Patent Application
20220341913
This invention relates to assays for screening for, or determining activity of (including determining the potency of a preparation of), an agonist of lymphocyte-activation gene 3 (LAG-3). The invention also relates to kits for carrying out the assays.
Stimulation of T cell function is initiated upon interaction of the T cell receptor (TCR) with short peptides presented by MHC class I or II molecules (MHC I for CD8 T cells, MHC II for CD4 T cells) on the surface of antigen-presenting cells (APCs). In primary T cell, the TCR by itself is not capable of activating downstream pathways to initiate T cell activation. This also requires co-receptors, such as CD4 for helper T cells, and CD8 for cytotoxic T cells. These co-receptors bind their respective MHC molecules and stabilise the interaction of T cells and APCs. In addition to TCR binding to antigen-loaded MHC, both helper T cells and cytotoxic T cells require a number of 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 the receptor for two molecules expressed on the APC (CD80 and CD86) and initiates T-cell proliferation, leading to the expansion of T cell clones specific for the antigen. Cytotoxic T cells are less reliant on CD28 for activation, but do require signals from other co-stimulatory molecules such as CD70 and CD137.
The TCR is located in close proximity to a complex of signaling molecules, which mediate T cell activation through multiple signaling cascades (see
Phosphorylated CD3 ITAMs recruit and activate the Syk family kinase zeta-activated protein 70 kDa (ZAP70). ZAP70 then phosphorylates a membrane associated scaffolding protein called Linker for Activation of T cells (LAT). LAT in turn recruits a second molecular scaffold, SH2-domain-containing Leukocyte Protein of 76 kDa (Slp-76). Slp-76 is then phosphorylated by ZAP70 and the resulting LAT-Slp-76 complex acts as a scaffold for the recruitment of signalling effector molecules. Interleukin-2 inducible tyrosine kinase (ITK) then interacts with the LAT-Slp-76 complex and becomes activated by auto-phosphorylation. This promotes 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 trisphosphate (IP3).
DAG, a membrane associated lipid, activates a number of downstream proteins, including various isoforms of protein kinase C (PKC) and RAS guanyl nucleotide-releasing protein (RasGRP). Upon activation by DAG, PKC-theta participates in activating the NF-κB pathway, whereas RasGRP is a crucial activator of MAPK signalling pathways. IP3 stimulates the efflux of Ca2+ from the endoplasmic reticulum to the cytoplasm. Elevated Ca2+ levels induce activation of the protein phosphatase, calcineurin, which then dephosphorylates the T cell transcription factor nuclear factor of activated T cells (NFAT). Dephosphorylated NFAT then migrates to the nucleus to join other transcription factors in inducing the transcription of specific genes.
Uncontrolled immune responses to pathogens or self-antigens can cause inflammatory tissue damage and autoimmune diseases. To prevent this, immune responses are regulated by a balance between co-stimulatory and inhibitory signals, collectively referred to as immune checkpoints, which are necessary for maintaining self-tolerance and protecting 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 modulate T cell responses to self proteins as well as to chronic infections and tumor antigens. Inhibitory immune checkpoint receptors are targets for cancer immunotherapy due to their potential for use in multiple types of cancers.
LAG-3 is a CD4 homolog type I membrane protein with four extracellular Ig superfamily domains. Similar to CD4, LAG-3 oligomerizes at the surfaces 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 at the cell surface and negatively regulates signal transduction. As a consequence, it negatively regulates T cell proliferation, function, and homeostasis. When recognition of the MHC class II-peptide complex by a specific TCR occurs, intracellular signals are transduced in the − cell through the TCR and in the APC through MHC class II molecules. The negative regulatory role of LAG-3 signalling into T cells operates in primary CD4+ and CD8 + human T-cell responses (Maçon-Lemaître, et al., Immunology. 2005 Jun; 115(2):170-178). However, the molecular mechanism by which LAG-3 negatively regulates signal transduction in T cells is not known. The inhibitory function of LAG-3 requires its intracellular (IC) region, but this region does not contain a typical signalling motif with a known signalling mechanism. Maeda et al. (J. Biol. Chem. 2019, RA119.007455) have recently reported that LAG-3 transduces two independent inhibitory signals through an FxxL motif in the membrane-proximal region and the C-terminal EX repeat. However, these motifs have not previously been reported for inhibitory co-receptors, and the molecular mechanism of the inhibitory signal transduced by LAG-3 remains elusive.
WO 2017/037203 describes antibodies (such as the humanized monoclonal antibody IMP761), and antigen-binding fragments thereof, that are agonists of LAG-3, and their use for the treatment of conditions associated with proliferation and/or activation of CD4+ and/or CD8+ T cells, in particular inflammatory and autoimmune disorders.
Conventional methods used to measure the activity of antibody and other drugs designed to target immune checkpoint receptors rely on primary human cells and measurement of functional endpoints, such as cell proliferation, cell surface marker expression, and cytokine production. These assays are laborious and highly variable due to their reliance on donor primary cells, complex assay protocols, and unqualified assay reagents. As a result, these assays are difficult to establish in quality-controlled drug development settings. To address these difficulties, cell-based bioluminescent reporter bioassays have been developed by Promega Corporation for individual and combination immune checkpoint immunotherapy targets (Cheng et al., Jun. 2016, Promega, “Quantitative Cell-Based Bioassays for Indivisual and Combination Immune Checkpoint Immunotherapy Targets”).
The bioassays developed by Promega include Blockade Bioassays for PD-1, CTLA-4, LAG-3, and TIGIT. These assays rely on use of Jurkat T cells, which have been genetically engineered to express the co-inhibitory receptor of interest on the surface, and which contain a firefly luciferase reporter gene under the control of an NFAT response element (NFAT-RE). These cells express endogenous TCR, CD3 and CD28 receptors. When the cells are engaged with an appropriate ligand, the TCR transduces intracellular signals resulting in increased NFAT-RE-mediated luminescence. The bioluminescent signal is detected and quantified using a luciferase substrate and a standard luminometer. The assays also use artificial antigen-presenting cells (aAPCs) expressing an engineered cell surface protein designed to activate TCRs in an antigen-independent manner, and expressing a natural ligand for the co-inhibitory receptor on their surface (for the PD-1, CTLA-4 and TIGIT Blockade Bioassays), or Raji cells in the presence of a Staphylococcal Enterotoxin E (SEE) Superantigen (for the LAG-3 Blockade Bioassay—Raji cells naturally express MHC class II, a LAG-3 ligand). TCR engagement on the Jurkat cells induces luciferase activity. Co-engagement of the co-inhibitory receptor with its natural ligand inhibits luciferase activity. Antibody-mediated blockade of binding of the co-inhibitory receptor to its natural ligand restores luciferase activity.
These bioassays demonstrate the performance required 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 antibody (for example, IMP761, described in WO 2017/037203). In particular, the presence of natural ligand for LAG-3 in the blockade bioassay (such as MHC class II expressed Raji cells) would interfere with testing of an agonist. There is, therefore, a need to provide in vitro assays for identifying and testing agonists of LAG-3.
It has now surprisingly been found that IMP761, through agonism of LAG-3, inhibits TCR signaling, in particular NFAT-regulated gene expression, in LAG-3 positive T-cells. The applicant has appreciated that this can form the basis of in vitro bioassays to determine activity of a LAG-3 agonist, or a preparation of a LAG-3 agonist, and to identify new LAG-3 agonists.
According to the invention there is provided an in vitro assay for determining activity of an agonist of lymphocyte-activation gene 3 (LAG-3), which comprises:
Assays of the invention for determining activity of the agonist include assays for determining the potency of a preparation of the agonist, for example, as part of a quality control step in production of the agonist (in particular, as a cell-based potency assay required for product release according to Good Manufacturing Practice, GMP), or for stability testing of a preparation of the agonist, for example, after a period of storage, or as a product characterization assay.
There is also provided according to the invention an in vitro assay for screening for an agonist of LAG-3, which comprises:
Assays of the invention for screening for an agonist of LAG-3 can be used to identify an agonist of LAG-3, for example, from a library of candidate agonists. Such candidate agonists may be drugs (e.g. synthetic small molecules), or biological agents, such as recombinant or natural proteins, antibodies, or fragments or derivatives thereof.
The reporter may be expressed in the effector T cells in the absence of the agonist, or candidate agonist.
Optionally expression of the reporter is reduced in the presence of the agonist, or candidate agonist, compared with expression of the reporter in the absence of the agonist, or candidate agonist.
For example, LAG-3-mediated inhibition of TCR signaling within the effector T cells may cause a reduction in a basal level of expression of the reporter in the effector T cells (see
The term “LAG-3-mediated inhibition of TCR signaling within the effector T cells” is used herein to mean that agonism of LAG-3 expressed on the surface of the effector T cells inhibits TCR-mediated signal transduction within the effector T cells, with a consequential alteration (i.e. an increase, or a decrease) in expression of the reporter. Any TCR-mediated signalling pathway within the effector T cells may be inhibited. For example, expression of the reporter gene may be under the control of a promoter or a response element (RE) which is responsive to binding of a transcription factor which is part of the signaling pathway. For example, the calcineurin/NFAT signaling pathway within the effector T cells may be inhibited. In particular, expression of the reporter gene may be under the control of an NFAT response element, such that a reduction in calcinuerin/NFAT signaling, following agonism of LAG-3, causes a reduction in expression of the reporter from the reporter gene.
Expression of the reporter may be altered (i.e. increased or decreased) in each effector T cell in response to activation of the effector T cell via the TCR. Optionally assays of the invention further comprise: 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; and determining the activity (including the potency of a preparation) of the agonist, or the candidate agonist, from the extent to which expression of the reporter, in response to activation of the effector T cells, is altered in the presence of the agonist, or candidate agonist, compared with expression of the reporter, in response to activation of the effector T cells, in the absence of the agonist, or candidate agonist.
Thus, according to the invention there is provided an in vitro assay for determining activity of an agonist of lymphocyte-activation gene 3 (LAG-3), which comprises:
According to the invention there is also provided an in vitro assay for screening for an agonist of LAG-3, which comprises:
Optionally expression of the reporter is increased in each effector T cell in response to activation of the effector T cell via the TCR, and is reduced in the presence of the agonist, or candidate agonist, as a result of LAG-3-mediated inhibition of TCR signaling within the effector T cell, and wherein the activity of the agonist, or candidate agonist, is determined from the extent to which expression of the reporter, in response to activation of the effector T cells, is reduced in the presence of the agonist, or candidate agonist, compared with 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 extent to which expression of the reporter is altered in response to activation of the effector T cells in the presence of the agonist, or the candidate agonist, compared with expression of the reporter in response to activation of the effector T cells in the absence of the agonist, or the candidate agonist, the greater the activity (or potency of a preparation) of the agonist, or the candidate agonist, for LAG-3.
Optionally the effector T-cells are activated by cell-free T-cell activation.
The term “cell-free T-cell activation” is used herein to refer to activation of the effector T cells by use of one or more cell-free T-cell activators in the absence of any cells, other than the effector T cells. In particular, cell-free T-cell activation occurs without use of an antigen-presenting cell (APC), an artificial APC (aAPC), or any other MHC class I-, or MHC class II-expressing cell, such as a Raji cell.
Cell-free activation of T cells is advantageous because cell-based reagents require special storage conditions. Cells are typically kept in frozen storage and must then be thawed before use.
Reference to “activation of the effector T cell via the TCR” is used herein to mean that upon engagement of the TCR by a T-cell activator, a signal is transduced by the TCR within the effector T cell, resulting in an alteration (i.e. an increase, or a decrease) in expression of the reporter within the effector T cell. For example, expression from the reporter gene may be under the control of a promoter or a response element (RE) which is responsive to an activation signal of the effector T cells. For example, the response element may be bound by one or more transcription factors following activation of the effector T cell via the TCR.
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.
There is also provided according to the invention a kit for carrying out an in vitro assay for determining activity of, or screening for, an agonist of LAG-3, which comprises: a plurality of effector T cells, each effector T cell expressing LAG-3 and a T-cell receptor (TCR) on its surface, and comprising a reporter gene encoding a reporter, wherein expression of the reporter is regulated by LAG-3-mediated inhibition of TCR signaling within the effector T cells, and wherein expression of the reporter is altered (i.e. increased or decreased) in each effector T cell in response to activation of the effector T cell via the TCR; and a T-cell activator capable of antigen-independent, MHC class II-independent, TCR-mediated activation of the effector T cells.
Antigen-independent, MHC class II-independent, T cell activators are distinguished from superantigens, such as Staphylococcal enterotoxins (SEs). SEs have binding regions for MHC Class II and the TCR. They bind first to MHC Class II, then to the variable alpha or beta chain of the TCR. Interaction with the T cell receptor allows the SE to act as a wedge between the TCR and the MHC. This displaces any antigenic peptide away from the TCR and circumvents the normal mechanism for T-cell activation. SEs are, therefore MHC class II-dependent T cell activators.
Optionally the effector T cells are contacted with a concentration of the T-cell activator at which maximal inhibition of expression of the reporter occurs in the presence of an excess of an agonist of LAG-3. Use of such a concentration of the T-cell activator optimises the accuracy of the assay.
The applicant has found that the concentration of the T-cell activator at which maximal inhibition of expression of the reporter occurs in the presence of an excess of an agonist of LAG-3 is a sub-optimal concentration of the T-cell activator (i.e. a concentration which is less than a concentration of the T-cell activator at which the greatest 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 the candidate agonist).
Optionally the T-cell activator is contacted with the effector T cells at a concentration which is less than a concentration of the T-cell activator at which the greatest 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 the candidate agonist.
It will be appreciated that antigen-dependent stimulation expands only antigen-specific T cells, whereas antigen-independent stimulation expands up to 100% of the effector T cells.
An example of a T-cell activator capable of antigen-independent, MHC class II-independent, TCR-mediated activation of the effector T cells is an anti-CD3 antibody. Anti-CD3 antibody binds to CD3 and activates TCR complex without antigenic peptide from an APC (i.e. antigen-independent, MHC class II-independent, TCR-mediated effector T-cell stimulation) (see
Optionally the T-cell activator comprises 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 an anti-CD3 antibody include OKT3 and UCHT1. Optionally the anti-CD3 antibody is OKT3.
Optionally the anti-CD3 antibody, or fragment or derivative thereof, is contacted with the effector T-cells at a concentration of ˜6-30×10−12 M (1-4 ng/ml for whole antibody, or molar equivalent for a fragment or derivative thereof).
Optionally a kit of the invention comprises 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 to allow its use in the assay at a concentration of ˜6-30×10−12 M (1-4 ng/ml for whole antibody). For example, the kit may comprise one or more aliquots of anti-CD3 antibody, or fragment or derivative thereof, at a concentration of ˜6-30×10−12M (1-4 ng/ml for whole antibody).
Optionally the effector T cells are contacted with several different concentrations of the agonist or the candidate agonist. For example, a plurality of different assays may be carried out in parallel, wherein the effector T cells for each different assay are contacted with a different concentration of the agonist or the candidate agonist.
Optionally an IC50 value of the candidate agonist or agonist for inhibition of expression of the reporter is determined. This can be done, for example, from a dose-response curve generated from the results obtained by contacting the effector T cells with several different concentrations of the candidate agonist or agonist.
Optionally an assay of the invention further comprises carrying out a negative control assay (for example, in parallel with the assay). For example, the effector T cells may be contacted with the T-cell activator under conditions for antigen-independent activation of the 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 which is known to lack agonist activity for LAG-3 (i.e. as a negative control).
For example, if the agonist or candidate agonist is an antibody, optionally the molecule for use as a negative control is also an antibody. Preferably the negative control antibody is an antibody of the same isotype as the agonist or candidate agonist antibody.
Optionally the T cell activator is a cell-free T-cell activator.
Optionally a kit of the invention does not comprise a cell expressing MHC class II molecules.
Optionally a kit of the invention does not comprise Raji cells.
Optionally a kit of the invention does not comprise APCs, aAPCs, or any other MHC class I-, or MHC class II-expressing cells, such as Raji cells.
Optionally the only cells in a kit of the invention are the effector T cells.
Optionally the effector T cells of a kit of the invention are frozen, for example “Thaw-and-Use” cells.
There is also provided according to the invention a kit of the invention for carrying out an assay of the invention.
Optionally the reporter gene is a heterologous reporter gene.
Genetic reporters are used widely as indicators to study gene expression and cellular events coupled to gene expression for pharmaceutical and biomedical research. Typically, a reporter gene encoding the reporter is cloned into an expression vector that is then transferred into cells. Following transfer, the cells are assayed for the presence of the reporter by directly measuring the reporter protein itself or the enzymatic activity of the reporter protein. Preferred reporters are those that can be identified easily and measured quantitatively when expressed in the effector T cells. Many suitable examples are known to those skilled in the art, including fluorescent and luminescent reporters. Optionally the reporter is a bioluminescent reporter, such as a luciferase.
Bioluminescence is a special form of chemiluminescence found in living organisms. This class of chemiluminescence is an enzyme-catalyzed process, where the high efficiency of photon emission is derived through natural evolution. The enzymes are called luciferases, and the photon-emitting substrates are luciferins. Bioluminescent chemistries have evolved from multiple independent origins and comprise many distinct molecular structures. Of the many natural forms, two have been used widely for genetic reporter assays: firefly luciferase and Renilla luciferase (Fan, and Wood (2007) Bioluminescent assays for high-throughput screening. Assay Drug Dev. Technol. 5, 127-36).
Bioluminescent reporter gene assays have a distinct advantage over fluorescent assays, such as green fluorescent protein (GFP), in that they can deliver 10- to 1,000-fold higher assay sensitivity. Both fluorescence and luminescence yield photons as a consequence of energy transitions from excited-state molecular orbitals to lower-energy orbitals. However, they differ in how the excited-state orbitals are created. In luminescence, the excited states are the product of exothermic chemical reactions, whereas in fluorescence the excited states are created by absorption of light. In reporter assays, bioluminescence is advantageous because photons are not required to create the excited states. Therefore, they do not constitute an inherent background when measuring photon efflux from a sample. The resulting low background permits precise measurement of small changes in light across four to eight orders of magnitude in the linear range of the assay.
Luciferase reporter technology is based on the interaction of the enzyme luciferase with a luminescent substrate luciferin, which releases light by the process of bioluminescence. Bioluminescence is found in a number of different organisms; however, it is firefly (Photinus pyralis) luciferase that is by far the most commonly used bioluminescent reporter due to both the sensitivity and convenience of the enzyme assay and to the tight coupling of protein synthesis with enzyme activity. This monomeric enzyme of 61 kDa catalyzes a two-step oxidation reaction to yield light, usually in the green to yellow region, typically 550-570 nm. The gene encoding firefly luciferase (luc) is a cDNA and does not require any post-translational modifications. This means it is available as a mature enzyme directly upon translation from its mRNA.
Suitable reporter genes encoding luciferase are available commercially from several companies, including Promega.
By coupling operative regulatory elements to expression of a luciferase gene, typically by placing the regulatory element just upstream of a gene encoding luciferase, activation of the effector T cells can be detected readily by a luminescent signal. Typically, reporter genes are downstream of cloned response elements.
Optionally the reporter gene is under control of a promoter or response element. A suitable response element comprises a NFAT (nuclear factor of activated T cells) response element (NFAT-RE).
As explained above, TCR stimulation induces intracellular calcium release and activation of calcineurin, which dephosphorylates cytoplasmic nuclear factor of activated T cells (NFAT). Dephosphorylated NFAT translocates to the nucleus and binds to NFAT-RE, inducing transcription of the reporter gene.
Optionally the effector T cells comprise heterologous nucleic acid comprising the reporter gene.
Optionally the effector T cells comprise heterologous nucleic acid encoding LAG-3.
The term “heterologous” reporter gene or nucleic acid is used herein to include reference to a reporter gene or nucleic acid which is not naturally present in the effector T cells, but which has been introduced into the effector T cells or into effector T cells from which the effector T cells are derived, for example by cloning, recombinant technology, or transfection (techniques which are well-known to the skilled person).
Optionally the effector T cells are double-transfected with heterologous nucleic acid comprising the reporter gene (optionally wherein the reporter gene is operably linked to a promoter or response element for directing expression of the reporter gene in the effector T cell), and heterologous nucleic acid encoding LAG-3.
Optionally a kit of the invention further comprises a molecule of the same type as the agonist or candidate agonist, but which is known to lack agonist activity for LAG-3, for use as a negative control.
For example, if the agonist or candidate agonist is an antibody, optionally the molecule for use as a negative control is also an antibody. Preferably the negative control antibody is an antibody of the same isotype as the agonist or candidate agonist antibody.
Optionally a kit of the invention further comprises a known agonist of LAG-3 for use as a positive control. For example, the known agonist may be an agonist antibody, or fragment or derivative thereof that retains agonist activity.
It will be appreciated that an assay of the invention should be carried out in the absence of a natural ligand for LAG-3 because the natural ligand would otherwise interfere with the results of the assay.
Optionally a kit of the invention does not include a natural ligand for LAG-3.
Effector T cells include several T cell types that actively respond to a stimulus, such as co-stimulation. They include CD4+, CD8+, and regulatory T cells. A suitable example is a Jurkat cell. Jurkat cells are immortalized T lymphocytes first derived from the peripheral blood of a child with T cell leukemia (Schneider et al., 1977, Int J Cancer 19 (5):621-6).
Optionally the agonist is an agonist anti-LAG-3 antibody, or a fragment or derivative thereof that retains anti-LAG-3 agonist activity.
Optionally the effector T cells comprise a T cell line that has been double-transfected with nucleic acid encoding LAG-3, and a reporter gene under the control of a promoter or response element, for example a Jurkat cell line double transfected with nucleic acid encoding LAG-3, and a NFAT/luciferase reporter gene (Jurkat LAG-3+/NFAT-luc cells). Jurkat LAG3+/NFAT-luc2 cells are available from Promega (ref: CS194801). Jurkat LAG3+/NFAT-luc cells are available from BPS Bioscience (Catalog #: 71278).
Optionally the agonist is an 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.
Optionally the agonist anti-LAG-3 antibody is an agonist anti-LAG-3 antibody described in WO 2017/037203. Agonist anti-LAG-3 antibodies decribed in WO 2017/037203 include mouse monoclonal antibody 13E2, and humanized 13E2-human IgG4 Fc antibody (referred to as IMP761). Antibodies 13E2 and IMP761 comprise VH CDR1-3 and VL CDR1-3 sequences as set out in Table 1 below:
Optionally the agonist anti-LAG-3 antibody, or fragment or derivative thereof, comprises VH CDR1-3 and VL CDR1-3 sequences of SEQ ID NOs:1-6, respectively, or VH CDR1-3 and VL CDR1-3 sequences of SEQ ID Nos:7-12, respectively:
Optionally the agonist anti-LAG-3 antibody is IMP761.
Embodiments of the invention are described below, by way of example only, with reference to the accompanying drawings in which:
Optimization of IMP761 Potency Assay Protocol
Jurkat Lag-3+/NFAT-luc2 effector cells were initially developed by Promega to determine antagonist anti-LAG-3 antibody activity after TCR activation through superantigen stimulation presented by MHC II molecules. Antagonist anti-LAG-3 antibody blockage 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 agonist anti-LAG-3 antibody activity requires a very different experimental set-up, as explained below.
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 Entereotoxin E or D (SEE or SED). Raji cells express endogenous MHC class II, a LAG-3 ligand. This is important to test the blocking activity of antagonist anti-LAG-3 antibody on the LAG-3/MHC II interaction. However, as no LAG-3/MHC II interaction is required for testing the potency of an agonist anti-LAG-3 antibody, there is no requirement for Raji cells nor the Staphylococcal Enterotoxin. A single cell-type assay was used with anti-CD3 antibodies to activate Jurkat Lag-3+/NFAT-luc2 effector cells through TCR signaling.
Anti-CD3 Concentration and LAG-3-Related Inhibition:
The effect on the cell potency assay of two different anti-CD3 antibodies (OKT3 and UCHT1) was tested at different antibody concentrations, ranging from 1 to 500 ng/ml.
Jurkat Lag-3+/NFAT-luc2 cells were incubated with 300 ng/ml of IMP761, or human-IgG4 (as a negative control), in the presence of different concentrations of OKT3 or UCHT1 for 24 hours. The average RLU values obtained for the different concentrations of anti-CD3 antibody are shown in
Luciferase activty is inhibited by IMP761 across the range of different concentrations of each anti-CD3 antibody.
There is a basal level of reporter expression in the Jurkat cell line without stimulation, so luciferase activity and inhibition of luciferase activity by IMP761 was also tested in the absence of anti-CD3 antibody. However, stimulation of the Jurkat cells with anti-CD3 antibody gives higher RLU values, so the inhibitory effect of IMP761 is more apparent in the presence of anti-CD3 antibody (in particular, a low concentration of anti-CD3 antibody). The percentage inhibition of luciferase activity for OKT3 antibody is shown in Table 3 below:
It was concluded that the maximum effect of IMP761 (inhibition of approximately 80%) was observed when a low concentration of OKT3 antibody (between 1 and 4 ng/ml) was used. Thus, an optimum potency assay includes stimulation of Jurkat cells with low concentrations of anti-CD3 antibody (for example, OKT3 antibody).
IMP761 Potency Assay
This example describes a potency assay, according to an embodiment of the invention, to measure the activity of the IMP761 monoclonal antibody in vitro. The method is based on the capacity of IMP761 to decrease the activation of a LAG-3 effector cell line induced by a low dose of an anti-CD3 antibody (OKT3 clone, 3 ng/ml), mimicking antigen-stimulation of T cells. The LAG-3 effector cell line is a Jurkat T cell line expressing LAG-3 at the surface, and containing the luciferase gene under the control of an NFAT (nuclear factor of activated T cells) response element (Jurkat Lag-3+/NFAT-luc2 effector cells, from Promega). After binding to its target, IMP761 triggers a downregulation of TCR-induced NFAT-regulated expression (illustrated schematically in
Jurkat LAG-3+/NFAT-luc2 effector cells (Promega, ref: CS194801)
RPMI 1640 (GIBCO, ref: 31870-025)
L-Glutamine (200 mM) (GIBCO, ref: 25030-024)
HEPES (1 M) (GIBCO, ref: 15630-080)
FCS (GIBCO, ref: 10270106)
IMP761 (2.06 mg/ml) (IMMUTEP, lot: 270416)
human IgG4, control (Biolegend, ref: 403402)
Anti-CD3 (OKT3) (eBioscience, ref: 16-0037-85)
Bio-Glo reagent (PROMEGA, ref: G7941)
White, 96-well solid flat-bottom microplates (COSTAR, ref: 3917)
Assay medium: RPMI 1640, L-Glutamine (2 mM), Hepes (10 mM), FCS 1%
Cell concentration: 1.33×106 cells/ml
1. Pass Jurkat LAG-3+/NFAT-luc2 effector cells at day −1 before assay in order to have a cell density around 1 Million/ml (between 0.8 and 1.2 Million/ml) at day 0.
2. Prepare assay medium if needed and pre-warm medium for 30 minutes at 37° C.:
3. Prepare IMP761 quality control (QC) stock solution if needed:
Make 24,000 ng/ml stock solution; for example:
10 μl of stock IMP761 (2.06 mg/ml)+848.3 μl Assay medium.
Store 25 μl aliquots in −80° C. freezer.
4. Prepare 3× solutions of IMP761 (batch 270416 at 2.06 mg/ml), human IgG4 negative control (or any other antibody).
Adapt the predilution steps according to the initial concentration of the antibody: the volume of assay medium (in μl) to be added to dilute 2μl of a stock solution in order to prepare a 100 μg/ml predilution is:
5. Prepare 3× IMP761 quality control (QC) solutions
Thaw stock IMP761 QC [24,000 ng/ml] aliquot and make serial dilutions
Very High (VH) QC: 20 μl [24,000 ng/ml]+380 μl=1,200 ng/ml (final concentration: 400 ng/ml)
High (H) QC: 150 μl [1,200 ng/ml]+225 μl=480 ng/ml (final concentration: 160 ng/ml)
Medium (M) QC: 150 μl [480 ng/ml]+225 μl=192 ng/ml (final concentration: 64 ng/ml)
Low (L) QC: 150 μl [192 ng/ml]+225 μl=76.8 ng/ml (final concentration: 25.6 ng/ml)
Very Low (VL) QC: 150 μl [25.6 ng/ml]+225 μl=10.24 ng/ml (final concentration: 3.4 ng/ml)
6. Prepare OKT3 reagent
3.6 μl stock+4 ml assay medium=0.9 μg/ml
150 μl of [0.9 μg/ml]+14,850 μl of assay medium=9 ng/ml (3×)
7. Prepare Jurkat LAG-3/NFAT-luc2 effector cells
At day 0, count cells using Trypan Blue staining
Centrifuge cells at 1200 rpm for 5 minutes
Aspirate media and resuspend cells at 3.75×106/ml in assay medium.
8. Distribute 3× solutions in 96 well plate
Do not use outer wells because of edge effect
Distribute 40 μl of 3× solutions of IMP761/QC/assay medium (0) into the corresponding wells of the assay plate in duplicate
Distribute 40 μl/well of 3× solution of anti-CD3/assay medium (Unstimulated control: unstim) into each well of the assay plate in duplicate. Final concentration: 3 ng/ml
Distribute 40 μl per well (0.15×106/well)
9. Incubate plate(s) at 37° C. in a humidified incubator, with 5% CO2, for 24 hours
10. Prepare BioGlo reagent
11. Equilibrate plate(s) at RT for 15 minutes
12. Add 120 μl /well of BioGlo, avoid/remove bubbles
13. Incubate at RT for 5 to 15 minutes
14. Measure luminescence (RLU), integration time=0.5 sec/well using PerkinElmer 2103 Multilabel reader Envision. 3 measures for each well are collected 45 seconds apart, calculate the average of 3 measures to obtain “RLU Average”.
Ability of IMP761 Potency Assay to Evaluate Denatured IMP761 Antibody
The capacity of the potency assay as described in Example 2 to evaluate the decreased efficacy of a denatured IMP761 antibody was tested by comparing the IC50 of a reference IMP761 stored at 4° C. with the IC50 of the same batch of IMP761 after temperature stress (10 minutes at 70° C. and 20 minutes at 70° C.). The results are shown in
Table 4: dataset (mean of duplicates) for a potency assay with a reference IMP761 (4° C.) and IMP761 after denaturing temperature stress (10 and 20 minutes at 70° C.), expressed as relative light units (RLU)
A five-parameter non-linear regression model determined that the IC50 of the denatured IMP761 antibodies (74 ng/ml and 81.5 ng/ml after 10 minutes and 20 minutes at 70° C., respectively) was higher than the IC50 (41 ng/ml) of the reference IMP761 antibody stored at 4° C.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1906127.4 | May 2019 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/062206 | 5/1/2020 | WO |
