determined

Abstract

A assay for screening or determining the activity of a lymphocyte-activation gene 3 (LAG-3) agonist is described. According to the assay, a plurality of effector T cells is 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 protein, wherein expression of the reporter protein is regulated by LAG-3-mediated inhibition of TCR signaling within the effector T cell. The activity of the agonist is determined according to the extent to which expression of the reporter protein is altered in the presence of the agonist as compared to expression of the reporter protein in the absence of the agonist. The assay can be used to determine the potency of a preparation of the agonist, as a quality control step in the production of the agonist, or for stability testing of a preparation of the agonist. A kit for performing the assay is also described.

Description

[0001] This invention relates to assays for screening agonists of lymphocyte activation gene 3 (LAG-3) or for determining their activity (including determining the potency of their formulations). The invention also relates to kits for performing said assays.

[0002] Stimulation of T cell function is initiated by the interaction of the T cell receptor (TCR) with short peptides presented by MHC class I or II molecules (MHC I for CD8 T cells and MHC II for CD4 T cells) on the surface of antigen-presenting cells (APCs). In primary T cells, the TCR itself cannot activate downstream pathways to initiate T cell activation. This requires co-receptors, such as CD4 for helper T cells and CD8 for cytotoxic T cells. These co-receptors bind to their respective MHC molecules and stabilize the interaction between T cells and APCs. In addition to the binding of the TCR to antigen-loaded MHC, both helper and cytotoxic T cells require several secondary signals to be fully activated and responsive. 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 and initiates T cell proliferation, leading to the expansion of antigen-specific T cell clones. Cytotoxic T cell activation is less dependent on CD28 but does require signaling from other co-stimulatory molecules such as CD70 and CD137.

[0003] The TCR is located near a complex of signal transduction molecules and mediates T cell activation through multiple signaling cascades (see overview of TCR signaling for details). Figure 1 These signaling molecules include the CD3 protein family. Once the TCR correctly binds to the peptide-MHC complex, it induces a conformational change in the associated CD3 chain, leading to its phosphorylation and association with downstream proteins. The TCRζ chain is also phosphorylated upon TCR binding. These molecules are phosphorylated by Src kinase, leukocyte-specific tyrosine kinase (LCK), and Fyn via their C-terminal immunoreceptor tyrosine-based activation motif (ITAM).

[0004] Phosphorylated CD3 ITAM recruits and activates the 70 kDa Syk family kinase ζ-activating protein (ZAP70). ZAP70 then phosphorylates a membrane-associated scaffold protein called the Linker for Activation of T Cell (LAT). LAT then recruits a second scaffold molecule, a 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 recruiting signaling effector molecules. Interleukin-2-inducible tyrosine kinase (ITK) then interacts with the LAT-Slp-76 complex and is activated via autophosphorylation. This promotes the phosphorylation of the effector molecule phospholipase Cγ (PLC-γ1). PLC-γ1 transduces TCR signaling by cleaving phosphatidylinositol triphosphate (PIP2) in the plasma membrane to generate the second messengers diacylglycerol (DAG) and inositol triphosphate (IP3).

[0005] DAG is a membrane-associated lipid that activates several downstream proteins, including protein kinase C (PKC) and various isoforms of RAS guanylate-releasing protein (RasGRP). After activation by DAG, PKC-θ participates in the activation of the NF-κB pathway, while RasGRP is a key activator of the MAPK signaling pathway. IP3 stimulates Ca... 2+ Flowing out of the endoplasmic reticulum into the cytoplasm. Ca 2+ Elevated levels induce activation of the protein phosphatase calmophosphatase, which then dephosphorylates T cell transcription factors, including nuclear factor-T (NFAT). The dephosphorylated NFAT then migrates to the nucleus, joins other transcription factors, and induces the transcription of specific genes.

[0006] Uncontrolled immune responses to pathogens or self-antigens can cause inflammatory tissue damage and autoimmune diseases. To prevent this, the immune response is regulated by a balance between co-stimulatory and inhibitory signals (collectively known as immune checkpoints), which are essential for maintaining self-tolerance and protecting the host from tissue damage. Activated T cells express a variety of 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 the T cell immunoglobulin and immune receptor tyrosine inhibitory motif [ITIM] domain (TIGIT). Inhibitory immune checkpoint receptors have been shown to regulate T cell responses to self-proteins as well as to chronic infections and tumor antigens. Inhibitory immune checkpoint receptors have become targets for cancer immunotherapy due to their potential use in various types of cancer.

[0007] LAG-3 is a CD4 homologous type I membrane protein with four extracellular Ig superfamily domains. Similar to 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-positive and CD8-positive T lymphocytes, where it associates with the CD3-TCR complex on the cell surface and negatively regulates signal transduction. Therefore, it negatively regulates T cell proliferation, function, and homeostasis. When a specific TCR recognizes an MHC class II peptide complex, intracellular signals are transduced in T cells via the TCR and in APCs via MHC class II molecules. The negative regulatory role of LAG-3 signaling in T cells is reflected in primary CD4... + and CD8 + Plays a role in human T cell response ( Maeda et al., Immunology. June 2005; 115(2):170-178). However, the molecular mechanism by which LAG-3 negatively regulates signal transduction in T cells remains unclear. LAG-3's inhibitory function requires its intracellular (IC) region, but this region does not contain typical signal transduction motifs with known signal transduction mechanisms. Maeda et al. (J. Biol. Chem. 2019, RA119.007455) recently reported that LAG-3 transduces two independent inhibitory signals via the FxxL motif in its juxtamembrane region and the EX repeat sequence at its C-terminus. However, these motifs have not been previously reported in inhibitory co-receptors, and the molecular mechanism of LAG-3-transduced inhibitory signals remains difficult to determine.

[0008] WO 2017 / 037203 describes antibodies (such as the humanized monoclonal antibody IMP761) as LAG-3 agonists, their antigen-binding fragments, and their therapeutic effects on CD4+. + and / or CD8 + Use in diseases related to T cell proliferation and / or activation, particularly inflammatory and autoimmune diseases.

[0009] Traditional methods for measuring the activity of antibodies and other drugs designed to target immune checkpoint receptors rely on primary human cells and measurements of functional endpoints such as cell proliferation, expression of cell surface markers, and cytokine production. These assays are laborious and highly variable due to their reliance on donor primary cells, complex assay protocols, and unqualified reagents. Consequently, they are difficult to establish in quality-controlled drug development environments. To address these challenges, Promega has developed cell-based bioluminescent reporter protein bioassays for individual and combination immune checkpoint immunotherapy targets (Cheng et al., June 2016, Promega, “Quantitative Cell-Based Bioassays for Indivisual and Combination Immune Checkpoint Immunotherapy Targets”).

[0010] Promega has developed bioassays including BlockadeBioassay for PD-1, CTLA-4, LAG-3, and TIGI. These assays rely on the use of Jurkat T cells, which are genetically engineered to express co-inhibitory receptors of interest on their surface and contain firefly luciferase reporter genes under the control of NFAT response elements (NFAT-RE). These cells express endogenous TCR, CD3, and CD28 receptors. When the cells bind to appropriate ligands, the TCR transduces intracellular signals, leading to an increase in NFAT-RE-mediated luminescence. The bioluminescent signals are detected and quantified using luciferase substrates and a standard photometer. The assays also used artificial antigen-presenting cells (aAPCs) expressing engineered cell surface proteins programmed to activate the TCR in an antigen-dependent manner and expressing the natural ligands of co-inhibitory receptors on their surfaces (for PD-1, CTLA-4, and TIGIT Blockade Bioassays), or Raji cells in the presence of staphylococcal enterotoxin E (SEE) superantigen (for LAG-3 Blockade Bioassays, Raji cells naturally express MHC class II (LAG-3 ligands)). TCR conjugation on Jurkat cells induced luciferase activity. Co-conjugation of the co-inhibitory receptor with its natural ligand inhibited luciferase activity. Antibody-mediated blockade of the binding of the co-inhibitory receptor with its natural ligand restored luciferase activity.

[0011] These bioassays demonstrate the performance required for antagonist antibody screening, potency testing, and stability studies. However, they are unsuitable for testing agonist antibodies, such as LAG-3 agonist antibodies (e.g., IMP761, described in WO2017 / 037203). In particular, the presence of the natural LAG-3 ligand in blocking bioassays (such as MHC class II expressing Raji cells) interferes with agonist testing. Therefore, an in vitro assay is needed for identifying and testing LAG-3 agonists.

[0012] It has now been surprisingly discovered that IMP761 inhibits TCR signaling in LAG-3-positive T cells, particularly NFAT-regulated gene expression, through its agonistic effect on LAG-3. The applicant recognizes that this could form the basis for in vitro bioassays to determine the activity of LAG-3 agonists or LAG-3 agonist formulations and to identify novel LAG-3 agonists.

[0013] According to the present invention, an in vitro assay for determining the activity of a lymphocyte activation gene 3 (LAG-3) agonist is provided, comprising:

[0014] Provided are multiple effector T cells, each effector T cell expressing LAG-3 and T cell receptor (TCR) on its surface and including a reporter gene encoding a reporter protein, wherein the expression of the reporter protein is regulated by LAG-3-mediated inhibition of TCR signaling within the effector T cell; and

[0015] The activity of the agonist is determined based on the degree to which the expression of the reporter protein changes (i.e., increases or decreases) in the presence of the agonist compared to the expression of the reporter protein in the absence of the agonist.

[0016] The assays of the present invention for determining agonist activity include the following assays for determining the potency of agonist formulations, for example as part of a quality control step in agonist production (in particular, as a cell-based potency assay required for product release according to Good Manufacturing Practice (GMP), or for, for example, stability testing of agonist formulations after a period of storage, or as a product characterization assay.

[0017] According to the present invention, an in vitro assay for screening LAG-3 agonists is also provided, comprising:

[0018] Provided are multiple effector T cells, each effector T cell expressing LAG-3 and T cell receptor (TCR) on its surface and including a reporter gene encoding a reporter protein, wherein the expression of the reporter protein is regulated by LAG-3-mediated inhibition of TCR signaling within the effector T cell; and

[0019] Whether a candidate agonist is a LAG-3 agonist is determined by measuring the degree to which the expression of the reporter protein changes (i.e., increases or decreases) in the presence of the candidate agonist compared to the expression of the reporter protein in the absence of the candidate agonist.

[0020] The assays of the present invention for screening LAG-3 agonists can be used to identify LAG-3 agonists, for example, from a library of candidate agonists. Such candidate agonists may be pharmaceuticals (e.g., synthetic small molecules) or biological agents (such as recombinant or natural proteins, antibodies, or fragments or derivatives thereof).

[0021] In the absence of agonists or candidate agonists, reporter proteins can be expressed in effector T cells.

[0022] Optionally, the expression of the reporter protein is reduced in the presence of the agonist or candidate agonist compared to the expression of the reporter protein in the absence of the agonist or candidate agonist.

[0023] For example, LAG-3-mediated inhibition of TCR signaling in effector T cells can lead to a decrease in the basal expression levels of reporter proteins in effector T cells (see [link to relevant documentation]). Figure 2 (a) Therefore, the activity of LAG-3 agonists (including the potency of LAG-3 agonist formulations) or the identification of LAG-3 agonists can be determined by determining the extent to which the basal expression level of the reporter protein changes in the presence of an agonist or candidate agonist compared to the absence of an agonist or candidate agonist.

[0024] The term "LAG-3-mediated inhibition of TCR signaling in effector T cells" is used herein to refer to the inhibition of TCR-mediated signal transduction within effector T cells by the agonist effect of LAG-3 expressed on the surface of effector T cells, resulting in altered (i.e., increased or decreased) reporter gene expression. Any TCR-mediated signaling pathway within effector T cells can be inhibited. For example, reporter gene expression can be controlled by promoters or response elements (REs) that are responsive to the binding of transcription factors as part of a signaling pathway. For instance, the calcineurin / NFAT signaling pathway within effector T cells can be inhibited. In particular, reporter gene expression can be controlled by NFAT response elements, such that a decrease in calcineurin / NFAT signaling following LAG-3 agonism results in a decrease in reporter protein expression.

[0025] In response to activation of effector T cells via the TCR, the expression of reporter proteins in each effector T cell may change (i.e., increase or decrease). Optionally, the assay of the present invention further includes: activating effector T cells by antigen-independent, MHC class II-independent, TCR-mediated T cell activation in the presence and absence of an agonist or candidate agonist; and determining the activity (including the potency of the formulation) of the agonist or candidate agonist based on the degree of change in the expression of reporter proteins in response to activation of effector T cells in the presence of an agonist or candidate agonist compared to the expression of reporter proteins in response to activation of effector T cells in the absence of an agonist or candidate agonist.

[0026] Therefore, according to the present invention, an in vitro assay for determining the activity of a lymphocyte activation gene 3 (LAG-3) agonist is provided, comprising:

[0027] Multiple effector T cells are provided, each effector T cell expressing LAG-3 and T cell receptor (TCR) on its surface and including a reporter gene encoding a reporter protein whose expression changes in response to activation of the effector T cell via the TCR, wherein the expression of the reporter protein is regulated by LAG-3-mediated inhibition of TCR signaling within the effector T cell.

[0028] Effector T cells are activated in the presence and absence of agonists via antigen-independent, MHC class II-independent, and TCR-mediated T cell activation; and

[0029] The activity of the agonist is determined by the degree to which the expression of reporter proteins in response to the activation of effector T cells changes in the presence of the agonist compared to the expression of reporter proteins in response to the activation of effector T cells in the absence of the agonist.

[0030] According to the present invention, an in vitro assay for screening LAG-3 agonists is also provided, comprising:

[0031] Multiple effector T cells are provided, each effector T cell expressing LAG-3 and T cell receptor (TCR) on its surface and including a reporter gene encoding a reporter protein whose expression changes in response to activation of the effector T cell via the TCR, wherein the expression of the reporter protein is regulated by LAG-3-mediated inhibition of TCR signaling within the effector T cell.

[0032] Effector T cells are activated through antigen-independent, MHC class II-independent, TCR-mediated T cell activation, both in the presence and absence of candidate agonists; and

[0033] The activity of a candidate agonist is determined by the degree to which the expression of reporter proteins in response to effector T cell activation changes in the presence of the candidate agonist compared to the expression of reporter proteins in response to effector T cell activation in the absence of the candidate agonist.

[0034] Optionally, due to the LAG-3-mediated inhibition of TCR signaling within effector T cells, the expression of reporter proteins in each effector T cell increases in response to activation of effector T cells via TCR and decreases in the presence of an agonist or candidate agonist, wherein the activity of the agonist or candidate agonist is determined based on the degree to which the expression of reporter proteins in response to activation of effector T cells in the presence of an agonist or candidate agonist is reduced compared to the expression of reporter proteins in response to activation of effector T cells in the absence of an agonist or candidate agonist.

[0035] The greater the change in the expression of reporter genes in response to effector T cell activation in the presence of an agonist or candidate agonist compared to the expression of reporter proteins in response to effector T cell activation in the absence of an agonist or candidate agonist, the greater the activity (or formulation potency) of the LAG-3 agonist or candidate agonist.

[0036] Optionally, effector T cells are activated by cell-free T cell activation.

[0037] The term "cell-free T cell activation" is used herein to refer to the activation of effector T cells using one or more cell-free T cell activators in the absence of any cells (other than effector T cells). Specifically, cell-free T cell activation occurs without the use of antigen-presenting cells (APCs), artificial APCs (aAPCs), or any other MHC class I or MHC class II expressing cells (such as Raji cells).

[0038] Cell-free activation of T cells is advantageous because cell-based reagents require special storage conditions. Cells are typically kept frozen and must then be thawed before use.

[0039] In this paper, "activation of effector T cells via TCR" refers to the transduction of signals by the TCR within effector T cells after the TCR is bound by T cell activators, resulting in altered (i.e., increased or decreased) expression of reporter proteins within the effector T cells. For example, the expression of reporter genes may be under the control of promoters or response elements (REs) that respond to activation signals from effector T cells. For instance, after activation of effector T cells via TCR, response elements may be bound by one or more transcription factors.

[0040] Optionally, effector T cells are activated by contacting them with an antigen-independent, MHC class II-independent T cell activator under conditions that allow effector T cells to be activated by the T cell activator antigen-independent, MHC class II-independent, TCR-mediated activation.

[0041] According to the present invention, a kit for performing an in vitro assay to determine the activity of a LAG-3 agonist or to screen for LAG-3 agonists is also provided, comprising: a plurality of effector T cells, each effector T cell expressing LAG-3 and a T cell receptor (TCR) on its surface and including a reporter gene encoding a reporter protein, wherein the expression of the reporter protein is regulated by LAG-3-mediated inhibition of TCR signaling within the effector T cells, and wherein the expression of the reporter protein in each effector T cell is altered (i.e., increased or decreased) in response to activation of the effector T cells via the TCR; and a T cell activator capable of activating effector T cells in an antigen-independent, MHC class II-independent, TCR-mediated manner.

[0042] Antigen-independent, MHC class II-independent T cell activators are distinguished from superantigens (such as staphylococcal enterotoxin (SE)). SEs possess binding regions for both MHC class II and the TCR. They bind first to MHC class II and then to a variable α or β chain of the TCR. This interaction with the T cell receptor allows SEs to act as a wedge between the TCR and MHC. This keeps any antigenic peptide away from the TCR and circumvents the normal mechanisms of T cell activation. Therefore, SEs are MHC class II-dependent T cell activators.

[0043] Optionally, effector T cells are contacted with a concentration of a T cell activator that, at which concentration, maximal inhibition of reporter protein expression is achieved in the presence of an excess of LAG-3 agonist. This concentration of T cell activator is used to optimize the accuracy of the assay.

[0044] The applicant found that, in the presence of an excess of LAG-3 agonist, the T cell activator concentration that produced the maximum inhibition of reporter protein expression was the second-best concentration of T cell activator (i.e., lower than the concentration of T cell activator observed to maximize reporter protein expression in response to activation of effector T cells by T cell activator in the absence of an agonist or candidate agonist).

[0045] Optionally, the T cell activator is contacted with effector T cells at a concentration lower than that observed when the expression of reporter proteins is maximized in response to activation of effector T cells by the T cell activator in the absence of an agonist or candidate agonist.

[0046] It should be understood that antigen-dependent stimulation only amplifies antigen-specific T cells, while antigen-independent stimulation amplifies up to 100% of effector T cells.

[0047] An example of a T cell activator capable of antigen-independent, MHC class II-independent, TCR-mediated activation of effector T cells is an anti-CD3 antibody. Anti-CD3 antibodies bind to CD3 and activate the TCR complex in the absence of an antigenic peptide from APCs (i.e., antigen-independent, MHC class II-independent, TCR-mediated effector T cell stimulation) (see [link to relevant documentation]). Figure 2 (b) Anti-CD3 antibodies can activate up to 100% of effector T cells.

[0048] Optionally, the T-cell activator 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 capabilities. Suitable examples of anti-CD3 antibodies include OKT3 and UCHT1. Optionally, the anti-CD3 antibody is OKT3.

[0049] Optionally, the concentration of the anti-CD3 antibody or its fragments or derivatives in contact with effector T cells is approximately 6-30 x 10⁻⁶. -12 M (1-4 ng / ml for intact antibody, or molar equivalents for its fragments or derivatives).

[0050] Optionally, the kit of the present invention comprises an anti-CD3 antibody or a fragment or derivative thereof that retains antigen-independent, MHC class II-independent, and TCR-mediated effector T cell activation capabilities, at a concentration permissible for use in the assay of approximately 6-30 x 10⁻⁶. -12 M (intact antibody at 1-4 ng / ml). For example, the kit may include one or more aliquots of anti-CD3 antibody or its fragments or derivatives at a concentration of approximately 6-30 x 10⁻⁶. -12 M (intact antibody is 1-4 ng / ml).

[0051] Optionally, effector T cells are exposed to several different concentrations of agonists or candidate agonists. For example, multiple different assays can be performed in parallel, with effector T cells in each assay exposed to different concentrations of agonists or candidate agonists.

[0052] Optionally, the IC50 of the agonist or candidate agonist is determined for the inhibition of reporter protein expression. 50 Values. This can be accomplished, for example, from dose-response curves generated by exposing effector T cells to several different concentrations of candidate agonists or agonists.

[0053] Optionally, the assay of the present invention further includes a negative control assay (e.g., performed in parallel with the assay). For example, in the absence of an 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 against LAG-3, effector T cells may be exposed to a T cell activator (i.e., as a negative control) under conditions of T cell activator antigen-independent activation of effector T cells.

[0054] For example, if the agonist or candidate agonist is an antibody, then the molecule optionally used as a negative control is also an antibody. Preferably, the negative control antibody is an antibody of the same type as the agonist or candidate agonist antibody.

[0055] Optionally, the T-cell activator is a cell-free T-cell activator.

[0056] Optionally, the kit of the present invention does not include cells expressing MHC class II molecules.

[0057] Optionally, the kit of the present invention does not include Raji cells.

[0058] Optionally, the kits of the present invention do not include APC, aAPC or any other MHC class I or MHC class II expressing cells, such as Raji cells.

[0059] Optionally, the only cells in the kit of the present invention are effector T cells.

[0060] Optionally, the effector T cells in the kit of the present invention are frozen, such as "thaw-and-use" cells.

[0061] According to the present invention, a kit for performing the determinations of the present invention is also provided.

[0062] Optionally, the reporter gene is a heterologous reporter gene.

[0063] Genetic reporter proteins are widely used in pharmaceutical and biomedical research as indicators of gene expression and related cellular events. Typically, a reporter gene encoding a reporter protein is cloned into an expression vector, which is then transferred to cells. After transfer, the presence of the reporter protein in the cell is determined by directly measuring the reporter protein itself or its enzymatic activity. Preferred reporter proteins are those that can be easily identified and quantified when expressed in effector T cells. Many suitable examples are known to those skilled in the art, including fluorescent reporter proteins and luminescent reporter proteins. Optionally, the reporter protein is a bioluminescent reporter protein, such as luciferase.

[0064] Bioluminescence is a special form of chemiluminescence present in living organisms. This type of chemiluminescence is an enzyme-catalyzed process that has evolved naturally to achieve high efficiency in photon emission. These enzymes are called luciferases, and the photon-emitting substrate is luciferin. Bioluminescence chemistry has evolved from several independent origins and includes many different molecular structures. Among the many natural forms, two have been widely used for the assay of genetic reporter proteins: firefly luciferase and kidney luciferase (Fan and Wood (2007) Bioluminescent assays for high-throughput screening. Assay Drug Dev. Technol. 5, 127–36).

[0065] Bioluminescence reporter gene assays offer significant advantages over fluorescence assays (such as green fluorescent protein (GFP)) because they can provide 10 to 1,000 times higher assay sensitivity. Both fluorescence and luminescence generate photons due to the energy conversion from excited-state molecular orbitals to lower-energy orbitals. However, they differ in how they generate excited-state orbitals. In luminescence, the excited state is a product of an exothermic chemical reaction, while in fluorescence, the excited state is generated by the absorption of light. Bioluminescence is advantageous in reporter protein assays because it does not require photons to generate the excited state. Therefore, the photons emitted when measuring the sample do not constitute an inherent background. The resulting low background allows for the precise measurement of minute changes in light within a linear range of four to eight orders of magnitude.

[0066] Luciferase reporter protein technology is based on the interaction between luciferase and the luminescent substrate luciferin, which emits light through a bioluminescent process. Bioluminescence exists in many different organisms; however, firefly (Photinus pyralis) luciferase is by far the most commonly used bioluminescent reporter protein due to the sensitivity and convenience of enzyme assays and the close relationship between protein synthesis and enzyme activity. This 61 kDa monomeric enzyme catalyzes a two-step oxidation reaction to produce light, typically in the green to yellow region, usually at 550–570 nm. The gene encoding firefly luciferase (luc) is a cDNA and does not require any post-translational modifications. This means that it can be used directly as a mature enzyme after translation from its mRNA.

[0067] Suitable reporter genes encoding luciferase are commercially available from several companies, including Promega.

[0068] By coupling a operative regulatory element to the expression of the luciferase gene, typically by placing the regulatory element only upstream of the gene encoding luciferase, the activation of effector T cells can be readily detected by a luminescent signal. Typically, the reporter gene is located downstream of the cloned response element.

[0069] Optionally, the reporter gene is under the control of a promoter or a response element. Suitable response elements include the NFAT (Activated T Nuclear Factor) Response Element (NFAT-RE).

[0070] As explained above, TCR stimulation induces intracellular calcium release and activation of calcineurin, thereby dephosphorylating activating T nuclear factor (NFAT) in the cytoplasm. Dephosphorylated NFAT translocates to the nucleus and binds to NFAT-RE, inducing transcription of reporter genes.

[0071] Optionally, effector T cells include heterologous nucleic acids containing reporter genes.

[0072] Optionally, effector T cells include heterologous nucleic acids encoding LAG-3.

[0073] The term "heterogeneous" reporter gene or nucleic acid is used herein to include reporter genes or nucleic acids that are not naturally present in effector T cells but have been introduced into effector T cells or effector T cells from which said effector T cells are derived, for example, by cloning, recombination, or transfection (techniques well known to those skilled in the art).

[0074] Optionally, effector T cells are double-transfected with a heterologous nucleic acid comprising a reporter gene (optionally, wherein the reporter gene is operatively linked to a promoter or response element to direct the expression of the reporter gene in effector T cells) and a heterologous nucleic acid encoding LAG-3.

[0075] Optionally, the kit of the present invention may further include a molecule of the same type as the agonist or candidate agonist but known to lack agonist activity against LAG-3, for use as a negative control.

[0076] For example, if the agonist or candidate agonist is an antibody, then the molecule optionally used as a negative control is also an antibody. Preferably, the negative control antibody is an antibody of the same type as the agonist or candidate agonist antibody.

[0077] Optionally, the kit of the present invention also includes a known LAG-3 agonist for use as a positive control. For example, a known agonist may be an agonist antibody or a fragment or derivative thereof that retains agonist activity.

[0078] It should be understood that the determination of this invention should be performed in the absence of natural ligands of LAG-3, because otherwise the natural ligands will interfere with the determination results.

[0079] Optionally, the kit of the present invention does not include a natural ligand for LAG-3.

[0080] Effector T cells include several types of T cells that actively respond to stimuli (such as co-stimuli). These include CD4+ T cells. + CD8 + And regulatory T cells. A suitable example is Jurkat cells. Jurkat cells are immortalized T lymphocytes, first derived from the peripheral blood of children with T-cell leukemia (Schneider et al., 1977, Int J Cancer 19(5):621–6).

[0081] Optionally, the agonist is an anti-LAG-3 agonist antibody or a fragment or derivative thereof that retains anti-LAG-3 agonist activity.

[0082] Optionally, effector T cells include T cell lines double-transfected with nucleic acids encoding LAG-3 and reporter genes under the control of promoters or response elements, such as the Jurkat cell line (Jurkat LAG-3) double-transfected with nucleic acids encoding LAG-3 and the NFAT / luciferase reporter gene. + Jurkat 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).

[0083] 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 include Jurkat LAG-3. + / NFAT-luc2 cells.

[0084] Optionally, the agonist anti-LAG-3 antibody is the agonist anti-LAG-3 antibody described in WO 2017 / 037203. The agonist anti-LAG-3 antibody described in WO 2017 / 037203 includes mouse monoclonal antibody 13E2 and humanized 13E2-human IgG4Fc antibody (referred to as IMP761). Antibodies 13E2 and IMP761 include the VH CDR1-3 and VLCDR1-3 sequences as shown in Table 1 below:

[0085] Table 1. CDR sequences of anti-LAG-3 agonist antibodies VH and VL

[0086]

[0087] Optionally, the agonist anti-LAG-3 antibody or fragments or derivatives thereof comprise the VHCDR1-3 and VL CDR1-3 sequences of SEQ ID NO:1-6 or the VH CDR1-3 and VL CDR1-3 sequences of SEQ ID NO:7-12, respectively.

[0088] Optionally, the agonist anti-LAG-3 antibody is IMP761.

[0089] The embodiments of the present invention are described below by way of example only, with reference to the accompanying drawings, in which:

[0090] Figure 1 A schematic diagram showing TCR signal transduction (from Belikov, Aleksey. (2016). The role of reactive oxygen species and mitochondria in T-cell activation. 10.13140 / RG.2.1.2916.0568);

[0091] Figure 2 The mechanism of action of the IMP761 potency assay according to an embodiment of the present invention is shown. 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 via TCR-mediated signal transduction (top figure). IMP761 binds to LAG-3 on the surface of reporter Jurkat cells, inhibiting TCR-mediated signal transduction, resulting in downregulation of luciferase expression from the NFAT / Luc reporter gene (bottom figure).

[0092] Figure 3The study showed the stimulatory effects of different concentrations of OKT3 and UCHT1 anti-CD3 antibodies on Jurkat LAG-3+ / NFAT-luc2 cells in the presence of 300 ng / ml IMP761 or human IgG4 negative control antibodies; and

[0093] Figure 4 Examples of efficacy assays of IMP761 relative to an IgG4 negative control, according to embodiments of the present invention, are shown; and

[0094] Figure 5 Examples of efficacy determination results comparing a reference IMP761 formulation (4°C) with an IMP761 formulation after denaturing temperature stress (10 or 20 minutes at 70°C) according to embodiments of the present invention are shown.

[0095] Example 1

[0096] Optimization of the IMP761 efficacy assay protocol

[0097] Jurkat Lag-3+ / NFAT-luc2 effector cells were originally developed by Promega to determine the activity of antagonist anti-LAG-3 antibodies following TCR activation via stimulation with a superantigen presented by MHC II molecules. Blockage of the LAG-3 / MHC II interaction antagonist anti-LAG-3 antibody leads to TCR activation and enhanced luciferase activity. Determining agonist anti-LAG-3 antibody activity using Jurkat Lag-3+ / NFAT-luc2 effector cells requires a very different experimental setup, as explained below.

[0098] Anti-CD3 antibodies as stimulants for Jurkat cells:

[0099] 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 (LAG-3 ligand). This is important for testing the blocking activity of antagonist anti-LAG-3 antibodies against LAG-3 / MHC II interaction. However, since testing the potency of agonist anti-LAG-3 antibodies does not require LAG-3 / MHC II interaction, Raji cells and staphylococcal enterotoxin are not necessary. A single-cell type assay is used in conjunction with an anti-CD3 antibody to activate Jurkat Lag-3+ / NFAT-luc2 effector cells via TCR signaling.

[0100] Anti-CD3 concentration and LAG-3-related inhibition:

[0101] The effects of two different anti-CD3 antibodies (OKT3 and UCHT1) on cell potency assays were tested at different antibody concentrations ranging from 1 to 500 ng / ml.

[0102] Jurkat Lag-3+ / NFAT-luc2 cells were incubated for 24 hours with 300 ng / ml IMP761 or human IgG4 (as a negative control) in the presence of different concentrations of OKT3 or UCHT1. The mean RLU values ​​obtained for different concentrations of anti-CD3 antibody are shown below. Figure 3 And as shown in Table 2 below:

[0103] Table 2

[0104]

[0105] Within different concentration ranges for each anti-CD3 antibody, luciferase activity was inhibited by IMP761.

[0106] In unstimulated Jurkat cell lines, basal levels of reporter protein expression exist, so luciferase activity and the inhibition of luciferase activity by IMP761 were tested in the absence of anti-CD3 antibody. However, stimulation of Jurkat cells with anti-CD3 antibody yielded higher RLU values, therefore the inhibitory effect of IMP761 was more pronounced in the presence of anti-CD3 antibody (especially low concentrations). The percentage inhibition of luciferase activity by OKT3 antibody is shown in Table 3 below:

[0107] Table 3

[0108]

[0109] The conclusion was that IMP761 exhibited the greatest effect (approximately 80% inhibition) when using low concentrations of OKT3 antibody (between 1 and 4 ng / ml). Therefore, optimal potency assays include stimulating Jurkat cells with low concentrations of anti-CD3 antibody (e.g., OKT3 antibody).

[0110] Example 2

[0111] IMP761 efficacy test

[0112] This embodiment describes a potency assay according to an embodiment of the present invention, which measures the activity of the IMP761 monoclonal antibody in vitro. The method is based on the ability of IMP761 to reduce the 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 is the Jurkat T cell line, which expresses LAG-3 on its surface and contains the luciferase gene (Jurkat Lag-3) under the control of NFAT (activated T cell nuclear factor) response elements. + / NFAT-luc2 effector cells, from Promega). After binding to its target, IMP761 triggers TCR-induced downregulation of NFAT expression (e.g., Figure 2 (See diagram below for schematic representation). The luciferase activity of the cell line was used to measure TCR-driven cell activation inhibited in the presence of IMP761 activity. Therefore, this assay measures the potency of IMP761 based on its ability to inhibit TCR signaling.

[0113] reagents

[0114] Jurkat LAG-3+ / NFAT-luc2 effector cells (Promega, reference: CS194801)

[0115] RPMI 1640 (GIBCO, Reference: 31870-025)

[0116] L-Glutamine (200mM) (GIBCO, Reference: 25030-024)

[0117] HEPES (1M) (GIBCO, Reference: 15630-080)

[0118] FCS (GIBCO, Reference: 10270106)

[0119] IMP761 (2.06 mg / ml) (IMMUTEP, batch number: 270416)

[0120] Human IgG4, control (Biolegend, reference: 403402)

[0121] Anti-CD3 (OKT3) (eBioscience, Reference: 16-0037-85)

[0122] Bio-Glo reagent (PROMEGA, reference: G7941)

[0123] White 96-hole solid flat-bottom microperforated plate (COSTAR, reference: 3917)

[0124] Assay medium: RPMI 1640, L-glutamine (2 mM), Hepes (10 mM), FCS 1%

[0125] Cell concentration: 1.33 x 10⁻⁶ 6 Cells / mL

[0126] plan

[0127] 1. On day -1 before assay, subculture Jurkat LAG-3+ / NFAT-luc2 effector cells to achieve a cell density of approximately 1 million cells / mL (between 80 and 1.2 million cells / mL) on day 0.

[0128] 2. If necessary, prepare the assay medium and preheat the medium at 37°C for 30 minutes:

[0129] FCS 0.5ml L-Glutamine (200mM) 0.5ml HEPES (1M) 0.5ml RPMI 1640 48.5ml

[0130] 3. If necessary, prepare IMP761 quality control (QC) stock solution:

[0131] Prepare a stock solution of 24,000 ng / ml; for example:

[0132] 10 μl of stock IMP761 (2.06 mg / ml) + 848.3 μl of assay medium.

[0133] Store 25 μl aliquots of the sample in a -80°C freezer.

[0134] 4. Prepare 3X solutions of IMP761 (batch 270416, 2.06 mg / ml), human IgG4 negative control (or any other antibody).

[0135] Adjust the pre-dilution steps according to the initial antibody concentration: The volume (in μl) of the assay medium to be added to dilute 2 μl of the stock solution to prepare a 100 μg / ml pre-diluted solution is:

[0136]

[0137]

[0138] 5. Preparation of 3X IMP761 quality control (QC) solution

[0139] Thaw the stock IMP761 QC [24,000 ng / ml] aliquots and prepare serially diluted solutions.

[0140] Very High (VH) QC: 20 μl [24,000 ng / ml] + 380 μl = 1,200 ng / ml (final concentration: 400 ng / ml)

[0141] High (H) QC: 150 μl [1,200 ng / ml] + 225 μl = 480 ng / ml (final concentration: 160 ng / ml)

[0142] Medium (M) QC: 150 μl [480 ng / ml] + 225 μl = 192 ng / ml (final concentration: 64 ng / ml)

[0143] Low (L) QC: 150 μl [192 ng / ml] + 225 μl = 76.8 ng / ml (final concentration: 25.6 ng / ml)

[0144] Very low (VL) QC: 150 μl [25.6 ng / ml] + 225 μl = 10.24 ng / ml (final concentration: 3.4 ng / ml)

[0145] 6. Preparation of OKT3 reagent

[0146] 3.6 μl stock solution + 4 ml assay medium = 0.9 μg / ml

[0147] 150 μl [0.9 μg / ml] + 14,850 μl assay medium = 9 ng / ml (3X)

[0148] 7. Preparation of Jurkat LAG-3 / NFAT-luc2 effector cells

[0149] On day 0, cells were counted using trypan blue staining.

[0150] Centrifuge the cells at 1200 rpm for 5 minutes.

[0151] Aspirate the culture medium and transfer the cells at a temperature of 3.75 x 10⁻⁶. 6 / ml resuspended in the assay medium

[0152] 8. Distribute 3X solution in a 96-well plate.

[0153] Due to edge effects, do not use external holes.

[0154] Dispense 40 μl of the 3X solution of IMP761 / QC / Assay Medium (0) into the corresponding wells of the assay plate, in duplicate.

[0155] Dispense 40 μl / well of the 3X solution of anti-CD3 / assay medium (unstimulated control) into each well of the assay plate, in duplicate. Final concentration: 3 ng / ml

[0156] Dispense 40 μl / well (0.15 x 10⁻⁶) 6 / hole)

[0157] Example of a board template (reference batch / unknown)

[0158]

[0159] 9. Incubate the plates at 37°C for 24 hours in a humidified incubator with 5% CO2.

[0160] 10. Preparation of BioGlo Reagent

[0161] a) Thaw frozen BioGlo reagent at room temperature (RT) for 3-6 hours before use.

[0162] b) Transfer the buffer solution (10 ml) to the substrate vial, mix, and store at room temperature protected from light until use.

[0163] 11. Balance the plate under RT for 15 minutes.

[0164] 12. Add 120 μl / well of BioGlo to avoid / remove air bubbles.

[0165] 13. Incubate at RT for 5 to 15 minutes.

[0166] 14. Use a PerkinElmer 2103 Multilabel reader Envision to measure light emission (RLU), with an integration time of 0.5 seconds per well. Collect three measurements for each well at 45-second intervals and calculate the average of the three measurements to obtain the "RLU average".

[0167] result

[0168] Figure 4 This example illustrates the results obtained from a potency assay using an IgG4 antibody as a negative control. Maximum activation is recorded as the activation observed in the absence of IMP761. Maximum inhibition is recorded as the activation observed at an IMP761 concentration of 1000 ng / ml. A five-parameter nonlinear regression model was used to determine the IC50 of IMP761. 50 It was 37 ng / ml.

[0169] Example 3

[0170] IMP761 potency assay assesses the ability of denatured IMP761 antibodies.

[0171] By comparing the IC of reference IMP761 stored at 4°C 50ICs from the same batch of IMP761 after temperature stress (10 minutes and 20 minutes at 70°C) 50 The ability of the denatured IMP761 antibody to assess reduced efficacy was tested using the potency assay described in Example 2. The results are shown in... Figure 5 And in Table 4 below:

[0172] Table 4: IMP761 after using reference IMP761 (4°C) and deformation temperature stress (10 and 20 minutes at 70°C) The efficacy measurement dataset (average of two copies) is expressed in relative light units (RLU).

[0173]

[0174] Outliers were removed from the dataset.

[0175] A five-parameter nonlinear regression model determined the IC50 of the denatured IMP761 antibody (74 ng / ml and 81.5 ng / ml, respectively, after 10 and 20 minutes at 70°C). 50 The IC50 value is higher than that of the reference IMP761 antibody stored at 4°C. 50 (41ng / ml). sequence list <110> Emutech Ltd. <120> Measurement <130> P / 80025.WO01 <140> PCT / EP2020 / 062206 <141> 2020-05-01 <150> GB 1906127.4 <151> 2019-05-01 <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR sequence <400> 1 Gly Phe Ser Leu Ser Thr Ser Gly Met Gly 1 5 10 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR sequence <400> 2 Ile Trp Trp Asp Asp Ile Lys 1 5 <210> 3 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> CDR sequence <400> 3 Ala Arg Ile Val Glu Gly Ser Tyr Ser Ser Ser Tyr Phe Asp Val 1 5 10 15 <210> 4 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> CDR sequence <400> 4 Gln Asp Val Ile Phe Asp 1 5 <210> 5 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> CDR sequence <400> 5 Ser Ala Ser 1 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR sequence <400> 6 Gln Gln His Tyr Ser Thr Pro Tyr Thr 1 5 <210> 7 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR sequence <400> 7 Thr Ser Gly Met Gly Leu Gly 1 5 <210> 8 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDR sequence <400> 8 His Ile Trp Trp Asp Asp Ile Lys Arg Tyr Asn Pro Asp Leu Arg Ser 1 5 10 15 <210> 9 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDR sequence <400> 9 Ile Val Glu Gly Ser Tyr Ser Ser Ser Tyr Phe Asp Val 1 5 10 <210> 10 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR sequence <400> 10 Lys Ala Ser Gln Asp Val Ile Phe Asp Val Ala 1 5 10 <210> 11 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR sequence <400> 11 Ser Ala Ser Ser Arg Val Ser 1 5 <210> 12 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR sequence <400> 12 Gln Gln His Tyr Ser Thr Pro Tyr Thr 1 5

Claims

1. An in vitro assay for determining the activity of a lymphocyte activation gene 3 (LAG-3) agonist, comprising: Multiple effector T cells are provided, each effector T cell expressing LAG-3 and T cell receptor (TCR) on its surface and including a reporter gene encoding a reporter protein, wherein the expression of the reporter protein is regulated by LAG-3-mediated inhibition of TCR signaling within the effector T cell; as well as The activity of the agonist is determined based on the degree to which the expression of the reporter protein changes in the presence of the agonist compared to the expression of the reporter protein in the absence of the agonist. The determination was performed in the absence of a natural ligand for LAG-3, wherein the natural ligand is an MHC class II ligand.

2. The assay according to claim 1 is used to determine the potency of a LAG-3 agonist formulation.

3. An in vitro assay for screening LAG-3 agonists, comprising: Provided are multiple effector T cells, each effector T cell expressing LAG-3 and T cell receptor (TCR) on its surface and including a reporter gene encoding a reporter protein, wherein the expression of the reporter protein is regulated by LAG-3-mediated inhibition of TCR signaling within the effector T cell; and Whether a candidate agonist is a LAG-3 agonist is determined by measuring the degree to which the expression of the reporter protein changes in the presence of the candidate agonist compared to the absence of the candidate agonist. The determination was performed in the absence of a natural ligand for LAG-3, wherein the natural ligand is an MHC class II ligand.

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

5. The assay according to claim 3, wherein the reporter gene is expressed at a basal level in the effector T cells in the absence of the candidate agonist.

6. The assay according to claim 1 or 3, wherein the expression of the reporter protein is reduced in the presence of the agonist or candidate agonist compared with the expression of the reporter protein in the absence of the agonist or candidate agonist.

7. The assay according to claim 1 or 3, wherein the expression of the reporter protein in each effector T cell is altered in response to activation of the effector T cells via the TCR, and the assay further comprises: The effector T cells are activated in the presence or absence of the agonist or candidate agonist via antigen-independent, MHC class II-independent, TCR-mediated T cell activation. as well as The activity of the agonist or candidate agonist is determined based on the degree to which the expression of the reporter protein in response to activation of the effector T cells in the presence of the agonist or candidate agonist changes compared to the expression of the reporter protein in response to activation of the effector T cells in the absence of the agonist or candidate agonist.

8. The assay according to claim 7, wherein the expression of the reporter protein in each effector T cell increases in response to activation of the effector T cell via the TCR due to LAG-3-mediated inhibition of TCR signaling in the effector T cell and decreases in the presence of the agonist or candidate agonist, and wherein the activity of the agonist or candidate agonist is determined based on the degree to which the expression of the reporter protein in response to activation of the effector T cell in the presence of the agonist or candidate agonist is reduced compared to the expression of the reporter protein in response to activation of the effector T cell in the absence of the agonist or candidate agonist.

9. The assay according to claim 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 the effector T cells to be activated by the T cell activator in an antigen-independent, MHC class II-independent, TCR-mediated manner.

10. The assay according to claim 9, wherein the effector T cell is in contact with the T cell activator, the concentration of the T cell activator being the concentration that produces maximum inhibition of the expression of the reporter protein in the presence of an excess of LAG-3 agonist.

11. The assay of claim 9, wherein the effector T cell is in contact with the T cell activator, and the concentration of the T cell activator is lower than the concentration of the T cell activator observed to be at which the expression of the reporter protein is maximized in response to the activation of the effector T cell by the T cell activator in the absence of the agonist or candidate agonist.

12. The assay according to claim 9, wherein the T cell activator comprises or is composed of the following: an anti-CD3 antibody or a fragment or derivative thereof that retains antigen-independent, MHC class II-independent, and TCR-mediated effector T cell activation capacity.

13. The assay according to claim 12, wherein the anti-CD3 antibody is OKT3.

14. The assay according to claim 12, wherein the concentration of the anti-CD3 antibody or a fragment or derivative thereof in contact with the effector T cells is 6-30 x 10⁻⁶. -12 M.

15. The assay according to claim 1 or 3, wherein the effector T cells are activated via cell-free, antigen-independent, MHC class II-independent, TCR-mediated T cell activation.

16. The assay according to claim 1 or 3, wherein the effector T cells are contacted with several different concentrations of the agonist or candidate agonist.

17. The assay of claim 16, further comprising determining the IC50 of the agonist or candidate agonist for inhibiting the expression of the reporter protein. 50 value.

18. The assay according to claim 1 or 3, wherein the effector T cell comprises a heterologous nucleic acid including the reporter gene.

19. The assay according to claim 1 or 3, wherein the reporter gene is under the control of a promoter or response element.

20. The assay according to claim 19, wherein the reaction element comprises the NFAT (Activated T Nuclear Factor) Reaction Element (NFAT-RE).

21. The assay according to claim 1 or 3, wherein the reporter protein comprises a bioluminescent reporter protein.

22. The assay according to claim 21, wherein the bioluminescent reporter protein is luciferase.

23. The assay according to claim 1 or 3, wherein the effector T cells comprise a heterologous nucleic acid encoding LAG-3.

24. The assay according to claim 1 or 3, further comprising 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 known to lack agonist activity against LAG-3.

25. The assay according to claim 1 or 3, 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.

26. The assay according to claim 1 or 3, wherein the effector T cells comprise Jurkat-derived cells.

27. The assay according to claim 1 or 3, wherein 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 comprise Jurkat LAG-3. + / NFAT-luc2 cells.

28. The assay according to claim 1 or 3, wherein the agonist is an anti-LAG-3 antibody or a fragment or derivative thereof comprising the VH CDR1-3 sequence and VL CDR1-3 sequence composed of SEQ ID NO: 1-6 or the VH CDR1-3 sequence and VLCDR1-3 sequence composed of SEQ ID NO: 7-12.

29. The assay according to claim 1 or 3, wherein the agonist is anti-LAG-3 antibody IMP761.

30. A kit for performing an in vitro assay to determine the activity of a LAG-3 agonist or to screen for LAG-3 agonists, comprising: Multiple effector T cells, each effector T cell expressing LAG-3 and T cell receptor (TCR) on its surface and including a reporter gene encoding a reporter protein, wherein the expression of the reporter protein is regulated by LAG-3-mediated inhibition of TCR signaling within the effector T cell, and wherein the expression of the reporter protein in each effector T cell is altered in response to activation of the effector T cell via the TCR; and T cell activators that can activate effector T cells in an antigen-independent, MHC class II-independent, and TCR-mediated manner; The kit does not include a natural ligand for LAG-3, which is an MHC class II ligand.

31. The kit of claim 30, wherein the kit does not contain cells expressing MHC class II molecules.

32. The kit of claim 30, wherein the expression of the reporter protein is increased in response to activation of the effector T cells via the TCR.

33. The kit of claim 30, wherein the effector T cells comprise heterologous nucleic acids including the reporter gene.

34. The kit of claim 30, wherein the reporter gene is under the control of a promoter or reaction element.

35. The kit according to claim 34, wherein the reaction element comprises the NFAT (nuclear factor for activating T cells) reaction element (NFAT-RE).

36. The kit of claim 30, wherein the reporter protein comprises a bioluminescent reporter protein.

37. The kit according to claim 36, wherein the bioluminescent reporter protein is luciferase.

38. The kit of claim 30, wherein the effector T cells comprise a heterologous nucleic acid encoding LAG-3.

39. The kit according to claim 30, wherein the T cell activator is a cell-free T cell activator.

40. The kit of claim 30, wherein the T cell activator comprises an anti-CD3 antibody or a fragment or derivative thereof that retains antigen-independent, MHC class II-independent, and TCR-mediated effector T cell activation capabilities.

41. The kit according to claim 40, wherein the anti-CD3 antibody is OKT3.

42. The kit of claim 40, wherein the concentration of the anti-CD3 antibody or a fragment or derivative thereof present allows it to be used in the assay at a concentration of 6-30 x 10⁻⁶. -12 M.

43. The kit of claim 30, further comprising a molecule of the same type as the agonist but known to lack agonist activity against LAG-3, for use as a negative control.

44. The kit of claim 30, further comprising a known LAG-3 agonist for use as a positive control.

45. The kit of claim 30, wherein the effector T cells comprise Jurkat-derived cells.

46. ​​The kit of claim 30, wherein the effector T cells comprise Jurkat LAG-3. + / NFAT-luc2 cells.

47. The kit of claim 30, wherein the kit does not include APC, aAPC, or MHC class I or MHC class II expressing cells.

48. The kit according to claim 30, wherein the only cells in the kit are the effector T cells.

49. The kit according to claim 30, used to determine the potency of a LAG-3 agonist formulation.

50. The kit according to claim 30, used for performing the assay according to any one of claims 1 to 27.

51. The kit according to claim 30 is used for determining the activity of a LAG-3 agonist, determining the potency of a LAG-3 agonist formulation, or screening for LAG-3 agonists.

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