ASSAYS

Abstract

Assays for screening for, or determining activity of, an agonist of lymphocyte-activation gene 3 (LAG-3) are described. According to the assays, 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, wherein expression of the reporter is regulated by LAG-3-mediated inhibition of TCR signaling within the effector T cells. Activity of the agonist is determined from the extent to which expression of the reporter is altered in the presence of the agonist compared with expression of the reporter in the absence of the agonist. The assays may be used for determining the potency of a preparation of the agonist as part of a quality control step in production of the agonist, or for stability testing of a preparation of the agonist. Kits for carrying out the assays are also described.

Claims

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

2. An assay according to claim 1, for determining the potency of a preparation of an agonist of LAG-3.

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

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

5. An assay according to claim 1, wherein 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.

6. An assay according to claim 1, wherein expression of the reporter is altered in each effector T cell in response to activation of the effector T cell via the TCR, and which further comprises: 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 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.

7. An assay according to claim 6, wherein 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.

8. An assay according to claim 6, wherein the effector T cells are activated by contacting the effector T cells with an antigen-independent, MHC class II-independent, T-cell activator under conditions for antigen-independent, MHC class II-independent, TCR-mediated activation of the effector T cells by the T-cell activator.

9. An assay according to claim 8, wherein the effector T cells are contacted with the T-cell activator at 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.

10. An assay according to claim 8, wherein the effector T cells are contacted with the T-cell activator at a concentration of the T-cell activator 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 candidate agonist.

11. An assay according to claim 8, wherein the T-cell activator comprises or consists of an anti-CD3 antibody, or a fragment or derivative thereof that retains antigen-independent, MHC class II-independent, TCR-mediated effector T-cell activation ability.

12. An assay according to claim 11, wherein the anti-CD3 antibody is OKT3.

13. An assay according to claim 11, wherein the anti-CD3 antibody, or fragment or derivative thereof, is contacted with the effector T-cells at a concentration of ˜6-30×10.sup.−12 M (1-4 ng/ml for whole antibody, or molar equivalent for fragment or derivative thereof).

14. An assay according to claim 1, wherein the effector T-cells are activated by cell-free, antigen-independent, MHC class II-independent, TCR-mediated T-cell activation.

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

16. An assay according to claim 15, which further comprises determining an IC.sub.50 value of the agonist, or candidate agonist, for inhibition of expression of the reporter.

17. An assay according to claim 1, wherein the effector T cells comprise heterologous nucleic acid comprising the reporter gene.

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

19. An assay according to claim 18, wherein the response element comprises a NFAT (nuclear factor of activated T cells) response element (NFAT-RE).

20. An assay according to claim 1, wherein the reporter comprises a bioluminescent reporter, such as a luciferase.

21. An assay according to claim 1, wherein the effector T cells comprise heterologous nucleic acid encoding LAG-3.

22. An assay according to claim 1, which further comprises carrying out a negative control assay, wherein the effector T cells are activated in the absence of the agonist or candidate agonist, but in the presence of a molecule of the same type as the agonist or candidate agonist, but which is known to lack agonist activity for LAG-3.

23. An assay according to claim 1, which is carried out in the absence of a natural ligand for LAG-3.

24. An assay according to claim 1, 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.

25. An assay according to claim 1, wherein the effector T cells comprise Jurkat-derived cells.

26. An assay according to claim 1, 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.sup.+/NFAT-luc2 cells.

27. An assay according to claim 1, wherein the agonist anti-LAG-3 antibody, or the fragment or derivative thereof, comprises VH CDR1-3 sequences and VL CDR1-3 sequences of SEQ ID NOs:1-6, respectively, or SEQ ID NOs:7-12, respectively.

28. An assay according to claim 1, wherein the agonist anti-LAG-3 antibody is IMP761.

29-50. (canceled)

Description

EXAMPLE 1

[0093] Optimization of IMP761 Potency Assay Protocol

[0094] 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.

[0095] Anti-CD3 Antibody as a Stimulator of Jurkat Cells:

[0096] 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.

[0097] Anti-CD3 Concentration and LAG-3-Related Inhibition:

[0098] 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.

[0099] 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 FIG. 3, and in Table 2 below:

TABLE-US-00002 TABLE 2 [anti CD3] ng/ml 0 1.95 3.91 7.81 15.63 31.25 62.50 125.00 250.00 500 OKT3 hIgG4 209832 469467 889256 1862472 3067845 4011104 4083312 2786384 2087877 1652699 IMP761 51635 89112 190253 597080 1399133 2178685 2124312 1405789 1086563 1073739 UCHT1 hIgG4 131512 138995 145635 178997 256723 497907 730163 814064 806883 827064 IMP761 56024 49957 47856 58803 79861 126859 228299 319683 453048 540205

[0100] Luciferase activty is inhibited by IMP761 across the range of different concentrations of each anti-CD3 antibody.

[0101] 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:

TABLE-US-00003 TABLE 3 anti-CD3 concentration (ng/ml) % Inhibition 0 75.4 1.95 81.0 3.91 78.6 7.81 67.9 15.63 54.4 31.25 45.7 62.5 48.0 125 49.5 250 48.0 500 35.0

[0102] 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).

Example 2

[0103] IMP761 Potency Assay

[0104] 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.sup.+/NFAT-luc2 effector cells, from Promega). After binding to its target, IMP761 triggers a downregulation of TCR-induced NFAT-regulated expression (illustrated schematically in FIG. 2, lower part). Luciferase activity of the cell line is used to measure TCR-driven cell activation which is dampened in the presence of the presence of IMP761 activity. Thus, the assay measures IMP761 potency in terms of its ability to inhibit TCR signaling.

Reagents

[0105] Jurkat LAG-3+/NFAT-luc2 effector cells (Promega, ref: CS194801)

[0106] RPMI 1640 (GIBCO, ref: 31870-025)

[0107] L-Glutamine (200 mM) (GIBCO, ref: 25030-024)

[0108] HEPES (1 M) (GIBCO, ref: 15630-080)

[0109] FCS (GIBCO, ref: 10270106)

[0110] IMP761 (2.06 mg/ml) (IMMUTEP, lot: 270416)

[0111] human IgG4, control (Biolegend, ref: 403402)

[0112] Anti-CD3 (OKT3) (eBioscience, ref: 16-0037-85)

[0113] Bio-Glo reagent (PROMEGA, ref: G7941)

[0114] White, 96-well solid flat-bottom microplates (COSTAR, ref: 3917)

[0115] Assay medium: RPMI 1640, L-Glutamine (2 mM), Hepes (10 mM), FCS 1%

[0116] Cell concentration: 1.33×10.sup.6 cells/ml

Protocol

[0117] 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.

[0118] 2. Prepare assay medium if needed and pre-warm medium for 30 minutes at 37° C.:

TABLE-US-00004 FCS 0.5 ml L-Glutamine (200 mM) 0.5 ml HEPES (1M) 0.5 ml RPMI 1640 48.5 ml 

[0119] 3. Prepare IMP761 quality control (QC) stock solution if needed:

Make 24,000 ng/ml stock solution; for example:

[0120] 10 μl of stock IMP761 (2.06 mg/ml)+848.3 μl Assay medium.

Store 25 μl aliquots in −80° C. freezer.

[0121] 4. Prepare 3× solutions of IMP761 (batch 270416 at 2.06 mg/ml), human IgG4 negative control (or any other antibody).

[0122] 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:

[00001]$\left(2\times \left(\frac{\mathrm{Initial}\mathrm{concentration}\left(\mathrm{in}\mathrm{mg}/\mathrm{ml}\right)}{0.1}\right)-2\right)\mathrm{.Math.l}\mathrm{of}\mathrm{assay}\mathrm{medium}$

TABLE-US-00005 IMP761 Dilution to prepare 3X working solutions Final Predilution concentration (100 μg/ml) 2 μl of stock (2.06 mg/ml) + 39.2 μl of assay medium (ng/ml) Serial 2000 ng/ml 10 μl of 100 μg/ml predilution + 490 μl of assay medium 666.7 Dilutions 1000 ng/ml 200 μl of 2000 ng/ml dilution + 200 μl of assay medium 333.3 500 ng/ml 200 μl of 1000 ng/ml dilution + 200 μl of assay medium 166.7 250 ng/ml 200 μl of 500 ng/ml dilution + 200 μl of assay medium 83.3 125 ng/ml 200 μl of 250 ng/ml dilution + 200 μl of assay medium 41.7 62.5 ng/ml 200 μl of 125 ng/ml dilution + 200 μl of assay medium 20.8 31.2 ng/ml 200 μl of 62.5 ng/ml dilution + 200 μl of assay medium 10.4 15.6 ng/ml 200 μl of 31.2 ng/ml dilution + 200 μl of assay medium 5.2 7.8 ng/ml 200 μl of 15.6 ng/ml dilution + 200 μl of assay medium 2.6

[0123] 5. Prepare 3× IMP761 quality control (QC) solutions

[0124] Thaw stock IMP761 QC [24,000 ng/ml] aliquot and make serial dilutions

[0125] Very High (VH) QC: 20 μl [24,000 ng/ml]+380 μl=1,200 ng/ml (final concentration: 400 ng/ml)

[0126] High (H) QC: 150 μl [1,200 ng/ml]+225 μl=480 ng/ml (final concentration: 160 ng/ml)

[0127] Medium (M) QC: 150 μl [480 ng/ml]+225 μl=192 ng/ml (final concentration: 64 ng/ml)

[0128] Low (L) QC: 150 μl [192 ng/ml]+225 μl=76.8 ng/ml (final concentration: 25.6 ng/ml)

[0129] Very Low (VL) QC: 150 μl [25.6 ng/ml]+225 μl=10.24 ng/ml (final concentration: 3.4 ng/ml)

[0130] 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×)

[0131] 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 3.75×10.sup.6/ml in assay medium.

[0132] 8. Distribute 3× solutions in 96 well plate

[0133] Do not use outer wells because of edge effect

[0134] Distribute 40 μl of 3× solutions of IMP761/QC/assay medium (0) into the corresponding wells of the assay plate in duplicate

[0135] 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

[0136] Distribute 40 μl per well (0.15×10.sup.6/well)

Example of Plate Template (Reference Batch/Unknown)

[0137]

TABLE-US-00006 666.7 333.3 166.7 83.3 41.7 20.8 10.4 5.2 2.6 0 666.7 333.3 166.7 83.3 41.7 20.8 10.4 5.2 2.6 0 666.7 333.3 166.7 83.3 41.7 20.8 10.4 5.2 2.6 0 666.7 333.3 166.7 83.3 41.7 20.8 10.4 5.2 2.6 0 QC QC QC QC QC UNSTIM VHIGH HIGH MID LOW VLOW QC QC QC QC QC UNSTIM VHIGH HIGH MID LOW VLOW

[0138] 9. Incubate plate(s) at 37° C. in a humidified incubator, with 5% CO.sub.2, for 24 hours

[0139] 10. Prepare BioGlo reagent [0140] a) Place frozen BioGlo reagent at room temperature (RT) 3-6 hours before use to allow thawing [0141] b) Transfer the buffer (10 ml) to substrate bottle, mix and keep at RT in the dark until use

[0142] 11. Equilibrate plate(s) at RT for 15 minutes

[0143] 12. Add 120 μl /well of BioGlo, avoid/remove bubbles

[0144] 13. Incubate at RT for 5 to 15 minutes

[0145] 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”.

Results

[0146] FIG. 4 shows an example of results obtained from the potency assay, using IgG4 antibody as a negative control. Maximum activation was recorded as the activation observed when no IMP761 was present. Maximum inhibition was recorded as the activation observed when IMP761 concentration was 1000 ng/ml. A five-parameter non-linear regression model determined that the IC.sub.50 of the IMP761 was 37 ng/ml.

Example 3

[0147] Ability of IMP761 Potency Assay to Evaluate Denatured IMP761 Antibody

[0148] 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 IC.sub.50 of a reference IMP761 stored at 4° C. with the IC.sub.50 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 FIG. 5, and in Table 4 below:

[0149] 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)

TABLE-US-00007 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) [IMP761] 70° C. ng/ml 4° C. 10 minutes 20 minutes 667 141272 139814 155807 333 139620 148490 163200 167 152260 178717 188419 83 196259 271611 292410 56 274748 365493 368244 37 316679 381826 425702 25 338896 420090 429251 12 409866 441327 430144 6.2  328883* 426592 450974 0 457495 468616 420222 *Outliers, removed from dataset

[0150] A five-parameter non-linear regression model determined that the IC.sub.50 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 IC.sub.50 (41 ng/ml) of the reference IMP761 antibody stored at 4° C.