Assay

Abstract

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

Description

[Technical Field]

[0001] The present invention relates to assays for screening for or determining the activity of lymphocyte activation gene 3 (LAG-3) agonists, including determining the efficacy of preparations thereof. The present invention also relates to kits for carrying out the assays.

[0002] Stimulation of T cell function is initiated by the interaction of the T cell receptor (TCR) with a short peptide presented by an MHC class I or II molecule (MHC1 for CD8 T cells and MHCII for CD4 T cells) on the surface of an antigen-presenting cell (APC). In primary T cells, the TCR itself cannot activate downstream 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 T cell–APC interaction. In addition to TCR binding to antigen-loaded MHC, both helper and cytotoxic T cells require many secondary signals to become fully activated and respond. In the case of helper T cells, the first of these is provided by CD28. This protein is a receptor for two molecules (CD80 and CD86) expressed on APCs, which initiate T cell proliferation and lead 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.

[0003] 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 family of proteins. Once the TCR properly engages a peptide-MHC complex, a conformational change in the associated CD3 chain is induced, resulting in phosphorylation and binding to downstream proteins. The TCR zeta chain is also phosphorylated upon TCR engagement. These molecules are phosphorylated by Src kinase, leukocyte-specific tyrosine kinase (LCK), and Fyn via C-terminal immunoreceptor tyrosine-based activation motifs (ITAMs).

[0004] Phosphorylated CD3 ITAM recruits and activates the Syk family kinase zeta-activating protein 70 kDa (ZAP70). ZAP70 then phosphorylates a membrane-bound scaffold protein called linker for activation of T cells (LAT). LAT, in turn, recruits a second molecular scaffold, the 76 kDa SH2 domain-containing leukocyte protein (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 becomes activated by autophosphorylation. This promotes the phosphorylation of the effector molecule phospholipase C gamma (PLC-γ1). PLC-γ1 transduces TCR signals by cleaving phosphatidylinositol triphosphate (PIP2) in the plasma membrane to generate the second messengers diacylglycerol (DAG) and inositol triphosphate (IP3).

[0005] DAG, a membrane-bound lipid, activates many downstream proteins, including various isoforms of protein kinase C (PKC) and RAS guanyl nucleotide-releasing protein (RasGRP). When activated by DAG, PKC theta is involved in the activation of the NF-κB pathway, whereas RasGRP is a critical activator of the MAPK signaling pathway. IP3 transports Ca from the endoplasmic reticulum to the cytoplasm. 2+ Stimulates the efflux of Ca 2+ Elevated levels induce activation of the protein phosphatase calcineurin, which in turn dephosphorylates the T cell transcription factor nuclear factor of activated T cells (NFAT). Dephosphorylated NFAT then translocates to the nucleus and joins other transcription factors in inducing the transcription of specific genes.

[0006] Uncontrolled immune responses to pathogens or self-antigens can lead to inflammatory tissue damage and autoimmune diseases. To prevent this, the immune response is regulated by a balance of costimulatory and inhibitory signals, collectively known as immune checkpoints, 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 immunoreceptor tyrosine-based inhibitory motif [ITIM] domain (TIGIT). Inhibitory immune checkpoint receptors have been shown to regulate T cell responses to self-proteins as well as chronic infections and tumor antigens. Inhibitory immune checkpoint receptors are targets for cancer immunotherapy due to their potential use in multiple types of cancer.

[0007] LAG-3 is a CD4 homolog type I membrane protein with four extracellular Ig superfamily domains. Like CD4, LAG-3 oligomerizes on the surface of T cells and binds to MHC class II molecules on APCs, but with significantly higher affinity than CD4. LAG-3 is expressed on activated CD4+ and CD8+ T lymphocytes, where it associates with the CD3 / TCR complex on the cell surface and negatively regulates signal transduction. As a result, it negatively regulates T cell proliferation, function, and homeostasis. Upon recognition of the MHC class II-peptide complex by a specific TCR, an intracellular signal is transmitted to the T cell via the TCR and to the APC via the MHC class II molecule. The negative regulatory role of LAG-3 signaling to T cells is demonstrated in primary CD4 + and CD8 + LAG-3 operates in human T cell responses (Macon-Lemaitre et al., Immunology. 2005 Jun;115(2):170-178). However, the molecular mechanism by which LAG-3 negatively regulates T cell signaling is unknown. The inhibitory function of LAG-3 requires its intracellular (IC) region, but this region does not contain typical signaling motifs with known signaling mechanisms. Maeda et al. (J. Biol. Chem. 2019, RA119.007455) recently reported that LAG-3 transmits two independent inhibitory signals via an FxxL motif in the membrane-proximal region and a C-terminal EX repeat. However, these motifs have not previously been reported for inhibitory coreceptors, and the molecular mechanism of the inhibitory signals transmitted by LAG-3 remains unclear.

[0008] WO2017 / 037203 discloses antibodies (such as humanized monoclonal antibody IMP761) that are agonists of LAG-3 and antigen-binding fragments thereof, as well as CD4 + and / or CD8 + Their use for the treatment of conditions associated with T cell proliferation and / or activation, particularly inflammatory and autoimmune diseases, is described.

[0009] Traditional 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 labor-intensive and subject to high variability due to their reliance on donor primary cells, complex assay protocols, and unqualified assay reagents. As a result, these assays are difficult to establish in a quality-controlled drug development environment. To address these challenges, cell-based bioluminescent reporter bioassays have been developed by Promega Corporation for individual and combination immune checkpoint immunotherapy targets (Cheng et al., June 2016, Promega, "Quantitative Cell-Based Bioassays for Individual and Combination Immune Checkpoint Immunotherapy Targets").

[0010] Bioassays developed by Promega include Blockade Bioassays for PD-1, CTLA-4, LAG-3, and TIGIT. These assays rely on the use of Jurkat T cells genetically engineered to express the co-inhibitory receptor of interest on their surface and containing a firefly luciferase reporter gene under the control of the NFAT response element (NFAT-RE). These cells express endogenous TCR, CD3, and CD28 receptors. When the cells engage with the appropriate ligand, the TCR transduces an intracellular signal, increasing light emission via the NFAT-RE. The bioluminescent signal is detected and quantified using a luciferase substrate and a standard luminometer. These assays use artificial antigen-presenting cells (aAPCs) that express engineered cell surface proteins designed to activate the TCR antigen-independently and express the natural ligand of the co-inhibitory receptor on their surface (PD-1, CTLA-4, and TIGIT Blockade Bioassays) or Raji cells (for the LAG-3 Blockade Bioassay—Raji cells naturally express MHC class II, the LAG-3 ligand) in the presence of the Staphylococcal Enterotoxin E (SEE) superantigen. TCR engagement on Jurkat cells induces luciferase activity. Co-engagement of the co-inhibitory receptor with its natural ligand inhibits luciferase activity. Antibody-mediated blockade of the co-inhibitory receptor's binding to its natural ligand restores luciferase activity.

[0011] These bioassays demonstrate the necessary performance for use in antagonist antibody screening, potency testing, and stability studies. However, they are not suitable for testing agonist antibodies, such as LAG-3 agonist antibodies (e.g., IMP761, described in WO2017 / 037203). In particular, the presence of natural ligands for LAG-3 in blocking bioassays (e.g., MHC class II-expressing Raji cells) interferes with agonist testing. Therefore, there is a need to provide an in vitro assay for identifying and testing LAG-3 agonists.

[0012] Surprisingly, IMP761 has been found to inhibit TCR signaling, particularly NFAT-regulated gene expression, in LAG-3-positive T cells via agonism of LAG-3. Applicant understands that this may form the basis of an in vitro bioassay for determining the activity of LAG-3 agonists or preparations of LAG-3 agonists and for identifying new LAG-3 agonists.

[0013] In accordance with the present invention, there is provided an in vitro assay for determining the activity of an agonist of lymphocyte activation gene 3 (LAG-3), the in vitro assay 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 expression of the reporter is regulated by LAG-3-mediated inhibition of TCR signaling in the effector T cells; and determining the activity of the agonist from the extent to which expression of the reporter is altered (i.e., increased or decreased) in the presence of the agonist compared to expression of the reporter in the absence of the agonist.

[0014] Assays of the invention for determining the activity of an agonist include assays for determining the potency of a preparation of an agonist, for example, as part of a quality control step in the production of the agonist (particularly as cell-based potency assays required for product release according to Good Manufacturing Practice (GMP)), or for stability testing of a preparation of the agonist, e.g., after a shelf life, or as a product characteristic assay.

[0015] The present invention also provides an in vitro assay for screening for an agonist of LAG-3, the in vitro assay 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, expression of the reporter being regulated by LAG-3-mediated inhibition of TCR signaling in the effector T cells; and determining whether the candidate agonist is an agonist of LAG-3 by determining the extent to which expression of the reporter is altered (i.e., increased or decreased) in the presence of the candidate agonist compared to expression of the reporter in the absence of the candidate agonist.

[0016] The assays of the present invention for screening LAG-3 agonists can be used, for example, to identify LAG-3 agonists from a library of candidate agonists. Such candidate agonists can be drugs (e.g., synthetic small molecules) or biological agents such as recombinant or natural proteins, antibodies, or fragments or derivatives thereof.

[0017] The reporter can be expressed in effector T cells in the absence of the agonist or candidate agonist.

[0018] Optionally, expression of the reporter is decreased in the presence of the agonist or candidate agonist compared to expression of the reporter in the absence of the agonist or candidate agonist.

[0019] For example, inhibition of TCR signaling in effector T cells via LAG-3 can cause a decrease in the basal level of reporter expression in the effector T cells (see Figure 2(a)). Thus, by determining the extent to which the basal level of reporter expression is reduced in the presence of an agonist or candidate agonist compared to the basal level of reporter expression in the absence of the agonist or candidate agonist, the activity of the LAG-3 agonist (including the efficacy of a LAG-3 agonist preparation) can be determined or a LAG-3 agonist can be identified.

[0020] The term "LAG-3-mediated inhibition of TCR signaling in effector T cells" is used herein to mean that agonism of LAG-3 expressed on the surface of effector T cells inhibits TCR-mediated signaling in the effector T cells, with a consequent change (i.e., increase or decrease) in reporter expression. A TCR-mediated signaling pathway in the effector T cells may be inhibited. For example, reporter gene expression may be under the control of a promoter or 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 in the effector T cells may be inhibited. In particular, reporter gene expression may be under the control of an NFAT response element, such that a decrease in calcineurin / NFAT signaling following agonism of LAG-3 causes a decrease in reporter expression from the reporter gene.

[0021] Expression of the reporter may change (i.e., increase or decrease) in each effector T cell in response to activation of the effector T cell via the TCR. Optionally, the assay of the invention further includes activating the effector T cell 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 of the agonist or candidate agonist (including the efficacy of the preparation) from the extent to which expression of the reporter changes in response to activation of the effector T cell in the presence of the agonist or candidate agonist, compared to expression of the reporter in response to activation of the effector T cell in the absence of the agonist or candidate agonist.

[0022] Thus, in accordance with the present invention, there is provided an in vitro assay for determining the activity of an agonist of lymphocyte activation gene 3 (LAG-3), the in vitro assay 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, expression of which is altered in response to activation of the effector T cell via the TCR and regulated by LAG-3-mediated inhibition of TCR signaling in said effector T cell; activating the effector T cells by antigen-independent, MHC class II-independent, TCR-mediated T cell activation in the presence and absence of the agonist; and determining the activity of the agonist from the extent to which expression of the reporter in response to activation of the effector T cell changes in the presence of the agonist compared to expression of the reporter in response to activation of the effector T cell in the absence of the agonist.

[0023] The present invention also provides an in vitro assay for screening for an agonist of LAG-3, the in vitro assay 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 and regulated by LAG-3-mediated inhibition of TCR signaling in said effector T cell; activating the effector T cells by antigen-independent, MHC class II-independent, TCR-mediated T cell activation in the presence and absence of the candidate agonist; determining the activity of the agonist or candidate agonist from the extent to which expression of the reporter changes in response to activation of the effector T cell in the presence of the candidate agonist compared to expression of the reporter in response to activation of the effector T cell in the absence of the agonist or candidate agonist.

[0024] Optionally, expression of a reporter increases in each effector T cell in response to activation of the effector T cell via the TCR and decreases in the presence of an agonist or candidate agonist as a result of LAG-3-mediated inhibition of TCR signaling in the effector T cell, and activity of the agonist or candidate agonist is determined from the extent to which expression of the reporter in response to activation of the effector T cell is decreased in the presence of the agonist or candidate agonist compared to expression of the reporter in response to activation of the effector T cell in the absence of the agonist or candidate agonist.

[0025] The greater the degree to which the expression of the reporter in response to activation of the effector T cells changes in the presence of the agonist or candidate agonist compared to the expression of the reporter in response to activation of the effector T cells in the absence of the agonist or candidate agonist, the greater the activity of the agonist or candidate agonist (or the efficacy of the preparation) against LAG-3.

[0026] Optionally, the effector T cells are activated by cell-free T cell activation.

[0027] The term "cell-free T cell activation" is used herein 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 any cells other than the effector T cells. In particular, the activation of cell-free T cells occurs without the use of other MHC class I- or MHC class II-expressing cells, such as antigen-presenting cells (APCs), artificial APCs (aAPCs), or Raji cells.

[0028] Cell-free activation of T cells is advantageous because cell-based reagents require special storage conditions: cells are typically maintained in cryopreservation and must be thawed before use.

[0029] References to "TCR-mediated activation of effector T cells" are used herein to mean that upon engagement of the TCR by a T cell activating factor, a signal is transduced by the TCR in the effector T cell, resulting in a change (i.e., an increase or decrease) in expression of a reporter in the effector T cell. For example, expression from a reporter gene can be under the control of a promoter or response element (RE) that responds to an activation signal in the effector T cell. For example, the response element can be bound by one or more transcription factors following activation of the effector T cell via the TCR.

[0030] Optionally, 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 for antigen-independent, MHC class II-independent, TCR-mediated activation of the effector T cells by the T cell activator.

[0031] The present invention also provides a kit for performing an in vitro assay to determine the activity of or screen for agonists of LAG-3, the kit comprising: a plurality of effector T cells, each effector T cell expressing LAG-3 and a T cell receptor (TCR) on its surface and containing a reporter gene encoding a reporter, expression of the reporter being regulated by LAG-3-mediated inhibition of TCR signaling in the effector T cell, and expression of the reporter being 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.

[0032] Antigen-independent, MHC class II-independent T cell activators are distinct from superantigens such as staphylococcal enterotoxins (SEs). SEs contain binding regions for MHC class II and TCR. They first bind to MHC class II and then to the variable alpha or beta chain of the TCR. Upon interaction with the T cell receptor, SEs can function as a wedge between the TCR and MHC. This separates antigenic peptides from the TCR, bypassing the normal mechanism of T cell activation. Therefore, SEs are MHC class II-dependent T cell activators.

[0033] Optionally, the effector T cells are contacted with a concentration of T cell activator that results in maximal inhibition of reporter expression in the presence of an excess of an agonist of LAG-3. Use of such a concentration of T cell activator optimizes the precision of the assay.

[0034] Applicants have discovered that the concentration of T cell activator at which maximal inhibition of reporter expression occurs in the presence of excess LAG-3 agonist is suboptimal (i.e., a concentration lower than the concentration of T cell activator at which maximal expression of the reporter is observed in response to activation of effector T cells by the T cell activator in the absence of the agonist or candidate agonist).

[0035] Optionally, the T cell activator is contacted with the effector T cell at a concentration of the T cell activator that is lower than the concentration at which maximal expression of the reporter is observed in response to activation of the effector T cell by the T cell activator in the absence of the agonist or candidate agonist.

[0036] It is understood that antigen-dependent stimulation expands only antigen-specific T cells, whereas antigen-independent stimulation expands up to 100% of effector T cells.

[0037] An example of a T cell activator capable of antigen-independent, MHC class II-independent, TCR-mediated activation of effector T cells is anti-CD3 antibody. Anti-CD3 antibody binds to CD3 and activates the TCR complex without antigenic peptide from an APC (i.e., antigen-independent, MHC class II-independent, TCR-mediated effector T cell stimulation) (see Figure 2(b)). Anti-CD3 antibody can activate up to 100% of effector T cells.

[0038] Optionally, the T cell activator comprises an anti-CD3 antibody, or a fragment or derivative thereof that retains antigen-independent, MHC class II-independent, TCR-mediated effector T cell activation ability. Suitable examples of anti-CD3 antibodies include OKT3 and UCHT1. Optionally, the anti-CD3 antibody is OKT3.

[0039] Optionally, the anti-CD3 antibody, or fragment or derivative thereof, is administered at a concentration of about 6-30×10 -12The antibody is contacted with effector T cells at a concentration of M (1 to 4 ng / ml for whole antibodies, or a molar equivalent thereof for fragments or derivatives thereof).

[0040] Optionally, the kit of the invention contains about 6-30 x 10 -12 The kit includes an anti-CD3 antibody, or a fragment or derivative thereof, that retains antigen-independent, MHC class II-independent, TCR-mediated effector T cell activation ability at a concentration that can be used in an assay at a concentration of about 6-30 x 10 M (1-4 ng / ml for whole antibody). For example, the kit may contain one or more aliquots of the anti-CD3 antibody, or a fragment or derivative thereof, at a concentration of about 6-30 x 10 -12 M (1 to 4 ng / ml for whole antibodies).

[0041] Optionally, the effector T cells are contacted with several different concentrations of the agonist or candidate agonist. For example, multiple different assays can be performed in parallel, with the effector T cells in each different assay being contacted with different concentrations of the agonist or candidate agonist.

[0042] Optionally, a candidate agonist or IC of the agonist to inhibit expression of the reporter 50 This can be done, for example, from a dose-response curve generated from the results obtained by contacting effector T cells with several different concentrations of the candidate agonist or agonists.

[0043] Optionally, the assay of the present invention further comprises (e.g., in parallel with) performing a negative control assay. For example, effector T cells can be contacted with a 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).

[0044] For example, if the agonist or candidate agonist is an antibody, optionally the molecule for use as a negative control is also an antibody. Preferably, the negative control antibody is of the same isotype as the agonist or candidate agonist antibody.

[0045] Optionally, the T cell activator is a cell-free T cell activator.

[0046] Optionally, the kit of the present invention does not include cells that express MHC class II molecules.

[0047] Optionally, the kit of the invention does not include Raji cells.

[0048] Optionally, the kits of the invention do not include APCs, aAPCs, or other MHC class I, or MHC class II expressing cells such as Raji cells.

[0049] Optionally, the only cells in the kits of the invention are effector T cells.

[0050] Optionally, the effector T cells of the kits of the invention are frozen, eg, "Thaw-and-Use" cells.

[0051] The present invention also provides kits of the invention for carrying out assays of the invention.

[0052] Optionally, the reporter gene is a heterologous reporter gene.

[0053] Gene reporters are widely used as indicators for studying gene expression and cellular events, in conjunction with gene expression for pharmaceutical and biomedical research. Typically, reporter genes encoding reporters are cloned into expression vectors and then transferred into cells. After transfer, cells are assayed for the presence of the reporter by directly measuring the reporter protein itself or the enzyme activity of the reporter protein. Preferred reporters are those that can be easily identified and quantitatively measured when expressed in effector T cells. Many suitable examples, including fluorescent and luminescent reporters, are known to those skilled in the art. Optionally, the reporter is a bioluminescent reporter, such as luciferase.

[0054] Bioluminescence is a specialized form of chemiluminescence found in living organisms. This class of chemiluminescence is an enzyme-catalyzed process, and the high efficiency of photon emission was achieved by natural evolution. The enzymes are called luciferases, and the photon-emitting substrates are luciferins. Bioluminescent chemistry has evolved from multiple independent origins and encompasses many different molecular structures. Of the many natural forms, two are widely used in gene reporter assays: firefly luciferase and Renilla luciferase (Fan and Wood (2007) Bioluminescent assays for high-throughput screening. Assay Drug Dev. Technol. 5, 127-36).

[0055] Bioluminescence reporter gene assays offer a distinct advantage over fluorescent assays, such as those using green fluorescent protein (GFP), by achieving 10- to 1,000-fold higher assay sensitivity. Both fluorescence and luminescence generate photons as a result of energy transfer from excited-state molecular orbitals to lower-energy orbitals. However, they differ in the way the excited-state orbitals are created. In luminescence, the excited state is the product of an exothermic chemical reaction, whereas in fluorescence, the excited state arises from the absorption of light. Bioluminescence is advantageous in reporter assays because no photons are required to create the excited state. Therefore, when measuring photon efflux from a sample, these do not constitute inherent background. The resulting low background allows for accurate measurement of small changes in light over 4-8 orders of magnitude in the linear range of the assay.

[0056] Luciferase reporter technology is based on the interaction of the enzyme luciferase with the luminescent substrate luciferin, which emits light through the process of bioluminescence. Bioluminescence is found in many different organisms. However, due to both the sensitivity and convenience of enzyme assays and the tight coupling of protein synthesis and enzyme activity, the most commonly used bioluminescent reporter to date is firefly (Photinus pyralis) luciferase. This 61 kDa monomeric enzyme catalyzes a two-step oxidation reaction to produce light usually in the green to yellow region, typically 550–570 nm. The gene encoding firefly luciferase (luc) is a cDNA, meaning no post-translational modifications are required. This means that the mature enzyme is available directly upon translation from mRNA.

[0057] Suitable reporter genes encoding luciferase are commercially available from several companies, including Promega.

[0058] By coupling the operative regulatory element with expression of the luciferase gene, typically by placing the regulatory element immediately upstream of the gene encoding luciferase, activation of effector T cells can be easily detected by a luminescent signal. A reporter gene is usually downstream of a cloned response element.

[0059] Optionally, the reporter gene is under the control of a promoter or response element. Suitable response elements include the NFAT (nuclear factor of activated T cells) response element (NFAT-RE).

[0060] As explained above, TCR stimulation induces intracellular calcium release and activation of calcineurin, which dephosphorylates the cytoplasmic nuclear factor of activated T cells (NFAT). Dephosphorylated NFAT translocates to the nucleus, binds to the NFAT-RE, and induces transcription of a reporter gene.

[0061] Optionally, the effector T cell comprises a heterologous nucleic acid comprising a reporter gene.

[0062] Optionally, the effector T cell comprises a heterologous nucleic acid encoding LAG-3.

[0063] The term "heterologous" reporter gene or nucleic acid is used herein to include reference to a reporter gene or nucleic acid that is not naturally present in the effector T cell, e.g., introduced by cloning, recombinant techniques, or transfection (techniques well known to those of skill in the art), but is not originally present in the effector T cell or the effector T cell from which the effector T cell is derived.

[0064] Optionally, the effector T cells are double-transfected with a heterologous nucleic acid comprising a reporter gene (optionally, the reporter gene is operably linked to a promoter or response element for directing expression of the reporter gene in the effector T cells) and a heterologous nucleic acid encoding LAG-3.

[0065] Optionally, the kit of the invention further comprises a molecule of the same type as the agonist or candidate agonist, but known to lack agonist activity towards LAG-3, for use as a negative control.

[0066] For example, if the agonist or candidate agonist is an antibody, optionally the molecule for use as a negative control is also an antibody. Preferably, the negative control antibody is of the same isotype as the agonist or candidate agonist antibody.

[0067] Optionally, the kit of the present invention further comprises a known agonist of LAG-3 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.

[0068] It is understood that the assays of the present invention should be performed in the absence of the natural ligand of LAG-3, as the natural ligand would otherwise interfere with the results of the assay.

[0069] Optionally, the kit of the invention does not include a natural ligand of LAG-3.

[0070] Effector T cells include several T cell types that respond positively to stimuli, including costimulation. + , CD8 +, and regulatory T cells. A suitable example is Jurkat cells, which are immortalized T lymphocytes originally derived from the peripheral blood of children with T-cell leukemia (Schneider et al., 1977, Int J Cancer 19(5):621-6).

[0071] Optionally, the agonist is an agonist anti-LAG-3 antibody, or a fragment or derivative thereof that retains anti-LAG-3 agonist activity.

[0072] Optionally, the effector T cells are a T cell line double-transfected with a nucleic acid encoding LAG-3 and a reporter gene under the control of a promoter or response element, e.g., a nucleic acid encoding LAG-3 and an NFAT / luciferase reporter gene (Jurkat LAG-3 + Jurkat cell lines include those double-transfected with LAG3 / NFAT-luc cells. Jurkat LAG3 / NFAT-luc2 cells are available from Promega (reference: CS194801). Jurkat LAG3 / NFAT-luc cells are available from BPS Bioscience (catalog number: 71278).

[0073] Optionally, the agonist is an agonist anti-LAG-3 antibody, or a fragment or derivative thereof that retains anti-LAG-3 agonist activity, and the effector T cells are Jurkat LAG-3 + / NFAT-luc2 cells.

[0074] Optionally, the agonist anti-LAG-3 antibody is an agonist anti-LAG-3 antibody described in WO 2017 / 037203. The agonist anti-LAG-3 antibodies described in WO 2017 / 037203 include the murine monoclonal antibody 13E2 and the humanized 13E2 human IgG4 Fc antibody (referred to as IMP761). Antibodies 13E2 and IMP761 comprise VH CDR1-3 and VL CDR1-3 sequences as shown in Table 1 below. JPEG0007785542000001.jpg57170

[0075] Optionally, the agonist anti-LAG-3 antibody, or fragment or derivative thereof, comprises the VH CDR1-3 and VL CDR1-3 sequences of SEQ ID NOs: 1-6, respectively, or the following VH CDR1-3 and VL CDR1-3 sequences of SEQ ID NOs: 7-12, respectively. Optionally, the agonist anti-LAG-3 antibody is IMP761.

[0076] Embodiments of the present invention will now be described, by way of example only, with reference to the following drawings, in which: [Brief explanation of the drawings]

[0077] [Figure 1] shows a schematic diagram of TCR signaling (Belikov,Aleksey.(2016).The role of reactive oxygen species and mitochondria in T-cell activation.110.13140 / RG.2.1.2916.0568); [Figure 2] 1 shows the mechanism of action of an IMP761 potency assay according to one embodiment of the present invention. In the presence of an anti-CD3 antibody, the anti-CD3 antibody binds to CD3 on the surface of reporter Jurkat cells, resulting in increased expression of luciferase from the NFAT / Luc reporter gene through TCR-mediated signaling (top). Binding of IMP761 to LAG-3 on the surface of reporter Jurkat cells inhibits TCR-mediated signaling, resulting in downregulation of luciferase expression from the NFAT / Luc reporter gene (bottom). [Figure 3] 1 shows the effect of stimulation of Jurkat LAG-3+ / NFAT-luc2 cells with various concentrations of OKT3 and UCHT1 anti-CD3 antibodies in the presence of 300 ng / ml IMP761 or a human IgG4 negative control antibody. [Figure 4]1 shows example results of a potency assay of IMP761 versus an IgG4 negative control, according to one embodiment of the present invention. [Figure 5] 1 shows example results of a potency assay using a reference preparation of IMP761 (4° C.) compared to a preparation of IMP761 after denaturing temperature stress (70° C. for 10 or 20 minutes), according to one embodiment of the present invention.

[0078] Example 1 Optimization of IMP761 potency assay protocol Jurkat Lag-3+ / NFAT-luc2 effector cells were initially developed by Promega to determine antagonistic anti-LAG-3 antibody activity following TCR activation through stimulation with a superantigen presented by MHC II molecules. Antagonistic anti-LAG-3 antibody blockade of the LAG-3 / MHC II interaction results in enhanced TCR activation and luciferase activity. The use of Jurkat Lag-3+ / NFAT-luc2 effector cells to determine agonistic anti-LAG-3 antibody activity requires a very different experimental setup, as described below.

[0079] Anti-CD3 antibody as a stimulator of Jurkat cells: In the Promega bioassay, Jurkat Lag-3+ / NFAT-luc2 effector cells are activated using Raji cells in the presence of staphylococcal enterotoxin E or D (SEE or SED). Raji cells express endogenous MHC class II, the ligand for LAG-3. This is important for testing the blocking activity of antagonist anti-LAG-3 antibodies against the LAG-3 / MHC II interaction. However, because LAG-3 / MHC II interaction is not required to test the efficacy of agonist anti-LAG-3 antibodies, neither Raji cells nor staphylococcal enterotoxins are required. A single-cell assay was used with an anti-CD3 antibody to activate Jurkat Lag-3+ / NFAT-luc2 effector cells through TCR signaling.

[0080] Anti-CD3 concentrations and LAG-3-associated inhibition The effect of two different anti-CD3 antibodies (OKT3 and UCHT1) on the cytocapacity assay was tested at different antibody concentrations ranging from 1 to 500 ng / ml.

[0081] Jurkat Lag-3+ / NFAT-luc2 cells were incubated with 300 ng / ml IMP761 or human IgG4 (as a negative control) in the presence of various concentrations of OKT3 or UCHT1 for 24 hours. The mean RLU values ​​obtained with various concentrations of anti-CD3 antibody are shown in Figure 3 and Table 2 below. JPEG0007785542000002.jpg30170

[0082] Luciferase activity is inhibited by IMP761 across a range of concentrations of each anti-CD3 antibody.

[0083] Because Jurkat cell lines without stimulation have a basal level of reporter expression, luciferase activity and the inhibition of luciferase activity by IMP761 were also tested in the absence of anti-CD3 antibody. However, stimulating Jurkat cells with anti-CD3 antibody resulted in higher RLU values, suggesting that the inhibitory effect of IMP761 is more pronounced in the presence of anti-CD3 antibody, especially at low concentrations. The inhibition rate of luciferase activity by OKT3 antibody is shown in Table 3 below. JPEG0007785542000003.jpg68170

[0084] It was concluded that the maximal effect of IMP761 (approximately 80% inhibition) was observed when low concentrations of OKT3 antibody (1-4 ng / ml) were used. Therefore, an optimal potency assay would involve stimulation of Jurkat cells with low concentrations of anti-CD3 antibody (e.g., OKT3 antibody).

[0085] Example 2 IMP761 potency assay This example describes a potency assay for measuring the activity of IMP761 monoclonal antibody in vitro, according to an embodiment of the present invention. The method is based on the ability of IMP761 to reduce activation of the LAG-3 effector cell line induced by a low dose of anti-CD3 antibody (OKT3 clone, 3 ng / ml), mimicking antigen stimulation of T cells. The LAG-3 effector cell line expresses LAG-3 on its surface and expresses the NFAT (nuclear factor of activated T cells) response element (Jurkat Lag-3). + IMP761 is a Jurkat T cell line containing a luciferase gene under the control of IMP761 / NFAT-luc2 effector cells (from Promega). After binding to its target, IMP761 triggers TCR-induced downregulation of NFAT-regulated expression (schematically depicted in Figure 2, bottom). The luciferase activity of the cell line is used to measure TCR-driven cellular activation, which is attenuated in the presence of IMP761 activity. Thus, this assay measures the potency of IMP761 in terms of its ability to inhibit TCR signaling.

[0086] reagent Jurkat LAG-3+ / NFAT-luc2 effector cells (Promega, reference: CS194801) RPMI 1640 (GIBCO, Reference: 31870-025) L-glutamine (200 mM) (GIBCO, reference: 25030-024) HEPES(1M) (GIBCO, Reference: 15630-080) FCS (GIBCO, reference: 10270106) IMP761 (2.06 mg / ml) (IMMUTEP, Lot: 270416) Human IgG4, control (Biolegend, reference: 403402) Anti-CD3 (OKT3) (eBioscience, Reference: 16-0037-85) Bio-Glo Reagent (PROMEGA, Reference: G7941) White 96-well solid flat-bottom microplate (COSTAR, reference: 3917) Assay medium: RPMI 1640, L-glutamine (2 mM), Hepes (10 mM), FCS 1% Cell concentration: 1.33 x 10 6 cells / ml

[0087] protocol Passage Jurkat LAG-3+ / NFAT-luc2 effector cells on day -1 prior to the assay to obtain a cell density of approximately 1 million / ml (0.8–1.2 million / ml) on day 1.0. 2. If necessary, prepare assay medium and pre-warm to 37°C for 30 minutes. JPEG0007785542000004.jpg281703. If needed, prepare IMP761 quality control (QC) stock solution. Make a stock solution of 24,000 ng / ml. For example, 10 μl stock IMP761 (2.06 mg / ml) + 848.3 μl assay medium. Store 25 μl aliquots in a −80° C. freezer. 4. Prepare a 3X solution of IMP761 (batch 270416 at 2.06 mg / ml), human IgG4 negative control (or other antibody). Adjust the predilution step depending on the initial antibody concentration. To prepare a 100 μg / ml predilution, add 2 μl of stock solution to the following volume of assay medium (μl):

number

[0088] result Figure 4 shows an example of results obtained from a potency assay using an IgG4 antibody as a negative control. Maximum activation was recorded as the activation observed in the absence of IMP761. Maximum inhibition was recorded as the activation observed at an IMP761 concentration of 1000 ng / ml. A five-parameter nonlinear regression model was used to calculate the IC of IMP761. 50 was determined to be 37 ng / ml.

[0089] Example 3 Ability of the IMP761 potency assay to evaluate modified IMP761 antibodies The ability of the potency assay as described in Example 2 to assess the loss of potency of denatured IMP761 antibodies was assessed using the IC of reference IMP761 stored at 4°C. 50 IC of IMP761 of the same batch after temperature stress (70℃ for 10 minutes, 70℃ for 20 minutes) 50 The results are shown in Figure 5 and Table 4 below. JPEG0007785542000008.jpg91170

[0090] A five-parameter nonlinear regression model was used to calculate the IC of the modified IMP761 antibody. 50(74 ng / ml and 81.5 ng / ml after 10 and 20 min at 70°C, respectively) compared with the IC of the reference IMP761 antibody stored at 4°C. 50 It was determined that the serum creatine phosphate (CPP) level was higher than that of the control (41 ng / ml). The claims at the time of filing are as follows: [Claim 1] 1. An in vitro assay for determining lymphocyte activation gene 3 (LAG-3) agonist activity, 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, expression of the reporter being regulated by LAG-3-mediated inhibition of TCR signaling in the effector T cells; determining the activity of the agonist from the extent to which expression of the reporter is altered in the presence of the agonist compared to expression of the reporter in the absence of the agonist; In vitro assays, including: [Claim 2] 10. The assay of claim 1 for determining the potency of a preparation of an agonist of LAG-3. [Claim 3] 1. An in vitro assay for screening for LAG-3 agonists, 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, expression of the reporter being regulated by LAG-3-mediated inhibition of TCR signaling in the effector T cells; determining whether the candidate agonist is an agonist of LAG-3 by determining the extent to which expression of the reporter changes in the presence of the candidate agonist compared to expression of the reporter in the absence of the candidate agonist; In vitro assays, including: [Claim 4] 3. The assay of claim 1 or 2, wherein the reporter is expressed at a basal level in effector T cells in the absence of the agonist, or the assay of claim 3, wherein the reporter is expressed at a basal level in effector T cells in the absence of the candidate agonist. [Claim 5] 5. The assay of claim 1, wherein expression of the reporter is decreased in the presence of the agonist or candidate agonist compared to expression of the reporter in the absence of the agonist or candidate agonist. [Claim 6] expression of the reporter is altered in each effector T cell in response to activation of the effector T cell via the TCR; activating the 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; determining the activity of the agonist or candidate agonist from the extent to which expression of the reporter changes in response to activation of the effector T cells in the presence of the agonist or candidate agonist compared to expression of the reporter in response to activation of the effector T cells in the absence of the agonist or candidate agonist; 6. The assay of claim 1, further comprising: [Claim 7] 7. The assay of claim 6, wherein expression of the reporter increases in each effector T cell in response to activation of the effector T cell via the TCR and decreases in the presence of the agonist or candidate agonist as a result of LAG-3-mediated inhibition of TCR signaling in the effector T cell, and the activity of the agonist or candidate agonist is determined from the extent to which expression of the reporter decreases in response to activation of the effector T cell in the presence of the agonist or candidate agonist compared to expression of the reporter in response to activation of the effector T cell in the absence of the agonist or candidate agonist. [Claim 8] 8. The assay of claim 6 or 7, wherein 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 for antigen-independent, MHC class II-independent TCR-mediated activation of the effector T cells by the activator. [Claim 9] 9. The assay of claim 8, wherein the effector T cells are contacted with the T cell activator at a concentration of the T cell activator that results in maximal inhibition of expression of the reporter in the presence of an excess of an agonist of LAG-3. [Claim 10] 10. The assay of claim 8 or 9, wherein 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 maximal expression of the reporter 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. [Claim 11] 11. The assay of any one of claims 8 to 10, wherein the T cell activating agent comprises or consists of an anti-CD3 antibody, or a fragment or derivative thereof that retains antigen-independent, MHC class II-independent, TCR-mediated effector T cell activation ability. [Claim 12] The assay of claim 11, wherein the anti-CD3 antibody is OKT3. [Claim 13] the anti-CD3 antibody, or fragment or derivative thereof, Approximately 6~30×10 -12 13. The assay of claim 11 or 12, wherein the antibody is contacted with the effector T cells at a concentration of M (1 to 4 ng / ml for whole antibodies, or a molar equivalent for fragments or derivatives thereof). [Claim 14] 14. The assay of claim 1, wherein the effector T cells are activated by cell-free, antigen-independent, MHC class II-independent, TCR-mediated T cell activation. [Claim 15] 15. The assay of any one of claims 1 to 14, wherein the effector T cells are contacted with several different concentrations of the agonist or candidate agonist. [Claim 16] the IC of the agonist or candidate agonist to inhibit expression of the reporter 50 16. The assay of claim 15, further comprising determining a value. [Claim 17] 17. The assay of claim 1, wherein the effector T cells comprise a heterologous nucleic acid comprising the reporter gene. [Claim 18] 18. The assay of claim 1, wherein the reporter gene is under the control of a promoter or response element. [Claim 19] 19. The assay of claim 18, wherein the response element comprises an NFAT (nuclear factor of activated T cells) response element (NFAT-RE). [Claim 20] 20. The assay of claim 1, wherein the reporter comprises a bioluminescent reporter such as luciferase. [Claim 21] 21. The assay of claim 1, wherein the effector T cells comprise a heterologous nucleic acid encoding LAG-3. [Claim 22] 22. The assay of any one of claims 1 to 21, further comprising the step of performing a negative control assay, in which 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 known to lack agonist activity for LAG-3. [Claim 23] 23. The assay of any one of claims 1 to 22, which is carried out in the absence of a natural ligand of LAG-3. [Claim 24] 24. The assay of any one of claims 1 to 23, wherein the agonist or candidate agonist is an anti-LAG-3 antibody, or a fragment or derivative thereof that retains anti-LAG-3 agonist activity. [Claim 25] 25. The assay of any one of claims 1 to 24, wherein the effector T cells comprise Jurkat-derived cells. [Claim 26] the agonist is an agonist anti-LAG-3 antibody, or a fragment or derivative thereof that retains anti-LAG-3 agonist activity, and the effector T cells are Jurkat LAG-3 + 26. The assay of claim 1, comprising / NFAT-luc2 cells. [Claim 27] 27. The assay of any one of claims 1 to 26, wherein the agonist anti-LAG-3 antibody, or fragment or derivative thereof, comprises the VH CDR1-3 sequences and VL CDR1-3 sequences of SEQ ID NOs: 1 to 6, respectively, or SEQ ID NOs: 7 to 12, respectively. [Claim 28] 28. The assay of any one of claims 1 to 27, wherein the agonist anti-LAG-3 antibody is IMP761. [Claim 29] 1. A kit for performing an in vitro assay for determining the activity of or screening for agonists of LAG-3, 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, expression of the reporter being regulated by LAG-3-mediated inhibition of TCR signaling in the effector T cell, and expression of the reporter being altered in each effector T cell in response to activation of the effector T cell via the TCR; a T cell activating factor capable of antigen-independent, MHC class II-independent, TCR-mediated activation of said effector T cells; Includes a kit. [Claim 30] 30. The kit of claim 29, wherein the kit does not include cells that express MHC class II molecules. [Claim 31] 31. The kit of claim 29 or 30, wherein expression of the reporter increases in response to activation of effector T cells via the TCR. [Claim 32] 32. The kit according to claim 29, wherein the effector T cells comprise a heterologous nucleic acid comprising the reporter gene. [Claim 33] 33. The kit according to any one of claims 29 to 32, wherein the reporter gene is under the control of a promoter or a response element. [Claim 34] 34. The kit of claim 33, wherein the response element comprises an NFAT (nuclear factor of activated T cells) response element (NFAT-RE). [Claim 35] 35. The kit according to claim 29, wherein the reporter comprises a bioluminescent reporter such as luciferase. [Claim 36] 36. The kit according to any one of claims 29 to 35, wherein the effector T cells comprise a heterologous nucleic acid encoding LAG-3. [Claim 37] 37. The kit according to claim 29, wherein the T cell activator is a cell-free T cell activator. [Claim 38] 38. The kit according to any one of claims 29 to 37, wherein the T cell activating factor comprises an anti-CD3 antibody, or a fragment or derivative thereof that retains antigen-independent, MHC class II-independent, TCR-mediated effector T cell activation ability. [Claim 39] 39. The kit of claim 38, wherein the anti-CD3 antibody is OKT3. [Claim 40] Anti-CD3 antibody, or a fragment or derivative thereof, is about 6 to 30 × 10 -12 40. The kit of claim 38 or 39, wherein the antibody is present in a concentration that can be used in an assay at a concentration of M (1 to 4 ng / ml in the case of a whole antibody, or a molar equivalent in the case of a fragment or derivative thereof). [Claim 41] 41. The kit of any one of claims 29 to 40, further comprising a molecule of the same type as the agonist but known to lack agonist activity for LAG-3, for use as a negative control. [Claim 42] 42. The kit of claim 29, further comprising a known agonist of LAG-3 for use as a positive control. [Claim 43] The kit according to any one of claims 29 to 42, which does not contain a natural ligand of LAG-3. [Claim 44] 44. The kit according to claim 29, wherein the effector T cells comprise Jurkat-derived cells. [Claim 45] The effector T cells are Jurkat LAG-3 + 45. The kit according to claim 29, comprising NFAT-luc2 cells. [Claim 46] The kit according to any one of claims 29 to 45, wherein the kit does not comprise APCs, aAPCs, or any other MHC class I or MHC class II expressing cells. [Claim 47] 47. The kit according to claim 29, wherein the only cells in the kit are the effector T cells. [Claim 48] 48. A kit according to any one of claims 29 to 47 for determining the efficacy of a preparation of an agonist of LAG-3. [Claim 49] A kit according to any one of claims 29 to 48 for carrying out an assay according to any one of claims 1 to 28. [Claim 50] Use of a kit according to any one of claims 29 to 47 for determining the activity of an agonist of LAG-3, for determining the efficacy of a preparation of an agonist of LAG-3, or for screening for agonists of LAG-3.

Claims

1. 1. An in vitro assay for determining lymphocyte activation gene 3 (LAG-3) agonist activity, 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, expression of the reporter being regulated by LAG-3-mediated inhibition of TCR signaling in the effector T cells; determining the activity of the agonist from the extent to which expression of the reporter is altered in the presence of the agonist compared to expression of the reporter in the absence of the agonist; Including, An in vitro assay performed in the absence of cells expressing MHC class II molecules.

2. 10. The assay of claim 1 for determining the potency of a preparation of an agonist of LAG-3.

3. 1. An in vitro assay for screening for an agonist of LAG-3, 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, expression of the reporter being regulated by LAG-3-mediated inhibition of TCR signaling in the effector T cells; determining whether the candidate agonist is an agonist of LAG-3 by determining the extent to which expression of the reporter changes in the presence of the candidate agonist compared to expression of the reporter in the absence of the candidate agonist; Including, An in vitro assay performed in the absence of cells expressing MHC class II molecules.

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

5. 5. The assay of claim 1, wherein expression of the reporter is decreased in the presence of the agonist or candidate agonist compared to expression of the reporter in the absence of the agonist or candidate agonist.

6. expression of the reporter is altered in each effector T cell in response to activation of the effector T cell via the TCR; activating the effector T cells by antigen-independent and MHC class II-independent TCR-mediated T cell activation in the presence and absence of the agonist or candidate agonist; determining the activity of the agonist or candidate agonist from the extent to which expression of the reporter changes in response to activation of the effector T cells in the presence of the agonist or candidate agonist compared to expression of the reporter in response to activation of the effector T cells in the absence of the agonist or candidate agonist; The assay of any one of claims 1 to 5, further comprising:

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

8. 8. The assay of claim 6 or 7, wherein the effector T cells are activated by contacting the effector T cells with an antigen-independent and MHC class II-independent T cell activator under conditions for antigen-independent and MHC class II-independent TCR-mediated activation of the effector T cells by the activator, and the T cell activator comprises or consists of an anti-CD3 antibody or a fragment thereof that retains the ability to activate antigen-independent and MHC class II-independent TCR-mediated effector T cells.

9. 9. The assay of claim 8, wherein the effector T cells are contacted with the T cell activator at a concentration of the T cell activator that results in maximal inhibition of expression of the reporter in the presence of an excess of an agonist of LAG-3.

10. 10. The assay of claim 8 or 9, wherein 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 maximal expression of the reporter 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 anti-CD3 antibody is OKT3, and / or The anti-CD3 antibody or a fragment thereof that retains the ability to activate TCR-mediated effector T cells in an antigen-independent and MHC class II-independent manner is 6 to 30 × 10 －１２ The assay of any one of claims 8 to 10, wherein the antibody is contacted with the effector T cells at a concentration of M (1 to 4 ng / ml for whole antibodies, or a molar equivalent for fragments thereof).

12. the effector T cells are activated by cell-free, antigen-independent and MHC class II-independent TCR-mediated T cell activation; and / or The assay of any one of claims 1 to 11, wherein the effector T cells are contacted with several different concentrations of the agonist or candidate agonist.

13. The assay determines the IC of the agonist or candidate agonist for inhibiting expression of the reporter. ５０ 13. The assay of claim 12, further comprising determining a value.

14. the effector T cells comprise a heterologous nucleic acid comprising the reporter gene; and / or 14. The assay of any one of claims 1 to 13, wherein the reporter gene is under the control of a promoter or response element.

15. the response element comprises an NFAT (nuclear factor of activated T cells) response element (NFAT-RE); and / or the reporter comprises a bioluminescent reporter such as luciferase; and / or the effector T cells comprise a heterologous nucleic acid encoding LAG-3; and / or the assay further comprises the step of performing a negative control assay, in which 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 known to lack agonist activity for LAG-3; and / or the agonist or candidate agonist is an anti-LAG-3 antibody, or a fragment thereof that retains anti-LAG-3 agonist activity, and / or the effector T cells comprise Jurkat-derived cells; and / or the agonist is an agonist anti-LAG-3 antibody, or a fragment thereof that retains anti-LAG-3 agonist activity, and the effector T cells are Jurkat LAG-3 ＋ / NFAT-luc2 cells, and / or the agonist is an anti-LAG-3 antibody, or a fragment thereof that retains anti-LAG-3 agonist activity, comprising the VH CDR1-3 and VL CDR1-3 sequences of SEQ ID NOs: 1-6, respectively, or SEQ ID NOs: 7-12, respectively; and / or The assay of claim 14, wherein the agonist is the anti-LAG-3 antibody IMP761.

16. 1. A kit for performing an in vitro assay for determining the activity of or screening for agonists of LAG-3, 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, expression of the reporter being regulated by LAG-3-mediated inhibition of TCR signaling in the effector T cell, and expression of the reporter being altered in each effector T cell in response to activation of the effector T cell via the TCR; a T cell activator capable of antigen-independent and MHC class II-independent TCR-mediated activation of said effector T cells; wherein the T cell activator comprises an anti-CD3 antibody or a fragment thereof that retains antigen-independent and MHC class II-independent TCR-mediated effector T cell activation ability, and the kit does not contain cells that express MHC class II molecules.

17. expression of the reporter increases in response to activation of effector T cells via the TCR; and / or the effector T cells comprise a heterologous nucleic acid comprising the reporter gene; and / or The kit of claim 16 , wherein the reporter gene is under the control of a promoter or response element.

18. the response element comprises an NFAT (nuclear factor of activated T cells) response element (NFAT-RE); and / or the reporter comprises a bioluminescent reporter, such as luciferase; and / or the effector T cells comprise a heterologous nucleic acid encoding LAG-3; and / or 18. The kit of claim 17, wherein the T cell activator is a cell-free T cell activator.

19. The kit according to any one of claims 16 to 18, wherein the anti-CD3 antibody is OKT3.

20. The anti-CD3 antibody or a fragment thereof that retains the ability to activate TCR-mediated effector T cells in an antigen-independent and MHC class II-independent manner is 6 to 30 × 10 －１２ M (1-4 ng / ml for whole antibodies, or the molar equivalent for fragments thereof) at a concentration that allows use in the assay; and / or the kit further comprises a molecule of the same type as the agonist but known to lack agonist activity for LAG-3 for use as a negative control; and / or the kit further comprises a known agonist of LAG-3 for use as a positive control; and / or the effector T cells comprise Jurkat-derived cells; and / or The effector T cells are Jurkat LAG-3 ＋ / NFAT-luc2 cells, and / or the kit does not include APCs, aAPCs, or any other MHC class I or MHC class II expressing cells; and / or The kit of any one of claims 16 to 19, wherein the only cells in the kit are the effector T cells.

21. A kit according to any one of claims 16 to 20 for determining the potency of a preparation of an agonist of LAG-3.

22. A kit according to any one of claims 16 to 21 for carrying out an assay according to any one of claims 1 to 15.

23. Use of the kit according to any one of claims 16 to 20 for determining the activity of an agonist of LAG-3, for determining the efficacy of a preparation of an agonist of LAG-3, or for screening for agonists of LAG-3.

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