can be administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly or intracranially.

According to another aspect of the present invention, the present invention provides a composition for detecting LAG-3.

In another aspect, the present invention provides a kit for diagnosing cancer or infection.

The antibody or antigen-binding fragment according to the present invention can bind to human LAG-3, monkey LAG-3, mouse LAG-3, or all of these. Furthermore, the antibody or antigen-binding fragment according to the present invention can inhibit binding of LAG-3 to MHC Class II molecules, inhibit binding of LAG-3 to B7-H3, and/or stimulate an antigen-specific T cell response.

Figure 1 is a schematic diagram for the construction of recombinant human LAG-3 full ECD, LAG-3-D1D2, and LAG-3-D3D4.
Figure 2 is a schematic diagram for the expression of membrane-bound LAG-3-mCHERRY constructs and the generation of membrane-bound human LAG-3 D1D2 and LAG-3 D3D4 domains.
Figure 3 is a schematic diagram illustrating antibody selection for LAG-3 using phage-display.
Figure 4 illustrates the binding of anti-LAG-3 antibodies to HEK293 cell lines overexpressing human LAG-3. (A) Results for HEK293 cells expressing human full-length LAG-3, and (B) results for HEK293 cells expressing human LAG-3 D1D2 domain.
Figure 5 shows the expression of LAG-3 in stimulated CD4+ and CD8+ T cells. No LAG-3 expression was observed in unstimulated CD4+ and CD8+ T cells (A), whereas PHA-L-stimulated T cells dramatically increased LAG-3 expression (B).
Figures 6a to 6g are graphs showing the binding of selected antibodies to human LAG-3 expressed on the surface of unstimulated or stimulated T cells measured by FACS: S2-5A1-A (Figure 6a), S1-2H6-A (Figure 6b), S2-2D11-C (Figure 6c), S2-1B6-C (Figure 6d), S3-1H9-C (Figure 6e), S2-4B3-A (Figure 6f), S1-2D1-A (Figure 6g) were measured for their binding ability to unstimulated or stimulated T cells.
Figure 7 is a graph showing the change in binding intensity of selected anti-LAG-3 antibodies depending on T cell activation.
Figure 8 shows the ELISA results for analyzing cross-reactivity.
Figure 9 shows the results of ELISA testing to demonstrate that binding of available human LAG-3 to B7-H3 is competitively inhibited by anti-LAG-3 antibodies.
Figure 10 shows the results of the HTRF assay, where binding of available human LAG3 to MHC II was blocked by anti-LAG-3 antibodies.
Figure 11 shows the results of measuring IFN-gamma production in human peripheral blood mononuclear cells (PBMCs) stimulated with mixed lymphocyte reaction (MLR) by anti-LAG-3 antibody treatment, as measured by Elisa. In addition, IFN-gamma production induced in the reaction by anti-PD-1 antibody treatment can be compared.
Figure 12 shows the results of Elisa measurement showing that IFN-gamma production in human peripheral blood mononuclear cells (PBMCs) stimulated with mixed lymphocyte reaction (MLR) is enhanced by co-treatment with anti-PD-1 antibody compared to anti-LAG-3 antibody alone.

The present invention provides antibodies or antigen-binding fragments thereof that specifically bind to human LAG-3. The antibodies or antigen-binding fragments thereof according to the present invention have binding to one or more LAG-3 selected from human LAG-3, monkey LAG-3 and mouse LAG-3, the ability to inhibit binding of LAG-3 to a B7-H3 molecule, the ability to inhibit binding of LAG-3 to an MHC II molecule, and/or the ability to stimulate an antigen-specific T cell response. The antibodies of the present invention can be used, for example, to detect LAG-3 protein, or to stimulate an antigen-specific T cell response, such as in a tumor-bearing subject. In addition, the antibodies of the present invention can be useful for treating various cancers and infectious diseases.

Definition of terms

In order to facilitate a better understanding of the present invention, terms used in this specification are defined. Unless specifically defined elsewhere in this specification, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which the present invention belongs.

Additionally, as used herein, including in the claims, the singular forms “a,” “an,” and “the” include their corresponding plural referents unless the context clearly dictates otherwise.

The terms "comprising," "comprises," or variations thereof, as used herein, are intended to be open-ended. As an example, an antibody or antigen-binding fragment thereof comprising a listed amino acid sequence may, whether inherently or otherwise, comprise additional amino acid sequences not listed.

The term "consisting substantially of" or variations thereof, as used herein, includes any of the recited elements, and allows for the presence of elements that do not substantially affect the basic, novel, or functional properties of the embodiment. As an example, an antibody or antigen-binding fragment thereof consisting essentially of the recited amino acid sequence may include substitutions of one or more amino acid residues that do not substantially affect the properties of the antibody or fragment thereof.

The term "consisting of" or variations thereof, as used herein, means that each component does not allow any elements not mentioned or listed in that description of the embodiment.

Each chain or variable region of another antibody or antigen-binding fragment thereof as defined herein may also comprise, consist essentially of, or consist of an amino acid sequence identical to the above examples.

The term "LAG-3" as used herein refers to lymphocyte activation gene-3. "LAG-3" includes variants, isoforms, homologs, orthologs, and paralogs. The anti-LAG-3 antibodies according to the present invention specifically bind to human LAG-3 protein, and in certain cases may cross-react with LAG-3 of other species than human, such as monkey LAG-3, mouse LAG-3, or both. Human LAG-3 may comprise the amino acid sequence of SEQ ID NO: 127, monkey LAG-3 may comprise the amino acid sequence of SEQ ID NO: 128, and mouse LAG-3 may comprise the amino acid sequence of SEQ ID NO: 129. Unless specifically stated herein, "LAG-3" refers to human LAG-3.

The anti-LAG antibody or antigen-binding fragment thereof of the present invention binds to the D1 region of human LAG-3 protein (SEQ ID NO: 130). In some embodiments, the anti-LAG antibody of the present invention binds to the D2 region of human LAG-3 protein (SEQ ID NO: 131). In some embodiments, the anti-LAG antibody of the present invention binds to the D3 region of human LAG-3 protein (SEQ ID NO: 132). In some embodiments, the anti-LAG antibody of the present invention binds to the D4 region of human LAG-3 protein (SEQ ID NO: 133). The epitope consists of one or more contiguous sequences of 3 to 10 amino acids in any one of the D1 to D4 regions of LAG-3 protein.

The term "antibody" as used herein is an immunoglobulin molecule capable of specifically binding to a target molecule via at least one antigen recognition site located in the variable region of an immunoglobulin molecule. The term "antibody" as used herein includes an intact whole antibody as well as any antigen-binding fragment thereof (i.e., an "antigen binding portion"), an antibody fragment, a fusion protein or a single chain comprising any other modified arrangement of an immunoglobulin molecule comprising an antigen recognition site. A whole antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains that are interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable domain (hereinafter abbreviated as "VH") and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains, namely, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (hereinafter abbreviated as "VL") and a light chain constant region (CL). The light chain constant region consists of a single domain, the CL. The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs).

The term "CDR" as used herein refers to the complementarity determining region amino acid sequence of an antibody or antigen-binding fragment. These are the hypervariable regions of immunoglobulin heavy and light chains. There are three heavy chain and three light chain CDRs in the variable portion of immunoglobulins, which are referred to herein as CDR-H1, CDR-H2, CDR-H3 and CDR-L1, CDR-L2, CDR-L3, respectively.

The terms "antibody recognizing an antigen" or "antibody specific for an antigen" are used interchangeably with the terms "antibody that binds specifically to an antigen."

The antibody of the present invention may be a chimeric antibody, a humanized antibody, or a human antibody.

The term "chimeric antibody" as used herein refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, e.g., the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody. Methods for making chimeric antibodies are known in the art. See, e.g., Morrison, Science 229:1202 (1985), which is incorporated herein by reference in its entirety.

The term "humanized antibody" as used herein refers to an antibody in which one or more CDR sequences derived from the germline of a non-human species, e.g., another mammalian species, e.g., mouse or chicken, are inserted into a framework sequence from a human immunoglobulin molecule. The framework sequences may further be modified, e.g., by mutation. Human Ig sequences can be found, e.g., in the NCBI Database (Entez Gene), etc. By using appropriate sequences, the immunogenicity of the antibody can be reduced, enhanced, or altered in avidity, affinity, on-rate, off-rate, affinity, specificity, half-life, or any other suitable characteristic.

The term "human antibody" as used herein refers to an antibody comprising a variable region in which both the framework and CDR regions are derived from human immunoglobulin sequences. The constant region of the antibody is also derived from human immunoglobulin sequences.

The term "antigen-binding fragment" as used herein refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a LAG-3 protein). Typically, it refers to antigen-binding fragments and antibody analogs that comprise at least a portion of the antigen-binding specificity of the parent antibody or a variable region (e.g., one or more CDRs). Antigen-binding fragments retain at least a portion of the binding specificity of the parent antibody. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', Fab'-SH, Fv, single chain antibodies (scFv), (Fab')2 fragments, single domain antibodies, diabodies (dAbs), or linear antibodies. Such antigen-binding fragments can be prepared using techniques known to those of ordinary skill in the art, and can be screened for utility in the same manner as antibodies.

Specifically, the Fab fragment refers to a monovalent fragment consisting of the VL, VH, CL, and CH1 domains.

The Fab' fragment differs from the Fab fragment in that it has several residues added at the carboxy terminus of the CH1 domain, including one or more cysteines from the antibody hinge region.

Fab'-SH refers to Fab' in which the cysteine residues in the constant domains bear a free thiol group.

F(ab')2 antibody fragments are produced as pairs of Fab' fragments joined by hinge cysteines between the Fab' fragments.

Fv is the minimum antibody fragment containing a complete antigen recognition site and antigen binding site. This fragment consists of a dimer of one heavy chain variable region and one light chain variable region, tightly non-covalently linked. These two regions fold to form six hypervariable loops (three loops each from the heavy and light chains), which provide amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable region has the ability to recognize and bind antigen, although with a lower affinity than the entire binding site.

A single-chain antibody (scFv) is an antibody fragment comprising VH and VL antibody domains linked by a single polypeptide chain. Preferably, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. Also referred to herein as scFv antibody fragment, antigen-binding fragment scFv, scFv antibody, antibody scFv, or simply scFv.

Diabodies refer to small antibody fragments prepared by constructing scFv fragments using a short linker (about 5-10 residues) between the VH and VL domains such that intrachain, rather than interchain, pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. Bispecific diabodies are heterodimers composed of two "bridged" scFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains.

An antibody "that specifically binds to human LAG-3" of the present invention means an antibody that binds to human LAG-3 protein (and possibly LAG-3 antibodies from one or more non-human species) but does not substantially bind to non-LAG-3 proteins. An anti-LAG-3 antibody or antigen-binding fragment of the present invention binds monomeric LAG-3 protein with high affinity. In certain embodiments, an anti-LAG-3 antibody or antigen-binding fragment of the present invention binds LAG-3 protein with a K _D of less than about 10 nM, preferably less than about 3 nM, preferably less than about 2 nM, more preferably less than about 1 nM, and most preferably less than about 0.1 nM, as measured by surface plasmon resonance.

In this specification, "Ka" represents the association rate of a specific antibody-antigen interaction and "Kd" represents the dissociation rate of a specific antibody-antigen interaction. The term "K _D " in this specification means the association-dissociation equilibrium constant obtained from Kd/Ka and expressed in molar concentration (M). The K _D value for an antibody can be determined using methods well-established in the art. Methods for measuring the K _D of an antibody are, for example, surface plasmon resonance (SRP), preferably using a biosensor system, such as the Biacore® system, or bio-layer interferometry (BLD), such as using the Octet® system.

The term "subject" as used herein includes a human or non-human animal. "Non-human animal" includes all vertebrates, including mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians and reptiles.

Binding to LAG-3

In one aspect, an anti-LAG-3 antibody is an antibody that specifically binds to LAG-3. The term "specific binding" is well known to those skilled in the art, and methods for determining specific binding between an antibody or antigen-binding fragment thereof and an antigen or epitope are also well known. In one embodiment, "specific binding" is understood to mean that the anti-LAG-3 antibody can bind to LAG-3 by an immunological reaction more rapidly and/or for a longer period of time with higher binding affinity and binding activity compared to when it binds to other target molecules. This does not mean that an antibody that specifically binds to LAG-3 does not bind to other target molecules. In another embodiment, "specific binding" can be exhibited by an antibody having a KD for LAG-3 of at least about 10 ^-7 M, or at least about 10 ^-8 M, or at least about 10 ^-9 M or less. Additionally, in one embodiment, the anti-LAG-3 antibody can specifically bind to LAG-3 and inhibit binding between LAG-3 and B7H3 and/or inhibit binding between LAG-3 and MHC II.

Anti-LAG-3 antibody or antigen-binding fragment

Preferred antibodies of the present invention are monoclonal antibodies S2-2D11-C, S3-1H9-C, S2-1B6-C, S1-2D1-A, S2-5A1-A, S1-2H6-A and S2-4B3-A, which are isolated and structurally characterized as described in the Examples.

In one aspect of the present invention, the present invention provides an antibody comprising CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 of S2-2D11-C, S3-1H9-C, S2-1B6-C, S1-2D1-A, S2-5A1-A, S1-2H6-A and S2-4B3-A, or a combination thereof. The amino acid sequences of CDR-H1 of the antibodies are disclosed in SEQ ID NOs: 1, 13, 25, 37, 49, 61 and 73. The amino acid sequences of CDR-H2 of the antibodies are disclosed in SEQ ID NOs: 3, 15, 27, 39, 51, 63 and 75. The amino acid sequences of CDR-H3 of the above antibodies are disclosed in SEQ ID NOs: 5, 17, 29, 41, 53, 65, and 77. The amino acid sequences of CDR-L1 of the above antibodies are disclosed in SEQ ID NOs: 7, 19, 31, 43, 55, 67, and 79. The amino acid sequences of CDR-L2 of the above antibodies are disclosed in SEQ ID NOs: 9, 21, 33, 45, 57, 69, and 81. The amino acid sequences of CDR-L3 of the above antibodies are disclosed in SEQ ID NOs: 11, 23, 35, 47, 59, 71, and 83.

Since the antigen-binding specificity of the above antibodies is mainly provided by the CDR regions, the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 sequences can be arbitrarily combined to produce anti-LAG-3 antibodies, which also fall within the scope of the present invention. The LAG-3 binding of these novel combination antibodies can be tested according to the binding assay test described in the Examples. Likewise, it will be apparent to those skilled in the art that modified anti-LAG-3 antibodies can be produced by replacing one or more V _H and/or V _L CDR sequences with the CDR sequences of antibodies S2-2D11-C, S3-1H9-C, S2-1B6-C, S1-2D1-A, S2-5A1-A, S1-2H6-A and S2-4B3-A disclosed herein or with V _H and/or V _L CDR sequences structurally similar thereto.

Therefore, according to another aspect, the present invention

(a) a heavy chain variable domain comprising a CDR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 13, 25, 37, 49, 61 and 73, a CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 15, 27, 39, 51, 63 and 75, and a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 17, 29, 41, 53, 65 and 77; and/or

(b) provides an antibody or an antigen-binding fragment thereof that specifically binds to human LAG-3, comprising a light chain variable domain comprising a CDR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 19, 31, 43, 55, 67 and 79, a CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 21, 33, 45, 57, 69 and 81, and a CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 23, 35, 47, 59, 71 and 83.

In a preferred embodiment, the anti-LAG-3 antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 3, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 5; and/or a light chain variable domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

In another preferred embodiment, the anti-LAG-3 antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 15, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, and/or a light chain variable domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 19, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 23.

In another preferred embodiment, the anti-LAG-3 antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 27, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 29, and/or a light chain variable domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 31, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 33, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 35.

In another preferred embodiment, the anti-LAG-3 antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 39, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 41, and/or a light chain variable domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 43, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 45, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 47.

In another preferred embodiment, the anti-LAG-3 antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 49, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 51, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 53, and/or a light chain variable domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 55, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 57, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 59.

In another preferred embodiment, the anti-LAG-3 antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 61, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 63, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 65, and/or a light chain variable domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 67, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 69, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 71.

In another preferred embodiment, the anti-LAG-3 antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 73, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 75, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 77, and/or a light chain variable domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 79, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 81, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 83.

The amino acid sequences of the heavy chain variable domains (VH) of preferred antibodies S2-2D11-C, S3-1H9-C, S2-1B6-C, S1-2D1-A, S2-5A1-A, S1-2H6-A and S2-4B3-A according to the present invention are disclosed in SEQ ID NOs: 85, 89, 93, 97, 101, 105 and 109, respectively. The amino acid sequences of the light chain variable domains (V _L ) of the above antibodies are disclosed in SEQ ID NOs: 87, 91, 95, 99, 103, 107 and 111, respectively.

As long as each of the above antibodies can bind to human LAG-3, another anti-LAG-3 binding molecule can be generated by any combination of the V _H and V _L sequences. When the V _H and V _L chains are combined, it is preferred that the V _H sequence from a particular V _H /V _L pair is replaced with a structurally similar V _H sequence, and it is preferred that the V _L sequence from a particular V _H /V _L pair is replaced with a structurally similar V _L sequence.

Thus, in one embodiment, the anti-LAG-3 antibody or antigen-binding fragment thereof according to the present invention

(a) a heavy chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 85, 89, 93, 97, 101, 105 and 109, and/or

(b) a light chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 87, 91, 95, 99, 103, 107 and 111.

In a preferred embodiment, the anti-LAG-3 antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 85 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 87.

In another preferred embodiment, the anti-LAG-3 antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 89 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 91.

In another preferred embodiment, the anti-LAG-3 antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 93 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 95.

In another preferred embodiment, the anti-LAG-3 antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 97 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 99.

In another preferred embodiment, the anti-LAG-3 antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 101 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 103.

In another preferred embodiment, the anti-LAG-3 antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 105 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 107.

In another preferred embodiment, the anti-LAG-3 antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 109 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 111.

In another embodiment, the antibodies or antigen-binding fragments of the present invention comprise heavy and light chain variable regions comprising amino acid sequences homologous to the amino acid sequences of the preferred antibodies disclosed herein, and these antibodies also retain the functional properties of the anti-LAG-3 antibodies according to the present invention, and in this respect, they are also within the scope of the present invention. For example, in one embodiment of the present invention,

(a) a heavy chain variable domain having at least 90%, at least 95%, or at least 98% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 85, 89, 93, 97, 101, 105, and 109, and/or

(b) provides an antibody or antigen-binding fragment thereof that specifically binds to human LAG-3, comprising a light chain variable domain having at least 90%, at least 95%, or at least 98% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 87, 91, 95, 99, 103, 107, and 111.

The % homology between two amino acid sequences is synonymous with the % identity between the two sequences. The % identity between two sequences is a function of the number of identical positions shared by the sequences, expressed as % homology = number of identical positions / total number of positions × 100. Taking into account the number of gaps that should be introduced for optimal alignment of the two sequences and the length of each gap, the comparison of sequences and determination of the % identity between the two sequences can be performed using algorithms and software known in the art.

In a preferred embodiment of the present invention, the antibody or antigen-binding fragment thereof of the present invention comprises heavy and light chain variable domains comprising amino acid sequences that are homologous to the amino acid sequences of the preferred antibodies disclosed herein, i.e., heavy and light chain variable domains having different sequences other than the CDRs. These antibodies also retain the functional properties of the anti-LAG-3 antibodies according to the present invention, and in this respect, they are also within the scope of the present invention.

Therefore, in a preferred embodiment, the antibody or antigen-binding fragment thereof according to the present invention

(a) a heavy chain variable domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 3; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 5, and having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 85, and/or

(b) a light chain variable domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9; and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 11, and having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 87.

In another embodiment, an antibody or antigen-binding fragment thereof according to the present invention

(a) a heavy chain variable domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 15; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17, and having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 89, and/or

(b) a light chain variable domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 19; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21; and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 23, and having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 91.

In another embodiment, an antibody or antigen-binding fragment thereof according to the present invention

(a) a heavy chain variable domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 27; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 29, and having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 93, and/or

(b) a light chain variable domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 31; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 33; and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 35, and having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 95.

In another embodiment, an antibody or antigen-binding fragment thereof according to the present invention

(a) a heavy chain variable domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 37; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 39; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 41, and having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 97, and/or

(b) a light chain variable domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 43; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 45; and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 47, and having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 99.

In another embodiment, an antibody or antigen-binding fragment thereof according to the present invention

(a) a heavy chain variable domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 49; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 51; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 53, and having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 101, and

(b) a light chain variable domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 55; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 57; and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 59, and having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 103.

In another embodiment, an antibody or antigen-binding fragment thereof according to the present invention

(a) a heavy chain variable domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 61; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 63; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 65, and having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 105, and/or

(b) a light chain variable domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 67; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 69; and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 71, and having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 107.

In another embodiment, an antibody or antigen-binding fragment thereof according to the present invention

(a) a heavy chain variable domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 73; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 75; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 77, and having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 109, and/or

(b) a light chain variable domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 79; a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 81; and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 83, and having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 111.

Conservative transformation

In certain embodiments, the antibodies of the invention comprise a heavy chain variable domain comprising CDR-H1, CDR-H2 and CDR-H3 sequences and a light chain variable domain comprising CDR-L1, CDR-L2 and CDR-L3 sequences, wherein one or more of these CDR sequences comprise a specific amino acid sequence of the preferred antibodies disclosed herein (e.g., S2-2D11-C, S3-1H9-C, S2-1B6-C, S1-2D1-A, S2-5A1-A, S1-2H6-A and S2-4B3-A) or conservative modifications thereof, which also retain the functional properties of the anti-LAG-3 antibodies of the invention. Conservative modifications are meant to be modified by, for example, substitutions, deletions, and/or additions of one, two or three amino acids, and include modifications within a CDR and/or modifications outside of a CDR, but which retain the biological characteristics of the antibody or antigen-binding fragment prior to modification, particularly the ability to specifically bind to LAG-3. In one embodiment, the modifications may be, but are not limited to, conservative substitutions as set forth in Table 1 below.

chain Member Hydrophobic Met, Ala, Val, Leu, Ile Neutral hydrophilic Cys, Ser, Thr Acidic Asp, Glu Alkalinity Asn, Gln, His, Lys, Arg Residues that affect chain orientation Gly, Pro Aromatic Trp, Tyr, Phe

Modification and manipulation of antibodies

The anti-LAG3 antibodies and antigen-binding fragments thereof disclosed herein (e.g., S2-2D11-C, S3-1H9-C, S2-1B6-C, S1-2D1-A, S2-5A1-A, S1-2H6-A, and S2-4B3-A) can also be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or effector function (e.g., antigen-dependent cellular cytotoxicity). Moreover, the antibodies and antigen-binding fragments thereof (e.g., S2-2D11-C, S3-1H9-C, S2-1B6-C, S1-2D1-A, S2-5A1-A, S1-2H6-A, and S2-4B3-A) can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or modified to alter its glycosylation, thereby further altering one or more functional properties of the antibody or fragment. Any of the anti-LAG3 antibodies or antigen-binding fragments thereof having the modifications (e.g., Fc modifications) and/or alterations discussed herein are part of the present invention.

The anti-LAG3 antibodies and antigen-binding fragments thereof disclosed herein (e.g., S2-2D11-C, S3-1H9-C, S2-1B6-C, S1-2D1-A, S2-5A1-A, S1-2H6-A, and S2-4B3-A) also include antibodies and fragments having an Fc region that has been modified (or blocked) to provide altered effector function (see, e.g., U.S. Patent Nos. 5,624,821; WO2003/086310; WO2005/120571; WO2006/0057702). Such modifications can be utilized to enhance or inhibit various responses of the immune system, with possible beneficial effects in diagnostics and therapy. Modifications of the Fc region include amino acid changes (substitutions, deletions, and insertions), glycosylation or deglycosylation, and multiple Fc additions. Changes to the Fc may also alter the half-life of the antibody in therapeutic antibodies, thereby increasing convenience and reducing the use of the agent by allowing for a reduction in the frequency of administration (see Presta (2005) J. Allergy Clin. Immunol. 116:731 at 734-35).

Another type of modification is to mutate amino acid residues within the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and/or CDR-L3 regions to improve one or more binding properties of the antibody of interest. Mutations can be introduced by site-directed mutagenesis or PCR-mediated mutagenesis, and their effect on antibody binding, and other functional properties, can be assessed by assays such as those described herein. Mutations can be amino acid substitutions, additions, or deletions, although substitutions are preferred. Typically, no more than one, two, three, four, or five residues within a CDR region are altered.

Method for producing antibodies and antigen-binding fragments thereof

The non-human antibodies in the present invention can be derived from, for example, any antibody-producing animal, such as a mouse, rat, rabbit, goat, donkey, or non-human primate (e.g., a monkey such as a cynomolgus or rhesus macaque) or ape (e.g., a chimpanzee). Non-human antibodies can be produced by immunizing an animal using methods known in the art. The antibodies can be polyclonal, monoclonal, or can be synthesized in a host cell by expressing recombinant DNA. Fully human antibodies can be prepared by administering an antigen to a transgenic animal containing human immunoglobulin loci, or by treating the antigen with a phage display library expressing a repertoire of human antibodies, and selecting the desired antibody.

Monoclonal antibodies (mAbs) can be produced by a variety of techniques, including conventional monoclonal antibody methods, for example, the standard somatic cell hybridization technique of the literature (reviewed in Kohler and Milstein, 1975, Nature 256:495).

The antibodies of the present invention can be produced by linking fragments of heavy and light chain variable domains (Fv regions) using amino acid bridges (short peptide linkers). The antibodies disclosed in the present invention include, but are not limited to, scFv comprising a combination of domains of the heavy and light chain variable regions, or a combination of light and heavy chain variable domains comprising the CDRs.

The antibodies of the present invention can also be changed into antibodies of different subtypes through subtype switching. Thus, for example, an IgG antibody can be derived from an IgM antibody, and vice versa.

antibody conjugate

The anti-LAG3 antibodies and antigen-binding fragments thereof of the present invention can be conjugated to a therapeutic agent to form an immunoconjugate, e.g., an antibody-drug conjugate (ADC). Suitable therapeutic agents include antimetabolites, alkylating agents, DNA minor groove binders, DNA intercalators, DNA cross-linkers, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and anti-mitotic agents. In the ADC, the antibody and therapeutic agent are preferably conjugated via a cleavable linker, e.g., a peptidyl, disulfide, or hydrazone linker. More preferably, the linker is a peptidyl linker, for example, Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu.

The anti-LAG3 antibodies and antigen-binding fragments thereof of the present invention may also be conjugated to a chemical moiety. Such conjugated antibodies and fragments are part of the present invention. The chemical moiety may be, in particular, a polymer, a radionuclide, or a cytotoxic agent. In a particular embodiment, the chemical moiety is a polymer that increases the half-life of the antibody or fragment in the body of the subject. Suitable polymers include, but are not limited to, hydrophilic polymers such as polyethylene glycol (PEG), dextran, and monomethoxypolyethylene glycol (mPEG). The cytotoxic factors may be, but are not limited to, diphtheria toxin, Pseudomonas aeruginosa exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins and compounds (e.g., fatty acids), dianthin proteins, Phytoiacca americana proteins PAPI, PAPII, and PAP-S, momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, mitogellin, restrictocin, phenomycin, and enomycin.

The anti-LAG3 antibodies and antigen-binding fragments thereof of the present invention can be conjugated with an isotope label, such as ⁹⁹ Tc, ⁹⁰ Y, ¹¹¹ In, ³² P, ¹⁴ C, ¹²⁵ I, ³ H, ¹³¹ I, ¹¹ C, ¹⁵ O, ¹³ N, ¹⁸ F, ³⁵ S, ⁵¹ Cr, ⁵⁷ To, ²²⁶ Ra, ⁶⁰ Co, ⁵⁹ Fe, ⁵⁷ Se, ¹⁵² Eu, ⁶⁷ CU, ²¹⁷ Ci, ²¹¹ At, ²¹² Pb, ⁴⁷ Sc, ¹⁰⁹ Pd, ²³⁴ Th, ⁴⁰ K, ¹⁵⁷ Gd, ⁵⁵ Mn, ⁵² Tr, or ⁵⁶ Fe.

The anti-LAG3 antibodies and antigen-binding fragments thereof of the present invention can also be pegylated, for example, to increase their biological (e.g., serum) half-life. To pegylate an antibody or fragment, the antibody or fragment is typically reacted with a reactive form of polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions such that one or more PEG groups become attached to the antibody or antibody fragment. In a particular embodiment, the pegylation is accomplished via an acylation reaction or alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" encompasses any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody or fragment to be pegylated is an aglycosylated antibody or fragment. Methods for PEGylating proteins are known in the art and can be applied to the antibody of the present invention.

Therapeutic Uses of Antibodies

The anti-LAG3 antibodies and antigen-binding fragments thereof of the present invention may be effective in the treatment or prevention of diseases or conditions associated with a suppressed immune system. Methods are provided for treating or preventing diseases or conditions associated with a suppressed immune system, comprising administering to a subject, e.g., a human subject, an effective amount of an anti-LAG3 antibody or antigen-binding fragment thereof disclosed herein (e.g., S2-2D11-C, S3-1H9-C, S2-1B6-C, S1-2D1-A, S2-5A1-A, S1-2H6-A, and S2-4B3-A).

The present invention also relates to inducing T cell activation using an anti-LAG3 antibody or an antigen-binding fragment thereof. In one embodiment, the present invention provides a method for inducing or enhancing T cell activation, comprising administering to a subject an effective amount of an anti-LAG3 antibody or an antigen-binding fragment thereof. In another embodiment, the present invention provides a use of an anti-LAG3 antibody or an antigen-binding fragment thereof for inducing or enhancing T cell activation. In another embodiment, the present invention provides a pharmaceutical composition for inducing or enhancing T cell activation, comprising an anti-LAG3 antibody or an antigen-binding fragment thereof.

Accordingly, the present invention relates to the prevention, improvement or treatment of diseases associated with a suppressed immune system using an anti-LAG3 antibody or an antigen-binding fragment thereof.

In certain embodiments of the present invention, the disease associated with the suppressed immune system is cancer or an infectious disease.

In one embodiment, the invention provides a method for preventing, ameliorating, or treating a disease associated with a suppressed immune system, comprising administering to a subject an effective amount of an anti-LAG3 antibody or an antigen-binding fragment thereof.

In another embodiment, the invention provides the use of an anti-LAG3 antibody or an antigen-binding fragment thereof for the prevention, amelioration or treatment of a disease associated with a suppressed immune system.

In another embodiment, the present invention provides a pharmaceutical composition for preventing, ameliorating or treating a disease associated with a suppressed immune system, comprising an anti-LAG3 antibody or an antigen-binding fragment thereof.

The anti-LAG3 antibody or antigen-binding fragment thereof of the present invention can activate T cells by blocking the binding of LAG3 protein and/or enhance the immune response against cancer cells in a cancer patient, thereby inhibiting the growth of cancer cells in vivo, and can thus be usefully used for preventing, improving or treating cancer.

In a specific embodiment of the present invention, the cancer is myeloma, osteosarcoma, head and neck cancer, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, Hodgkin's lymphoma, non-Hogdkin's lymphoma, squamous cell carcinoma, urothelial carcinoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, basal-cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous carcinoma, papillary cancer, papillary adenocarcinoma, cystadenocarcinoma, medullary thyroid cancer, bronchial cancer, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, spermocytoma, Wilm's cancer, bone cancer, cervical cancer, testicular cancer, colorectal cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder carcinoma, colon cancer, stomach cancer, kidney cancer, epithelial cell carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealocytoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia or metastatic lesions of the above cancers, but are not limited thereto. Doesn't.

Additionally, in certain embodiments of the present invention, the infectious disease is a viral infection, a bacterial infection, a parasitic infection, or a fungal infection.

The viral infection may be, but is not limited to, an infection by a virus selected from the group consisting of, for example, human immunodeficiency virus (HIV), Ebola virus, hepatitis virus (type A, type B, or type C), herpes virus (e.g., VZV, HSV-I, HAV-6, HSV-II, and CMV, Epstein Barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papilloma virus, molluscum virus, poliovirus, rabies virus, JC virus, or arboviral encephalitis virus.

The above bacterial infections include Chlamydia, Rickettsia bacteria, Mycobacteria, Staphylococcus, Streptococcus, Pneumonococcus, Meningococcus, Gonococcus, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Corynebacterium diphtheriae, Salmonella, Bacillus, Vibrio cholerae, Clostridium tetani, Clostridium botulinum, Bacillus anthracis. An infection caused by a bacteria selected from the group consisting of, but not limited to, Mycobacterium anthricis, Yersinia pestis, Mycobacterium leprae, Mycobacterium lepromatosis, and Borriella.

The above fungal infection may be an infection caused by a fungus selected from the group consisting of Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), Mucorales (mucor, absidia, rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum. It is not limited to these.

The above parasitic infections are Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba, Giardia lambia, Cryptosporidium, Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, Nippostrongylus brasiliensis. brasiliensis).

In certain embodiments of the present invention, a method is provided for treating or preventing a disease or condition associated with a suppressed immune system, by administering to a subject, e.g., a human subject, an effective amount of an anti-LAG3 antibody or antigen-binding fragment thereof according to the present invention and one or more additional immune stimulatory antibodies.

The above additional immune stimulating antibodies include anti-PD-1 antibodies such as pembrolizumab, nivolumab, cemiplimab, dostarlimab, vopratelimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, INCMGA00012, AMP-224 or AMP-514, atezolizumab, avelumab, durvalumab, KN035, cosibelimab, AUNP12, CA-170, BMS-9861899, STI-1014, CX-072, LY3300054 or CK-301; and anti-CTLA-4 antibodies such as ipilimumab, but are not limited thereto.

Pharmaceutical composition

According to another aspect of the present invention, a pharmaceutical composition comprising an anti-LAG3 antibody or an antigen-binding fragment thereof according to the present invention as an active ingredient together with a pharmaceutically acceptable carrier is provided. The pharmaceutical composition may comprise one or more additional active ingredients, such as another antibody or drug. Furthermore, the pharmaceutical composition according to the present invention may be administered simultaneously or sequentially in combination therapy with, for example, another immunostimulatory antibody, an anticancer agent, an antiviral agent or a vaccine.

Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active ingredients may be coated in a material that protects the active ingredients from the action of acids and other natural conditions that may inactivate the active ingredients. "Parenteral administration" means a mode of administration other than enteral and topical administration, and generally includes intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, the antibodies of the present invention may be administered, for example, by topical, epidermal or mucosal administration routes, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

The amount of active ingredient that can be combined with the carrier material to produce a single dosage form will vary depending on the subject to be treated and the particular mode of administration, and is generally an amount capable of producing a therapeutic effect. Typically, when combined with a pharmaceutically acceptable carrier, the amount is from about 0.01% to about 99%, preferably from about 0.1% to about 70%, and most preferably from about 1% to about 30%.

The antibody dosage ranges from about 0.0001 to about 100 mg/kg, preferably from about 0.01 to about 5 mg/kg (based on subject body weight). More specifically, the dosage can be 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 5 mg/kg or 10 mg/kg, or can be within the range of 1-10 mg/kg.

Diagnostic Uses of Antibodies

The antibodies of the present invention can be used for diagnostic purposes to detect, diagnose, or monitor diseases and/or symptoms associated with LAG-3. In one embodiment, a method according to the present invention is a method for diagnosing LAG-3 in a subject in need of diagnosis of LAG-3, comprising the steps of: measuring a concentration or intracellular localization of LAG-3 in the subject with an antibody or antigen-binding fragment according to the present invention; and comparing the concentration or intracellular localization of the LAG-3 aggregates measured in the subject with the results of a control sample, wherein similarity or difference from the results of the control is indicative of LAG-3 protein expression in the subject.

The above method can be performed by In vivo imaging. In vivo imaging can be performed using, for example, positron emission tomography (PET), single photon emission tomography (SPECT), near infrared (NIR) optical imaging or magnetic resonance imaging (MRI). The above method can be performed in vitro. The above method can be performed using Western blotting, immunoprecipitation, Enzyme Linked Immuno Sorbent Assay (ELISA), Radio Immuno Assay (RIA) or immunohistochemical methods which are well known to those skilled in the art (e.g., Tijssen, 1993, Practice and Theory of Enzyme Immunoassays, Vol 15 (Eds R.H. Burdon and P.H. van Knippenberg, Elsevier, Amsterdam); Zola, 1987, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc.); Jalkanen et al., 1985, J. Cell. Biol. 101:976-985; Jalkanen et al., 1987, J. Cell Biol. 105:3087-3096).

For diagnostic purposes, antibodies will typically be labeled with a detectable label. Suitable labels include, but are not limited to, radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorescent substances (e.g., FITC, rhodamine, lanthanide fluorophores), enzymes (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinyl groups, or polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, the labeling agent may be coupled to the antibody via spacer arms of varying lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art and can be applied herein.

In another aspect, the antibodies of the present invention can be used to identify tissues comprising LAG-3. In certain embodiments, the antibodies are labeled with a labeling agent, and binding of the labeled antibody to LAG-3 is detected. In one embodiment, binding of the antibody to LAG-3 is detected in vivo. In another aspect, the present invention discloses detecting or selecting for a test agent that competes for binding to LAG-3 with an antibody disclosed herein. For example, the method comprises detecting the amount of free antibody in a solution comprising LAG-3 in the presence or absence of the test agent. An increase in the concentration of free antibody, i.e., antibody that is not bound to LAG-3, will indicate that the test molecule can compete with the antibody for binding to LAG-3. In one embodiment, the antibody is labeled with a labeling group. Alternatively, the test agent is labeled, and the amount of free test agent is monitored in the presence or absence of the antibody.

In addition, the antibodies or antigen-binding fragments disclosed herein have various utilities, such as for specific binding assays, affinity-based LAG-3 purification, or screening methods for identifying LAG-3 antagonists.

For LAG-3 detection and immune response modulation

The antibodies, antibody compositions, and methods of the present invention have many in vitro and in vivo utilities, including, for example, enhancing an immune response by detecting LAG-3 or blocking LAG-3. In a preferred embodiment, the antibodies of the present invention are human antibodies. For example, these molecules can be administered to cells in culture, in vitro, or ex vivo, or to human subjects, for example, in vivo, to enhance immunogenicity in a variety of situations. Accordingly, the present invention provides a method of modifying an immune response in a subject, comprising administering to the subject an antibody or antigen-binding fragment thereof of the present invention. Preferably, the immune response is enhanced, stimulated, or upregulated. Preferred subjects include human patients in need of enhancing an immune response. The methods are particularly suitable for treating human patients having a disease that can be treated by enhancing an immune response (e.g., a T-cell mediated immune response). In certain embodiments, the methods are particularly suitable for treating cancer in vivo. To achieve antigen-specific enhancement of immunity, the anti-LAG-3 antibody may be administered together with the antigen of interest, or the antigen may already be present in the subject to be treated (e.g., a tumor-bearing or virus-bearing subject). When the antibody to LAG-3 is administered together with another agent, the two agents may be administered in any order or simultaneously.

The present invention further provides a method of detecting the presence of human LAG-3 antigen in a sample or measuring the amount of human LAG-3 antigen in a sample, comprising contacting the sample and a control sample with a human monoclonal antibody or an antigen-binding portion thereof that specifically binds human LAG-3 under conditions that allow for complex formation between the antibody or portion thereof and human LAG-3. The complex formation is then detected, wherein a difference in complex formation between the sample as compared to the control sample is indicative of the presence of human LAG-3 antigen in the sample. Additionally, the anti-LAG-3 antibodies of the present invention can be used to purify human LAG-3 via immunoaffinity purification.

In addition, given the ability of the anti-LAG-3 antibodies of the invention to inhibit binding of LAG-3 to MHC class II molecules and to stimulate antigen-specific T cell responses, the invention provides methods of using the antibodies of the invention in vitro and in vivo to stimulate, enhance, or upregulate antigen-specific T cell responses. For example, the invention provides a method of stimulating an antigen-specific T cell response, comprising contacting a T cell with an antibody of the invention such that an antigen-specific T cell response is stimulated. Any suitable marker of an antigen-specific T cell response can be used to measure the antigen-specific T cell response. Non-limiting examples of such suitable markers include increased T cell proliferation in the presence of the antibody and/or increased cytokine production in the presence of the antibody. In a preferred embodiment, interferon gamma (IFNγ) production by the antigen-specific T cell is stimulated.

The present invention also provides a method of stimulating an immune response (e.g., an antigen-specific T cell response) in a subject, comprising administering to the subject an antibody of the present invention such that an immune response (e.g., an antigen-specific T cell response) is stimulated in the subject. In a preferred embodiment, the subject is a tumor-bearing subject, and an immune response against the tumor is stimulated. In another preferred embodiment, the subject is a subject having an infectious disease, and an immune response against the infection is stimulated.

Hereinafter, the present invention will be described in more detail through examples. These examples are only examples for explaining the present invention more specifically, and the scope of the present invention is not limited by these examples.

Example

Example 1. Preparation of anti-LAG-3 antibody

1.1 Preparation of antigen

For the production of anti-LAG-3 antibodies, the antigen was prepared as follows. An antigen containing the human LAG-3 extracellular domain (ECD) in the amino acid sequence of human LAG-3 (NP_002277.4) sequence was used.

Specifically, recombinant human LAG-3 fused to the Fc region of human IgG1 or murine IgG2a at the C-terminus of human LAG-3 ECD was prepared as follows. Human LAG-3 ECD DNA was obtained from the human LAG-3 plasmid (HG16498-G) purchased from Sinobiological by PCR (polymerase chain reaction), and the obtained DNA fragment was cloned into the pCEP4 vector for animal cell expression using AgeI and Aflll restriction enzymes. In addition, a plasmid in the form of a SBP-tag fused to the C-terminus of the human LAG-3 gene was prepared using the pCEP4-human IgG-Fc-SBP backbone vector (Fig. 1). In addition, the D1D2 domain (Ig-V like type I and Ig-C like type 1) and D3D4 domain (Ig-C like type Ⅱ and Ig-C like type Ⅲ) of human LAG-3 were synthesized and PCR-generated, respectively, and cloned into an animal cell expression vector using the same method as above. All completed human LAG-3 plasmids were transformed into E. coli DH5a and stored.

The constructed vector was transformed into HEK293F animal cells, and human LAG-3 protein was purified by affinity chromatography using a MabSelect SuRe column (Cytiva).

To determine cross-reactivity between species, monkey (Rhesus) LAG-3 His tag (9992-L3, R&D systems), mouse LAG-3 His tag (LA3-M52H2, Acrobiosystems), and human LAG-3 His tag (LA3-H5222, Acrobiosystems) were used.

In addition, in order to select specific antibodies against native human LAG-3 expressed on the cell surface, the human LAG-3 sequence was cloned into the mCherry fluorescent pTK162 expression vector using the same method as above. In addition, the D1D2 domain or D3D4 domain expression vector was produced by inducing silent mutations by introducing HindⅢ or KpnI sites using the previously produced vectors (Fig. 2).

1.2 Library phage preparation and phage display panning

For various antigens, the binding library phage was prepared as follows. Helper phage (10 MOI) was added to the cultured phage library and infected in a 37℃ incubator. The infected cells were replaced with a medium containing kanamycin and ampicillin but not glucose, and phage particles were produced overnight. The produced culture medium was centrifuged at 4,500 rpm at 4℃ for 15 min, and PEG/NaCl (20% (w/v) polyethylene glycol 8000, 2.5 M NaCl) was added to the supernatant, mixed, and incubated on ice for 1 h. Then, after centrifugation at 10,000 × g for 30 minutes at 4°C, the pellet was suspended in 10 mM Tis-HCl pH7.4, and the obtained phage was treated with PEG/NaCl once again and incubated on ice for at least 2 hours, and the concentrated phage particles were secured in the same manner as above.

To select antibodies having specific binding affinity to human LAG-3, bio-panning was performed using the secured phage library as follows. Specifically, to remove antibodies with non-specific binding, BSA (bovine serum albumin), human IgG, and streptavidin were each coated on an immunotube at 4°C overnight, and the following day, 3% (w/v) skimmed milk/PBS was treated, blocked at 37°C for 1 hour, and 5x10 ¹¹ CFU of the phage library obtained in Example 1.2 were sequentially treated to the protein-coated immunotube, and incubated for 1 hour each to remove non-specific antibodies. The phage library from which non-specific binding antibodies had previously been removed was treated with biotinylated human LAG-3, reacted for 1 hour at room temperature, and phage antibodies specific to biotinylated LAG-3 were attracted by binding to streptavidin-magnetic particles. After washing several times with PBS-T (phosphate buffered saline-0.1% Tween 20) solution, phage antibodies were eluted with 100 mM glycine-HCL. The eluted phage antibodies were neutralized with 1 M Tris-HCl, infected with TG1 E. coli, and the recovery process was repeated three times to perform biopanning (Fig. 3).

Next, flow cytometry/separation was performed to select antibodies specific for the human LAG-3 domain expressed on the cell surface. The mCherry fusion-human LAG-3 expression vector produced in Example 1.1 was transiently transfected into HEK293F cells for 48 hours to prepare cell lines expressing LAG-3-full ECD or LAG3-D1D2 domains conjugated with mCherry fluorescent domain. The prepared ^4x106 cells were treated with ^1x1011 CFU phage antibody, incubated at 4°C for 1 hour, washed 7 times with PBS containing 1% BSA, and then separated with a FACS Aria (BD bioscience) device.

1.3 Single clone screening

To select a monoclonal phage antibody that specifically binds to LAG-3 from the phage pool secured by biopanning, antibody screening was performed. Specifically, the phage pool secured in Example 1.2 was spread on 2xYT agar solid medium to secure a single colony and individually cultured in a 96-well microplate. When the OD600 reached 0.6, 10 MOI helper phage was infected and phage particles (phage antibodies) were secured in the same manner as in Example 1.2. To select a monoclonal antibody specific for human LAG-3, phage-ELISA was performed using the secured phage antibody. A 96-well ELISA plate coated with 50 ng of human recombinant LAG-3 was treated with the monoclonal phage antibody and incubated at room temperature for 1 hour. After washing the plate six times with PBS-T (0.05% T20), the plate was treated with anti-M13-phage antibody conjugated with horseradish peroxidase (HRP) and incubated for 1 hour at room temperature. Then, the reaction was started by treating ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) substrate and the substrate reaction was stopped by treating 1% SDS (sodium dodecyl sulfate). The absorbance was measured at 405 nm using a macroplate reader.

The amino acid sequence of the antibody heavy chain variable region and the amino acid sequence of the light chain variable region were analyzed from the nucleic acid sequence encoding the selected antibody, and the complementarity determining region (CDR) was determined according to the AbM definition. The determined CDR amino acid and nucleic acid sequences (N->C) of the heavy and light chains are shown in Tables 2 and 3, respectively.

Antibody Sequence number CDR-H1 CDR-H2 CDR-H3 VH CDR-L1 CDR-L2 CDR-L3 VL S2-2D11-C 1 3 5 85 7 9 11 87 S3-1H9-C 13 15 17 89 19 21 23 91 S2-1B6-C 25 27 29 93 31 33 35 95 S1-2D1-A 37 39 41 97 43 45 47 99 S2-5A1-A 49 51 53 101 55 57 59 103 S1-2H6-A 61 63 65 105 67 69 71 107 S2-4B3-A 73 75 77 109 79 81 83 111

Antibody Sequence number CDR-H1 CDR-H2 CDR-H3 VH CDR-L1 CDR-L2 CDR-L3 VL S2-2D11-C 2 4 6 86 8 10 12 88 S3-1H9-C 14 16 18 90 20 22 24 92 S2-1B6-C 26 28 30 94 32 34 36 96 S1-2D1-A 38 40 42 98 44 46 48 100 S2-5A1-A 50 52 54 102 56 58 60 104 S1-2H6-A 62 64 66 106 68 70 72 108 S2-4B3-A 74 76 78 110 80 82 84 112

1.4 Production of anti-LAG-3 scFv-Fc antibodies from selected anti-LAG-3 phage antibodies

In Example 1.3, a plasmid having a nucleic acid sequence encoding the selected antibody was extracted from E. coli (according to the GeneAll® Exprep™ Plasmid SV User Manual) and prepared, and cloned into a vector for animal cell expression (pCEP4/maxi-v3b harboring hIgG1 Fc).

The anti-LAG-3 scFv-Fc antibody obtained by cloning was expressed according to the ExpiCHO Expression System. Specifically, ExpiCHO-S cells were cultured in ExpiCHO ^TM Expression medium, transfected with the vector of the antibody extracted with high purity and ExpiFectamine CHO reagent, and treated with ExpiCHO enhancer the next day. Antibody expression was performed for up to 12 days under the conditions of 32℃, 5% CO2 _, 120 rpm.

Purification of the expressed antibody was performed by affinity chromatography using a MabSelect SuRe column (Cytiva), and the antibody bound to the column was eluted with a solution of 45 mM NaOAc (pH 3.2) and 20 mM NaCl, followed by neutralization with 2 M Tris-HCl (pH 8.0). The antibody buffer was then exchanged with PBS, and all antibodies were stored at -20°C until use.

Example 2. Confirmation of binding properties of LAG-3 monoclonal antibody

2.1 Binding of anti-LAG-3 antibodies to human LAG-3 expressing cells

To confirm the binding properties of anti-LAG-3 antibodies to LAG-3 expressed on the cell surface, the following generated expression vectors were used.

The total DNA corresponding to human LAG-3 (NP_002277.4) was amplified by PCR and subcloned into the pTK162 plasmid containing an mCherry fluorescent tag to generate pTK162-Full-LAG-3-mCheery vector for expressing human LAG-3 on the cell surface with a fluorescent tag in the cytoplasm. For the human LAG-3 D1D2-mCherry expression vector, silent mutations were introduced into the parental LAG-3 construct (pTK162-Full-LAG-3-mCheery) to generate a LAG-3 D1D2 domain construct with a fluorescent mCherry tag at the C-terminus.

Human LAG-3 and LAG-3-D1D2 expressing cells were generated by transient transfection of HEK293 with LAG-3 expression vector, and cell binding of anti-LAG-3 antibodies was analyzed using flow cytometry. 5 μg/mL of scFv-Fc was added to 5 × 10 ⁵ LAG-3 expressing cells and incubated for 1 h at 4°C. Cells were washed three times with ice-cold wash buffer (PBS containing 3% BSA and 0.03% NaN3). scFv-Fc was captured with FITC anti-human IgG Fc antibody (Jackson Immuneresearch). After the final wash, cells were analyzed by FACScalibur (BD bioscience).

Results showed that anti-LAG-3 antibodies bound to human LAG-3 full-form expressing cells. Similarly, antibody binding was observed in LAG-3-D1D2 expressing cells, and S2-2E11-B, which targets LAG-3-D3D4, did not bind well to LAG-3-D1D2 expressing cells, as expected (Fig. 4, Table 4).

Ratio of MFI
(anti-LAG3 antibodies/hIgG) A Human LAG3-full+HEK293 Human LAG3-D1D2+HEK293 S2-2D11-C 23.7 37.4 S3-1H9-C 16.7 28.9 S2-1B6-C 28.0 40.8 S1-2D1-A 22.0 36.7 S2-5A1-A 28.1 51.0 S1-2H6-A 27.3 52.7 S2-4B3-A 3.3 3.9 S3-2E11-B (D3D4 Ab) 14.1 2.7

2.2 LAG-3 expression and antibody binding to stimulated CD4+ and CD8+ T cells

The specific binding capacity of anti-LAG-3 antibodies to LAG-3 on the surface of PHA-L-stimulated T cells expressing LAG-3 was investigated.

T cells were obtained from peripheral blood mononuclear cells (PBMCs). PBMCs were purified and isolated from whole blood obtained from a healthy donor (Donor: APH30478) using a density gradient centrifugation method according to the instructions of Ficoll-Paque™ PLUS Media (Cytiva, 17144002). Isolated PBMCs were stimulated with 5 μg/mL PHA-L (Invitrogen, 00-4977-93) for 24 h. T cells for analysis were incubated in the presence of recombinant human IL-7, IL-15, and IL-2 (Peprotech).

LAG-3 expression in unstimulated and stimulated T cells (day 2 of culture) was evaluated by flow cytometry. BV421 conjugated anti-LAG-3 antibody or the corresponding isotype control (BioLegend, clone T47-530) was incubated with the isolated T cells. Anti-human CD3-APC-eFluorTM 780 (Invitrogen, clone SK7) antibody, mouse anti-human CD8-FITC (BD, RPA-T8) antibody were used as phenotypic markers. The results of this study are shown in Fig. 5 . No LAG-3 expression was observed in unstimulated CD4+ and CD8+ T cells. However, PHA-L stimulated T cells dramatically increased LAG-3 expression (Fig. 5 ).

The cell binding ability of S2-5A1-A (Fig. 6a), S1-2H6-A (Fig. 6b), S2-2D11-C (Fig. 6c), S2-1B6-C (Fig. 6d), S3-1H9-C (Fig. 6e), S2-4B3-A (Fig. 6f), and S1-2D1-A (Fig. 6g) was investigated in detail (Fig. 7). Stimulated CD4+ and CD8+ T cells and unstimulated CD4+ and CD8+ T cells were analyzed using LAG-3 mAb clone and fluorescently conjugated secondary antibody (PE conjugated anti-human IgG Fc) (BioLegend, clone M1310G05). Anti-human CD3-APC-eFluorTM 780 (Invitrogen, clone SK7) antibody, mouse anti-human CD8-FITC (BD, RPA-T8) antibody were used as phenotypic markers. Binding was measured on a BD FACSCanto™ II Flow Cytometry System, and data were analyzed using Flow Jo software version 10.0.6 (Tree Star, Inc.).

As a result, the binding of each anti-LAG-3 antibody clone to stimulated CD4+, CD8+ T cells was measured, but no binding was observed to unstimulated CD4+, CD8+ T cells. These findings demonstrate that LAG-3 expression is upregulated in stimulated T cells and that the anti-LAG-3 antibodies of the present invention specifically bind to stimulated T cells.

2.3 Recombinant LAG-3 binding (cross-reactivity)

To check cross-reactivity between species, 96-well microplates were coated overnight at 4°C using cynomolgus LAG-3 His tag (9992-L3, R&D systems), mouse LAG-3 His tag (LA3-M52H2, Acrobiosystems), and human LAG-3 His tag (LA3-H5222, Acrobiosystems). After washing three times, the plates were blocked with ELISA blocking buffer (3% skimmed milk in PBST (0.05% T20)) for 1 h at 37°C. Anti-LAG-3 antibody at 1 μg/mL was added to the blocked plates, and the plates were incubated for 1 h at room temperature. Anti-LAG-3 antibody was detected using peroxidase-conjugated anti-human IgG secondary antibody (Jackson Immunoresearch) with TMB (Sigma-Aldrich) as a substrate for peroxidase. Absorbance at 450 nm was measured using a Synergy H1 plate reader (BioTeK). Relative binding of LAG-3 to each protein was analyzed by comparison with the background control signal for BSA.

All antibodies tested were observed to bind to both human and cynomolgus LAG-3. In particular, antibodies S2-1B6-C, S2-5A1-A, S2-2D11-C, and S2-4B3-A exhibited absorbance signals confirming binding to human, mouse, and cynomolgus LAG-3 (Figure 8).

Example 3. Confirmation of binding kinetics of anti-LAG-3 antibodies to recombinant human LAG-3

The binding kinetics of S2-2D11-C, S3-1H9-C, S2-1B6-C, S1-2D1-A, S2-5A1-A, and S1-2H6-A antibodies were measured by surface plasmon resonance (SPR) using a Biacore T200 (Cytiva, Sweden).

The pH for antibody immobilization was set using four types of pH scouting buffers (pH 4.0, 4.5, 5.0, 5.5, GE Healthcare). Antibody samples were prepared using acetate buffer, and the antibodies were immobilized on the Series S Sensor Chip CM5 (GE Healthcare, BR100530) using the Amine Coupling Kit (GE Healthcare, BR100050). The running buffer for immobilization was HBS-EP+ (GE Healthcare, BR100669), and the target immobilization value of the ligand was set to 50 RU. Recombinant human LAG-3 tagged with human Fc (Acrobiosystems, LA3-H5222) in the range of 25 nM to 0.78 nM was injected at a flow rate of 30 ㎍/mL onto the immobilized anti-LAG-3 antibody surface. The contact time was set to 2 min, and the dissociation time was set to 20 min for S2-2D11-C, S3-1H9-C, S2-1B6-C, S1-2D1-A, S2-5A1-A, S2-4B3-A, and S1-2H6-A antibodies. The running buffer for binding analysis was HBS-EP+ (GE Healthcare, BR100669), and the regeneration buffer was a 10 mM glycine solution, pH 2.0 (GE Healthcare, BR100356). The Biacore T200 control software (v. 3.2) was used for operation of the Biacore T200 surface plasmon resonance instrument, and the Biacore T200 evaluation software (v. 3.2) was used for data analysis. The measured kinetic association (Ka), kinetic dissociation (Kd), and association-dissociation equilibrium constants (KD) of S2-2D11-C, S3-1H9-C, S2-1B6-C, S1-2D1-A, S2-5A1-A, S2-4B3-A, and S1-2H6-A antibodies are shown in Table 5 below.

Analyte Ligand
(Amine coupling) Ka (1/Ms) Kd (1/s) KD (M) HumanLAG-3/CD223
(dimer) S2-2D11-C 4.52E+06 4.50E-04 9.96E-11 S3-1H9-C 9.82E+06 4.80E-03 5.17E-10 S2-5A1-A 2.18E+07 3.94E-04 1.81E-11 S2-4B3-A 2.06E+06 3.65E-03 1.77E-09 S1-2D1-A 3.85E+05 6.30E-04 1.64E-10 S2-1B6-C 2.86E+07 1.40E-03 4.90E-11 S1-2H6-A 1.39E+07 3.59E-04 2.58E-11

Example 4. Blockade of the interaction between LAG-3 and B7H3 by anti-LAG-3 antibodies

To determine the ability of anti-LAG-3 antibodies to block the interaction between LAG-3 and B7H3, an ELISA-based protein-protein interaction assay was performed using the following procedure.

96-well microplates were coated with human B7H3 recombinant protein (R&D Systems, 2318-B3) overnight at 4°C. After washing three times with phosphate-buffered saline (PBST) containing 0.05% Tween 20, the plates were blocked with 3% BSA in PBST for 1 h at 37°C. Serially diluted anti-LAG-3 antibodies and biotinylated recombinant LAG-3 protein (Acrobiosystems, LA3-H82E5) were preincubated for 1 h at room temperature, and the preincubated mixture was added to the 3% BSA-blocked 96-well plates and incubated for 2 h at room temperature. After washing six times, biotinylated LAG-3 protein bound to B7H3 protein was detected using peroxidase-conjugated streptavidin (Pierce, 21130) together with TMB (Thermo Fisher Scientific, 34021) as a substrate for peroxidase. The absorbance at 450 nm was measured using a Synergy H1 plate reader (BioTeK). The data calculated as the percentage binding of LAG-3 protein to B7H3 are shown in Fig. 9, and 100% B7H3 binding of LAG-3 is based on when anti-LAG-3 antibody is 0 nM. The IC50 values are summarized in Table 6.

The results showed that anti-LAG-3 antibodies of the tested S2-2D11-C, S3-1H9-C, S2-1B6-C, S1-2D1-A, S2-5A1-A, S1-2H6-A and S2-4B3-A clones blocked the binding of LAG-3 protein to immobilized B7H3 protein. In contrast, an irrelevant antibody was found not to block the binding between the two proteins.

IC50
(nM) Inhibition (%) S1-2D1-A 44.93 46.7 S2-1B6-C 26.07 44.5 S2-2D11-C 35.05 63.2 S1-2H6-A 88.2 62.7 S2-5A1-A 96.01 50.4 S2-4B3-A 102.7 62.5 S3-1H9-C 66.79 66.8 Irrelevant Ab (IgG1-maxi control Ab) N/A 0

Example 5. Blockade of the interaction between LAG-3 and MHC II

To measure the blocking ability of antibodies to LAG-3 protein of the present invention for the binding between LAG-3 protein and MHC II protein, a LAG-3/MHC II binding assay kit (CISBIO, 64LAG3PEG) was used. This kit utilizes the HTRF (Homogeneous Time-resolved Fluorescence) technology and allows simple and rapid characterization of compounds and antibody blockers in a high-throughput format. In this kit, the interaction between LAG3 and MHC II is detected using anti-Tag1 labeled with Europium (HTRF donor) and anti-Tag2 labeled with d2 (HTRF acceptor). When the donor and acceptor antibodies come into proximity due to binding of LAG-3 and MHC II, excitation of the donor antibody induces fluorescence resonance energy transfer (FRET) toward the acceptor antibody, which in turn emits specific fluorescence at 665 nm. Since this specific signal is directly proportional to the degree of LAG-3/MHC II interaction, compounds or antibodies that block LAG-3/MHC II interaction will decrease the HTRF signal. This kit provides Tag1-LAG-3, Tag2-MHCII, Anti-Tag1 Eu Cryptate reagent, Anti-Tag2 d2 antibody, and PPI Europium Detection Buffer. To activate the provided reagents, 100 μL of Tag1-LAG-3 stock solution was mixed with 1900 μL of PPI Europium Detection Buffer to prepare a 20-fold dilution of Tag1-LAG-3 stock solution, 50 μL of Anti-Tag1 Eu Cryptate reagent stock solution was mixed with 2450 μL of PPI Europium Detection Buffer to prepare a 50-fold dilution of Anti-Tag1 Eu Cryptate reagent stock solution, and 50 μL of Anti-Tag2 d2 antibody stock solution was mixed with 2450 μL of PPI Europium Detection Buffer to prepare a 50-fold dilution of Anti-Tag2 d2 antibody stock solution. Anti-LAG-3 antibodies of the S2-2D11-C, S3-1H9-C, S2-1B6-C, S1-2D1-A, S2-5A1-A, S1-2H6-A and S2-4B3-A clones of the present invention and IgG4 and IgG1_Maxi antibodies used as negative controls were prepared at concentrations of 4000 nM, 3000 nM, 2000 nM, 1000 nM, 500 nM, 100 nM, 10 nM and 1 nM, respectively. First, 4 μl of Tag1-LAG-3, Tag2-MHC II reagents, which were diluted 20-fold as many times as the number of times to be measured for the blocking power of the antibodies of the concentrations described above for the binding between LAG-3 protein and MHC II protein, were dispensed into each well of HTRF 96well low volume plates (CISBIO, 66PL96025). And 2μl of the antibodies described above at the prepared concentrations were added to each well. And 5μl of Anti-Tag1 Eu Cryptate reagent and Anti-Tag2 d2 antibody diluted 50-fold were added to each well. At this time, since the anti-LAG-3 antibodies at concentrations of 4000nM, 3000nM, 2000nM, 1000nM, 500nM, 100nM, 10nM, and 1nM were diluted 10-fold, the blocking power was measured at the final concentrations of 400nM, 300nM, 200nM, 100nM, 50nM, 10nM, 1nM, and 0.1nM. The measurement of the blocking power of all antibodies at each prepared concentration was performed in triplicate. The plate was sealed with a 96-well plate lid and incubated at room temperature for 3 hours. The lid was removed, and the fluorescence from the wells of the HTRF 96-well low volume plate was measured using a SPARK microplate reader (TECAN). The measurement conditions of the SPARK microplate reader for measuring the fluorescence from the donor antibody were as follows. Mode: TR Fluorescence Intensity, Excitation: Monochromator, Excitation wavelength [nm]: 340, Excitation bandwidth [nm]: 20, Emission: Monochromator, Emission wavelength [nm]: 620, Emission bandwidth [nm]: 20, Gain: optimal gain, Mirror: Dichroic 510, Number of flashes: 50, Integration time [μs]: 400, Lag time [μs]: 100, Settle time [ms]: 0, Z-Position mode: From well giving the highest signal. The measurement conditions of the SPARK microplate reader for measuring fluorescence from the recipient antibody are as follows. Mode: TR Fluorescence Intensity, Excitation: Monochromator, Excitation wavelength [nm]: 340, Excitation bandwidth [nm]: 20, Emission: Monochromator, Emission wavelength [nm]: 665, Emission bandwidth [nm]: 20, Gain: optimal gain, Mirror: Dichroic 510, Number of flashes: 50, Integration time [μs]: 400, Lag time [μs]: 100, Settle time [ms]: 0, Z-Position mode: From well giving the highest signal. Using these measurement values, the HTRF ratio value can be derived by calculating Signal 665 nm / Signal 620 nm * 10000. A higher HTRF value indicates that more Tag1-LAG3 and Tag2-MHC II proteins are bound. These results are derived from the IC 50 values using the Nonlinear regression (curve fit), log (inhibitor) vs. response (three parameters) of the PRISM8 program.

The results are shown in Fig. 10 and Table 7, and the assay results showed that anti-LAG-3 antibodies of the tested S2-2D11-C, S3-1H9-C, S2-1B6-C, S1-2D1-A, S2-5A1-A, S1-2H6-A and S2-4B3-A clones blocked the binding of LAG-3 protein to MHC II protein. In contrast, an irrelevant antibody was found not to block the binding between the two proteins.

Antibody IC50 IgG4 Not converged Relatrlimab 4.098 S2-2D11-C 5.436 S3-1H9-C ~ 690696 S2-1B6-C 12.86 S1-2D1-A 11.67 S2-5A1-A 3.214 S1-2H6-A 19.56 S2-4B3-A 23.7 IgG1-Maxi control 205.2

Example 6. T cell activation effect of anti-LAG-3 antibody

The activity of anti-LAG-3 antibodies was tested in PBMC (Donor 1: APH26067, Donor 2: APH30478) using a mixed lymphocyte reaction (MLR) assay. Specifically, the anti-LAG-3 antibody clones were evaluated in a two-way MLR assay. In a two-way MLR assay, both lymphocyte populations can be stimulated by an allogeneic lymphocyte population. This occurs due to recognition of HLA mismatch between two unrelated donors. In this MLR assay, PBMC from different donors were cultured for 72 hours in the presence of 5 μg/mL of anti-LAG-3 antibody clones or isotype control and IFN-gamma secretion was measured to determine T cell activation.

As shown in Figure 11, several types of anti-LAG-3 antibody clones increased IFN-gamma production. Furthermore, this effect was further enhanced when anti-LAG-3 antibodies were co-treated with anti-PD-1 antibodies (Proteogenix, cat. PX-TA1007), as shown in Figure 12. These data demonstrate that blockade of LAG-3 alone or co-blockade of LAG-3 and PD-1 with anti-PD-1 antibodies can lead to improved T cell activation.

Attach electronic file of sequence list

Claims (33)

(a) a heavy chain variable domain comprising a CDR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 13, 25, 37, 49, 61 and 73, a CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 15, 27, 39, 51, 63 and 75, and a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 17, 29, 41, 53, 65 and 77; and/or
(b) a light chain variable domain comprising a CDR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 19, 31, 43, 55, 67 and 79, a CDR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 21, 33, 45, 57, 69 and 81, and a CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 23, 35, 47, 59, 71 and 83;
An antibody or antigen-binding fragment thereof that specifically binds to human LAG-3. In the first paragraph,
(1) CDR-H1 comprising the amino acid sequence of sequence number 1;
CDR-H2 comprising the amino acid sequence of sequence number 3; and
A heavy chain variable domain comprising a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 5; and/or
CDR-L1 comprising the amino acid sequence of sequence number 7;
CDR-L2 comprising the amino acid sequence of sequence number 9; and
A light chain variable domain comprising a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 11; or
(2) CDR-H1 comprising the amino acid sequence of sequence number 13;
CDR-H2 comprising the amino acid sequence of SEQ ID NO: 15; and
A heavy chain variable domain comprising a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17; and/or
CDR-L1 comprising the amino acid sequence of SEQ ID NO: 19;
CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21; and
A light chain variable domain comprising a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 23; or
(3) CDR-H1 comprising the amino acid sequence of sequence number 25;
CDR-H2 comprising the amino acid sequence of SEQ ID NO: 27; and
A heavy chain variable domain comprising a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 29; and/or
CDR-L1 comprising the amino acid sequence of SEQ ID NO: 31;
CDR-L2 comprising the amino acid sequence of SEQ ID NO: 33; and
A light chain variable domain comprising a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 35; or
(4) CDR-H1 comprising the amino acid sequence of sequence number 37;
CDR-H2 comprising the amino acid sequence of SEQ ID NO: 39; and
A heavy chain variable domain comprising a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 41; and/or
CDR-L1 comprising the amino acid sequence of SEQ ID NO: 43;
CDR-L2 comprising the amino acid sequence of SEQ ID NO: 45; and
A light chain variable domain comprising a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 47; or
(5) CDR-H1 comprising the amino acid sequence of sequence number 49;
CDR-H2 comprising the amino acid sequence of SEQ ID NO: 51; and
A heavy chain variable domain comprising a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 53; and/or
CDR-L1 comprising the amino acid sequence of SEQ ID NO: 55;
CDR-L2 comprising the amino acid sequence of SEQ ID NO: 57; and
A light chain variable domain comprising a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 59; or
(6) CDR-H1 comprising the amino acid sequence of sequence number 61;
CDR-H2 comprising the amino acid sequence of SEQ ID NO: 63; and
A heavy chain variable domain comprising a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 65; and/or
CDR-L1 comprising the amino acid sequence of SEQ ID NO: 67;
CDR-L2 comprising the amino acid sequence of SEQ ID NO: 69; and
A light chain variable domain comprising a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 71; or
(7) CDR-H1 comprising the amino acid sequence of sequence number 73;
CDR-H2 comprising the amino acid sequence of SEQ ID NO: 75; and
A heavy chain variable domain comprising a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 77; and/or
CDR-L1 comprising the amino acid sequence of SEQ ID NO: 79;
CDR-L2 comprising the amino acid sequence of SEQ ID NO: 81; and
Comprising a light chain variable domain comprising a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 83;
An antibody or an antigen-binding fragment thereof. In the first paragraph,
(a) a heavy chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 85, 89, 93, 97, 101, 105 and 109, and/or
(b) a light chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 87, 91, 95, 99, 103, 107 and 111;
An antibody or an antigen-binding fragment thereof. In the second paragraph,
(1) a heavy chain variable domain having at least 90%, at least 95%, or at least 98% homology with the amino acid sequence of sequence number 85, and
A light chain variable domain having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 87; or
(2) a heavy chain variable domain having at least 90%, at least 95%, or at least 98% homology with the amino acid sequence of sequence number 89, and
A light chain variable domain having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 91; or
(3) a heavy chain variable domain having at least 90%, at least 95%, or at least 98% homology with the amino acid sequence of sequence number 93, and
A light chain variable domain having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 95; or
(4) a heavy chain variable domain having at least 90%, at least 95%, or at least 98% homology with the amino acid sequence of sequence number 97, and
A light chain variable domain having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 99; or
(5) a heavy chain variable domain having at least 90%, at least 95%, or at least 98% homology with the amino acid sequence of sequence number 101, and
A light chain variable domain having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 103; or
(6) a heavy chain variable domain having at least 90%, at least 95%, or at least 98% homology with the amino acid sequence of sequence number 105, and
A light chain variable domain having at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 107; or
(7) a heavy chain variable domain having at least 90%, at least 95%, or at least 98% homology with the amino acid sequence of sequence number 109, and
Comprising a light chain variable domain having at least 90%, at least 95%, or at least 98% homology to the amino acid sequence of SEQ ID NO: 111;
An antibody or an antigen-binding fragment thereof. In the third paragraph,
(1) a heavy chain variable domain comprising the amino acid sequence of sequence number 85, and
A light chain variable domain comprising the amino acid sequence of SEQ ID NO: 87; or
(2) a heavy chain variable domain comprising the amino acid sequence of sequence number 89, and
A light chain variable domain comprising the amino acid sequence of SEQ ID NO: 91; or
(3) a heavy chain variable domain comprising the amino acid sequence of sequence number 93, and
A light chain variable domain comprising the amino acid sequence of SEQ ID NO: 95; or
(4) a heavy chain variable domain comprising the amino acid sequence of sequence number 97, and
A light chain variable domain comprising the amino acid sequence of SEQ ID NO: 99; or
(5) a heavy chain variable domain comprising the amino acid sequence of sequence number 101, and
A light chain variable domain comprising the amino acid sequence of SEQ ID NO: 103; or
(6) a heavy chain variable domain comprising the amino acid sequence of sequence number 105, and
A light chain variable domain comprising the amino acid sequence of SEQ ID NO: 107; or
(7) a heavy chain variable domain comprising the amino acid sequence of sequence number 109, and
Comprising a light chain variable domain comprising the amino acid sequence of sequence number 111;
An antibody or an antigen-binding fragment thereof. In the second paragraph,
A sequence having at least 90%, at least 95%, or at least 98% homology with the amino acid sequence of sequence number 113;
A sequence having at least 90%, at least 95%, or at least 98% homology with the amino acid sequence of sequence number 115,
A sequence having at least 90%, at least 95%, or at least 98% homology with the amino acid sequence of sequence number 117,
A sequence having at least 90%, at least 95%, or at least 98% homology with the amino acid sequence of sequence number 119;
A sequence having at least 90%, at least 95%, or at least 98% homology with the amino acid sequence of sequence number 121;
A sequence having at least 90%, at least 95%, or at least 98% homology with the amino acid sequence of SEQ ID NO: 123, or
Comprising a sequence having at least 90%, at least 95%, or at least 98% homology with the amino acid sequence of SEQ ID NO: 125;
An antibody or an antigen-binding fragment thereof. An antibody or an antigen-binding fragment thereof, comprising the amino acid sequence of SEQ ID NO: 113, the amino acid sequence of SEQ ID NO: 115, the amino acid sequence of SEQ ID NO: 117, the amino acid sequence of SEQ ID NO: 119, the amino acid sequence of SEQ ID NO: 121, the amino acid sequence of SEQ ID NO: 123, or the amino acid sequence of SEQ ID NO: 125, in claim 6. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof specifically binds to monkey LAG-3, mouse LAG-3, or both. In claim 8, the antibody or antigen-binding fragment is a monoclonal antibody, a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fv, a diabody or a scFv. In claim 9, the monoclonal antibody is an antibody or an antigen-binding fragment thereof of the IgG1, IgG2, IgG3 or IgG4 type. An antibody or antigen-binding fragment thereof, in claim 8, wherein the antibody or antigen-binding fragment is a bispecific antibody or antigen-binding fragment capable of simultaneously binding to LAG-3 and another epitope. In claim 8, the antibody or antigen-binding fragment thereof reduces binding between a major histocompatibility complex (MHC) class II molecule or a cell expressing an MHC class II molecule and LAG-3. In claim 8, the antibody or antigen-binding fragment thereof reduces binding between a B7-H3 molecule or a cell expressing a B7-H3 molecule and LAG-3. In claim 8, the antibody or antigen-binding fragment thereof is capable of stimulating a T cell-mediated immune response. An antibody or an antigen-binding fragment thereof, wherein the T cell-mediated immune response in claim 14 is an anti-cancer immune response. A pharmaceutical composition for treating a disease or condition associated with a suppressed immune system, comprising an antibody or antigen-binding fragment according to any one of claims 1 to 7. A pharmaceutical composition according to claim 16, wherein the disease or condition associated with the suppressed immune system is cancer or infection. In claim 17, the cancer is myeloma, osteosarcoma, head and neck cancer, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, Hodgkin's lymphoma, non-Hogdkin's lymphoma, squamous cell carcinoma, urothelial carcinoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, basal-cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous carcinoma, papillary cancer, papillary adenocarcinoma, cystadenocarcinoma, medullary thyroid cancer, bronchial cancer, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, spermocytoma, Wilm's cancer, bone cancer, cervical cancer, testicular A pharmaceutical composition comprising: cancer, colorectal cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder carcinoma, colon cancer, stomach cancer, kidney cancer, epithelial cell carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealocytoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia or a metastatic lesion of the above cancers. A pharmaceutical composition according to claim 17, wherein the infection is a viral infection, a bacterial infection, a parasitic infection, or a fungal infection. A pharmaceutical composition in claim 19, wherein the viral infection is an infection by a virus selected from the group consisting of human immunodeficiency virus (HIV), Ebola virus, hepatitis virus (type A, type B, or type C), herpes virus (e.g., VZV, HSV-I, HAV-6, HSV-II, and CMV, Epstein Barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papilloma virus, molluscum virus, poliovirus, rabies virus, JC virus, or arboviral encephalitis virus. In claim 19, the bacterial infection is selected from the group consisting of Chlamydia, Rickettsia bacteria, Mycobacteria, Staphylococcus, Streptococcus, Pneumonococcus, Meningococcus, Gonococcus, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Corynebacterium diphtheriae, Salmonella, Bacillus, Vibrio cholerae, Clostridium tetani, Clostridium botulinum, Bacillus A pharmaceutical composition, wherein the infection is caused by a bacteria selected from the group consisting of Bacillus anthricis, Yersinia pestis, Mycobacterium leprae, Mycobacterium lepromatosis, and Borriella. In claim 19, the fungal infection is selected from the group consisting of Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), Mucorales (mucor, absidia, rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum. A pharmaceutical composition for treating an infection caused by a fungus. In claim 19, the parasitic infection is Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba, Giardia lambia, Cryptosporidium, Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, Nippostrongylus A pharmaceutical composition, wherein the infection is caused by a parasite selected from the group consisting of Nippostrongylus brasiliensis. A pharmaceutical composition for treating a disease or condition associated with a suppressed immune system, comprising an antibody or antigen-binding fragment according to any one of claims 1 to 7, and at least one additional immunostimulatory antibody. A pharmaceutical composition according to claim 24, wherein the additional immune-stimulating antibody is at least one selected from an anti-PD-1 antibody, an anti-PD-L1 antibody, and an anti-CTLA-4 antibody. In claim 25, the additional immune stimulating antibody is pembrolizumab, nivolumab, cemiplimab, dostarlimab, vopratelimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, INCMGA00012, AMP-224, AMP-514, atezolizumab, avelumab, durvalumab, KN035, cosibelimab, AUNP12, CA-170, BMS-9861899, STI-1014, CX-072. A pharmaceutical composition comprising at least one selected from the group consisting of LY3300054, CK-301, and ipilimumab. An isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment according to any one of claims 1 to 7. A vector comprising the isolated nucleic acid molecule of claim 27, wherein said vector further comprises an expression regulator. A host cell comprising a nucleotide sequence encoding an antibody or antigen-binding fragment according to any one of claims 1 to 7. A method for producing an antibody or an antigen-binding fragment thereof, comprising providing a host cell according to claim 29, culturing the host cell under conditions suitable for expression of the antibody or antigen-binding fragment, and isolating the produced antibody or antigen-binding fragment. A composition for detecting LAG-3 comprising the antibody of claim 1 or an antigen-binding fragment thereof or the nucleic acid molecule of claim 27. A kit for diagnosing cancer or infection comprising the antibody or antigen-binding fragment thereof of claim 1 or the nucleic acid molecule of claim 27. A method for inhibiting or reducing binding between LAG-3 and B7H3 by binding an anti-LAG-3 antibody to LAG-3.