IN VITRO ASSAYS AND KITS FOR SCREENING AND DETERMINING THE ACTIVITY OF LAG-3 AGONISTS

Abstract

Assays for screening or determining the activity of a lymphocyte activation gene 3 (LAG-3) agonist are described. According to the assays, a plurality of effector T cells are provided, each effector T cell expressing LAG-3 and a T cell receptor (TCR) on its surface, and comprising a reporter gene encoding a reporter gene, wherein reporter expression is regulated by LAG-3-mediated inhibition of TCR signaling within the effector T cells. Agonist activity is determined from the degree to which reporter expression is altered in the presence of the agonist compared to reporter expression in the absence of the agonist. The assays can be used to determine the potency of an agonist preparation as part of a quality control step in agonist production, or for stability testing of an agonist preparation. Kits for performing the assays are also described.

Description

ESSAYS FIELD OF INVENTION This invention relates to assays for screening or determining the activity of (including determining the potency of a preparation of) a lymphocyte activation gene 3 (LAG-3) agonist. The invention also relates to kits for performing the assays. BACKGROUND OF THE INVENTION Stimulation of T cell function begins after the T cell receptor (TCR) interacts with short peptides presented by MHC class I or II molecules (MHC I for CD8 T cells, MHC II for CD4 T cells) on the surface of antigen-presenting cells (APCs). In primary T cells, the TCR alone is not capable of activating the subsequent pathways to initiate T cell activation. This also requires coreceptors, such as CD4 for helper T cells and CD8 for cytotoxic T cells. These coreceptors bind to their respective MHC molecules and stabilize the interaction between T cells and APCs. In addition to TCR binding to the antigen-laden MHC, both helper and cytotoxic T cells require a series of secondary signals to become fully activated and responsive. In the case of helper T cells, the first of these signals is provided by CD28.This protein is the receptor for two molecules expressed on the APC (CD80 and CD86) and initiates T cell proliferation, leading to the expansion of antigen-specific T cell clones. Cytotoxic T cells are less dependent on CD28 for activation but require signals from other costimulatory molecules such as CD70 and CD137. The TCR is located in close proximity to a complex of signaling molecules that mediate T cell activation through multiple signaling cascades (see Figure 1 for an overview of TCR signaling). These signaling molecules include the CD3 protein family. Once the TCR is properly coupled to the peptide-MHC complex, conformational changes are induced in the associated CD3 chains, leading to their phosphorylation and association with downstream proteins. The TCR zeta chain is also phosphorylated upon TCR docking. These molecules are phosphorylated by the kinases Src, leukocyte-specific tyrosine kinase (LCK), and Fyn, via their C-terminal immunoreceptor tyrosine-based activation motifs (ITAMs). Phosphorylated CD3 ITAMs recruit and activate the 70 kDa zeta-activated protein of the Syk family kinase (ZAP70). ZAP70 then phosphorylates a membrane-associated scaffold protein called link for T cell activation (LAT). LAT, in turn, recruits a second molecular scaffold, a leukocyte protein containing the 76 kDa SH2 domain (Slp-76). Slp-76 is then phosphorylated by ZAP70, and the resulting LAT-Slp-76 complex acts as a scaffold for the recruitment of signaling effector molecules. Interleukin-2-inducible tyrosine kinase (ITK) then interacts with the LAT-Slp-76 complex and is activated by autophosphorylation. This promotes the phosphorylation of the effector molecule phospholipase C gamma (PLC-y1). PLC-y1 transduces TCR signals by cleaving phosphatidylinositol triphosphate (PIP2) in the plasma membrane to generate the second messengers diacylglycerol (DAG) and inositol triphosphate (IP3). DAG, a membrane-associated lipid, activates a series of downstream proteins, including several isoforms of protein kinase C (PKC) and the guanylate nucleotide-releasing protein RAS (RasGRP). Following activation by DAG, PKC-theta participates in the activation of the NF-κB pathway, while RasGRP is a crucial activator of MAPK signaling pathways. IP3 stimulates the efflux of Ca2+ from the endoplasmic reticulum into the cytoplasm. Elevated Ca2+ levels induce the activation of the protein phosphatase calcineurin, which then dephosphorylates the T-cell transcription factor nuclear factor activated T cells (NFAT). The dephosphorylated NFAT then migrates to the nucleus to bind to other transcription factors to induce the transcription of specific genes. Uncontrolled immune responses to pathogens or self-antigens can cause inflammatory tissue damage and autoimmune diseases. To prevent this, immune responses are regulated by a balance between costimulatory and inhibitory signals, collectively called immune checkpoints, which are necessary to maintain self-tolerance and protect the host from tissue damage. Activated T cells express multiple co-inhibitory receptors, such as lymphocyte activation gene 3 (LAG-3), programmed cell death protein 1 (PD-1), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), and T-cell immunoglobulin and tyrosine receptor-based inhibitory motif domain [ITIM] (TIGIT). Inhibitory immune checkpoint receptors have been shown to modulate T-cell responses to self-proteins, as well as to chronic infections and tumor antigens.Inhibitory immune checkpoint receptors are targets of cancer immunotherapy due to their potential for use in multiple types of cancer. LAG-3 is a type I membrane protein homologous to CD4 with four extracellular Ig superfamily domains. Similar to CD4, LAG-3 oligomerizes on T cell surfaces and binds to MHC class II molecules on APCs, but with significantly higher affinity than CD4. LAG-3 is expressed on activated CD4-positive and CD8-positive T lymphocytes, where it associates with the CD3-TCR complex on the cell surface and negatively regulates signal transduction. Consequently, it negatively regulates T cell proliferation, function, and homeostasis. When the MHC class II peptide complex is recognized by a specific TCR, intracellular signals are transduced in the T cell via the TCR and in the APC via MHC class II molecules. The negative regulatory role of LAG-3 signaling in T cells operates in the primary responses of human CD4+ and CD8+ T cells (Magon-Lemaitre, et al., Immunology.June 2005; 115 (2): 170-178). However, the molecular mechanism by which LAG-3 negatively regulates signal transduction in T cells is unknown. The inhibitory function of LAG-3 requires its intracellular (IC) region, but this region does not contain a typical signaling motif with a known signaling mechanism. Maeda et al. (J. Biol. Chem. 2019, RA119.007455) recently reported that LAG-3 transduces two independent inhibitory signals through an FxxL motif in the proximal membrane region and the C-terminal EX repeat. However, these motifs have not been previously reported for inhibitory coreceptors, and the molecular mechanism of the inhibitory signal transduced by LAG-3 remains elusive. WO 2017 / 037203 describes antibodies (such as the humanized monoclonal antibody IMP761) and their antigen-binding fragments, which are LAG-3 agonists, and their use for the treatment of conditions associated with the proliferation and / or activation of CD4+ and / or CD8+ T lymphocytes, particularly inflammatory and autoimmune disorders. Conventional methods used to measure the activity of antibodies and other drugs designed to target immune checkpoint receptors rely on primary human cells and the measurement of functional endpoints, such as cell proliferation, cell surface marker expression, and cytokine production. These assays are laborious and highly variable due to their dependence on donor primary cells, complex assay protocols, and unqualified assay reagents. As a result, these assays are difficult to establish in quality-controlled drug development environments. To address these challenges, Promega Corporation has developed cell-based bioluminescent reporter bioassays for single and combined immune checkpoint immunotherapy targets (Cheng et al.)., Junio de 2016, Promega, “Quantitative Cell-Based Bioassays for Indivisual and Combination Immune Checkpoint Immunotherapy Targets”). The bioassays developed by Promega include blocking bioassays for PD-1, CTLA4, LAG-3, and TIGIT. These assays are based on the use of Jurkat T cells, which have been genetically modified to express the co-inhibitory receptor of interest on their surface and contain a firefly luciferase reporter gene under the control of an NFAT response element (NFAT-ER). These cells express endogenous TCR, CD3, and CD28 receptors. When the cells are coupled to an appropriate ligand, the TCR transduces intracellular signals, resulting in an increase in NFAT-ER-mediated luminescence. The bioluminescent signal is detected and quantified using a luciferase substrate and a standard luminometer.The assays also utilize artificial antigen-presenting cells (aAPCs) that express a cell surface protein designed to activate TCRs in an antigen-independent manner and that express a natural ligand for the co-inhibitory receptor on their surface (for the PD-1, CTLA-4, and TIGIT blockade bioassays), or Raji cells in the presence of a staphylococcal enterotoxin E (SEE) superantigen (for the LAG-3 blockade bioassay: Raji cells naturally express MHC class II, a LAG-3 ligand). TCR engagement in Jurkat cells induces luciferase activity. Co-binding of the co-inhibitory receptor with its natural ligand inhibits luciferase activity. Antibody-mediated blockade of co-inhibitory receptor binding to its natural ligand restores luciferase activity. These bioassays demonstrate the required performance for use in the detection of antagonist antibodies, potency testing, and stability studies. However, they are not suitable for testing agonist antibodies, such as the LAG-3 agonist antibody (e.g., IMP761, described in WO 2017 / 037203). In particular, the presence of a natural ligand for LAG-3 in the blocking bioassay (such as MHC class II-expressing Raji cells) would interfere with the testing of an agonist. Therefore, there is a need to provide in vitro assays to identify and test LAG-3 agonists. It has now been surprisingly discovered that IMP761, through LAG-3 agonism, inhibits TCR signaling, particularly NFAT-regulated gene expression, in anrr in / ι zoz / e / yl-positive T cells LAG-3. The applicant has appreciated that this can form the basis of in vitro bioassays to determine the activity of a LAG-3 agonist, or a preparation of a LAG-3 agonist, and to identify new LAG-3 agonists. BRIEF DESCRIPTION OF THE INVENTION According to the invention, an in vitro assay is provided for determining the activity of a lymphocyte activation gene 3 (LAG-3) agonist, comprising: providing a plurality of effector T cells, each effector T cell expressing LAG-3 and a T cell receptor (TCR) on its surface, and comprising a reporter gene encoding a reporter, wherein reporter expression is regulated by LAG-3-mediated inhibition of TCR signaling within the effector T cells; and determining agonist activity from the degree to which reporter expression is altered (i.e., increased or decreased) in the presence of the agonist compared to reporter expression in the absence of the agonist. The assays of the invention for determining agonist activity include assays to determine the potency of an agonist preparation, for example, as part of a quality control step in the production of the agonist (in particular, as a cell-based potency assay required for product release in accordance with Good Manufacturing Practices, GMP), or for stability testing of an agonist preparation, for example, after a period of storage, or as a product characterization assay. Also provided according to the invention is an in vitro assay for the detection of a LAG-3 agonist, comprising: providing a plurality of effector T cells, each effector T cell expressing LAG-3 and a T cell receptor (TCR) on its surface, and comprising a reporter gene encoding a reporter, wherein reporter expression is regulated by LAG-3-mediated inhibition of TCR signaling within the effector T cells; and determining whether a candidate agonist is a LAG-3 agonist by determining the extent to which reporter expression is altered (i.e., increased or decreased) in the presence of the candidate agonist compared to reporter expression in the absence of the candidate agonist. The assays of the invention for screening a LAG-3 agonist can be used to identify a LAG-3 agonist, for example, from a gene library of candidate agonists. Such candidate agonists may be drugs (for example, synthetic small molecules) or biological agents, such as recombinant or natural proteins, antibodies, or fragments or derivatives thereof. The reporter can be expressed in effector T cells in the absence of the agonist or candidate agonist. Optionally, reporter expression is reduced in the presence of the agonist or candidate agonist, compared to reporter expression in the absence of the agonist or candidate agonist. For example, inhibition of LAG-3-mediated TCR signaling within effector T cells can cause a reduction in the basal level of reporter expression in effector T cells (see Figure 2A). Therefore, the activity of a LAG-3 agonist (including the potency of a LAG-3 agonist preparation) can be determined, or a LAG-3 agonist can be identified, by determining the degree to which the basal level of reporter expression is reduced in the presence of the agonist, or candidate agonist, compared to the basal level of reporter expression in the absence of the agonist or candidate agonist. The term “LAG-3-mediated inhibition of TCR signaling within effector T cells” is used in this document to mean that LAG-3 agonism expressed on the surface of effector T cells inhibits TCR-mediated signal transduction within effector T cells, with a consequent alteration (i.e., an increase or decrease) in reporter gene expression. Any TCR-mediated signaling pathway within effector T cells may be inhibited. For example, reporter gene expression may be under the control of a promoter or a response element (RE) that responds to the binding of a transcription factor that is part of the signaling pathway. For example, the calcineurin / NFAT signaling pathway within effector T cells may be inhibited.In particular, reporter gene expression may be under the control of an NFAT response element, such that a reduction in calcineurin / NFAT signaling, following LAG-3 agonism, causes a reduction in reporter gene expression. Reporter expression can be altered (i.e., increased or decreased) in each effector T cell in response to effector T cell activation via the TCR. Optionally, the assays of the invention further comprise: activating effector T cells by antigen-independent, MHC class II-independent TCR-mediated T cell activation in the presence and absence of the agonist or candidate agonist; and determining the activity (including the potency of a preparation) of the agonist or candidate agonist from the degree to which reporter expression, in response to effector T cell activation, is altered in the presence of the agonist or candidate agonist compared to reporter expression, in response to effector T cell activation, in the absence of the agonist or candidate agonist. Therefore, according to the invention, an in vitro assay is provided for determining the activity of a lymphocyte activation gene 3 (LAG-3) agonist, comprising: providing a plurality of effector T cells, each effector T cell expressing LAG-3 and a T cell receptor (TCR) on its surface, and comprising a reporter gene encoding a reporter, the expression of which is altered in response to activation of the effector T cell via the TCR, wherein the reporter expression is regulated by LAG-3-mediated inhibition of TCR signaling within the effector T cells; to activate effector T cells by TCR-mediated, antigen-independent, MHC class II-independent T cell activation in the presence and absence of the agonist; and to determine agonist activity from the degree to which reporter expression, in response to activation of effector T cells, is altered in the presence of the agonist compared to reporter expression, in response to activation of effector T cells, in the absence of the agonist. Qbrri n / ι znz / E / Yl· According to the invention, an in vitro assay for screening a LAG-3 agonist is also provided, comprising: providing a plurality of effector T cells, each effector T cell expressing LAG-3 and a T cell receptor (TCR) on its surface, and comprising a reporter gene encoding a reporter, the expression of which is altered in response to activation of the effector T lymphocyte via the TCR, wherein the reporter expression is regulated by LAG-3-mediated inhibition of TCR signaling within the effector T lymphocytes; to activate effector T cells by TCR-mediated, antigen-independent, MHC class II-independent T cell activation in the presence and absence of the candidate agonist; and to determine the activity of the candidate agonist from the degree to which reporter expression, in response to activation of effector T cells, is altered in the presence of the candidate agonist compared to reporter expression, in response to activation of effector T cells, in the absence of the candidate agonist. Optionally, reporter expression increases in each effector T cell in response to activation of the effector T cell via the TCR, and is reduced in the presence of the agonist, or candidate agonist, as a result of TCR signaling-mediated inhibition of LAG-3 within the effector T cells, and wherein the activity of the agonist, or candidate agonist, is determined from the degree to which reporter expression, in response to activation of effector T cells, is reduced in the presence of the agonist, or candidate agonist, compared to reporter expression, in response to activation of effector T cells, in the absence of the agonist, or candidate agonist. The greater the degree to which reporter expression is altered in response to activation of effector T cells in the presence of the agonist, or candidate agonist, compared to reporter expression in response to activation of effector T cells in the absence of the agonist, or candidate agonist, the greater the activity (or potency of a preparation) of the agonist, or candidate agonist, for LAG-3. Optionally, effector T cells are activated by cell-free T cell activation. The term “cell-free T cell activation” is used in this document to refer to the activation of effector T cells by the use of one or more cell-free T cell activators in the absence of cells other than effector T cells. In particular, cell-free T cell activation occurs without the use of an antigen-presenting cell (APC), an artificial APC (aAPC), or any other cell expressing MHC class I or MHC class II, such as a Raji cell. Cell-free T-cell activation is advantageous because cell-based reagents require special storage conditions. Typically, cells are stored frozen and must then be thawed before use. The reference to “activation of the effector T cell via the TCR” is used herein to mean that upon binding of the TCR by a T cell activator, the TCR transduces a signal within the effector T cell, resulting in an alteration (i.e., an increase or decrease) in reporter gene expression within the effector T cell. For example, reporter gene expression may be under the control of a promoter or a response element (RE) that responds to an activation signal from the effector T cell. For example, the response element may be bound by one or more transcription factors after activation of the effector T cell via the TCR. Optionally, effector T cells are activated by contacting effector T cells with an antigen-independent, MHC class II-independent T cell activator under conditions for antigen-independent, MHC class II-independent, TCR-mediated activation of effector T cells by the T cell activator. Also provided according to the invention is a kit for carrying out an in vitro assay to determine the activity of, or screen, a LAG-3 agonist, comprising: a plurality of effector T cells, each effector T cell expressing LAG-3 and a T cell receptor (TCR) on its surface, and comprising a reporter gene encoding a reporter, wherein the expression of the reporter is regulated by LAG-3-mediated inhibition of TCR signaling within the effector T cells, and wherein the expression of the reporter is altered (i.e., increased or decreased) in each effector T cell in response to activation of the effector T cell via the TCR; and a T cell activator capable of antigen-independent, MHC class II-independent, TCR-mediated activation of the effector T cells. Antigen-independent, MHC class II-independent T cell activators are distinct from superantigens, such as staphylococcal enterotoxins (SEs). SEs have binding regions for both MHC class II and the T cell receptor (TCR). They bind first to MHC class II, then to the alpha or beta variable chain of the TCR. This interaction with the T cell receptor allows the SE to act as a wedge between the TCR and the MHC. This displaces any antigenic peptide away from the TCR and bypasses the normal T cell activation mechanism. SEs are, therefore, MHC class II-dependent T cell activators. Optionally, effector T cells are exposed to a concentration of T cell activator at which maximum inhibition of reporter expression occurs in the presence of an excess of a LAG-3 agonist. The use of such a concentration of T cell activator optimizes the precision of the assay. The applicant has discovered that the concentration of T cell activator at which maximum inhibition of reporter expression occurs in the presence of an excess of a LAG-3 agonist is a suboptimal concentration of T cell activator (i.e., a concentration that is lower than a concentration of T cell activator at which maximum reporter expression is observed in response to activation of effector T cells by the T cell activator in the absence of the agonist or candidate agonist). Optionally, the T cell activator is made contact with effector T cells at a concentration that is lower than the concentration of T cell activator at which the highest reporter expression is observed in response to activation of effector T cells by T cell activator in the absence of the agonist or candidate agonist. It will be observed that antigen-dependent stimulation expands only antigen-specific T cells, whereas antigen-independent stimulation expands up to 100% of effector T cells. An example of a T cell activator capable of TCR-mediated, antigen-independent, MHC class II-independent activation of effector T cells is an anti-CD3 antibody. The anti-CD3 antibody binds to CD3 and activates the TCR complex without an antigenic peptide on an APC (i.e., TCR-mediated, antigen-independent, MHC class II-independent, TCR-mediated effector T cell stimulation) (see Figure 2B). Anti-CD3 antibodies can activate up to 100% of effector T cells. Optionally, the T cell activator comprises an anti-CD3 antibody, or a fragment or derivative thereof, that retains the antigen-independent, MHC class II-independent, TCR-mediated activation capacity of effector T cells. Suitable examples of an anti-CD3 antibody include OKT3 and UCHT1. Optionally, the anti-CD3 antibody is OKT3. Optionally, the anti-CD3 antibody, or a fragment or derivative thereof, is made in contact with effector T cells at a concentration of -6-30 x 1012M (1-4 ng / mL for the complete antibody, or molar equivalent for a fragment or derivative thereof). Optionally, a kit of the invention comprises an anti-CD3 antibody, or a fragment or derivative thereof, that retains the antigen-independent, MHC class II-independent, TCR-mediated activation capability of effector T cells, at a concentration to permit its use in the assay at a concentration of -6-30 x 10-12M (1-4 ng / mL for the full antibody). For example, the kit may comprise one or more aliquots of anti-CD3 antibody, or a fragment or derivative thereof, at a concentration of -6-30 x 10-12M (1-4 ng / mL for the full antibody). Optionally, effector T cells are exposed to several different concentrations of the agonist or agonist candidate. For example, multiple different assays can be performed in parallel, where the effector T cells for each different assay are exposed to a different concentration of the agonist or agonist candidate. Optionally, an IC50 value for the agonist or candidate agonist is determined for the inhibition of reporter expression. This can be done, for example, from a dose-response curve generated from the results obtained by exposing effector T cells to several different concentrations of the agonist or candidate agonist. Optionally, a trial of the invention further comprises performing a negative control trial (e.g., in parallel with the trial). For example, effector T cells may be brought into contact with the T cell activator under conditions for antigen-independent activation of effector T cells by the T cell activator in the absence of the agonist or candidate agonist, but in the presence of a molecule of the same type as the agonist or candidate agonist, but known to lack agonist activity for LAG-3 (i.e., as a negative control). For example, if the agonist or candidate agonist is an antibody, the molecule to be used as a negative control can optionally also be an antibody. Preferably, the negative control antibody is an antibody of the same isotype as the agonist or candidate agonist antibody. Optionally, the T cell activator is a cell-free T cell activator. Optionally, a kit of the invention does not comprise a cell expressing MHC class II molecules. Optionally, a kit of the invention does not comprise Raji cells. Optionally, a kit of the invention does not comprise APCs, aAPCs, or any other cell expressing MHC class I or MHC class II, such as Raji cells. Optionally, the only cells in a kit of the invention are effector T cells. Optionally, the effector T cells of a kit of the invention are frozen, e.g., cells for “Thaw and Use”. A kit of the invention for conducting a trial of the invention is also provided in accordance with the invention. Optionally, the reporter gene is a heterologous reporter gene. BRIEF DESCRIPTION OF THE FIGURES The embodiments of the invention are described below, by way of example only, with reference to the accompanying figures where: Figure 1 shows a schematic representation of TOR signaling (from Belikov, Aleksey. (2016). The role of reactive oxygen species and mitochondria in T-cell activation. 10.13140 / RG .2.1.2916.0568). Figures 2A and 2B show the mode of action of an IMP761 potency assay according to one embodiment of the invention. In the presence of anti-CD3 antibody, the anti-CD3 antibody binds to CD3 on the surface of the Jurkat reporter cell, leading to increased expression of luciferase from the NFAT / Luc reporter gene via TCR-mediated signal transduction (Figure 2A). The binding of IMP761 to LAG-3 on the surface of the Jurkat reporter cell inhibits TCR-mediated signal transduction, leading to the downregulation of luciferase expression from the NFAT / Luc reporter gene (Figure 2B). Figures 3A and 3B show the effect of stimulation of Jurkat LAG-3+ / NFAT-luc2 cells by different concentrations of anti-CD3 antibody OKT3 and UCFIT1 in the presence of 300 ng / mL of IMP761, or human IgG4 negative control antibody. Figures 4A and 4B show an example of power test results for IMP761 against a negative control of lgG4, according to one embodiment of the invention. Figure 5 shows an example of potency test results with a reference preparation of IMP761 (4°C), compared with a preparation of IMP761 after temperature stress denaturation (10 or 20 minutes at 70°C), according to one embodiment of the invention. DETAILED DESCRIPTION OF THE INVENTION Genetic reporters are widely used to study gene expression and cellular events coupled to gene expression in pharmaceutical and biomedical research. Typically, a reporter gene encoding the reporter gene is cloned into an expression vector. Qbrri n / ι znz / E / Yl· is then transferred to the cells. After transfer, the cells are analyzed for the presence of the reporter by directly measuring the reporter protein itself or the enzymatic activity of the reporter protein. Preferred reporters are those that can be easily identified and quantitatively measured when expressed in effector T cells. Those skilled in the technique are familiar with many suitable examples, including fluorescent and luminescent reporters. Optionally, the reporter can be a bioluminescent reporter, such as a luciferase. Bioluminescence is a special form of chemiluminescence found in living organisms. This type of chemiluminescence is an enzyme-catalyzed process where the high efficiency of photon emission is derived from natural evolution. The enzymes are called luciferases, and the photon-emitting substrates are luciferins. Bioluminescent chemistries have evolved from multiple independent origins and comprise many distinct molecular structures. Of the many natural forms, two have been widely used for genetic reporter assays: firefly luciferase and Renilla luciferase (Fan and Wood (2007) Bioluminescent assays for high-throughput screening. Assay Drug Dev. Technol. 5, 127-36). Bioluminescent reporter gene assays have a distinct advantage over fluorescent assays, such as green fluorescent protein (GFP), as they can provide assay sensitivity 10 to 1,000 times greater. Both fluorescence and luminescence produce photons as a consequence of energy transitions from excited molecular orbitals to lower-energy orbitals. However, they differ in how the excited orbitals are created. In luminescence, excited states are the product of exothermic chemical reactions, while in fluorescence, excited states are created by light absorption. In reporter assays, bioluminescence is advantageous because photons are not required to create the excited states. Therefore, it does not constitute an inherent background when measuring the photon flux of a sample.The resulting low background allows for the precise measurement of small changes in light over four to eight orders of magnitude in the linear range of the test. Luciferase reporter technology is based on the interaction of the enzyme luciferase with the luminescent substrate luciferin, which releases light through bioluminescence. Bioluminescence is found in several different organisms; however, firefly luciferase (Photinus pyralis) is by far the most widely used bioluminescent reporter due to both the sensitivity and convenience of the enzyme assay and the close coupling of protein synthesis with enzyme activity. This 61 kDa monomeric enzyme catalyzes a two-step oxidation reaction to produce light, generally in the green-to-yellow region, typically 550–570 nm. The gene encoding firefly luciferase (luc) is cDNA and does not require post-translational modifications. This means it is available as a mature enzyme directly after translation of its mRNA. The appropriate reporter genes that encode luciferase are commercially available from several companies, including Promega. By coupling operational regulatory elements to the expression of a luciferase gene, typically by placing the regulatory element just upstream of a gene encoding luciferase, the activation of reporter T cells can be readily detected by a luminescent signal. Reporter genes are typically located downstream of the cloned response elements. Optionally, the reporter gene is under the control of a promoter or response element. A suitable response element comprises an NFAT (nuclear factor of activated T cells) response element (NFAT-RE). As explained previously, TCR stimulation induces the release of intracellular calcium and the activation of calcineurin, which dephosphorylates the nuclear cytoplasmic factor of activated T cells (NFAT). The dephosphorylated NFAT translocates to the nucleus and binds to NFAT-ER, inducing transcription of the reporter gene. Optionally, effector T cells comprise heterologous nucleic acid comprising the reporter gene. Optionally, effector T cells comprise heterologous nucleic acid encoding LAG-3. The term “heterologous” reporter gene or nucleic acid is used herein to include a reference to a reporter gene or nucleic acid that is not naturally present in effector T cells, but has been introduced into effector T cells or effector T cells from which effector T cells are derived, for example, by cloning, recombinant technology, or transfection (techniques that are well known to experts). Optionally, effector T cells are subjected to double transfection with heterologous nucleic acid comprising the reporter gene (optionally wherein the reporter gene is operatively linked to a promoter or response element to direct reporter gene expression in the effector T cell), and heterologous nucleic acid encoding LAG-3. Optionally, a kit of the invention further comprises a molecule of the same type as the agonist or candidate agonist, but known to lack agonist activity for LAG-3, for use as a negative control. For example, if the agonist or candidate agonist is an antibody, the molecule used as a negative control can optionally also be an antibody. Preferably, the negative control antibody is an antibody of the same isotype as the agonist or candidate agonist antibody. Optionally, a kit of the invention further comprises a known LAG-3 agonist for use as a positive control. For example, the known agonist may be an agonist antibody, or a fragment or derivative thereof that retains agonist activity. It will be appreciated that a trial of the invention should be carried out in the absence of a natural ligand for LAG-3 because the natural ligand would otherwise interfere with the trial results. Optionally, a kit of the invention does not include a natural ligand for LAG-3. Effector T cells include several types of T cells that actively respond to a stimulus, such as costimulation. These include CD4+, CD8+, and regulatory T cells. A suitable example is a Jurkat cell. Jurkat cells are immortalized T lymphocytes derived first from the peripheral blood of a child with T-cell leukemia (Schneider et al., 1977, Int J Cancer 19 (5): 621-6). Optionally, the agonist is an anti-LAG-3 agonist antibody, or a fragment or derivative thereof that retains anti-LAG-3 agonist activity. Optionally, effector T cells comprise a T cell line that has been double-transfected with nucleic acid encoding LAG-3 and a reporter gene under the control of a promoter or response element, for example, a Jurkat cell line double-transfected with nucleic acid encoding LAG-3 and an NFAT / luciferase reporter gene (Jurkat LAG-3+ / NFAT-luc cells). Jurkat LAG3+ / NFAT-luc2 cells are available from Promega (ref: CS194801). Jurkat LAG3+ / NFAT-luc cells are available from BPS Bioscience (Catalog #: 71278). Optionally, the agonist is an anti-LAG-3 agonist antibody, or a fragment or derivative thereof that retains anti-LAG-3 agonist activity, and the effector T cells comprise LAG-3+ / NFAT-luc2 Jurkat cells. Optionally, the anti-LAG-3 agonist antibody is an anti-LAG-3 agonist antibody described in WO 2017 / 037203. The anti-LAG-3 agonist antibodies described in WO 2017 / 037203 include the mouse monoclonal antibody 13E2 and the humanized Fe IgG4 antibody 13E2 (designated IMP761). The 13E2 and IMP761 antibodies comprise the VH CDR1-3 and VL CDR1-3 sequences as indicated in Table 1 below: αίτΓΓίη / ίζηζ / Ε / γι Table 1. CDR VH and VL sequences of the anti-LAG-3 agonist antibody Antibody: 13E2; IMP761 CDR-1 CDR-2 CDR-3 VH (IMGT numbering) GFSLSTSGMG (SEQ ID NO:1) IWWDDIK (SEQ ID NO:2) ARIVEGSYSSSYFDV (SEQ ID NO:3) VL (IMGT numbering) QDVIFD (SEQ ID NO:4) SAS (SEQ ID NO:5) QQHYSTPYT (SEQ ID NO:6) VH (Kabat numbering) TSGMGLG (SEQ ID NO:7) HIWWDDIKRYNPDLRS (SEQ ID NO:8) IVEGSYSSSYFDV (SEQ ID NO:9) VL (Kabat numbering) KASQDVIFDVA (SEQ ID NQ:10) SASSRVS (SEQ ID NO:11) QQHYSTPYT (SEQ ID NO:12) Optionally, the anti-LAG-3 agonist antibody, or a fragment or derivative thereof, comprises VH CDR1-3 and VL CDR1-3 sequences from SEQ ID Nos: 1-6, respectively, or VH CDR1-3 and VL CDR1-3 sequences from SEQ ID Nos: 7-12, respectively: Optionally, the anti-LAG-3 agonist antibody is IMP761. Example 1 Optimization of the IMP761 power test protocol The Jurkat Lag-3+ / NFAT-luc2 effector cells were initially developed by Promega to determine the activity of the anti-LAG-3 antagonist antibody after TCR activation via superantigen stimulation presented by MHC II molecules. Blocking the LAG-3 / MHC II interaction with the anti-LAG-3 antagonist antibody results in enhanced TCR activation and luciferase activity. Using Jurkat Lag-3+ / NFAT-luc2 effector cells to determine the activity of the anti-LAG-3 agonist antibody requires a very different experimental setup, as explained below. Anti-CD3 antibody as a Jurkat cell stimulator: In the Promega bioassay, Jurkat Lag-3+ / NFAT-luc2 effector cells are activated using Raji cells in the presence of Staphylococcal Enterococcal Enterococcal E or D (SEE or SED). Raji cells express endogenous MHC class II, a LAG-3 ligand. This is important for testing the blocking activity of the anti-LAG-3 antagonist antibody on the LAG-3 / MHC II interaction. However, since the LAG-3 / MHC II interaction is not required to test the potency of an anti-LAG-3 agonist antibody, there is no requirement for Raji cells or the staphylococcal enterotoxin. A single-cell-type assay with anti-CD3 antibodies was used to activate Jurkat Lag-3+ / NFAT-luc2 effector cells via TCR signaling. Anti-CD3 concentration and LAG-3 related inhibition: The effect on the cell potency assay of two different anti-CD3 antibodies (OKT3 and UCHT1) was tested at different antibody concentrations, ranging from 1 to 500 ng / mL. Jurkat Lag-3+ / NFAT-luc2 cells were incubated with 300 ng / mL of IMP761, or human IgG4 (as a negative control), in the presence of different concentrations of OKT3 or UCHT1 for 24 hours. The mean RLU values ​​obtained for the different anti-CD3 antibody concentrations are shown in Figures 3A and 3B and in Table 2 below: αίτΓΓίη / ίζηζ / Ε / γι Table 2 [anti CD3] ng / ml 0 1.95 3.91 7.81 15.63 31.25 62.50 125.00 250.00 500 OKT3 hlgG4 209832 469467 889256 1862472 3067845 4011104 4083312 2786384 2087877 1652699 IMP761 51635 89112 190253 597080 1399133 2178685 2124312 1405789 1086563 1073739 UCHT1 hlgG4 131512 138995 145635 178997 256723 497907 730163 814064 806883 827064 IMP761 56024 49957 47856 58803 79861 126859 228299 319683 453048 540205 Luciferase activity is inhibited by IMP761 across a range of different concentrations of each anti-CD3 antibody. There is a baseline level of reporter expression in the unstimulated Jurkat cell line; therefore, luciferase activity and inhibition of luciferase activity by IMP761 were also tested in the absence of anti-CD3 antibody. However, stimulation of Jurkat cells with anti-CD3 antibody yields higher RLU values, so the inhibitory effect of IMP761 is more pronounced in the presence of anti-CD3 antibody (particularly at a low concentration). The percentage of inhibition of luciferase activity for the OKT3 antibody is shown in Table 3 below. Table 3 Anti-CD3 concentration (ng / mL) % Inhibition 0 75.4 1.95 81.0 3.91 78.6 7.81 67.9 15.63 54.4 31.25 45.7 62.5 48.0 125 49.5 250 48.0 500 35.0 QfrCPLn / LZnZ / E / Yl· It was concluded that the maximum effect of IMP761 (inhibition of approximately 80%) was observed when a low concentration of OKT3 antibody was used (between 1 and 4 ng / mL). Therefore, an optimal potency assay includes stimulation of Jurkat cells with low concentrations of anti-CD3 antibody (e.g., OKT3 antibody). Example 2 IMP761 Power Test This example describes a potency assay, according to one embodiment of the invention, for measuring the activity of the monoclonal antibody IMP761 in vitro. The method is based on the ability of IMP761 to decrease the activation of a LAG-3 effector cell line induced by a low dose of an anti-CD3 antibody (clone OKT3, 3 ng / mL), mimicking antigenic stimulation of T cells. The LAG-3 effector cell line is a Jurkat T cell line that expresses LAG-3 on its surface and contains the luciferase gene under the control of an NFAT (nuclear factor of activated T cells) response element (Jurkat Lag-3+ / NFAT-elector cells iuc2, from Promega). After binding to its target, IMP761 triggers TCR-induced downregulation of NFAT-regulated expression (illustrated schematically in Figure 2B).The luciferase activity of the cell line is used to measure TCR-driven cell activation that is dampened in the presence of IMP761 activity. Therefore, the assay measures the potency of IMP761 in terms of its ability to inhibit TCR signaling. Reagents Jurkat LAG-3+ / NFAT-luc2 effector cells (Promega, ref: CS194801) RPMI 1640 (GIBCO, ref: 31870-025) L-glutamine (200 mM) (GIBCO, ref: 25030-024) HEPES (1 M) (GIBCO, ref: 15630-080) FCS (GIBCO, ref: 10270106) IMP761 (2.06 mg / mL) (IMMUTEP, lot: 270416) human lgG4, control (Biolegend, ref: 403402) Anti-CD3 (OKT3) (eBioscience, ref: 16-0037-85) Bio-Glo Reagent (PROMEGA, ref: G7941) Solid white flat bottom microplates with 96 wells (COSTAR, ref: 3917) Assay medium: RPMI 1640, L-glutamine (2 mM), Hepes (10 mM), FCS 1% Cell concentration: 1.33 x 106 cells / mL Protocol 1. Pass Jurkat LAG-3+ / NFAT-luc2 effector cells on day -1 before the assay to have a cell density of around 1 million / mL (between 0.8 and 1.2 million / mL) on day 0. 2. Prepare the test medium if necessary and preheat the medium for 30 minutes at 37°C: οήγγ Ln / Lznz / E / Yi FCS 0.5 ml L-Glutamine (200mM) 0.5 ml HEPES (1M) 0.5 ml RPMI 1640 48.5 ml 3. Prepare the IMP761 quality control (QC) stock solution, if necessary: ​​Prepare a 24,000 ng / mL stock solution; for example: 10 mL of stock IMP761 (2.06 mg / mL) + 848.3 pL of test medium. Store 25 pL aliquots in a freezer at -80°C. 4. Prepare 3X solutions of IMP761 (lot 270416 at 2.06 mg / mL), negative control of human lgG4 (or any other antibody). Adjust the pre-dilution steps according to the initial antibody concentration: the volume of assay medium (in pL) to be added to dilute 2 mL of a stock solution to prepare a pre-dilution of 100 pg / mL is: 2) ÍA of the test medium / CawísSwws ¡«έσέα.ί í«j \ k ai / IMP761 Dilution to Prepare 3X Working Solutions Pre-dilution (100 pg / mL) 2 pL of stock (2.06 mg / mL) + 39.2 pL of test medium Final Concentration (ng / mL) Serial Dilutions 2000 ng / mL 10 pL of 100 pg / mL pre-dilution + 490 pL of test medium 666.7 1000 ng / mL 200 pL of 2000 ng / mL dilution + 200 pL of test medium 333.3 500 ng / mL 200 pL of 1000 ng / mL dilution + 200 pL of test medium 166.7 250 ng / mL 200 pL of 500 ng / mL dilution + 200 pL of Test medium 83.3 125 ng / mL 200 pL of 250 ng / mL dilution + 200 pL of test medium 41.7 62.5 ng / mL 200 pL of 125 ng / mL dilution + 200 pL of test medium 20.8 31.2 ng / mL 200 pL of 62.5 ng / mL dilution + 200 pL of test medium 10.4 15.6 ng / mL 200 pL of 31.2 ng / mL dilution + 200 pL of test medium 5.2 7.8 ng / mL 200 pL of 15.6 ng / mL dilution + 200 pL of test medium 2.6 5. Prepare quality control (QC) solutions 3X IMP761 Thaw the stock aliquot of IMP761 QC [24,000 ng / mL] and perform serial dilutions Very high QC (VH): 20 pL [24,000 ng / mL] + 380 pL = 1,200 ng / mL (final concentration: 400 ng / mL) High QC (H): 150 pL [1,200 ng / mL] + 225 pL = 480 ng / mL (final concentration: 160 ng / mL) QC Medium (M): 150 pL [480 ng / mL] + 225 pL = 192 ng / mL (final concentration: 64 ng / mL) QC Low (L): 150 pL [192 ng / mL] + 225 pL = 76.8 ng / mL (final concentration: 25.6 ng / mL) Very low QC (VL): 150 pL [25.6 ng / mL] + 225 pL = 10.24 ng / mL (final concentration: 3.4 ng / mL) 6. Prepare the OKT3 reagent 3.6 pL of reserve + 4 mL of test medium = 0.9 mg / mL 150 pL of [0.9 pg / mL] + 14,850 pL of test medium = 9 ng / mL (3X) 7. Prepare Jurkat LAG-3 / NFAT-luc2 effector cells On day 0, count the cells stained with trypan blue. Centrifuge the cells at 1200 rpm for 5 minutes. Aspirate the medium and resuspend the cells at 3.75 x 106 / mL in the test medium. 8. Distribute the 3X solutions onto a 96-well plate Do not use external wells due to the rim effect Distribute 40 mL of 3X solutions of IMP761 / QC / test medium (0) into the corresponding wells of the test plate in duplicate Dispense 40 pL / well of 3X anti-CD3 solution / assay medium (unstimulated control: unstim) into each well of the assay plate in duplicate. Final concentration: 3 ng / mL Distribute 40 pL per well (0.15 x 106 / well) αίτΓΓίη / ίζηζ / Ε / γι Example plate template (reference / unknown batch) 666.7 333.3 166.7 83.3 41.7 20.8 10.4 5.2 2.6 0 666.7 333.3 166.7 83.3 41.7 20.8 10.4 5.2 2.6 0 666.7 333.3 166.7 83.3 41.7 20.8 10.4 5.2 2.6 0 666.7 333.3 166.7 83.3 41.7 20.8 10.4 5.2 2.6 0 QC VHIGH QC HIGH QC MID QC LOW QC VLOW UNSTIM QC VHIGH QC HIGH QC MID QC LOW QC VLOW UNSTIM 9. Incubate the plate(s) at 37°C in a humidified incubator, with 5% CO2, for 24 hours. 10. Prepare the BioGlo reagent a) Place the frozen BioGlo reagent at room temperature (RT) 3-6 hours before use to allow thawing b) Transfer the buffer solution (10 mL) to the substrate bottle, mix, and keep RT in the dark until use 11. Equalize the plate(s) at RT for 15 minutes 12. Add 120 pL / well of BioGlo, avoid / remove bubbles 13. Incubate at RT for 5 to 15 minutes. 14. Measure the luminescence (RLU), integration time = 0.5 sec / well with the PerkinElmer 2103 Multilabel Envision reader. 3 measurements are collected for each well with a 45-second difference, calculate the average of 3 measurements to obtain the “Average RLU”. Results Figures 4A and 4B show an example of the results obtained from the potency assay, using IgG4 antibody as a negative control. Maximum activation was recorded as the activation observed when IMP761 was absent. Maximum inhibition was recorded as the activation observed when the IMP761 concentration was 1000 ng / mL. A five-parameter nonlinear regression model determined that the IC50 of IMP761 was 37 ng / mL. Example 3 Capability of the IMP761 potency assay to evaluate the denatured IMP761 antibody The ability of the potency assay as described in Example 2 to assess the diminished efficacy of a denatured IMP761 antibody was tested by comparing the ICso of a reference IMP761 stored at 4°C with the ICso of the same lot of IMP761 after temperature stress (10 minutes at 70°C and 20 minutes at 70°C). The results are shown in Figure 5 and Table 4 below. Table 4: Data set (mean of duplicates) for a power test with a reference IMP761 (4°C) and IMP761 after denaturing temperature stress (10 and 20 minutes at 70°C), expressed as relative light units (RLU) αίτΓΓίη / ίζηζ / E / γι 70°C [IMP761] ng / mL 4°C 10 minutes 20 minutes 667 141272 139814 155807 333 139620 148490 163200 167 152260 178717 188419 83 196259 271611 292410 56 274748 365493 368244 37 316679 381826 425702 25 338896 420090 429251 12 409866 441327 430144 6.2 328883* 426592 450974 0 457495 468616 420222 ★Outliers, remove from dataset A five-parameter nonlinear regression model determined that the ICso of denatured IMP761 antibodies (74 ng / mL and 81.5 ng / mL after 10 minutes and 20 minutes at 70°C, respectively) was higher than the ICso (41 ng / mL) of the reference IMP761 antibody stored at 4°C.

Claims

1. An in vitro assay for determining the activity of a lymphocyte activation gene 3 (LAG-3) agonist, characterized in that it comprises: providing a plurality of effector T cells, each effector T cell expressing LAG-3 and a T cell receptor (TCR) on its surface, and comprising a reporter gene encoding a reporter, wherein the reporter expression is regulated by LAG-3-mediated inhibition of TCR signaling within the effector T cells; and determining the agonist activity from the degree to which the reporter expression is altered in the presence of the agonist compared to the reporter expression in the absence of the agonist.

2. The test according to claim 1, further characterized in that it is for determining the potency of a LAG-3 agonist preparation.

3. An in vitro assay for the detection of a LAG-3 agonist, further characterized in that it comprises: providing a plurality of effector T cells, each effector T cell expressing LAG-3 and a T cell receptor (TCR) on its surface, and comprising a reporter gene encoding a reporter, wherein the reporter expression is regulated by LAG-3-mediated inhibition of TCR signaling within the effector T cells; and determining whether a candidate agonist is a LAG-3 agonist by determining the extent to which the reporter expression is altered in the presence of the candidate agonist compared to the reporter expression in the absence of the candidate agonist.

4. The assay according to claim 1 or 2, further characterized in that the reporter is expressed at a basal level in effector T cells in the absence of the agonist, or an assay according to claim 3, wherein the reporter is expressed at a basal level in effector T cells in the absence of the candidate agonist.

5. The test in accordance with any of the preceding claims, further characterized in that the reporter expression is reduced in the presence of the agonist or candidate agonist, compared to the reporter expression in the absence of the agonist or candidate agonist.

6. The assay according to any of the preceding claims, further characterized in that it further comprises the reporter expression being altered in each effector T cell in response to activation of the effector T cell via the TCR, and further comprising: activating the effector T cells by TCR-mediated, MHC class II-independent, antigen-independent T cell activation in the presence and absence of the agonist, or candidate agonist; and determining the activity of the agonist, or candidate agonist, from the degree to which the reporter expression, in response to activation of the effector T cells, is altered in the presence of the agonist or candidate agonist, compared to the reporter expression, in response to activation of the effector T cells, in the absence of the agonist, or candidate agonist.

7. The assay according to claim 6, further characterized in that reporter expression increases in each effector T cell in response to activation of the effector T cell via the TCR, and is reduced in the presence of the agonist, or candidate agonist, as a result of LAG-3-mediated inhibition of TCR signaling within the effector T cell, and wherein the activity of the agonist, or candidate agonist, is determined from the degree to which reporter expression, in response to activation of effector T cells, is reduced in the presence of the agonist, or candidate agonist, compared to reporter expression, in response to activation of effector T cells, in the absence of the agonist, or candidate agonist.

8. The assay according to claim 6 or 7, further characterized in that the effector T cells are activated by contacting the effector T cells with an antigen-independent, MHC class II-independent T cell activator, under conditions of antigen-independent, MHC class II-independent TCR-mediated activation of the effector T cells by the T cell activator.

9. The assay according to claim 8, further characterized in that the effector T cells are contacted with the T cell activator at a concentration of the T cell activator at which maximum inhibition of reporter expression occurs in the presence of an excess of a LAG-3 agonist.

10. The assay according to claim 8 or 9, further characterized in that the effector T cells are contacted with the T cell activator at a concentration of the T cell activator that is lower than the concentration of the T cell activator at which the greatest reporter expression is observed in response to activation of the effector T cells by the T cell activator in the absence of the agonist or candidate agonist.

11. The assay according to any of claims 8 to 10, further characterized in that the T cell activator comprises or consists of an anti-CD3 antibody, or a fragment or derivative thereof, retaining the antigen-independent, MHC class II-independent, TCR-mediated effector T cell activation capability.

12. The test according to claim 11, further characterized in that the anti-CD3 antibody is OKT3.

13. The assay according to claim 11 or 12, further characterized in that the anti-CD3 antibody, or a fragment or derivative thereof, is brought into contact with effector T cells at a concentration of -6-30 x 10-12M (1-4 ng / mL for the complete antibody, or molar equivalent for the fragment or derivative thereof).

14. The assay according to any preceding claim, further characterized in that the effector T cells are activated by TCR-mediated, cell-free, antigen-independent, MHC class II-independent, TCR-mediated T cell activation.

15. The assay in accordance with any of the preceding claims, further characterized in that the effector T cells are brought into contact with several different concentrations of the agonist or candidate agonist.

16. The assay according to claim 15, further characterized in that it additionally comprises determining an IC50 value of the agonist, or candidate agonist, for the inhibition of reporter expression.

17. The assay in accordance with any of the preceding claims, further characterized in that the effector T cells comprise heterologous nucleic acid comprising the reporter gene.

18. The assay in accordance with any of the preceding claims, further characterized in that the reporter gene is under the control of a promoter or response element.

19. The test according to claim 18, further characterized in that the response element comprises an NFAT (nuclear factor of activated T cells) response element (NFAT-RE).

20. The test in conformity with any of the preceding claims, further characterized in that the reporter comprises a bioluminescent reporter, such as a luciferase.

21. The assay in accordance with any of the preceding claims, further characterized in that the effector T cells comprise heterologous nucleic acid encoding LAG-3.

22. The assay according to any preceding claim, further characterized in that it additionally comprises performing a negative control assay, wherein the effector T cells are activated in the absence of the agonist or candidate agonist, but in the presence of a molecule of the same type as the agonist or candidate agonist, but which is known to lack agonist activity for LAG-3.

23. The test in accordance with any of the preceding claims, further characterized in that it is carried out in the absence of a natural ligand for LAG-3.

24. The assay in accordance with any of the preceding claims, further characterized in that the agonist or candidate agonist is an anti-LAG-3 antibody, or a fragment or derivative thereof that retains anti-LAG-3 agonist activity.

25. The assay in accordance with any of the preceding claims, further characterized in that the effector T cells comprise Jurkat-derived cells.

26. The assay according to any of the preceding claims, further characterized in that the agonist is an anti-LAG-3 agonist antibody, or a fragment or derivative thereof retaining anti-LAG-3 agonist activity, and the effector T cells comprise Jurkat LAG3+ / NFAT-luc2 cells.

27. The assay according to any of the preceding claims, further characterized in that the anti-LAG-3 agonist antibody, or the fragment or derivative thereof, comprises VH CDR1-3 sequences and VL CDR1-3 sequences of SEQ ID NO: 1-6, respectively, or SEQ ID NO: 7-12, respectively.

28. The assay in accordance with any of the preceding claims, further characterized in that the anti-LAG-3 agonist antibody is IMP761.

29. A kit for performing an in vitro assay to determine the activity of, or screen, a LAG-3 agonist, characterized in that it comprises: a plurality of effector T cells, each effector T cell expressing LAG-3 and a T cell receptor (TCR) on its surface, and comprising a reporter gene encoding a reporter, wherein the reporter expression is regulated by LAG-3-mediated inhibition of TCR signaling within the effector T cells, and wherein the reporter expression is altered in each effector T cell in response to activation of the voter T cell via the TCR; and a T cell activator capable of antigen-independent, MHC class II-independent, TCR-mediated activation of the effector T cells.

30. The kit according to claim 29, further characterized in that the kit does not comprise a cell expressing MHC class II molecules.

31. The kit according to claim 29 or 30, further characterized in that reporter expression increases in response to activation of effector T cells via the TCR.

32. The kit according to any of claims 29 to 31, further characterized in that the effector T cells comprise heterologous nucleic acid comprising the reporter gene.

33. The kit according to any of claims 29 to 32, further characterized in that the reporter gene is under the control of a promoter or response element.

34. The kit according to claim 33, further characterized in that the response element comprises an NFAT (nuclear factor of activated T cells) response element (NFATRE).

35. The kit according to any of claims 29 to 34, further characterized in that the reporter comprises a bioluminescent reporter, such as a luciferase.

36. The kit according to any of claims 29 to 35, further characterized in that the effector T cells comprise heterologous nucleic acid encoding LAG-3.

37. The kit in accordance with any of claims 29 to 36, further characterized in that the T cell activator is a cell-free T cell activator.

38. The kit according to any of claims 29 to 37, further characterized in that the T cell activator comprises an anti-CD3 antibody, or a fragment or derivative thereof, retaining the antigen-independent, MHC class II-independent TCR-mediated effector T cell activation capability.

39. The kit according to claim 38, further characterized in that the anti-CD3 antibody is OKT3.

40. The kit according to claim 38 or 39, further characterized in that the anti-CD3 antibody, or a fragment or derivative thereof, is present in a concentration to permit its use in the assay at a concentration of -6-30 x 1012M (1-4 ng / mL for the complete antibody, or molar equivalent for the fragment or derivative thereof).

41. The kit according to any of claims 29 to 40, further characterized in that it additionally comprises a molecule of the same type as the agonist, but which is known to lack agonist activity for LAG-3, for use as a negative control.

42. The kit according to any of claims 29 to 41, further characterized in that it additionally comprises a known LAG-3 agonist for use as a positive control.

43. The kit in accordance with any of claims 29 to 42, further characterized in that it does not include a natural ligand for LAG-3.

44. The kit according to any of claims 29 to 43, further characterized in that the effector T cells comprise Jurkat-derived cells.

45. The kit according to any of claims 29 to 44, further characterized in that the effector T cells comprise Jurkat LAG-3+ / NFAT-luc2 cells.

46. ​​The kit in accordance with any of claims 29 to 45, further characterized in that the kit does not comprise APC, aAPC or any other cell expressing MHC class I or MHC class II.

47. The kit in accordance with any of claims 29 to 46, further characterized in that the only cells in the kit are effector T cells.

48. The kit according to any of claims 29 to 47, further characterized in that it is for determining the potency of a LAG-3 agonist preparation.

49. The kit in accordance with any of claims 29 to 48, further characterized in that it is for carrying out a test in accordance with any of claims 1 to 28.

50. Use of a kit according to any of claims 29 to 47 to determine the activity of a LAG-3 agonist, to determine the potency of a LAG-3 agonist preparation, or to screen a LAG-3 agonist.