Combined preparations for treating cancer or infection

Abstract

To provide combined preparations for treating cancers or infections.SOLUTION: Disclosed herein are a combined preparation and a pharmaceutical composition, both comprise: (a) an LAG-3 protein or derivative thereof that can bind to MHC class II molecule; and (b) a programmed cell death protein-1 (PD-1) pathway inhibitor. PD-1 pathway inhibitors, e.g., anti-PD-1 antibodies or anti-PD-L1 antibodies and soluble derivatives of LAG-3 that act as an APC activator, together synergistically activate T cells (in particular, CD8+T cells). Also disclosed are use of the combined preparations and compositions as medicaments, in particular use for treating cancer or infection, as well as a method for treating cancer or infections.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to combinatorial preparations and pharmaceutical compositions, and their use as medicaments, in particular for the treatment of cancer or infectious diseases, and to methods for the treatment of cancer or infectious diseases.

Background Art

[0002] Once out of the thymus, naive T cells circulate in the blood through the lymph nodes, searching for foreign (“non-self”) antigens presented by specific antigen-presenting cells (APCs), typically dendritic cells. T cells can recognize not only pathogen-associated antigens but also abnormally expressed self-proteins - those presented by mutated or transformed tumorigenic cells - as “non-self”. When T cells encounter their specific antigens in the context of appropriate co-stimulatory molecules, the cells are activated and upregulate activation and homing molecules. These T cells, named effector T cells, can enter inflamed tissues in search of infected or cancerous cells. Among several functions, effector T cells can produce inflammatory cytokines and / or cytolytic granules and can lead to apoptosis or necrosis of infected or tumor cells.

[0003] Throughout the course of the immune response, local and systemic downregulatory forces minimize damage to healthy cells and tissues. These can include immunosuppressive cytokines, regulatory T cells (Tregs) and negative signaling from other cells. Tumor antigen-specific T cells exhibit an exhausted phenotype characterized by impaired effector function, as well as reduced production of pro-inflammatory cytokines and hyporesponsiveness to antigenic restimulation. This is mediated by inhibitory molecules upregulated by extracellular mechanisms, such as regulatory T cells (Tregs), and cell-intrinsic mechanisms, such as exhausted tumor-infiltrating lymphocytes (TILs).

[0004] The immune checkpoint pathway strongly downregulates T cell activation in order to suppress the developing T cell response and reduce the potential for immune attack on normal tissues. However, during tumor formation, cancer cells can exploit these co-inhibitory pathways in order to resist detection by the adaptive immune system or avoid elimination thereby. Programmed cell death protein-1 (PD-1) is an essential checkpoint molecule expressed by T cells as a result of activation. The PD-1 checkpoint pathway is thought to act primarily in peripheral tissues in order to dampen ongoing immune responses and / or prevent damage to self-tissues. PD-1 is expressed by B cells, natural killer (NK) cells, dendritic cells and activated monocytes in addition to T cells. PD-1 ligands - including among others PD-L1 and PD-L2 - are expressed by macrophages and monocytes and can be induced in a number of cell types within the inflammatory milieu.

[0005] As one way to avoid immune attack, the ability of non-immune cells expressing the ligand for PD-1, mainly PD-L1, is exploited by tumors. Tumor cells can also downregulate antigen expression in order to avoid detection. Furthermore, the production of immunosuppressive mediators and the retention of Tregs and immune suppressor cells in the tumor microenvironment can weaken the anti-tumor immune response.

[0006] Figure 1 (Harvey, Clinical Pharmacology & Therapeutics, 2014, 96(2 (taken from pages 214 - 223) represents the role of the PD-1 pathway in tumor immune evasion and the mechanism of action of PD-1 pathway blockade: (a) PD-1 in T cell activation. T cells are activated by (i) the binding of MHC and peptide on APCs to the TCR, followed by (ii) the binding of APC CD80 / 86 to T cell CD28. In cancer patients, tumor cells can also act as APCs. As a result of T cell activation, PD-1 expression is induced; (b) PD-1 in T cell exhaustion. In the context of chronic infection or persistent stimulation, to minimize damage to healthy tissues, PD-L1 signals through T cell PD-1 to "turn off" T cells (blocking activation signaling). Tumor cells can upregulate PD-L1 and "block" T cells that can destroy the cells. (c) Blockade of the PD-1 / PD-L1 signaling pathway enables T cells to maintain their effector functions. In cancer patients, activated tumor-specific T cells can kill tumor cells and secrete cytokines that activate / mobilize other immune cells to participate in the anti-tumor response. PD-L1 can upregulate and "block" T cells that can destroy the cells. (c) Blockade of the PD-1 / PD-L1 signaling pathway enables T cells to maintain their effector functions. In cancer patients, activated tumor-specific T cells can kill tumor cells and secrete cytokines that activate / mobilize other immune cells to participate in the anti-tumor response.

[0007] The cloning of PD-1 was described by Ishida et al. (The EMBO Journal (1992), 11(11), 3887 - 3895). The sequence of human PD-1 cDNA is recorded under GenBank accession number NM_005018. Human PD-L1 The sequence of cDNA is given by GenBank accession number AF233516, and the sequence of human PD-L2 cDNA is given by GenBank accession number NM_025239.

[0008] In September 2014, the US Food and Drug Administration (FDA) granted accelerated approval to Keytruda (pembrolizumab) for the treatment of patients with advanced or metastatic melanoma who are no longer responding to other drugs. Keytruda (Merck & Co.) is a humanized monoclonal IgG4 antibody against PD-1. It contains the variable region sequences of a murine anti-human PD-1 antibody with very high affinity that has been grafted onto human IgG4 immunoglobulin with modifications to increase stability. Keytruda blocks the binding of PD-1 to PD-L1 and PD-L2.

[0009] In December 2014, the US FDA similarly granted accelerated approval to Opdivo (nivolumab), a new treatment for patients with unresectable or metastatic melanoma who are no longer responding to other drugs. Opdivo (Bristol-Myers Squibb) is a fully human monoclonal IgG4 antibody against PD-1 that blocks the binding of PD-1 to PD-L1 and PD-L2.

[0010] Nivolumab has received the most extensive clinical evaluation in lung cancer among PD-1 pathway inhibitors. Evidence of activity has been demonstrated in NSCLC patients both as monotherapy and in combination with conventional chemotherapy in squamous and non-squamous non-small cell lung cancer (NSCLC). Pembrolizumab is being evaluated in ongoing clinical trials in NSCLC patients (NCT01295827). Several other promising agents (PD-1 pathway inhibitors) targeting the PD-1 pathway are in clinical development (see Table 1.1 below):

Table 1.1

[0011] ADCC, antibody-dependent cell-mediated cytotoxicity; IgG, immunoglobulin G; PD-1, programmed death-1; PD-L1, PD ligand 1.

[0012] Additional PD-1 pathway inhibitors in clinical development are avelumab (also known as MSB0010718C), a fully human anti-PD-L1 IgG1 monoclonal antibody, being co-developed by Merck KGaA and Pfizer.

[0013] Despite the recent FDA approvals of Keytruda and Opdivo for the treatment of advanced melanoma and promising results in clinical trials for NSCLC from drugs targeting the PD-1 pathway, there remains a need to provide more effective cancer treatments, treatments that are effective for a broader number of cancer patients, treatments effective for other cancers, and effective cancer treatments with reduced side effects.

[0014] Lymphocyte activation gene 3 (LAG-3) is a CD4 homolog type I membrane protein with four extracellular immunoglobulin superfamily domains. Similar to CD4, LAG-3 oligomerizes on the surface of T cells and binds to MHC class II molecules on antigen-presenting cells (APCs) with a significantly higher affinity than CD4. LAG-3 associates with the activated CD4 + and CD8 + T lymphocytes expressed on the surface and negatively regulates signaling. As a result, it negatively regulates T cell proliferation, function, and homeostasis. LAG-3 is upregulated in exhausted T cells compared to effector or memory T cells. LAG-3 is also upregulated in tumor-infiltrating lymphocytes (TILs), and blockade of LAG-3 using anti-LAG-3 antibodies can enhance anti-tumor T cell responses.

[0015] Blackburn et al. (Nat Immunol. 2009;10(1):29-37) described the co-regulation of CD8 + T cell exhaustion during chronic viral infection by multiple inhibitory receptors. Using a mouse model of chronic lymphocytic choriomeningitis virus (LCMV), the authors showed that exhausted antigen-specific CD8 + T cells are +Demonstrate that the expression of up to seven inhibitory receptors (PD-1, LAG3, 2B4, CD160, CTLA-4, PIR-B, and GP49) was increased compared to T cells. Co-expression of multiple distinct inhibitory receptors was associated with greater T cell exhaustion and more severe infection. Blockade of the T cell inhibitory receptors PD-1 and LAG-3 (using anti-PD-L1 and anti-LAG-3 antibodies) improved the T cell response and reduced the in vivo viral load.

[0016] Woo et al. (Cancer Research 2011; 72(4): 917 - 927) described the co-expression of PD-1 and LAG-3 on tumor-infiltrating CD4 T cells and CD8 + T cells in transplantable tumors. Treatment with a dual anti-LAG-3 / anti-PD-1 antibody cured most mice with established tumors that were highly resistant to single antibody treatment. + Based on the immunomodulatory role of LAG-3 in T cell function in chronic infection and cancer, the predicted mechanism of action of LAG-3-specific monoclonal antibodies is to inhibit the negative regulation of tumor-specific effector T cells.

[0017] LAG-3 also encodes an alternative splice variant that is converted to a soluble form of LAG-3 (sLAG-3). As a soluble molecule, LAG-3 activates antigen-presenting cells (APCs) through MHC class II signaling, resulting in an increase in antigen-specific T cell responses in vivo (Triebel, Trends Immunol., 2003, 24: 619 - 622

[0018] pages).

[0019] ​The major anti-tumor immune responses are mediated through the activation of type 1 cytotoxic (Tc1) CD8 T cells, NK cells and monocytes / macrophages. In short-term ex vivo assays, the soluble form of the LAG-3 protein (IMP321) induces an appropriate cytotoxic response in peripheral blood mononuclear cells (PBMC) (Brignone et al., Journal of Immunology, 2007 year, 179:4202-4211). IMP321 binds to a minority of MHC class II + cells in PBMC including all myeloid dendritic cells and a very small fraction of monocytes. Four hours after the addition of IMP321 to PBMC, these myeloid cells produce TNF-α and CCL4. Eighteen hours later, 1% of CD8 + T cells and 3.7% of NK cells produce Tc1 cytokines such as IFN-α and / or TNF-α. Since IMP321 cannot activate pure sorted CD8 + T cells, initial APC activation by IMP321 is required for this Tc1 type of activation. Only fully differentiated granzyme + CD8 T cells (effector and effector memory T cells but neither naive nor central memory T cells) that have experienced antigen are induced by IMP321 to full Tc1 activation.

[0020] PD-1 pathway inhibitors (anti-PD-1 antibody or anti-PD-L1 antibody) and the soluble derivative of LAG-3 (IMP321) that acts as an APC activator have now been found to synergistically activate T cells (especially CD8 + T cells) together in vitro. This synergistic activation of T cells is surprising. In the dual anti-LAG-3 / anti-PD-1 antibody treatment described by Woo et al. (supra), the anti-LAG-3 antibody is thought to inhibit the negative regulation of tumor-specific effector T cells by LAG-3, while the soluble derivative of LAG-3 (IMP321) is thought to act as an APC activator through a different mechanism.

Prior Art Documents

Non-Patent Literature

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Summary of the Invention

Means for Solving the Problems

[0022] The present invention provides a combined preparation comprising (a) an LAG-3 protein or a derivative thereof capable of binding to an MHC class II molecule; and (b) a PD-1 pathway inhibitor.

[0023] As used herein, the term "combination preparation" refers to a "kit of parts" in the sense that the combination components (a) and (b) defined above can be administered by the use of the distinct amounts of the combination components (a) and (b) either independently or in various fixed combinations. The components may be administered simultaneously or sequentially. When the components are administered sequentially, preferably the time interval between administrations is selected such that the therapeutic effect of the combination is greater than the effect obtained by the use of only any one of the combination components (a) and (b).

[0024] The components of the combination preparation may be present in one combination unit dosage form or as a first unit dosage form of component (a) and a separate second unit dosage form of component (b). The ratio of the total amounts of combination component (a) and combination component (b) administered in the combination preparation can be varied to address the requirements of the subpopulation of patients being treated or the requirements of an individual patient, which may be due to, for example, a particular disease, age, sex or weight of the patient.

[0025] Preferably, there is at least one beneficial effect, for example, enhancement of the effect of a PD-1 pathway inhibitor, or enhancement of the effect of an LAG-3 protein or its derivative, or mutual enhancement of the effects of combination components (a) and (b), such as an effect exceeding an additive effect, an additional advantageous effect, fewer side effects, less toxicity, or a combined therapeutic effect, compared to the effective dosage amount of one or both of combination components (a) and (b), and most preferably there is a synergistic effect between combination components (a) and (b).

[0026] The combination preparation of the present invention can be provided as a pharmaceutical combination preparation for administration to a mammal, preferably a human. The LAG-3 protein or its derivative may optionally be provided together with a pharmaceutically acceptable carrier, excipient or diluent, and / or the PD-1 pathway inhibitor may optionally be provided together with a pharmaceutically acceptable carrier, excipient or diluent.

[0027] LAG-3 or a derivative thereof may be present in an amount that is a molar equivalent of 0.25 to 30 mg, 1 to 30 mg, or 6 to 30 mg of the LAG-3 derivative LAG-3Ig fusion protein IMP321. A dose of 6 to 30 mg per subcutaneous (s.c.) injection of IMP321 has been shown to be safe and provides an acceptable systemic exposure based on the results of pharmacokinetic data obtained in patients with metastatic renal cell carcinoma. Blood concentrations of IMP321 exceeding 1 ng / ml for at least 24 hours after s.c. injection are obtained in patients injected with IMP321 doses exceeding 6 mg.

[0028] The combination preparation of the present invention may contain multiple doses of the LAG-3 protein or a derivative thereof.

[0029] The PD-1 pathway inhibitor may be an agent that inhibits the binding of PD-1 to PD-L1 and / or PD-L2. In particular, the agent can inhibit the binding of human PD-1 to human PD-L1 and / or human PD-L2. The agent can inhibit the binding of PD-1 to PD-L1 and / or PD-L2 by at least 50%, 60%, 70%, 80% or 90%. Assays suitable for determining the binding of PD-1 to PD-L1 or PD-L2 by surface plasmon resonance (SPR) analysis or flow cytometry analysis are described by Ghiotto et al. (Int. Immunol. August 2010; 22(8): 651-660 pages). The agent can inhibit the binding of PD-1 to PD-L1 and / or PD-L2, for example, by binding to PD-1, PD-L1 or PD-L2. The agent may be an antibody, preferably a monoclonal antibody, such as a human or humanized monoclonal antibody. The agent may also be a fragment or derivative of an antibody that retains the ability to inhibit the binding of PD-1 to PD-L1 and / or PD-L2.

[0030] Examples of anti-PD-1 antibodies suitable for use according to the present invention include the following: pembrolizumab (MK-3475), a humanized monoclonal IgG4 antibody; nivolumab, a fully human monoclonal IgG4 antibody; pidilizumab (CT-011), a humanized IgG1 monoclonal antibody. An example of a PD-1 pathway inhibitor that binds to PD-1 but is not an antibody is AMP-224. AMP-224 is a recombinant fusion protein of the extracellular domain of PD-L2 and the Fc region of human IgG. AMP-224 causes the elimination of PD-1 highly expressing T cells. Examples of anti-PD-L1 antibodies suitable for use according to the present invention include the following: BMS-936559, a fully human IgG4 monoclonal antibody; MEDI4736 (durvalumab), a fully human monoclonal antibody; MPDL3280A, a human monoclonal antibody containing an engineered IgG Fc domain to prevent ADCC; avelumab (also known as MSB0010718C), a fully human anti-PD-L1 IgG1 monoclonal antibody.

[0031] The dosage of the PD-1 pathway inhibitor depends on the particular PD-1 pathway inhibitor used. Generally, a typically prescribed dosage of a PD-1 pathway inhibitor for a human subject may be 0.1 to 10 mg / kg, for example 0.1 to 1 mg / kg or 1 to 10 mg / kg. The term "typically prescribed dosage" as used herein includes a dosage that is safe and therapeutically effective for administration to a subject (preferably a human subject) as monotherapy, or a dosage that is the same as or within the dosage range of a dosage approved by the regulatory agency in charge for administration to a subject (preferably a human subject) as monotherapy. Examples of typically prescribed dosages for humans of known PD-1 pathway inhibitors when used as monotherapy include the following:

[0032] Pembrolizumab (MK-3475): 2 to 10 mg / kg every 2 or 3 weeks. For example, the US FDA has approved the administration of 2 mg / kg of Keytruda (pembrolizumab) as a 30-minute intravenous infusion every 3 weeks; Nivolumab: 0.1 to 10 mg / kg every two weeks. For example, the US FDA has approved the administration of 3 mg / kg of Opdivo (nivolumab) as a 60-minute intravenous infusion every two weeks; BMS-936559: 0.3 to 10 mg / kg every two weeks.

[0033] PD-1 pathway inhibitors can be administered by any suitable route, for example parenterally (including subcutaneous, intravenous or intramuscular injection). Currently approved or in development PD-1 pathway inhibitors are administered as intravenous infusions.

[0034] The combination preparations of the present invention may comprise multiple doses of a PD-1 pathway inhibitor.

[0035] The LAG-3 protein may be an isolated native or recombinant LAG-3 protein. The LAG-3 protein may comprise the amino acid sequence of a LAG-3 protein from any suitable species such as a primate or mouse LAG-3 protein, but is preferably the amino acid sequence of a human LAG-3 protein. The amino acid sequences of human and mouse LAG-3 proteins are provided in FIG. 1 of Huard et al. (Proc. Natl. Acad. Sci. USA, Vol. 11: pp. 5744-5749, 1997). The sequence of the human LAG-3 protein is repeated in FIG. 15 below (SEQ ID NO: 1). The amino acid sequences of the four extracellular Ig superfamily domains (D1, D2, D3 and D4) of human LAG-3 are also the amino acid residues: 1 to 149 (D1); 150 to 239 (D2); 240 to 330 (D3); and 331 to 412 (D4) identified in FIG. 1 of Huard et al.

[0036] Derivatives of the LAG-3 protein include soluble fragments, variants or mutants of the LAG-3 protein that can bind to MHC class II molecules. Some derivatives of the LAG-3 protein are known to be able to bind to MHC class II molecules. Numerous examples of such derivatives are described by Huard et al. (Proc. Natl. Acad. Sci. USA, Vol. 11: pp. 5744-5749, 1997). This document describes the characterization of the MHC class II binding site on the LAG-3 protein. Methods for making mutants of LAG-3 and a quantitative cell adhesion assay for determining the ability of LAG-3 mutants to bind to class II positive Daudi cells are described. The binding of some different mutants of LAG-3 to MHC class II molecules was determined. Some mutations were able to reduce class II binding, while other mutations increased the affinity of LAG-3 for class II molecules. Many of the residues essential for binding to the MHC class II protein are clustered at the base of a large 30 amino acid extra-loop structure in the LAG-3 D1 domain. The amino acid sequence of the extra-loop structure of the D1 domain of the human LAG-3 protein is GPPAAAPGHPLAPGPHPAAPSSWGPRPRRY (SEQ ID NO: 2), the underlined sequence in Figure 15.

[0037] The LAG-3 protein derivative may include the 30 amino acid extra-loop sequence of the human LAG-3 D1 domain, or a variant of such a sequence containing one or more conservative amino acid substitutions. The variant may include an amino acid sequence having at least 70%, 80%, 90% or 95% amino acid identity with the 30 amino acid extra-loop sequence of the human LAG-3 D1 domain.

[0038] The derivative of the LAG-3 protein may include the amino acid sequences of the LAG-3 protein, preferably the D1 domain and optionally the D2 domain of the human LAG-3 protein.

[0039] Derivatives of the LAG-3 protein may comprise an amino acid sequence having at least 70%, 80%, 90% or 95% amino acid identity with the LAG-3 protein, preferably domain D1 of the human LAG-3 protein, and / or domains D1 and D2.

[0040] Derivatives of the LAG-3 protein may comprise the amino acid sequences of domains D1, D2, D3 and optionally D4 of the LAG-3 protein, preferably the human LAG-3 protein.

[0041] Derivatives of the LAG-3 protein may comprise an amino acid sequence having at least 70%, 80%, 90% or 95% amino acid identity with the LAG-3 protein, preferably domains D1, D2 and D3 of human LAG-3, and / or domains D1, D2, D3 and D4.

[0042] Sequence identity between amino acid sequences can be determined by comparing the sequence alignments. When corresponding positions in the sequences being compared are occupied by the same amino acid, the molecules are thereby identical at that position. Scoring of the alignment as a percentage of identity is a function of the number of identical amino acids at positions shared by the sequences being compared. When comparing sequences, the optimal alignment may require gaps to be introduced into one or more of the sequences to account for possible insertions and deletions in the sequences. Sequence comparison methods may use gap penalties for the same number of identical molecules in the sequences being compared, and a sequence alignment containing as few gaps as possible reflects a higher relatedness between the two sequences being compared and achieves a higher score than one containing a large number of gaps. Calculation of the maximum percent identity involves creating an optimal alignment that takes into account gap penalties.

[0043] Computer programs suitable for performing array comparisons are widely available commercially and in the public sector. Examples are MatGat (Campanella et al., 2003, BMC Bioinformatics 4:29; the program is available from http: / / bitincka.com / ledion / matgat), Gap (Needleman and Wunsch, 1970, J. Mol. Biol. 48:44 3-453), FASTA (Altschul et al., 1990, J. Mol. Biol. 215:403-410; the program is available from http: / / www.ebi.ac.uk / fasta), Clust al W 2.0 and X 2.0 (Larkin et al., 2007, Bioinformatics 23: 2947-2948; the program is available from http: / / www.ebi.ac.uk / tools / clustalw2 if possible) and EMBOSS Pairwise Alignment Algorithms (Needleman and Wunsch, 1970, supra; Kruskal, 1983, Time warps, string edits and macromolecules: the theory and practice of sequence comparison, Sankoff and Kruskal (eds.), 1-44, Addison Wesley; the program is available from http: / / www.ebi.ac.uk / tools / emboss / align). All programs can be run using the initial parameters.

[0044] For example, sequence comparison may be performed using the "needle" method of the EMBOSS Pairwise Alignment Algorithms, which determines the optimal alignment (including gaps) of two sequences when providing a percentage identity score considering their entire lengths. Initial parameters (the "protein molecule" option) for amino acid sequence comparison may be: gap extension penalty: 0.5, gap opening penalty: 10.0, matrix: Blosum 62.

[0045] Sequence comparison may be performed over the entire length of the reference sequence.

[0046] The LAG-3 protein derivative may be fused to an immunoglobulin Fc amino acid sequence, preferably a human IgG1 Fc amino acid sequence, optionally by a linker amino acid sequence.

[0047] The ability of a derivative of the LAG-3 protein to bind to MHC class II molecules can be determined using the quantitative cell adhesion assay described by Huard et al. ( supra). The affinity of a derivative of the LAG-3 protein for MHC class II molecules may be at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the affinity of the human LAG-3 protein for class II molecules. Preferably, the affinity of a derivative of the LAG-3 protein for MHC class II molecules is at least 50% of the affinity of the human LAG-3 protein for class II molecules. Examples of suitable derivatives of the LAG-3 protein that can bind to MHC class II molecules are: amino acid residues 23 to 448 of the human LAG-3 sequence; the amino acid sequences of domains D1 and D2 of LAG-3; The following positions: position 73 where ARG is replaced by GLU; position 75 where ARG is replaced by ALA or GLU; position 76 where ARG is replaced by GLU; position 30 where ASP is replaced by ALA; position 56 where HIS is replaced by ALA; position 77 where TYR is replaced by PHE; position 88 where ARG is replaced by ALA; position 103 where ARG is replaced by ALA; position 109 where ASP is replaced by GLU; position 115 where ARG is replaced by ALA, the amino acid sequences of domains D1 and D2 of LAG-3 having amino acid substitutions at one or more of these positions; The amino acid sequence of domain D1 of LAG-3 having a deletion of amino acid residues 54 to 66; Recombinant soluble human LAG-3Ig fusion protein (IMP321) - 200 kDa dimer produced by Chinese hamster ovary cells transfected with a plasmid encoding the extracellular domain of hLAG-3 fused to human IgG1 Fc Derivatives including the same are included. The sequence of IMP321 is given by SEQ ID NO: 17 in US Patent Application Publication No. 2011 / 0008331.

[0048] The present invention also provides a pharmaceutical composition comprising (a) an LAG-3 protein, or a derivative thereof capable of binding to an MHC class II molecule; (b) a PD-1 pathway inhibitor; and (c) a pharmaceutically acceptable carrier, excipient or diluent.

[0049] The present invention further provides a combination preparation or pharmaceutical composition of the present invention for use as a medicament.

[0050] The present invention also provides a combination preparation or pharmaceutical composition of the present invention for preventing, treating or recovering from cancer.

[0051] The present invention further provides the use of a combination preparation or pharmaceutical composition of the present invention in the manufacture of a medicament for preventing, treating or recovering from cancer.

[0052] A method for preventing, treating or restoring cancer according to the present invention, comprising administering to a subject in need of such prevention, treatment or restoration a LAG-3 protein or a derivative thereof capable of binding to an MHC class II molecule and an anti-PD-1 pathway inhibitor is also provided.

[0053] The inventors have fully understood that the combination preparations and compositions of the present invention can also be used for the prevention, treatment or restoration of infections, particularly chronic or persistent infections.

[0054] During acute infection, activated pathogen-specific cytotoxic CD8 T lymphocytes (CTLs) proliferate and acquire effector functions, such as cytokine production and cytotoxic ability, that enable them to effectively eliminate the infection. After elimination, a small pool of pathogen-specific memory T cells remains, which have the ability to reactivate very rapidly after re-exposure to the same pathogen and acquire their killing functions. However, during chronic infection, this does not occur as pathogen-specific CTLs have been found to have functional defects and are unable to eliminate the infection. These exhausted CTLs are defined by their impaired proliferative capacity, loss of cytokine production and cytotoxic ability (see Figure 1(b) and review by Hofmeyer et al., Journal of Biomedicine and Biotechnology, 2011, article ID 451694).

[0055] This phenomenon was originally defined using a well-established mouse model of chronic viral infection of mice with lymphocytic choriomeningitis virus (LCMV) (Zajac et al., The Journal of Experimental Medicine, Vol. 188, No. 12, pp. 2205-2213, 1998; Gallimore et al., The Journal of Experimental Medicine, Vol. 187, No. 9, pp. 1383-1393, 1998). The Armstrong strain of LCMV causes an acute infection, which is cleared by the immune system to generate robust CTL memory. In contrast, the Clone 13 strain of LCMV establishes a chronic infection in mice, exhausting CTLs and preventing the clearance of infection. Furthermore, exhausted CTLs have metabolic defects, as well as changes in the expression of genes involved in chemotaxis, adhesion, and migration, compared to normal T cells (Wherry et al., Immunity, Vol. 27, No. 4, pp. 670-684, 2007).

[0056] In a study conducted to clarify the mechanism leading to exhaustion, the gene profile of exhausted CTLs from chronic LMCV infection was compared with the gene profile of functional CTLs responding to acute LCMV infection (Barber et al., Nature, Vol. 439, No. 7077, pp. 682-687, 2006). Exhausted CTLs were found to have significant overexpression of PD-1, while functional LCMV-specific CTLs had no detectable expression of PD-1. Expression of PD-1 was found to correlate with the defined functional impairment seen in exhausted T cells and, in turn, with a higher viral load. Blockade of the PD-1 / PD-L1 pathway with anti-PD-L1 antibody in chronically infected mice led to an enhanced CTL response that caused a decrease in viral load. Since the loss of presentation of specific epitopes during chronic infection results in functional restoration and a decrease in PD-1 expression on epitope-specific CTLs, PD-1 expression by exhausted CTLs depends on persistent antigen-specific stimulation (Blattman et al., Journal of Virology, Vol. 83, No. 9, pp. 4386-4394, 2009). Persistence during chronic viral infection Antigenic stimulation has a progressive effect on the loss of CTL function and the correlated increase in PD-1 expression, which means that more exhausted CTLs (PD-1 hi ) are less sensitive to functional rescue by PD-1 blocking than others (PD-1 int ) (Blackburn et al., Proceedings of the National Academy of Sciences of the United States of America, Vol. 105, No. 39, pp. 15016-15021, 2008).

[0057] The present invention further provides a combined preparation or pharmaceutical composition of the present invention for use in the prevention, treatment or recovery from an infection.

[0058] The present invention also provides the use of the combined preparation or pharmaceutical composition of the present invention in the manufacture of a medicament for the prevention, treatment or recovery from an infection.

[0059] The present invention also provides a method for preventing, treating or recovering from an infection, the method comprising administering to a subject in need of such prevention, treatment or recovery an LAG-3 protein, or a derivative thereof capable of binding to an MHC class II molecule, and a PD-1 pathway inhibitor.

[0060] In certain embodiments, the infection is a chronic or persistent infection. As used herein, the term "chronic or persistent infection" is used to refer to an infection by a pathogen that has induced a classical CTL response in an infected subject, but the infection has not been eliminated and results in the presence of exhausted pathogen-specific CTLs expressing PD-1, accompanied by a loss of impaired proliferative capacity, cytokine production and cytotoxic ability.

[0061] Examples of infectious diseases that can be treated by the present invention include viral, bacterial, fungal or protozoal infectious diseases, particularly chronic or persistent viral, bacterial, fungal or protozoal infectious diseases.

[0062] Viral infections can be caused by, for example, adenovirus, adeno-associated virus, B virus (Macacine herpesvirus I), BK virus, bunyavirus, chikungunya virus, coxsackievirus, coronavirus, cytomegalovirus, eastern equine encephalitis virus, Ebola virus, enterovirus, Epstein-Barr virus, hantavirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, herpesvirus, herpes simplex virus 1, herpes simplex virus 2, human foamy virus, human herpesvirus 3, human herpesvirus 5, human herpesvirus 6, human herpesvirus 7, human immunodeficiency virus, human papillomavirus, human beta-lymphotropic virus, human T-cell leukemia virus I, human T-cell leukemia virus II, influenza virus, JC virus, JEV, Kaposi's sarcoma-associated herpesvirus, Lassa virus, lymphocytic choriomeningitis virus, Marburg virus, measles virus, mumps virus, Nipah virus, norovirus, Norwalk virus, orthoreovirus, parainfluenza virus, parvovirus, poliovirus, rabies virus, reovirus, respiratory syncytial virus, rhinovirus, Rift Valley fever virus, rotavirus, rubella virus, smallpox virus, St. Louis encephalitis virus, variola major virus, variola minor virus, varicella-zoster virus, West Nile virus, western equine encephalitis virus or yellow fever virus. In certain embodiments, the viral infection is caused by a hepatitis virus (e.g., hepatitis B virus, hepatitis C virus), a lentivirus (e.g., human immunodeficiency virus) or a herpesvirus (e.g., herpes simplex virus 1, herpes simplex virus 2).

[0063]

[0064] ​Bacterial infections can be caused by, for example, Escherichia coli, Clostridium difficile, Salmonella thyphimurium, Pseudomonas aeruginosa, Vibrio cholerae, Neisseria gonorrhoeae, Helicobacter pylori, Hemophilus influenzae, Shigella dysenteriae, Staphylococcus aureus, Mycobacterium tuberculosis, Streptococcus pneumonia or Chlamydia trachomatis.

[0065] Fungal infections can be caused by, for example, Candida, Aspergillus, Cryptococcus, Coccidioides, Histoplasma, Pneumocystis or Stachybotrys.

[0066] Protozoal infections can be caused by, for example, Amoebozoa, Excavata, Chromalveolata, Entamoeba, Plasmodium, Giardia, Trypanosoma, Coccidia, Besnoitia, Dicrocoelium or Leishmania.

[0067] The present invention further provides a combination preparation or pharmaceutical composition of the present invention for use in the prevention, treatment or recovery of a disease, disorder or condition that can be prevented, treated or recovered by activation of T cells, particularly by activation of CD8-positive T cells.

[0068] The present invention also provides the use of a combination preparation or pharmaceutical composition of the present invention in the manufacture of a medicament for the prevention, treatment or recovery of a disease, disorder or condition that can be prevented, treated or recovered by activation of T cells, particularly by activation of CD8-positive T cells.

[0069] A method for preventing, treating, or restoring a disease, disorder, or condition that can be prevented, treated, or restored by activation of T cells, particularly activation of CD8-positive T cells, the method comprising administering to a subject in need of such prevention, treatment, or restoration an LAG-3 protein, or a derivative thereof that can bind to an MHC class II molecule, and a PD-1 pathway inhibitor is also provided.

[0070] In some embodiments, the disease, disorder, or condition that can be prevented, treated, or restored by activation of T cells can exclude cancer.

[0071] The present invention also provides a combination preparation or pharmaceutical composition of the present invention for use in enhancing a T cell-mediated immune response, particularly a CD8-positive T cell-mediated immune response.

[0072] The present invention also provides the use of the combination preparation or pharmaceutical composition of the present invention in the manufacture of a medicament for enhancing a T cell-mediated immune response, particularly a CD8-positive T cell-mediated immune response.

[0073] The present invention further provides a method for enhancing a T cell-mediated immune response, particularly a CD8-positive T cell-mediated immune response, the method comprising administering to a subject in need of such enhanced T cell-mediated immune response an LAG-3 protein, or a derivative thereof that can bind to an MHC class II molecule, and a PD-1 pathway inhibitor.

[0074] In some embodiments, enhancement of the T cell-mediated immune response or the CD8-positive T cell-mediated immune response can exclude prevention, treatment, or restoration of cancer.

[0075] The LAG-3 protein or its derivative, and the PD-1 pathway inhibitor can be administered to the subject sequentially, i.e., the LAG-3 protein or its derivative can be administered before, together with, or after the PD-1 pathway inhibitor.

[0076] The LAG-3 protein or a derivative thereof and the PD-1 pathway inhibitor may be administered to a subject within 96 hours, 72 hours, 48 hours, 24 hours, or 12 hours of each other.

[0077] Alternatively, the LAG-3 protein or a derivative thereof and the PD-1 pathway inhibitor may be co-administered to a subject, for example, as a composition comprising the LAG-3 protein or a derivative thereof and the PD-1 pathway inhibitor, or by simultaneous administration of separate doses of the LAG-3 protein or a derivative thereof and the PD-1 pathway inhibitor.

[0078] According to some embodiments, multiple doses of the LAG-3 protein or a derivative thereof and / or multiple doses of the PD-1 pathway inhibitor are administered to the subject.

[0079] According to some embodiments, the dose of the LAG-3 protein or a derivative thereof is administered before, together with, or after each administration of 2 or more doses of the PD-1 pathway inhibitor.

[0080] For example, the dose of the LAG-3 protein or a derivative thereof may be administered within 96 hours, 72 hours, 48 hours, 24 hours, or 12 hours of each administration of 2 or more doses of the PD-1 pathway inhibitor.

[0081] The selection of appropriate dosages of the components used in the combination therapy according to the present invention can be determined and optimized by one of ordinary skill in the art, for example, based on the overall health of the patient and the patient's findings including response to the combination therapy. Optimization may be necessary, for example, if it is determined that the patient is not showing the desired therapeutic effect, or conversely, if the patient is experiencing undesirable or harmful side effects that are excessive in number or of a severity that is troublesome.

[0082] The dosages of the components used in the combination therapy according to the present invention should be selected to provide a therapeutically effective amount of the components in the combination.

[0083] The "effective amount" of the combination therapy can be an amount that results in a reduction of at least one pathological parameter related to cancer. For example, in some embodiments, the effective amount of the combination therapy is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% reduction in the pathological parameter compared to the reduction expected in the parameter related to cancer without the combination therapy, and is an amount effective to achieve such reduction. For example, the pathological parameter may be tumor growth or tumor growth rate.

[0084] Alternatively, the "effective amount" of the combination therapy may be an amount that results in an increase in the clinical benefit associated with cancer treatment. For example, in some embodiments, the "effective amount" of the combination therapy is an amount effective to achieve at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% increase in the clinical benefit compared to the clinical benefit expected without the combination therapy. For example, the clinical benefit may be tumor response rate, progression-free survival, overall survival or sensitization to subsequent treatment.

[0085] Alternatively, the "effective amount" of the combination therapy may be an amount that results in a change in at least one beneficial parameter associated with cancer treatment. For example, in some embodiments, the "effective amount" of the combination therapy is an amount effective to achieve at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% change in the parameter compared to the expected change in the parameter related to cancer treatment without the combination therapy. For example, the parameter may be an increase in the number of circulating tumor antigen-specific CD8 + T cells, or a decrease in the number of tumor antigen-specific regulatory T cells, or an increase in the number of activated T cells, particularly activated CD8 + T cells, a decrease in the number of exhausted antigen-specific CD8 + T cells, or an increase in the number of circulating functional (i.e., non-exhausted) antigen-specific CD8 + T cells.

[0086] In embodiments related to the treatment of an infection, the "effective amount" of the combination therapy may be an amount that results in a reduction of at least one pathological parameter related to the infection. For example, in some embodiments, the effective amount of the combination therapy is an amount effective to achieve at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% reduction in a pathological parameter as compared to the expected reduction of the parameter related to the infection without the combination therapy. For example, the pathological parameter may be viral load (e.g., the number of viral particles or the amount of viral DNA per 1 ml of blood) or bacterial load (e.g., the amount of bacterial DNA per 1 ml of blood or the number of bacterial colonies after a 1 - 21 day growth period on different agar plates).

[0087] Suitable methods for measuring viral and bacterial loads are well known to those of skill in the art. For example, a method for measuring viral load by ELISA has been compared by Goldschmidt et al. (Clinical and Diagnostic Laboratory Immunology, July 1998, pages 513 - 518). Methods for measuring viral load using different commercially available assays for the detection of viral nucleic acids have been compared by Holguin et al. (Eur J Clin Microbiol Infect Dis. April 1999; 18(4):256 - 9) and Swenson et al. (J. Clin. Microbiol. February 2014; 52(2):517 - 523). An example of a paper describing the measurement of bacterial load by real - time PCR is Nadkarni et al. (Microbiology (2002), 148, 257 - 266). This paper cites Bergey's Manual of Determinative Bacteriology, which has now been replaced by Bergey's Manual of Systematic Bacteriology, 2nd Edition. A molecular bacterial load assay has been described by Honeyborne et al. (J. Clin. Microbiol. 2011, 49:3905 - 3911 and J. Clin. Microbiol. August 2014; 52 It is described in Volume (Issue 8): 3064 - 3067. A list of FDA - approved screening assays for measuring viral load and bacterial load can be found on the following FDA website:

[0088] www.fda.gov / BiologicsBloodVaccines / BloodBloodProducts / ApprovedProducts / LicensedProductsBLAs / BloodDonorScreening / InfectiousDisease / ucm080466.htm.

[0089] Alternatively, an "effective amount" of the combination therapy may be an amount that results in an increase in the clinical benefit associated with the treatment of the infection. For example, in some embodiments, an "effective amount" of the combination therapy is an amount effective to achieve at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% increase in clinical benefit as compared to the clinical benefit expected without the combination therapy.

[0090] Alternatively, an "effective amount" of the combination therapy may be an amount that results in a change in at least one beneficial parameter associated with the treatment of the infection. For example, in some embodiments, an "effective amount" of the combination therapy is an amount effective to achieve at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% change in the parameter as compared to the expected change in the parameter associated with treatment without the combination therapy. For example, the parameter may be an increase in the number of activated T cells, particularly activated CD8 + T cells, an increase in the number of circulating functional (i.e., non - exhausted) antigen - specific CD8 + T cells, or a decrease in the number of exhausted antigen - specific CD8 + T cells or a decrease in the number of antigen - specific regulatory T cells.

[0091] According to the present invention, combination therapy can be used to increase the therapeutic effect of a PD-1 pathway inhibitor or an LAG-3 protein or its derivative as compared to the effect of the PD-1 pathway inhibitor or the LAG-3 protein or its derivative as a monotherapy, or to prevent or further reduce the risk of unwanted or harmful side effects of the individual components, and at the same time to reduce the dose of the individual components in the resulting combination.

[0092] In one embodiment, the LAG-3 protein or its derivative and the PD-1 pathway inhibitor are each defined at a dose within the typical defined dose range for each compound as a monotherapy. The compounds may be defined as separate dosages or as a combined dosage. Such a combination provides an increase in efficacy as compared to the effect of either compound as a monotherapy.

[0093] In another embodiment, the LAG-3 protein or its derivative and the PD-1 pathway inhibitor are each defined at a dose lower than the typical defined dose for each component as a monotherapy, but at a dose that is therapeutically effective in combination. The components may be defined as separate dosages or as a combined dosage. The dosages of the components in the combination provide a level of therapeutic efficacy similar to that of the LAG-3 protein or its derivative or the PD-1 pathway inhibitor as a monotherapy, but the lower dosages of the LAG-3 protein or its derivative and the PD-1 pathway inhibitor may be selected to have the advantage of reducing the risk of harmful side effects as compared to the defined dosage of each compound as a monotherapy.

[0094] In another embodiment, the defined dosage of the PD-1 pathway inhibitor is within the typical defined dose range for monotherapy, and the LAG-3 protein or its derivative is defined at a dosage lower than the typical defined dose for monotherapy.

[0095] In a further embodiment, the defined dosage of the PD-1 pathway inhibitor is lower than the typical defined dosage for monotherapy, and the LAG-3 protein or its derivative is defined at a dosage within the typical defined dosage range for monotherapy.

[0096] A preferred dosage lower than the typical defined dosage for monotherapy is a dosage that is up to 50% or up to 25% of the typical defined dosage. For example, a dosage less than the dosage typically prescribed for monotherapy may be a dosage of 1-50%, 1-25%, 1-10%, 2-50%, 2-25%, 2-10% of the typically prescribed dosage of the PD-1 pathway inhibitor and / or the LAG-3 protein or its derivative.

[0097] The typically prescribed dosage of the LAG-3 protein or its derivative for monotherapy in a human subject may be a dosage that is the molar equivalent of 0.25-30 mg, 1-30 mg or 6-30 mg of the LAG-3 derivative LAG-3Ig fusion protein IMP321.

[0098] The typically prescribed dosage of the PD-1 pathway inhibitor for monotherapy in a human subject may be 0.1-10 mg / kg, 0.1-1 mg / kg or 1-10 mg / kg. For example, the typically prescribed dosage of pembrolizumab for monotherapy in a human subject may be 2-10 mg / kg, such as 2 mg / kg, the typically prescribed dosage of nivolumab for monotherapy in a human subject may be 0.1-10 mg / kg, such as 3 mg / kg, and the typically prescribed dosage of BMS-936559 for monotherapy in a human subject may be 0.3-10 mg / kg.

[0099] In certain embodiments of the combination preparation or composition of the present invention, the prescribed dosage of the PD-1 pathway inhibitor is less than the dosage typically prescribed for monotherapy, for example, 1 to 50%, 1 to 25%, 1 to 20%, 1 to 10%, 2 to 50%, 2 to 25%, 2 to 20%, 2 to 10%, 0.1 to 50%, 0.1 to 25%, 0.1 to 20%, 0.1 to 10%, less than 20%, less than 10%, less than 0.1 to 20%, less than 0.1 to 10%, less than 0.01 to 20% or less than 0.01 to 10% of the dosage typically prescribed for the PD-1 pathway inhibitor. Examples of suitable dosages of the PD-1 pathway inhibitor and the LAG-3 protein or its derivative according to the present invention are shown in Table 1.2 below:

Table 1.2-1

Table 1.2-2

Table 1.2-3

Table 1.2-4

Table 1.2-5

[0100] The LAG-3 derivative may be any of the LAG-3 derivatives described above or shown in Figure 7. In certain embodiments, the LAG-3 derivative is IMP321.

[0101] When administered at separate dosages, the LAG-3 protein or its derivative and the PD-1 pathway inhibitor may be administered substantially simultaneously (e.g., within about 60 minutes, about 50 minutes, about 40 minutes, about 30 minutes, about 20 minutes, about 10 minutes, about 5 minutes or about 1 minute of each other), or about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, about 72 hours or about 96 hours or more apart in time.

[0102] One of ordinary skill in the art can determine and optimize the time course suitable for sequential administration according to a particular combination of an LAG-3 protein or its derivative and a PD-1 pathway inhibitor. The selection of the time course is preferably such that at least one beneficial effect is achieved, such as enhancement of the effect of the LAG-3 protein or its derivative or the PD-1 pathway inhibitor, or mutual enhancement of the effects of the combination components, such as an effect exceeding an additive effect, an additional advantageous effect, fewer side effects, less toxicity, or a combined therapeutic effect as compared to ineffective dosages of one or both of the combination components, and most preferably such that synergy of the combination components is observed.

[0103] It is understood that the optimal time course depends on factors such as the time it takes to reach the peak plasma concentration of the compound after administration and the elimination half-life of each compound. Preferably, the difference in time is less than the half-life of the first component administered.

[0104] One of ordinary skill in the art can also determine the appropriate timing for administration. In certain embodiments, the PD-1 pathway inhibitor may be administered in the morning, and the LAG-3 protein or its derivative may be administered at least once during the day. In other embodiments, the PD-1 pathway inhibitor and the LAG-3 protein or its derivative may be administered substantially at the same time.

[0105] In some embodiments, the PD-1 pathway inhibitor may be administered to the subject, for example, by a physician, and the subject may be provided with a dosage of the LAG-3 protein or its derivative, for example, in a pre-filled syringe, for administration later (e.g., during the day or the next day).

[0106] The PD-1 pathway inhibitor and the LAG-3 protein or its derivative can be administered daily, weekly, every two weeks, every three weeks, monthly, every two months, every three months, every four months, every five months, every six months, every seven months, every eight months, every nine months, every ten months, every eleven months, every year, every two years, every three years, every four years, every five years or more frequently.

[0107] Subjects may receive doses of a PD-1 pathway inhibitor and an LAG-3 protein or derivative thereof over a period of weeks, months or years. For example, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years or more.

[0108] The subject may be a mammalian subject, preferably a human subject.

[0109] Cancers that can be treated by the present invention include cancers in which cancer tumor cells express PD-L1 and / or PD-L2 (i.e., cancers that are PD-L1 and / or PD-L2 positive).

[0110] PD-L1 expression was detected in lung cancer, ovarian cancer, renal cancer and colon cancer as well as malignant melanoma, but not in normal tissues including lung, uterus, kidney, colon or skin (Benson et al., Blood 116, pp. 2286-2294 (2010); Blank et al., Int. J. Cancer 119, pp. 317-327 (2006); Dong et al., Nat. Med. 8, pp. 793-800 (2002)). PD-L1 expression by tumor cells is associated with a worse prognosis in breast cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma, malignant melanoma, ovarian cancer, pancreatic cancer, renal cell carcinoma and urothelial carcinoma (Zou & Chen, Nat. Rev. Immunol. 8, pp. 467-477 (2008)).

[0111] There is similarly evidence that human tumors can express PD-L2 (Rozali et al., Clin. Dev. Immunol. 2012, 656340 (2012); Karim et al., Clin. Cancer Res. 15, pp. 6341-6347 (2009)). Non-small cell lung cancer-(NSCL C-) -associated fibroblasts constitutively express PD-L1 and PD-L2. A decreased survival in patients with esophageal cancer, ovarian cancer or hepatocellular cancer that is PD-L2 positive (versus PD-L2 negative) has also been described.

[0112] The cancers that can be treated by the present invention include tumor-infiltrating lymphocytes (TILs), particularly CD8 + cancers in which TILs express PD-1, or cancers in which TILs express higher levels of PD-1 than circulating lymphocytes.

[0113] In both NSCLC and melanoma patients, higher levels of PD-1 were observed in TILs than in circulating lymphocytes (Blank et al., Int. J. Cancer 119, pp. 317 - 327 (2006 year); Zhang et al., Cell. Mol. Immunol. 7, pp. 389 - 395 (2010)). In the peripheral blood of vaccinated melanoma patients, both melanoma antigen-specific cytotoxic lymphocytes and Tregs expressed PD-1 (Wang et al., Int. Immunol. 21, pp. 1065 - 1077 (2009)). In esophageal cancer, there was also a negative correlation between tumor PD-L2 expression and the presence of CD8 + TILs (Rozali et al., Clin. Dev. Immunol. 2012, 65 6340 (2012)).

[0114] CD8 + TILs isolated from NSCLC had increased expression of PD-1 and impaired functional responses (in vitro proliferation and inflammatory cytokine production) compared to circulating CD8 + T cells or CD8 + T cells from healthy volunteers. The addition of anti-PD-L1 antibody restored the ability of CD8 +The ability of TILs was significantly improved (Zhang et al., Cell. Mol. Immunol. 7, 389-395 (2010)). In a similar study using cultures of tumor-derived dendritic cells and TILs from ovarian cancer patients, the addition of anti-PD-L1 antibody significantly increased interferon-γ production by TILs in response to tumor antigens. When these TILs were transferred into immunodeficient mice bearing ovarian tumors, reduced tumor growth was observed compared to that in the control group of mice (Curiel et al., Nat. Med. 9, 562-567 (2003)).

[0115] In particular, cancers that can be treated by the present invention include skin cancer, lung cancer (especially squamous or non-squamous NSCLC), ovarian cancer, kidney cancer, colon cancer, colorectal cancer, breast cancer, gastric cancer, esophageal cancer, pancreatic cancer, bladder cancer, urothelial cancer, and liver cancer.

[0116] Other examples of cancers that can be treated by the present invention include melanoma (e.g., metastatic malignant melanoma), prostate cancer (e.g., hormone-refractory adenocarcinoma of the prostate), head and neck cancer (e.g., squamous cell carcinoma of the head and neck), cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma (e.g., B-cell lymphoma), adrenal cancer, AIDS-related cancer, alveolar soft part sarcoma, astrocytic tumor, bone cancer, spinal cord cancer, metastatic brain tumor, carotid body tumor, chondrosarcoma, chordoma, chromophobic renal cell carcinoma, clear cell carcinoma, benign fibrous histiocytoma of the skin, desmoplastic small round cell tumor, epithelioma, Ewing tumor, extraskeletal myxoid chondrosarcoma, osteofibrous dysplasia, fibrous dysplasia of bone, gallbladder or bile duct cancer, gestational trophoblastic disease, germ cell tumor, hematological malignancy, hepatocellular carcinoma, islet cell tumor, Kaposi sarcoma, kidney cancer, lipoma / benign lipomatoid tumor, liposarcoma / malignant lipomatoid tumor, medulloblastoma, meningioma, Merkel cell carcinoma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, papillary thyroid carcinoma, parathyroid tumor, pediatric cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, rare hematological disorders, kidney metastatic cancer, rhabdoid tumor, rhabdomysarcoma , including sarcoma, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, metastatic cancer of the thyroid or uterine cancer.

[0117] Generally, the components of the combination of the present invention or the composition of the present invention may be administered by any suitable route in any suitable formulation by known means. In some embodiments, the LAG-3 protein or its derivative is administered parenterally (e.g., by subcutaneous, intravenous or intramuscular injection). In some embodiments, the PD-1 pathway inhibitor is administered intravenously. In certain embodiments, the LAG-3 protein or its derivative is administered subcutaneously and the PD-1 pathway inhibitor is administered intravenously.

[0118] Suitable pharmaceutical compositions and dosage forms can be prepared using conventional methods known to those skilled in the art of pharmaceutical formulations and described in related texts and literature, such as Remington: The Science and Practice of Pharmacy (Easton, Pa.: Mack Publishing Co., 1995). It may be prepared using.

[0119] It is particularly advantageous to formulate the combination or composition of the present invention into unit dosage forms for ease of administration and uniformity of dosage. As used herein, the term "unit dosage form" refers to physically discrete units suitable as unit dosages for the individual to be treated. That is, the composition is formulated into discrete dosage units each containing a predetermined "unit dose" of the active agent calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications of the unit dosage forms of the present invention depend on the unique characteristics of the active agent to be delivered. The dosage may be further determined with reference to the usual dosage and the mode of administration of the components. It should be noted that in some cases, two or more individual dosage units in the combination can provide a therapeutically effective amount of the active agent, for example, two tablets or capsules taken together can provide a therapeutically effective dosage, such that the unit dose in each tablet or capsule is approximately 50% of the therapeutically effective amount.

[0120] The preparations according to the invention for parenteral administration include sterile aqueous and non-aqueous solutions, suspensions and emulsions. An aqueous injection solution contains the active agent in a water-soluble form. Examples of non-aqueous solvents or vehicles include fatty oils such as olive oil and corn oil, synthetic fatty acid esters such as ethyl oleate or triglycerides, low molecular weight alcohols such as propylene glycol, synthetic hydrophilic polymers such as polyethylene glycol, liposomes and the like. Parenteral formulations may also contain adjuvants such as solubilizing agents, preservatives, wetting agents, emulsifying agents, dispersing agents and stabilizing agents, and aqueous suspensions may contain substances that increase the viscosity of the suspension such as sodium carboxymethylcellulose, sorbitol, and dextran. Injectable formulations may be sterilized by incorporation of a sterilizing agent, filtration through a bacteria-retaining filter, irradiation or heating. They may be manufactured using a sterile injectable medium. The active agent may be in a dry form, for example a lyophilized form, which can be rehydrated with a suitable vehicle immediately prior to administration by injection.

[0121] In addition to the formulations already described, the active agent may be formulated as a depot preparation for the controlled release, preferably sustained release, of the active agent over a long period of time. These sustained release forms are generally administered by implantation (e.g., subcutaneously or intramuscularly or by intramuscular injection).

[0122] The combination preparations of the present invention may be packaged together with instructions for the administration of the components in the combination. The instructions may be recorded on a suitable recording medium or substrate. For example, the instructions may be printed on a substrate such as paper or plastic. The instructions may be present as an accompanying document on the label of the container or its components (i.e., associated with the packaging or sub-packaging). In other embodiments, the instructions are present as an electronic storage data file on a suitable computer-readable storage medium, such as a CD-ROM, diskette. Some or all of the components of the combination preparation may be packaged in a suitable package to maintain sterility. Embodiments of the present invention will be described below by way of example only with reference to the accompanying drawings. The present invention provides, for example, the following items. (Item 1) (a) A LAG-3 protein or a derivative thereof capable of binding to an MHC class II molecule; and (b) A combination preparation comprising a programmed cell death protein-1 (PD-1) pathway inhibitor. (Item 2) (a) A LAG-3 protein or a derivative thereof capable of binding to an MHC class II molecule; (b) A PD-1 pathway inhibitor; and (c) A pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient or diluent. (Item 3) The combination preparation according to Item 1 or the pharmaceutical composition according to Item 2 for co-administration or sequential administration of the LAG-3 protein or its derivative and the PD-1 pathway inhibitor. (Item 4) The combination preparation or pharmaceutical composition according to any one of the preceding items, wherein the LAG-3 protein or its derivative is separated from the PD-1 pathway inhibitor. (Item 5) The combination preparation or pharmaceutical composition according to any one of the preceding items, wherein the LAG-3 protein or its derivative is present in a dose that is 0.25 to 30 mg molar equivalent of the LAG-3Ig fusion protein IMP321. (Item 6) The combination preparation or pharmaceutical composition according to any one of the preceding items, comprising a plurality of doses of the LAG-3 protein or its derivative. (Item 7) The combination preparation or pharmaceutical composition according to any one of the preceding items, comprising a plurality of doses of the PD-1 pathway inhibitor. (Item 8) The combination preparation or pharmaceutical composition according to any one of the preceding items, wherein the PD-1 pathway inhibitor inhibits the binding of PD-1 to PD-L1 and / or PD-L2. (Item 9) The combination preparation or pharmaceutical composition according to any one of the preceding items, wherein the PD-1 pathway inhibitor comprises an anti-PD-1 antibody, or a derivative or fragment thereof that retains the ability to inhibit the binding of PD-1 to PD-L1 and / or PD-L2. (Item 10) The combination preparation or pharmaceutical composition according to any one of the preceding items, wherein the PD-1 pathway inhibitor is pembrolizumab or nivolumab or pidilizumab. (Item 11) The combination preparation or pharmaceutical composition according to any one of the preceding items, wherein the PD-1 pathway inhibitor comprises an anti-PD-L1 antibody, or a derivative or fragment thereof that retains the ability to inhibit the binding of PD-L1 to PD-1. (Item 12) The combination preparation or pharmaceutical composition according to any one of the preceding items, wherein the PD-1 pathway inhibitor is BMS-936559, MEDI4736, MPDL3280A or MSB0010718C. (Item 13) The combination preparation or pharmaceutical composition according to any one of the preceding items, wherein the PD-1 pathway inhibitor is present in a dose that is up to 50%, 1-50%, 1-25% or 1-10% of the typically prescribed dose of the PD-1 pathway inhibitor as a monotherapy. (Item 14) The combination preparation or pharmaceutical composition according to any one of the preceding items, wherein the PD-1 pathway inhibitor is present in a dose that is 0.1-50%, 0.1-25%, 0.1-20%, 0.1-10%, less than 20%, less than 10%, less than 0.1-20%, less than 0.1-10%, less than 0.01-20% or less than 0.01-10% of the typically prescribed dose of the PD-1 pathway inhibitor as a monotherapy. (Item 15) The combination preparation or pharmaceutical composition according to any one of the preceding items, wherein the LAG-3 protein or its derivative, and the PD-1 pathway inhibitor are present in any of the dosage combinations shown in Table 1.2. (Item 16) The combination preparation or pharmaceutical composition according to any of the preceding items, wherein the derivative of the LAG-3 protein comprises an amino acid sequence having at least 70% amino acid identity with the LAG-3 protein, preferably domain D1 of the human LAG-3 protein, and optionally domain D2. (Item 17) The combination preparation or pharmaceutical composition according to any of the preceding items, wherein the derivative of the LAG-3 protein comprises an amino acid sequence having at least 70% amino acid identity with the LAG-3 protein, preferably domains D1, D2, D3, and optionally D4 of the human LAG-3 protein. (Item 18) The combination preparation or pharmaceutical composition according to any of the preceding items, wherein the derivative of the LAG-3 protein is fused to an immunoglobulin Fc sequence. (Item 19) The combination preparation or pharmaceutical composition according to any of the preceding items, wherein the derivative of the LAG-3 protein is the recombinant soluble human LAG-3Ig fusion protein IMP321. (Item 20) The combination preparation or pharmaceutical composition according to any of the preceding items for use as a medicament. (Item 21) The combination preparation or pharmaceutical composition according to any of items 1 to 19 for use in the prevention, treatment or recovery of cancer. (Item 22) Use of the combination preparation or pharmaceutical composition according to any of items 1 to 19 in the manufacture of a medicament for the prevention, treatment or recovery of cancer. (Item 23) The combination preparation or pharmaceutical composition according to item 21, or use of the combination preparation or pharmaceutical composition according to item 22, wherein the cancer is a PD-L1 positive or PD-L2 positive cancer. (Item 24) wherein the cancer is skin cancer, lung cancer (especially squamous or non-squamous non-small cell lung cancer, NSCLC), ovarian cancer, kidney cancer, colon cancer, colorectal cancer, breast cancer, gastric cancer, esophageal cancer, pancreatic cancer, bladder cancer, urothelial cancer or liver cancer, or melanoma (e.g., metastatic malignant melanoma), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), head and neck cancer (e.g., squamous cell carcinoma of the head and neck), cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma (e.g., B-cell lymphoma), adrenal cancer, AIDS-related cancer, alveolar soft part sarcoma, astrocytic tumor, bone cancer, cerebrospinal cancer, metastatic brain tumor, carotid body tumor, chondrosarcoma, chordoma, chromophobe renal cell carcinoma, clear cell carcinoma, benign fibrous histiocytoma of the skin, desmoplastic small round cell tumor, epithelioma, Ewing tumor, extraskeletal myxoid chondrosarcoma, osteofibrous dysplasia, fibrous dysplasia of bone, gallbladder or bile duct cancer, gestational trophoblastic disease, germ cell tumor, hematological malignancy, hepatocellular carcinoma, islet cell tumor, Kaposi sarcoma, kidney cancer, lipoma / benign lipomatous tumor, liposarcoma / malignant lipomatous tumor, medulloblastoma, meningioma, Merkel cell carcinoma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, papillary thyroid cancer, parathyroid tumor, pediatric cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, rare hematological disorders, renal metastatic cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, metastatic cancer of the thyroid or uterine cancer, use of the combination preparation or pharmaceutical composition according to item 21, or use of the combination preparation or pharmaceutical composition according to item 22. (Item 25) The combination preparation or pharmaceutical composition according to any one of items 1 to 19 for use in preventing, treating or recovering from an infection. (Item 26) Use of the combination preparation or pharmaceutical composition according to any one of items 1 to 19 in the manufacture of a medicament for preventing, treating or recovering from an infection. (Item 27) The combination preparation according to item 25 or the use according to item 26, wherein the infection is a chronic or persistent infection. (Item 28) The combination preparation according to item 25 or 27, or the use according to item 26 or 27, wherein the infection is a viral, bacterial, fungal or protozoal infection. (Item 29) The combination preparation or use according to item 28, wherein the viral infection is caused by adenovirus, adeno-associated virus, B virus (Macacine herpesvirus I), BK virus, bunyavirus, chikungunya virus, coxsackievirus, coronavirus, cytomegalovirus, eastern equine encephalitis virus, Ebola virus, enterovirus, Epstein-Barr virus, hantavirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, herpesvirus, herpes simplex virus 1, herpes simplex virus 2, human foamy virus, human herpesvirus 3, human herpesvirus 5, human herpesvirus 6, human herpesvirus 7, human immunodeficiency virus, human papillomavirus, human beta-lymphotropic virus, human T-cell leukemia virus I, human T-cell leukemia virus II, influenza virus, JC virus, JEV, Kaposi's sarcoma-associated herpesvirus, Lassa virus, lymphocytic choriomeningitis virus, Marburg virus, measles virus, mumps virus, Nipah virus, norovirus, Norwalk virus, orthoreovirus, parainfluenza virus, parvovirus, poliovirus, rabies virus, reovirus, respiratory syncytial virus, rhinovirus, Rift Valley fever virus, rotavirus, rubella virus, smallpox virus, St. Louis encephalitis virus, variola major virus, variola minor virus, varicella-zoster virus, West Nile virus, western equine encephalitis virus or yellow fever virus. (Item 30) The bacterial infection is Escherichia coli, Clostridium difficile, Salmonella thyphimurium, Pseudomonas aeruginosa, Vibrio cholerae, Neisseria Neisseria gonorrhoeae, Helicobacter pylori, Haemophilus influenzae, Shigella dysenteriae, Staphylococcus aureus, Mycobacterium tuberculosis, Streptococcus pneumoniae or Chlamydia The combination preparation or use according to item 28, which is caused by Chlamydia trachomatis. (Item 31) The combination preparation or use according to item 28, wherein the fungal infection is caused by Candida, Aspergillus, Cryptococcus, Coccidioides, Histoplasma, Pneumocystis or Stachybotrys. (Item 32) The combination preparation or use according to item 28, wherein the protozoal infection is caused by Amoebozoa, Excavata, Chromalveolata, Entamoeba, Plasmodium, Giardia, Trypanosoma, Coccidia, Besnoitia, Dicrocoelium or Leishmania. (Item 33) A method for preventing, treating or restoring cancer, comprising administering to a subject in need of such prevention, treatment or restoration, an LAG-3 protein, or a derivative thereof capable of binding to an MHC class II molecule, and a PD-1 pathway inhibitor. (Item 34) A method for preventing, treating or restoring an infection, comprising administering to a subject in need of such prevention, treatment or restoration, an LAG-3 protein, or a derivative thereof capable of binding to an MHC class II molecule, and a PD-1 pathway inhibitor. (Item 35) The method according to item 33 or 34, wherein the LAG-3 protein or its derivative, and the PD-1 pathway inhibitor are sequentially administered to the subject. (Item 36) The method according to item 33 or 34, wherein the LAG-3 protein or a derivative thereof is administered after the PD-1 pathway inhibitor. (Item 37) The method according to item 35 or 36, wherein the LAG-3 protein or a derivative thereof and the PD-1 pathway inhibitor are each administered to the subject within 96 hours of each other. (Item 38) The method according to item 33 or 34, wherein the LAG-3 protein or a derivative thereof and the PD-1 pathway inhibitor are co-administered to the subject. (Item 39) The method according to any one of items 33 to 38, wherein the LAG-3 protein or a derivative thereof is administered to the subject at a dose that is a molar equivalent of 0.25 to 30 mg of the LAG-3Ig fusion protein IMP321. (Item 40) The method according to any one of items 33 to 39, wherein a plurality of doses of the LAG-3 protein or a derivative thereof are administered to the subject. (Item 41) The method according to any one of items 33 to 40, wherein a plurality of doses of the PD-1 pathway inhibitor are administered to the subject. (Item 42) The method according to item 40 or 41, wherein the dose of the LAG-3 protein or a derivative thereof is administered before, together with, or after each administration of 2 or more doses of the PD-1 pathway inhibitor. (Item 43) The method according to any one of items 33 to 42, wherein the PD-1 pathway inhibitor inhibits the binding of PD-1 to PD-L1 and / or PD-L2. (Item 44) The method according to any one of items 33 to 43, wherein the PD-1 pathway inhibitor comprises an anti-PD-1 antibody or a derivative or fragment thereof that retains the ability to inhibit the binding of PD-1 to PD-L1 and / or PD-L2. (Item 45) The method according to any one of items 33 to 44, wherein the PD-1 pathway inhibitor is pembrolizumab or nivolumab or pidilizumab. (Item 46) The method according to any one of Items 33 to 43, wherein the PD-1 pathway inhibitor comprises an anti-PD-L1 antibody, or a derivative or fragment thereof that retains the ability to inhibit the binding of PD-L1 to PD-1. (Item 47) The method according to any one of Items 33 to 43 or 46, wherein the PD-1 pathway inhibitor is BMS-936559, MEDI4736, MPDL3280A or MSB0010718C. (Item 48) The method according to any one of Items 33 to 47, wherein the PD-1 pathway inhibitor is administered to the subject at a dose that is up to 50%, 1 - 50%, 1 - 25% or 1 - 10% of the typically prescribed dose of the PD-1 pathway inhibitor as a monotherapy. (Item 49) The method according to any one of Items 33 to 48, wherein the PD-1 pathway inhibitor is administered to the subject at a dose that is 0.1 - 50%, 0.1 - 25%, 0.1 - 20%, 0.1 - 10%, less than 20%, less than 10%, less than 0.1 - 20%, less than 0.1 - 10%, less than 0.01 - 20% or less than 0.01 - 10% of the typically prescribed dose of the PD-1 pathway inhibitor as a monotherapy. (Item 50) The method according to any one of Items 33 to 48, wherein the LAG-3 protein or its derivative, and the PD-1 pathway inhibitor are administered in any of the dosing combinations shown in Table 1.2. (Item 51) The method according to any one of Items 33 to 50, wherein the derivative of the LAG-3 protein comprises an amino acid sequence having at least 70% amino acid identity with the LAG-3 protein, preferably domain D1 of the human LAG-3 protein, and optionally domain D2. (Item 52) The method according to any one of Items 33 to 51, wherein the derivative of the LAG-3 protein comprises an amino acid sequence having at least 70% amino acid identity with the LAG-3 protein, preferably domains D1, D2, D3 of the human LAG-3 protein, and optionally D4. (Item 53) The method according to any one of items 33 to 52, wherein the derivative of the LAG-3 protein is fused to an immunoglobulin Fc sequence. (Item 54) The method according to any one of items 33 to 53, wherein the derivative of the LAG-3 protein is the recombinant soluble human LAG-3Ig fusion protein IMP321. (Item 55) The method according to any one of items 33 or 35 to 54, wherein the cancer is a PD-L1 positive or PD-L2 positive cancer. (Item 56) The cancer is skin cancer, lung cancer (especially squamous or non-squamous non-small cell lung cancer, NSCLC), ovarian cancer, kidney cancer, colon cancer, colorectal cancer, breast cancer, gastric cancer, esophageal cancer, pancreatic cancer, bladder cancer, urothelial cancer or liver cancer, or melanoma (e.g., metastatic malignant melanoma), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), head and neck cancer (e.g., squamous cell carcinoma of the head and neck), cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma (e.g., B cell lymphoma), adrenal cancer, AIDS-related cancer, alveolar soft part sarcoma, astrocytic tumor, bone cancer, spinal cord cancer, metastatic brain tumor, carotid body tumor, chondrosarcoma, chordoma, chromophobic renal cell carcinoma, clear cell carcinoma, benign fibrous histiocytoma of the skin, desmoplastic small round cell tumor, epithelioblastoma, Ewing tumor, extraskeletal myxoid chondrosarcoma, osteofibrous dysplasia, fibrous dysplasia of bone, gallbladder or bile duct cancer, gestational trophoblastic disease, germ cell tumor, hematological malignancy, hepatocellular carcinoma, islet cell tumor, Kaposi sarcoma, kidney cancer, lipoma / benign lipomatous tumor, liposarcoma / malignant lipomatous tumor, medulloblastoma, meningioma, Merkel cell carcinoma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, papillary thyroid cancer, parathyroid tumor, pediatric cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, rare hematological disorders, renal metastatic cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, metastatic cancer of the thyroid or uterine cancer, the method according to any one of items 33 or 35 to 55. (Item 57) The method according to any one of items 34 to 54, wherein the infection is a chronic or persistent infection. (Item 58) The method according to any one of items 34 to 54, or 57, wherein the infection is a viral, bacterial, fungal or protozoal infection. (Item 59) The method according to item 58, wherein the viral infection is caused by adenovirus, adeno-associated virus, B virus (Macacine herpesvirus I), BK virus, bunyavirus, chikungunya virus, coxsackievirus, coronavirus, cytomegalovirus, eastern equine encephalitis virus, Ebola virus, enterovirus, Epstein-Barr virus, hantavirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, herpesvirus, herpes simplex virus 1, herpes simplex virus 2, human foamy virus, human herpesvirus 3, human herpesvirus 5, human herpesvirus 6, human herpesvirus 7, human immunodeficiency virus, human papillomavirus, human beta-lymphotropic virus, human T-cell leukemia virus I, human T-cell leukemia virus II, influenza virus, JC virus, JEV, Kaposi's sarcoma-associated herpesvirus, Lassa virus, lymphocytic choriomeningitis virus, Marburg virus, measles virus, mumps virus, Nipah virus, norovirus, Norwalk virus, orthoreovirus, parainfluenza virus, parvovirus, poliovirus, rabies virus, reovirus, respiratory syncytial virus, rhinovirus, Rift Valley fever virus, rotavirus, rubella virus, smallpox virus, St. Louis encephalitis virus, variola major virus, variola minor virus, varicella-zoster virus, West Nile virus, western equine encephalitis virus or yellow fever virus. (Item 60) The method according to item 58, wherein the bacterial infection is caused by Escherichia coli, Clostridium difficile, Salmonella thyphimurium, Pseudomonas aeruginosa, Vibrio cholerae, Neisseria gonorrhoeae, Helicobacter pylori, Hemophilus influenzae, Shigella dysenteriae, Staphylococcus aureus, Mycobacterium tuberculosis, Streptococcus pneumonia or Chlamydia trachomatis. (Item 61) The method according to item 58, wherein the fungal infection is caused by Candida, Aspergillus, Cryptococcus, Coccidioides, Histoplasma, Pneumocystis or Stachybotrys. (Item 62) The method according to item 58, wherein the protozoan infection is caused by Amoebozoa, Excavata, Chromalveolata, Entamoeba, Plasmodium, Giardia, Trypanosoma, Coccidia, Besnoitia, Dicrocoelium or Leishmania. (Item 63) The method according to any one of items 33 to 62, wherein the subject is a human subject.

Brief Description of the Drawings

[0123]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6-1

Figure 6-2

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16-1

Figure 16-2

Figure 17

Figure 18

Figure 19

Mode for Carrying Out the Invention

[0124] In the following examples, tables, and figures, the term "anti-PD1 antibody" is used synonymously with "anti-PD-1 antibody", and the term "anti-PDL1 antibody" is used synonymously with "anti-PD-L1 antibody".

Examples

[0125] (Example 1) Effect of LAG-3Ig and anti-PD1 antibody on IFN-γ secretion induced by antigen stimulation This example demonstrates the effect of a soluble derivative of LAG-3 (LAG-3Ig, also known as IMP321) and an anti-PD1 antibody on in vitro T cell activation using an IFN-γ secretion assay. Peripheral blood mononuclear cells (PBMC) include lymphocytes (T cells, B cells, and NK cells), monocytes, and dendritic cells. IFN-γ is mainly secreted by activated CD4+ and CD8+ memory and effector T cells, as well as by NK cells after activation. After restimulation with specific antigen in vitro, secretion of IFN-γ is induced.

[0126] PBMC from three healthy donors (cell number 0.2×10 6 / well, Complete Roswell Park Memorial Institute (RPMI) + 10% fetal bovine serum (FBS) at 1×10 6In the presence or absence of 30 ng / ml of LAG-3Ig and the indicated concentrations of anti-PD1 mAb (clone EH12.1, BD biosciences, catalog #562138), three replicates were incubated with a pool of peptides containing the sequence of human cytomegalovirus (CMV) pp65 (PepTivator® CMV pp65, Miltenyi Biotec, catalog #130-093-435). The peptide pool consisted mainly of a 15-mer sequence with 11 amino acid overlaps, containing the complete sequence of the pp65 protein of the human CMV strain AD169 (Swiss-Prot accession number P06725).

[0127] T cell responses were evaluated by measuring the concentration of IFN-γ in the cell supernatant 2 days after stimulation using a BD cytometric bead array.

[0128] The concentration of IFN-γ present in the pooled triplicates for each donor was recorded in Table 2 below. Figure 2 shows the concentration of IFN-γ plotted against the concentration of anti-PD1 mAb for each donor.

Table 2

[0129] The results show that the secretion of IFN-γ increased dramatically when PBMCs were incubated in the presence of 30 ng / ml of LAG-3Ig and lower concentrations of anti-PD1 antibody compared to the anti-PD1 antibody alone. For example, as shown in Table 3 below, for each donor, the increase in the concentration of IFN-γ above the background level (i.e., the concentration of IFN-γ in the absence of anti-PD1 and LAG-3Ig) in the presence of 30 ng / ml of LAG-3Ig and 30 ng / ml of anti-PD1 antibody was greater than the sum of the corresponding increases in the presence of 30 ng / ml of LAG-3Ig alone and 30 ng / ml of anti-PD1 antibody alone. Thus, the effect of the combination of LAG-3Ig and anti-PD1 antibody for each donor was synergistic.

Table 3

[0130] The results also show that the secretion of IFN-γ induced by the combination of LAG-3Ig and a relatively low concentration of anti-PD1 antibody (30 ng / ml) was equivalent to the secretion of IFN-γ induced by anti-PD1 antibody alone at much higher concentrations (300 ng / ml to 1000 ng / ml, 10 to 30 times higher). In the case of donors No. 1 and 3, similar concentrations of IFN-γ were secreted when PBMCs were incubated with 30 ng / ml of anti-PD1 and 30 ng / ml of LAG-3Ig, compared to 1000 ng / ml of anti-PD1 antibody alone. In the case of donor No. 2, similar concentrations of IFN-γ were secreted when PBMCs were incubated with 30 ng / ml of anti-PD1 and 30 ng / ml of LAG-3Ig, compared to 300 ng / ml and 1000 ng / ml of anti-PD1 antibody alone.

[0131] From these results, it was concluded that the in vitro T cell response (measured by IFN-γ secretion) induced by a relatively low dose of anti-PD1 antibody was synergistically increased by the soluble LAG-3 derivative (by approximately 7.5-fold, 1.5-fold, and 2-fold in donors No. 1, 2, and 3, respectively). Furthermore, it was concluded that if the anti-PD1 antibody was combined with the soluble LAG-3 derivative, an equivalent in vitro T cell response could be obtained using approximately one-tenth to one-thirtieth of the anti-PD1 antibody. (Example 2) Effect of LAG-3Ig and anti-PD1 antibody on the secretion of IFN-γ induced by antigen stimulation

[0132] This example demonstrates the effect of the soluble derivative of LAG-3 (LAG-3Ig) and anti-PD1 antibody on in vitro T cell activation using an IFN-γ secretion assay.

[0133] PBMC from 10 healthy donors (cell number 0.2×10 6 / Wells, complete RPMI + 10% FBS at 1 × 10 6 M / ml) were incubated in triplicate without any additives (media) with a pool of peptides containing the sequence of CMV pp65 (PepTivator® CMV pp65, Miltenyi Biotec, catalog #130 - 093 - 435), with 30 ng / ml or 1000 ng / ml of anti - PD1 mAb (clone EH12.1, BD biosciences catalog #562138), with 30 ng / ml of LAG - 3Ig, or with 30 ng / ml of LAG - 3Ig and 30 ng / ml of anti - PD1 mAb.

[0134] T - cell responses were evaluated by measuring the concentration of IFN - γ in the cell supernatant 2 days after stimulation using a BD Cytometry Bead Array.

[0135] The concentration of IFN - γ in the pooled triplicates for each stimulation condition for each donor was recorded in Table 4 below. The mean of the results obtained for 10 donors is shown in Table 5. The results for each donor were also plotted in Figures 3 and 4. Statistical differences ( * p < 0.05) are shown in black in Figure 3.

Table 4

[0136]

Table 5

[0137] The results show that when PBMC were incubated in the presence of 30 ng / ml of LAG-3Ig and 30 ng / ml of anti-PD1 antibody, the secretion of IFN-γ was much higher in each donor as compared to 30 ng / ml of LAG-3Ig or 30 ng / ml of anti-PD1 antibody alone. Table 5 shows that the increase in the mean concentration of IFN-γ above the mean background level (i.e., the mean concentration of IFN-γ in the absence of anti-PD1 and LAG-3Ig) in the presence of 30 ng / ml of LAG-3Ig and 30 ng / ml of anti-PD1 antibody was greater than the sum of the corresponding increases in the presence of 30 ng / ml of LAG-3Ig alone and 30 ng / ml of anti-PD1 antibody alone (i.e., 765>239 + 172). Thus, the effect of the combination of LAG-3Ig and anti-PD1 antibody was synergistic.

[0138] The results also show that the secretion of IFN-γ induced by the combination of LAG-3Ig and a relatively low concentration of anti-PD1 antibody (30 ng / ml) was equivalent to the secretion of IFN-γ induced by anti-PD1 antibody alone at a much higher concentration (1000 ng / ml, 30-fold higher).

[0139] From these results, it was concluded that the in vitro T cell response (measured by IFN-γ secretion) induced by a relatively low dose of anti-PD1 antibody was synergistically increased (on average approximately 2-fold) by the soluble LAG-3 derivative. Furthermore, it was concluded that if anti-PD1 antibody was combined with the soluble LAG-3 derivative, an equivalent in vitro T cell response could be obtained using less than one-thirtieth of the anti-PD1 antibody. (Example 3) Effect of LAG-3Ig and anti-PD1 antibody on the secretion of TNF-α, IL-6, and RANTES induced by antigen stimulation

[0140] This example demonstrates the effect of the soluble derivative of LAG-3 (LAG-3Ig) and anti-PD1 antibody on T cell activation in vitro by measuring the secretion of TNF-α, IL-6, and RANTES (CCL5).

[0141] PBMC from 10 healthy donors (0.2 × 10 cells per well, in complete RPMI + 10% FBS at 1 × 10 6 / well, in complete RPMI + 10% FBS at 1 × 10 6 M / ml) were incubated in triplicate without any additives (media) with a pool of peptides containing the sequence of CMV pp65 (PepTivator® CMV pp65, Miltenyi Biotec, catalog #130 - 093 - 435), with 30 ng / ml or 1000 ng / ml of anti - PD1 mAb (clone EH12.1, BD biosciences catalog #562138), with 30 ng / ml of LAG - 3Ig, or with 30 ng / ml of LAG - 3Ig and 30 ng / ml of anti - PD1 mAb.

[0142] T - cell responses were evaluated by measuring the concentrations of TNF - α, IL - 6, and RANTES (CCL5) in the cell supernatants 2 days after stimulation using a BD Cytometry Bead Array.

[0143] The concentrations of cytokines / chemokines in the pooled triplicates for each stimulation condition for each donor were recorded in Tables 6 - 8 below. The mean of the results obtained from 10 donors is shown in Table 9, and the mean increases above the mean background are shown in Table 10. The results for each donor were also plotted in Figure 5, and the statistical differences ( * p < 0.05) are shown in black.

Table 6

[0144]

Table 7

[0145]

Table 8

[0146]

Table 9

[0147]

Table 10

[0148] The results show that when PBMCs were incubated in the presence of 30 ng / ml of LAG-3Ig and 30 ng / ml of anti-PD1 antibody, the secretion of IL-6 was much higher in each donor compared to incubation with 30 ng / ml of LAG-3Ig or 30 ng / ml of anti-PD1 antibody alone. Table 10 shows that the increase in the mean concentration of IL-6 above the mean background level (i.e., the mean concentration of IL-6 in the absence of anti-PD1 and LAG-3Ig) in the presence of 30 ng / ml of LAG-3Ig and 30 ng / ml of anti-PD1 antibody was greater than the sum of the corresponding increases in the presence of 30 ng / ml of LAG-3Ig alone and 30 ng / ml of anti-PD1 antibody alone (i.e., 8594>732 + 2964). Thus, the effect of the combination of LAG-3Ig and anti-PD1 antibody was synergistic.

[0149] The results also show that the secretion of IL-6 induced by the combination of LAG-3Ig and a relatively low concentration of anti-PD1 antibody (30 ng / ml) was equivalent to the secretion of IL-6 induced by anti-PD1 antibody alone at a much higher concentration (1000 ng / ml, 30-fold higher).

[0150] From these results, it was concluded that the in vitro T cell response (measured by IL-6 secretion) induced by a relatively low dose of anti-PD1 antibody was synergistically increased (more than 2.3-fold on average) by the soluble LAG-3 derivative. (Example 4) Effect of LAG-3Ig and anti-PD1 antibody on the expression of activation markers induced by antigen stimulation

[0151] This example demonstrates the effect of the soluble derivative of LAG-3 (LAG-3Ig) and anti-PD1 antibody on the expression of T cell activation markers.

[0152] PBMCs from 7 healthy donors (cell number 0.2×10 6 / well, in complete RPMI + 10% FBS at 1×10 6 M / ml) were incubated in triplicate without any additives (medium), with a pool of peptides containing the sequence of CMV pp65 (PepTivator® CMV pp65, Miltenyi Biotec, catalog #130-093-435), with 30 ng / ml or 1000 ng / ml of anti-PD1 mAb (clone EH12.1, BD biosciences catalog #562138), with 30 ng / ml of LAG-3Ig, or with 30 ng / ml of LAG-3Ig and 30 or 1000 ng / ml of anti-PD1 mAb.

[0153] The T cell response was evaluated by analyzing the cell phenotype for the expression of three activation markers (LAG-3, CD69, and CD25) by flow cytometry 2 days after stimulation.

[0154] The percentage of CD8 cells expressing at least one of the three activation markers (LAG-3, CD69, or CD25), or all three activation markers (LAG-3, CD69, and CD25), in the pooled triplicates for each stimulation condition was recorded in Tables 11 - 15 below. The average of the results obtained from 7 donors is shown in Table 16, and the average increase above the mean background is shown in Table 17. The results for each donor were also plotted in Figure 6, and the statistical differences ( * p < 0.05) are shown in black.

Table 11

[0155]

Table 12

[0156]

Table 13

[0157]

Table 14

[0158]

Table 15

[0159]

Table 16

[0160]

Table 17

[0161] The results show that stimulation with 30 ng / ml anti-PD-1 antibody and 30 ng / ml LAG-3Ig or 1000 ng / ml anti-PD-1 antibody and 30 ng / ml LAG-3Ig resulted in a multiplicative increase in the mean percentage of CD8 cells expressing any or all three of the activation markers.

[0162] The results also show that stimulation with 30 ng / ml anti-PD-1 antibody and 30 ng / ml LAG-3Ig resulted in a significantly higher mean percentage of CD8 cells expressing any or all three of the activation markers than stimulation with 1000 ng / ml anti-PD-1 antibody.

[0163] From these results, in vitro CD8 induced by relatively low doses of anti-PD1 antibody +The T cell response (measured by the expression of T cell activation markers) was concluded to be synergistically increased by the soluble LAG-3 derivative. Furthermore, if an anti-PD1 antibody is combined with the soluble LAG-3 derivative, a dramatic improvement in the in vitro CD8 + T cell response was concluded to be obtained using less than one-thirtieth of the anti-PD1 antibody.

[0164] PD-1 pathway inhibitors (such as Keytruda and Opdivo) are known to activate CD8 + T cells, and since this activation is associated with an anti-cancer effect, the results shown in the above examples provide evidence that an improved anti-cancer effect can be obtained by co-administering a PD-1 pathway inhibitor with an LAG-3 protein or a derivative thereof that can bind to an MHC class II molecule. Alternatively, a similar anti-cancer effect can be achieved by co-administering a PD-1 pathway inhibitor with an LAG-3 protein (or a derivative thereof that can bind to an MHC class II molecule) at a lower dose of the PD-1 pathway inhibitor (e.g., one-tenth to one-thirtieth of the dose) compared to the administration of the PD-1 pathway inhibitor as a monotherapy. Such co-administration is expected to reduce the side effects caused by the PD-1 pathway inhibitor.

[0165] Similarly, CD8 +T cell activation is also known to be effective against infections, including chronic or persistent infections. Thus, the results shown in the above examples provide evidence that the co - administration of a PD - 1 pathway inhibitor with an LAG - 3 protein or its derivative capable of binding to an MHC class II molecule can be used to more effectively prevent, treat, or recover from an infection. Alternatively, co - administration of a PD - 1 pathway inhibitor with an LAG - 3 protein (or its derivative capable of binding to an MHC class II molecule) at a lower dose of the PD - 1 pathway inhibitor (e.g., 1 / 30 to 1 / 100 of the dose) compared to the administration of the PD - 1 pathway inhibitor as monotherapy can achieve a similar effect against infection. Such co - administration is expected to reduce the side effects caused by the PD - 1 pathway inhibitor. (Example 5) Binding of LAG - 3 derivatives to MHC class II - positive cells The ability of several derivatives of LAG - 3 to bind to MHC class II - positive cells was examined: i) Domains D1 - D4 of LAG - 3 linked to an immunoglobulin Fc (Ig Fc) sequence by a first linker (LAG - 3 D1D4 - linker1 - Ig, sLAG - 3 D1D4 - Ig, LAG - 3Ig or IMP321); ii) Domains D1 - D4 of LAG - 3 linked to an Ig Fc sequence by a second linker (LAG - 3 D1D4 - linker2 - Ig or sLAG - 3 D1D4 - linkerB - Ig); iii) Domains D1 and D2 of LAG - 3 linked to an Ig Fc sequence by a second linker (LAG - 3 D1D2 - linker2 - Ig or sLAG - 3 D1D2 - linkerB - Ig); and

[0166] (iv) Domains D1 - D4 of LAG-3 linked to the Ig Fc sequence by the first linker, but having a mutation that enhances binding to MHC class II molecules up to 3-fold or more at position R75 (R75A) in the MHC class II binding site of the D1 domain of LAG-3 (Huard et al., Proc. Natl. Acad. Sci. USA, 1997, 94:5744) (IMP321 R75A).

[0167] The derivative is illustrated in Figure 7.

[0168] MHC class II+ Raji cells were incubated with various concentrations of the LAG-3 derivative or with human IgG1 antibody (hIgG1) as a negative control for 45 minutes at 4°C. LAG-3 molecules bound to the cell surface were revealed with FITC-conjugated goat anti-mouse Ig (Coulter). The cells were analyzed by flow cytometry. The results, expressed as fluorescence intensity units, are shown in Figure 8. The results indicate that all LAG-3 derivatives bound to MHC class II positive cells. (Example 6) Inhibition of binding of the LAG-3 derivative IMP321 to MHC class II positive cells by an antibody that blocks the binding of LAG-3 to MHC class II molecules

[0169] 17B4 and 11E3 are anti-LAG-3 monoclonal antibodies known to block the binding of LAG-3 to MHC class II molecules. The binding of IMP321-conjugate (LAG-3Ig-Alexa488) to MHC class II positive B cells (Raji cells) was determined following pre-incubation (4 μg / ml, 4°C) with the 17B4 or 11E3 blocking antibody or an isotype matched negative control monoclonal antibody (mIgG1). Analysis of cell-bound fluorescence was performed using fluorescence-activated cell sorting (FACS). The results are shown in Figure 9.

[0170] The results indicate that the binding of IMP321 to Raji cells was inhibited by a LAG-3-specific monoclonal antibody that blocks the binding of LAG-3 to MHC class II molecules. (Example 7) Activation of monocytes by LAG-3 derivatives

[0171] THP-1 cells were incubated with the LAG-3 derivatives illustrated in FIG. 5 or with human IgG1 as a negative control for 4 hours at 4° C. The amounts of chemokine CCL4 and cytokine tumor necrosis factor-α, TNF-α secreted by the THP-1 cells were determined and used as measurements of monocyte activation. CCL4 and TNF-α secretion were quantified in cell supernatants using a Cytometric Beads Array. The results of the CCL4 determination are shown in FIG. 10 and the results of the TNF-α determination are shown in FIG. 11.

[0172] The results indicate that all of the LAG-3 derivatives were able to activate THP-1 monocyte cells. (Example 8) Inhibition of IMP321-induced monocyte activation by an antibody that blocks the binding of LAG-3 to MHC class II molecules

[0173] IMP321 (20 ng / ml) was pre-incubated (for 5 minutes at 37° C.) with 17B4 or 11E3 antibodies prior to a 4-hour incubation at 37° C. of the mixture with THP-1 cells. The amount of CCL4 secreted by the THP-1 cells was used to determine the level of monocyte activation. The results of two experiments are shown in FIG. 12.

[0174] The results demonstrate that IMP321-induced monocyte activation is inhibited by the blocking anti-LAG-3 mAbs 17B4 and 11E3. This indicates that the ability of IMP321 to activate monocytes is dependent on the binding of IMP321 to MHC class II molecules. (Example 9) Activation of primary antigen-presenting cells (APCs) by LAG-3 derivatives

[0175] Human peripheral blood mononuclear cells (PBMCs) were incubated at 37°C for 4 hours in the presence of the LAG-3 derivatives exemplified in FIG. 7, or human IgG1 as a negative control, and the secretion inhibitor brefeldin. The cytokine responses of APCs present in the PBMCs were determined by intracellular staining for CCL4 (a chemokine known to support Th1 and CD8-positive responses) and TNF-α (a multifunctional cytokine that directly inhibits tumor formation). The results were analyzed by cytometry. The results are represented by the percentage of cells expressing CCL4 and / or TNF-α in MHC class II-positive cells and are shown in FIG. 13.

[0176] The results show that all LAG-3 derivatives tested in primary APCs induced the production of CCL4 and TNF-α. (Example 10) Activation of CD8 + T cells by LAG-3 derivatives

[0177] Human PBMCs were incubated for 18 hours with the LAG-3 derivatives exemplified in FIG. 7, or human IgG1 as a negative control. Brefeldin was present during the last 16 hours of incubation. The cytokine responses of CD8 + T cells after 18-hour exposure to the LAG-3 derivatives were followed by intracellular staining for CCL4, IFN-γ, and TNF-α and analyzed by cytometry. The results are represented as the percentage of cells expressing CCL4, IFN-γ, and / or TNF-α in CD3 + / CD8 + T cells and are shown in FIG. 14.

[0178] The results show that all LAG-3 derivatives tested induced the activation of type 1 cytotoxic CD8-positive T cells (Tc1 cells). Through binding to MHC class II molecules expressed by APCs, it can be concluded that the LAG-3 derivatives induced the activation of Tc1 cells. The activation of Tc1 cells forms a major anti-tumor immune response. (Example 11) Effect of LAG-3Ig and anti-PD-L1 on the expression of activation markers induced by antigen stimulation

[0179] This example demonstrates the effect of a soluble derivative of LAG-3 (LAG-3Ig) and an anti-PD-L1 antibody on the expression of T cell activation markers.

[0180] PBMCs from 12 healthy donors (0.2 × 10 6 / well, in complete RPMI + 10% FBS at 1 M / ml) were incubated in triplicate with a pool of peptides containing the sequence of CMV pp35, without any additives (medium), with 30 ng / ml or 3000 ng / ml of anti-PD-L1 humanized antibody (BPS Bioscience, catalog #71213), with 30 ng / ml of LAG-3Ig, or with 30 ng / ml of LAG-3Ig and 30 ng / ml of anti-PD-L1 antibody.

[0181] T cell responses were evaluated by analyzing the cell phenotype for the expression of three activation markers (LAG-3, CD69, and CD25) by flow cytometry 3 days after stimulation.

[0182] The percentage of CD8 cells expressing at least one of the three activation markers (LAG-3, CD69, or CD25), or all three activation markers (LAG-3, CD69, and CD25) in the pooled triplicates for each stimulation condition was recorded in Tables 18 - 22 below. The mean of the results obtained from 12 donors is shown in Table 23, and the mean increase above the mean background is shown in Table 24. The results for each donor were also plotted in Figure 16, and the statistical differences ( * p < 0.05) are shown in black.

Table 18

[0183]

Table 19

[0184]

Table 20

[0185]

Table 21

[0186]

Table 22

Table 23

[0187]

Table 24

[0188] The results show that stimulation with 30 ng / ml of anti-PD-L1 antibody and 30 ng / ml of LAG-3Ig resulted in a multiplicative increase in the mean percentage of CD8 cells expressing any or all three of the activation markers.

[0189] The results also show that stimulation with 30 ng / ml of anti-PD-L1 antibody and 30 ng / ml of LAG-3Ig resulted in a significantly higher mean percentage of CD8 cells expressing any or all three of the activation markers than stimulation with 3000 ng / ml of anti-PD-L1 antibody alone.

[0190] From these results, it was concluded that the in vitro CD8 + T cell response (measured by the expression of T cell activation markers) induced by a relatively low dose of anti-PD-L1 antibody is multiplicatively increased by a soluble LAG-3 derivative. Furthermore, if anti-PD-L1 antibody is combined with a soluble LAG-3 derivative, a dramatic improvement in the in vitro CD8 +It was concluded that a T cell response was obtained.

[0191] PD-1 pathway inhibitors (such as Keytruda and Opdivo) are known to activate CD8 + T cells, and since this activation is associated with an anti-cancer effect, the results shown in the above examples provide evidence that an improved anti-cancer effect can be obtained by co-administering a PD-1 pathway inhibitor with an LAG-3 protein or a derivative thereof that can bind to an MHC class II molecule. Alternatively, a similar anti-cancer effect can be achieved by co-administering a PD-1 pathway inhibitor with an LAG-3 protein (or a derivative thereof that can bind to an MHC class II molecule) at a lower dose of the PD-1 pathway inhibitor (e.g., 1 / 30 to 1 / 100 of the dose) compared to administration of the PD-1 pathway inhibitor as a monotherapy. Such co-administration is expected to reduce the side effects caused by the PD-1 pathway inhibitor.

[0192] Similarly, activation of CD8 + T cells is also known to be effective against infections including chronic or persistent infections. Thus, the results shown in the above examples also provide evidence that co-administration of a PD-1 pathway inhibitor with an LAG-3 protein or a derivative thereof that can bind to an MHC class II molecule can be used to more effectively prevent, treat or recover from an infection. Alternatively, a similar effect against infection can be achieved by co-administering a PD-1 pathway inhibitor with an LAG-3 protein (or a derivative thereof that can bind to an MHC class II molecule) at a lower dose of the PD-1 pathway inhibitor (e.g., 1 / 30 to 1 / 100 of the dose) compared to administration of the PD-1 pathway inhibitor as a monotherapy. Such co-administration is expected to reduce the side effects caused by the PD-1 pathway inhibitor. (Example 12) Effect of LAG-3Ig and various anti-PD-1 antibodies or anti-PD-L1 antibodies on IFN-γ and TNF-α production induced by antigen stimulation

[0193] This example demonstrates the effect of the soluble derivative of LAG-3 (LAG-3Ig) and various different anti-PD-1 antibodies or anti-PD-L1 antibodies on in vitro T cell activation using IFN-γ and TNF-α secretion assays.

[0194] PBMCs from healthy donors (0.2 × 10 6 / well, in complete RPMI + 10% FBS at 1M / ml) were incubated in triplicate with a pool of peptides containing the sequence of CMV pp35, with no additives (medium), with 30 ng / ml or 1000 ng / ml of an anti-PD-1 antibody (Ab1 or Ab2) or an anti-PD-L1 antibody (Ab3, Ab4, Ab5 or Ab6), with 10 or 30 ng / ml of LAG-3Ig, or with 10 or 30 ng / ml of LAG-3Ig and 30 ng / ml of an anti-PD-1 or anti-PD-L1 antibody.

[0195] T cell responses were evaluated by measuring the concentrations of IFN-γ and TNF in the cell culture supernatant 3 days after stimulation using a BD Cytometry Bead Array. Anti-PD-1: Ab1 (clone MIH4 from BD Pharmingen, catalog #557823) and Ab2 (humanized anti-PD-1 from BPS bioscience, catalog #71120); Anti-PD-L1: Ab3 (clone MIH1 from eBioscience, catalog #16-5983-82), Ab4 (clone MIH5 from eBioscience, catalog #16-5982-81), Ab5 (clone 1-111A from eBioscience, catalog #14-9971-81) and Ab6 (humanized anti-PD-L1 from BPS bioscience, catalog #71213).

[0196] The concentrations of IFN-γ and TNF-α in the pooled triplicates under each stimulation condition for the anti-PD-1 antibody are recorded in Table 25. The results are plotted in Figure 17.

Table 25

[0197] The results show that, for each anti-PD-1 antibody, when PBMCs were incubated in the presence of LAG-3Ig and a lower concentration of the anti-PD-1 antibody, the secretion of IFN-γ increased compared to the anti-PD-1 antibody alone. For example, the increase in the concentration of IFN-γ exceeding the background level (i.e., the concentration of IFN-γ in the absence of anti-PD-1 and LAG-3Ig) in the presence of 30 ng / ml of LAG-3Ig and 30 ng / ml of Ab1 anti-PD-1 antibody, or 10 ng / ml of LAG-3Ig and 30 ng / ml of Ab2 anti-PD-1 antibody, was greater than the sum of the corresponding increases in the presence of LAG-3Ig alone and 30 ng / ml of the anti-PD-1 antibody alone (i.e., for Ab1, 235.1 > -53.3 + 144.6; for Ab2, 273.8 > 176.1 + 18.2). Thus, the effects of the combination of LAG-3Ig and each different anti-PD-1 antibody were synergistic.

[0198] The results also show that the secretion of IFN-γ induced by the combination of LAG-3Ig and a relatively low concentration of anti-PD-1 antibody (30 ng / ml) was much higher than the secretion of IFN-γ induced by the anti-PD-1 antibody alone at a much higher concentration (1000 ng / ml, 30-fold higher) (i.e., for Ab1, 235.1 > 107.7; for Ab2, 273.8 > 27.3).

[0199] Regarding TNF-α secretion, neither the single anti-PD-1 antibody (at relatively low or high concentrations) had a significant effect on TNF-α secretion. However, with each anti-PD-1 antibody, when PBMCs were incubated in the presence of LAG-3Ig and a lower concentration of the anti-PD-1 antibody compared to the single anti-PD-1 antibody, the secretion of TNF-α increased. For example, the increase in the concentration of TNF-α above the background level (i.e., the concentration of TNF-α in the absence of anti-PD-1 and LAG-3Ig) in the presence of 30 ng / ml of LAG-3Ig and 30 ng / ml of Ab1 anti-PD-1 antibody, or 10 ng / ml of LAG-3Ig and 30 ng / ml of Ab2 antibody, was greater than the sum of the corresponding increases in the presence of single LAG-3Ig and single 30 ng / ml anti-PD-1 antibody (i.e., for Ab1, 118.7 > 0.9 + 82.2; for Ab2, 12.778 > 2.563 + 9.858). Thus, the effects of the combination of LAG-3Ig and each different anti-PD-1 antibody were synergistic.

[0200] The results also show that the secretion of TNF-α induced by the combination of LAG-3Ig and a relatively low concentration of anti-PD-1 antibody (30 ng / ml) was dramatically higher than the secretion of TNF-α induced by the anti-PD-1 antibody alone at a much higher concentration (1000 ng / ml, 30-fold higher) (i.e., for Ab1, 118.7 > 1.6; for Ab2, 12.778 > 2.494).

[0201] From these results, it was concluded that the in vitro T cell responses (measured by the secretion of IFN-γ and TNF-α) induced by relatively low doses of anti-PD-1 antibodies are synergistically increased by the soluble LAG-3 derivative. Furthermore, it was concluded that if the anti-PD-1 antibody is combined with the soluble LAG-3 derivative, a significantly greater in vitro T cell response can be obtained using less than one-thirtieth of the anti-PD-1 antibody. These effects were seen with different anti-PD-1 antibodies.

[0202] Record the concentrations of IFN-γ and TNF-α in the pooled three replicates under each stimulation condition for the anti-PD-L1 antibody in Table 26. Plot the results in Figure 18.

Table 26

[0203] The results show that for each anti-PD-L1 antibody, when PBMCs were incubated in the presence of LAG-3Ig and a lower concentration of the anti-PD-L1 antibody compared to the individual anti-PD-L1 antibody alone, the secretion of IFN-γ increased. For example, the increase in the concentration of IFN-γ above the background level (i.e., the concentration of IFN-γ in the absence of anti-PD-L1 and LAG-3Ig) in the presence of 10 or 30 ng / ml of LAG-3Ig and 30 ng / ml of the anti-PD-L1 antibody was greater than the sum of the corresponding increases in the presence of 10 or 30 ng / ml of LAG-3Ig alone and 30 ng / ml of the anti-PD-L1 antibody alone (i.e., for Ab3, 226.5 > 28.5 + 79.1; for Ab4, 126.03 > 2.31 + 8.00; for Ab5, 180.34 > 19.84 + 30.11; for Ab6, 95.14 > -16.51 + 19.84). Thus, the effect of the combination of LAG-3Ig and each different anti-PD-L1 antibody was synergistic.

[0204] The results also show that the secretion of IFN-γ induced by the combination of LAG-3Ig and a relatively low concentration of the anti-PD-L1 antibody (30 ng / ml) was dramatically higher than the secretion of IFN-γ induced by the anti-PD-L1 antibody alone at a much higher concentration (1000 ng / ml, 30 times higher) (i.e., for Ab3, 226.5 > 55.5; for Ab4, 126.03 > -10.66; for Ab5, 180.34 > 10.89; for Ab6, 95.14 > -49.61).

[0205] Regarding TNF-α secretion, in the presence of anti-PD-L1 antibodies Ab3, Ab4, and Ab5, when PBMCs were incubated in the presence of LAG-3Ig and a lower concentration of anti-PD-L1 antibody compared to the individual anti-PD-L1 antibody, TNF-α secretion increased. For example, the increase in TNF-α concentration above the background level (i.e., the concentration of TNF-α in the absence of anti-PD-L1 and LAG-3Ig) in the presence of 10 or 30 ng / ml of LAG-3Ig and 30 ng / ml of Ab3, Ab4, or Ab5 anti-PD-L1 antibody was greater than the sum of the corresponding increases in the presence of LAG-3Ig alone and 30 ng / ml of Ab3, Ab4, or Ab5 anti-PD-L1 antibody alone (i.e., for Ab3, 9.0 > -1.8 + 2.4; for Ab4, 80.34 > 2.08 + 58.71; for Ab5, 137.84 > 5.53 + 84.21). Thus, the effects of LAG-3Ig and the combination of these different anti-PD-L1 antibodies were synergistic.

[0206] No synergistic effect on TNF-α secretion was observed with anti-PD-L1 antibody Ab6 combined with LAG-3Ig, which may be due to the high level of TNF-α secretion in the presence of this antibody alone. Nevertheless, the level of TNF-α secretion in the presence of the combination of Ab6 and LAG-3Ig was higher than in the presence of Ab6 antibody alone (30 ng / ml and 1000 ng / ml).

[0207] The results also show that the secretion of TNF-α induced by the combination of LAG-3Ig and a relatively low concentration of anti-PD-L1 antibody (30 ng / ml) was dramatically higher than the secretion of TNF induced by anti-PD-L1 antibody alone at a much higher concentration (1000 ng / ml, 30 times higher) (i.e., for Ab3, 9.0 > 0.6; for Ab4, 80.34 > 2.50; for Ab5, 137.84 > 4.00; for Ab6, 100.99 > 47.81).

[0208] From these results, it was concluded that the in vitro T cell responses (measured by the secretion of IFN-γ and TNF) induced by relatively low doses of anti-PD-L1 antibody are synergistically increased by the soluble LAG-3 derivative. Furthermore, it was concluded that if the anti-PD-L1 antibody is combined with the soluble LAG-3 derivative, a significantly greater in vitro T cell response can be obtained using less than one-thirtieth of the anti-PD-L1 antibody. These effects were seen with different anti-PD-L1 antibodies. (Example 13) Effect of LAG-3 derivative and anti-PD-1 antibody on IFN-γ production induced by antigen stimulation

[0209] This example demonstrates the effect of various different soluble derivatives of LAG-3 (derivatives (i), (ii) and (iv) described in Example 5 and illustrated in Figure 7) and anti-PD-1 antibody on T cell activation in vitro using an IFN-γ secretion assay.

[0210] PBMC from healthy donors (0.2 × 10 6 / well, in complete RPMI + 10% FBS at 1 M / ml) were incubated in triplicate with a pool of peptides containing the sequence of CMV pp35, without any additives (medium), with 30 ng / ml or 1000 ng / ml of anti-PD-1 antibody (EH12 clone), with 30 ng / ml of LAG-3 derivative (IMP321, IMP321 R75A or LAG3 D1D4-linker2-Ig), or with 30 ng / ml of LAG-3 derivative and 30 ng / ml of anti-PD-1.

[0211] The T cell response was evaluated by measuring the concentration of IFN-γ in the cell culture supernatant 3 days after stimulation using a BD Cytometry Bead Array.

[0212] The concentration of IFN-γ in the pooled triplicates for each stimulation condition was recorded in Table 27. The results were plotted in Figure 19.

Table 27

[0213] The results show that for each LAG-3 derivative, when PBMCs were incubated in the presence of 30 ng / ml of the LAG-3 derivative and 30 ng / ml of the anti-PD-1 antibody, the secretion of IFN-γ increased compared to 30 ng / ml of the LAG-3 derivative or 30 ng / ml of the anti-PD-1 antibody alone. For example, the increase in the concentration of IFN-γ exceeding the background level (i.e., the concentration of IFN-γ in the absence of anti-PD-1 and the LAG-3 derivative) in the presence of 30 ng / ml of the LAG-3 derivative and 30 ng / ml of the anti-PD-1 antibody was greater than the sum of the corresponding increases in the presence of 30 ng / ml of the LAG-3 derivative alone and 30 ng / ml of the anti-PD-1 antibody alone (i.e., for IMP321, 572.3 > 249.7 + 22.3; for IMP321 R75A, 511.2 > 317.3 + 22.3; for LAG3 D1D4-linker2-Ig, 520.7 > 258.0 + 22.3). Thus, the effect of the combination of the anti-PD-1 antibody and each different LAG-3 derivative was synergistic.

[0214] The results also show that the secretion of IFN-γ induced by the combination of each LAG-3 derivative and a relatively low concentration of the anti-PD-1 antibody (30 ng / ml) was dramatically higher than the secretion of IFN-γ induced by the anti-PD-1 antibody alone at a much higher concentration (1000 ng / ml, 30-fold higher) (i.e., for IMP321, 572.3 > 85.9; for IMP321 R75A, 511.2 > 85.9; for LAG3 D1D4-linker2-Ig, 520.7 > 85.9).

[0215] From these results, it was concluded that in vitro T cell responses (measured by IFN-γ secretion) induced by relatively low doses of anti-PD-1 antibody are synergistically increased by various different soluble LAG-3 derivatives, each of which retains the ability to bind to MHC class II positive cells. Furthermore, it was concluded that if the anti-PD-1 antibody is combined with any of the soluble LAG-3 derivatives, significantly larger in vitro T cell responses can be obtained using less than one-thirtieth of the anti-PD-1 antibody.

Claims

【Claim 1】 The composition described in the specification.

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