BINDING ASSAY

Abstract

Methods for determining MHC class II binding activity of a preparation comprising lymphocyte activation gene-3 (LAG-3) protein, or a fragment, derivative, or analogue thereof, is described. The methods comprise determining binding of the LAG-3 protein, fragment, derivative, or analogue to MHC class II molecules using bio-layer interferometry (BLI). Such methods can be used as a quality control assay in good manufacturing practice (GMP) grade production of such compounds. Probes and kits for carrying out the methods are also described.

Claims

1. A method for determining MHC class II binding activity of a preparation comprising lymphocyte activation gene-3 (LAG-3) protein, or a fragment, derivative, or analogue thereof, wherein the method comprises determining binding of the LAG-3 protein, fragment, derivative, or analogue to MHC class II molecules using bio-layer interferometry (BLI).

2. A method according to claim 1, which comprises determining binding of the LAG-3 protein, fragment, derivative, or analogue to MHC class II molecules present on MHC class II-expressing cells.

3. A method according to claim 2, wherein the LAG-3 protein, fragment, derivative, or analogue is immobilised to a reagent layer of a BLI probe, and the MHC class II-expressing cells are in solution.

4. A method according to claim 3, wherein the MHC class H-expressing cells are present at a density of at least 1E6/mL, preferably at least 4E6/mL or 8E6/mL.

5. A method according to claim 3 or 4, wherein the reagent layer has been pre-treated with a blocking reagent to minimise non-specific binding of the MHC class II-expressing cells to the reagent layer.

6. A method according to claim 5, wherein the blocking reagent comprises albumin, preferably bovine serum albumin (BSA).

7. A method according to any of claims 2 to 6, wherein the MHC class H-expressing cells are Raji cells.

8. A method according to any of claims 2 to 7, wherein the MHC class H-expressing cells are thawed, ready-to-use cells obtained from a frozen stock solution.

9. A method according to any preceding claim, which comprises determining a rate of binding of the LAG-3 protein, fragment, derivative, or analogue, to the MHC class II molecules for a plurality of different concentrations of the LAG-3 protein, fragment, derivative, or analogue, and generating a dose-response curve for the rates of binding.

10. A method according to any preceding claim, which further comprises determining MHC class H binding activity of a reference sample of LAG-3 protein, or a fragment, derivative, or analogue thereof, by determining binding of the LAG-3 protein, fragment, derivative, or analogue of the reference sample to MHC class H molecules using BLI, under the same conditions used for determining binding of the LAG-3 protein, fragment, derivative, or analogue of the preparation, and comparing the MHC class II binding activity determined for the reference sample with the MHC class II binding activity determined for the preparation.

11. A method according to claim 10, wherein the MHC class II binding activity of the reference sample is set at 100%.

12. A method according to claim 10 or 11, wherein the reference sample comprises a LAG-3 protein, or a fragment, derivative, or analogue thereof, that has been treated to reduce its MHC class II binding activity.

13. A method according to claim 12, wherein the LAG-3 protein, fragment, derivative, or analogue, of the reference sample has been deglycosylated, stored at 370 C. for at least 12 days, oxidised, denatured by acid or alkali treatment, or exposed to light for at least 5 days.

14. A BLI probe for determining MHC class II binding activity of LAG-3 protein, or a fragment, derivative, or analogue thereof, which comprises a reagent layer to which the LAG-3 protein, or fragment, derivative, or analogue thereof, is immobilised.

15. A probe according to claim 14, wherein the reagent layer has been pre-treated with a blocking reagent to minimise non-specific binding of the MHC class H-expressing cells to the reagent layer.

16. A probe according to claim 15, wherein the blocking reagent comprises albumin, preferably BSA.

17. A kit for determining MHC class II binding activity of LAG-3 protein, or a fragment, derivative, or analogue thereof, which comprises a BLI probe having reagent layer to which the LAG-3 protein, or fragment, derivative, or analogue thereof, is immobilised, and MHC class II-expressing cells.

18. A kit according to claim 17, wherein the reagent layer of the BLI probe has been pre-treated with a blocking reagent to minimise non-specific binding of the MHC class H-expressing cells to the reagent layer.

19. A kit according to claim 18, wherein the blocking reagent comprises albumin, preferably BSA.

20. A kit according to any of claims 17 to 19, wherein the MHC class H-expressing cells are frozen cells.

21. A kit according to any of claims 17 to 20, wherein the cells are Raji cells.

22. A kit according to any of claims 17 to 21, wherein the cells are present at a density of at least 1E6/mL, preferably at least 4E6/mL or 8E6/mL.

23. A kit according to any of claims 17 to 22, which further includes a reference sample comprising LAG-3 protein, or a fragment, derivative, or analogue thereof.

24. A kit according to claim 23, wherein the MHC class H binding activity of the reference sample is known.

Description

[0065] Embodiments of the invention are described below, by way of example only, with reference to the following drawings in which:

[0066] FIG. 1 shows operation of the probe used to determine MHC class II binding activity of LAG-3 protein, or fragments, derivatives, or analogues thereof, according to an embodiment of the invention (Figure taken from U.S. Pat. No. 5,804,453);

[0067] FIG. 2 shows the results of a FACS assay to determine binding of IMP321 to Raji cells;

[0068] FIG. 3 shows schematically a MesoScale Discovery (MSD) electrochemiluminescent (ECL) assay to determine binding of IMP321 to Raji cells;

[0069] FIG. 4(a) shows a plot of the ECL signal obtained for an MSD assay at different concentrations of IMP321 in the presence and absence of Raji cells; FIG. 4(b) shows a plot of the ECL signal obtained for an MSD assay at different concentrations of Rituxan in the presence and absence of Raji cells;

[0070] FIG. 5(a) shows a plot of the OD signal obtained for an ELISA at different concentrations of IMP321 following blocking of the ELISA plate with 5% BSA or 10% FBS; FIG. 5(b) shows a plot of the OD signal obtained for an ELISA at different concentrations of IMP321 or Rituxan following blocking of the ELISA plate with 30% FBS in PBS; FIG. 5(c) shows a plot of the OD signal obtained for an ELISA at different concentrations of IMP321 or Rituxan following blocking of the ELISA plate with 5% BSA in RPIM1640;

[0071] FIG. 6(a) shows a plot of the OD signal obtained for an ELISA at different concentrations of IMP321 or Rituxan following blocking of the ELISA plate with different blocking reagents (1% nonfat milk, 3% nonfat milk, Casein); FIG. 6(b) shows a plot of the OD signal obtained for an ELISA at different concentrations of IMP321 or Rituxan following blocking of the ELISA plate with different blocking reagents (1% gelatin, 3% gelatin, or PBS);

[0072] FIG. 7(a) shows a plot of the raw ECL signal obtained for an MSD assay at different concentrations of IMP321 for different seeding densities of Raji cells; FIG. 7(b) shows a plot of the specific ECL signal obtained for an MSD assay at different concentrations of IMP321 for different seeding densities of Raji cells;

[0073] FIG. 8 shows a plot of the ECL signal obtained for an MSD assay for binding of different concentrations of IMP321 to Raji cells or HLA-DR.sup.dim L929 cells following bocking of the MSD plate with casein:

[0074] FIG. 9 shows schematically, on the left, a BLI probe with a protein A-conjugated sensor and IMP321 immobilised to the distal tip of the optical fibre of the sensor, with the tip of the sensor immersed in a sample solution containing Raji cells. The basic steps of the method are set out on the right of the figure;

[0075] FIG. 10(a) shows a plot of the binding signal obtained in a BLI assay for dose-dependent binding of immobilised IMP321 to Raji cells in solution in the association step; FIG. 10(b) shows a standard curve of IMP321 dose-dependent binding to Raji cells in the BLI assay;

[0076] FIG. 11(a) shows the association and dissociation curves for binding of immobilised IMP321 to different concentrations of Raji cells (which are MHC class II-expressing) or Jurkat cells (which are not MHC class II-expressing) in solution in a BLI assay; FIG. 11(b) shows a graph of the binding signal obtained for the different Raji cell concentrations;

[0077] FIG. 12(a) shows the association and dissociation curves for binding of immobilised IMP321, Humira, or Avastin, to Raji cells in solution in a BLI assay; FIG. 12(b) shows a graph of the binding signal obtained for the different immobilised proteins;

[0078] FIG. 13 shows a plot of the percentage binding potency, measured by BLI assay, for binding of different immobilised preparations of IMP321 to Raji cells in solution versus their expected potency;

[0079] FIG. 14(a) shows a plot of the binding signal obtained by BLI assay for binding of different concentrations of immobilised IMP321 to previously cultured Raji cells in solution;

[0080] FIG. 14(b) shows a plot of the binding signal obtained by BLI assay for binding of different concentrations of immobilised IMP321 to previously frozen Raji cells in solution;

[0081] FIG. 15(a) shows a plot of the downstream CCL4 release obtained by cell-based assay for binding of different concentrations of immobilised IMP321, or deglycosylated IMP321, to Raji cells;

[0082] FIG. 15(b) shows a plot of the binding signal obtained by BLI assay for binding of different concentrations of immobilised IMP321, or deglycosylated IMP321, to Raji cells;

[0083] FIG. 16 shows plots of the signal for binding of different concentrations of immobilised IMP321, or IMP321 stored inappropriately (at 37 C. for 12 days) to Raji cells. The results shown in FIG. 16(a) were obtained by cell-based assay measuring CCL4 release, and the results shown in FIG. 16(b) were obtained by BLI assay;

[0084] FIG. 17 shows plots of the signal for binding of different concentrations of immobilised IMP321, or IMP321 stored inappropriately (at 37 C. for 1 month) to Raji cells. The results shown in FIG. 17(a) were obtained by cell-based assay measuring CCL4 release, and the results shown in FIG. 17(b) were obtained by BLI assay;

[0085] FIG. 18 shows plots of the signal obtained by cell-based assay measuring CCL4 release (FIG. 18a), or by BLI assay (FIG. 18b), for binding of different concentrations of immobilised IMP321 untreated, or oxidised IMP321 (with 1% hydrogen peroxide), to Raji cells;

[0086] FIG. 19 shows plots of the signal obtained by cell-based assay measuring CCL4 release (FIG. 19a), or by BLI assay (FIG. 19b), for binding of different concentrations of immobilised IMP321 untreated, or oxidised IMP321 (with 0.1% hydrogen peroxide), to Raji cells;

[0087] FIG. 20 shows plots of the signal obtained by cell-based assay measuring CCL4 release (FIG. 20a), or by BLI assay (FIG. 20b), for binding of different concentrations of immobilised IMP321 untreated, or acid-treated (at pH 3.0), to Raji cells;

[0088] FIG. 21 shows plots of the signal obtained by cell-based assay measuring CCL4 release (FIG. 21a), or by BLI assay (FIG. 21b), for binding of different concentrations of immobilised IMP321 untreated, or acid-treated (at pH 3.1, or pH 3.6), to Raji cells;

[0089] FIG. 22 shows plots of the signal obtained by cell-based assay measuring CCL4 release (FIG. 22a), or by BLI assay (FIG. 22b), for binding of different concentrations of immobilised IMP321 untreated, or base-treated (at pH 9.2 or pH 9.75), to Raji cells;

[0090] FIG. 23 shows plots of the signal obtained by cell-based assay measuring CCL4 release (FIG. 23a), or by BLI assay (FIG. 23b), for binding of different concentrations of immobilised IMP321 untreated, or light-exposed (at 25 C. for 5 days), to Raji cells;

[0091] FIG. 24 shows plots of the signal obtained by cell-based assay measuring CCL4 release (FIG. 24a), or by BLI assay (FIG. 24b), for binding of different concentrations of immobilised IMP321 untreated, or light-exposed (at 25 C. for 10 days); and

[0092] FIG. 25 shows amino acid sequence of mature human LAG-3 protein. The four extracellular Ig superfamily domains are at amino acid residues: 1-149 (D1); 150-239 (D2); 240-330 (D3); and 331-412 (D4). The amino acid sequence of the extra-loop structure of the D1 domain of human LAG-3 protein is shown underlined in bold.

[0093] Examples 1 to 5 below describe evaluation of various different binding assays to determine whether they are suitable for use as quality control assays for GMP grade production of the recombinant LAG-3 protein derivative IMP321. None of the assays were found to be suitable. Examples 6 to 11 describe cell-based BLI methods, and demonstration of their suitability for determining MHC class II binding activity of preparations of IMP321.

EXAMPLE 1

Evaluation of Use of a Fluorescence-Activated Cell Sorting (FACS) Assay to Determine Binding of IMP321 to Raji Cells

[0094] A FACS assay was carried out to determine binding of IMP321 to Raji cells. IMP321 samples with 100%, 75%, and 50% MHC class II binding activity were tested. The sample with 100% activity was a reference sample with known MHC class II binding activity at a predetermined concentration. The samples with 75% and 50% activity were prepared by dilution of the reference sample.

[0095] The binding curves obtained are shown in FIG. 2. They show that no upper plateaus were reached, so there was no parallelism between the binding curve of the reference sample with 100% activity and the other samples. This prevented calculation of the relative potency of the different samples.

EXAMPLE 2

Evaluation of Use of a Meso Scale Discovery (MSD) Assay to Determine Binding of IMP321 to Raji Cells

[0096] This example describes evaluation of a Meso Scale Discovery (MSD) assay to determine binding of IMP321 to Raji cells.

[0097] The Meso Scale Discovery platform (MSD-ECL) uses electrochemiluminescent labels that are conjugated to detection antibodies. These labels generate light when stimulated by electricity in the appropriate chemical environment, which can then be used to measure key proteins and molecules.

[0098] Electricity is applied to the plate electrodes by the Meso Scale Discovery platform (MSD-ECL), leading to light emission by the labels. Light intensity is then measured to quantify analytes in the sample.

[0099] The detection process is initiated at electrodes located in the bottom of the Meso Scale Discovery (MSD-ECL)'s microplates, and only labels near the electrode are excited and detected. The system employs buffers with high concentrations of Tripropylamine as a catalyst for a dual redux reaction with Ruthenium, emitting light at 620 nm.

[0100] The MSD assay used is shown schematically in FIG. 3. Briefly, approximately 210.sup.4 cells per well of Raji cells in PBS were seeded into a Single-SPOT 96-well MSD plate (Meso Scale Discovery, Gaithersburg, Md.) at 25 uL/well. The plate was incubated at room temperature for 1-1.5 hours before being blocked with blocking buffer (25 uL/well). Then serial dilutions of IMP321 reference standard, or samples, were loaded into duplicate wells at 50 uL/well. After about 1 hour of incubation at room temperature, bound IMP321 was detected using ruthenium-conjugated anti-human Fc at 50 uL/well.

[0101] Electrochemiluminescence signal was acquired using MSD read buffer without surfactant. ECL counts should be proportional to IMP321 binding onto the cell surface within the assay range.

[0102] High binding carbon electrodes in the bottom of microplates allow for easy attachment of Raji cells. The assay uses electrochemiluminescent labels that are conjugated to anti-IMP321 antibodies. Electricity is applied to the plate electrodes by an MSD instrument leading to light emission by the labels. Light intensity is then measured to quantify the presence of IMP321 bound to MHC class molecules on the surface of the immobilised Raji cells.

[0103] The results obtained for samples containing IMP321 with and without Raji cells are shown in FIG. 4(a), and for samples containing Rituxan with and without Raji cells, is shown in FIG. 4(b).

[0104] The results show that non-specific binding of IMP321 to MSD plates was observed in the absence of Raji cells. By comparison, specific binding of Rituxan to Raji cells was observed.

[0105] Raji cells are cells of a cell line derived from the B-lymphocyte of an 11-year-old Nigerian Burkitt's lymphoma male patient in 1963. Rituxan (Rituximab) is a chimeric monoclonal antibody against the protein CD20, which is primarily found on the surface of B cells.

EXAMPLE 3

Evaluation of Non-Specific Binding of IMP321 to ELISA Plates

[0106] This example describes evaluation of non-specific binding of IMP321 and Rituxan to plates used for Enzyme-Linked Immunosorbent Assays (ELISAs) using different blocking reagents.

[0107] Briefly, microplates were blocked with blocking reagent at 25 C. for 2 hours. Samples and rituxan control were diluted with dilution buffer to 2 g/ml then further diluted by two-fold serial dilution. Microplates were washed and well-drained before and after adding the diluted samples and incubation. After incubation with secondary antibody, the signal was measured by a spectrometry assay using SpectraMax M2 (450-650 nm).

TABLE-US-00001 Condition Plate ELISA plate (Costar) Coating reagent None Blocking reagent/dilution buffer 5% BSA in RPIM 1640 Medium 30% FBS in PBS/10% FBS in PBS IMP321 or Rituxan concentration 0~2 g/ml

[0108] The results are shown in FIG. 5. FIG. 5(a) shows the results of ELISA using increasing concentrations of IMP321 and ELISA plates blocked with 5% BSA or 10% FBS. FIG. 5(b) shows the results of ELISA using increasing concentrations of IMP321 or Rituxan and ELISA plates blocked with 30% FBS in PBS. FIG. 5(c) shows the results of ELISA using increasing concentrations of IMP321 or Rituxan and ELISA plates blocked with 5% BSA in RPIM 1640.

[0109] The results show that there was severe non-specific binding of IMP321, but not Rituxan, to ELISA plates when using BSA or FBS as blocking reagents.

[0110] Various different types of blocking agents were then tested with IMP321 or Rituxan to see if the non-specific binding of IMP321 to ELISA plates could be eliminated.

TABLE-US-00002 Condition Plate ELISA high bind plate Coating reagent None Blocking reagent/dilution buffer 1% non-fat milk in PBS 3% non-fat milk in PBS Blocker Casein Blocking Buffers (Thermo) 1% gelatin (from bovine skin) 3% gelatin (from bovine skin) PBS (control) IMP321 or Rituxan concentration 0~8 g/ml

[0111] The results are shown in FIG. 6. FIG. 6(a) shows the results for IMP321 or Rituxan using 1% non-fat milk, 3% non-fat milk, or Blocker Casein Blocking Buffers (Thermo) as blocking reagent. FIG. 6(b) shows the results for IMP321 or Rituxan using 1% gelatin, 3% gelatin, or PBS as blocking reagent.

[0112] The results show that Casein was the best blocking reagent to reduce non-specific binding of IMP321 to ELISA plates.

EXAMPLE 4

[0113] Evaluation of Use of Meso Scale Discovery (MSD) Assay, with Casein Blocking Buffer, to Determine Binding of IMP321 to Raji Cells

[0114] This example describes evaluation of an MSD assay to determine binding of IMP321 to Raji cells at different seeding densities using casein blocking buffer.

[0115] An MSD assay was carried out, similar to that described in Example 2, to evaluate whether the non-specific binding of IMP321 to the MSD plate observed in that example could be minimized using Casein blocking buffer.

TABLE-US-00003 Condition Plate MSD high bind plate Cell density 5E4/well, 2.5E4/well, 5E3/well, 1E3/well Blocking reagent/dilution Blocker Casein Blocking Buffers (Thermo) buffer IMP321 concentration 0~8 g/ml in casein blocking buffer Goat anti-human antibody, 500 ng/ml in casein blocking buffer SULFO-TAG labelled

[0116] The results are shown in FIG. 7. FIG. 7(a) shows the results of binding of IMP321 to different seeding densities of Raji cells (0-510.sup.4 cells/well) at different concentrations of IMP321. The results show a cell density-dependent increase of maximal IMP321 binding. FIG. 7(b) shows the results of specific binding of IMP321 to different seeding densities of Raji cells (110.sup.3-510.sup.4 cells/well). The results show a cell density-dependent increase of specific IMP321 binding.

[0117] Binding of IMP321 to Raji cells was compared with binding of IMP321 to HLA-DR.sup.dim L929 cells (these cells do not express MHC class II), at different concentrations of IMP321, using the MSD assay with casein blocking buffer. L929 is a fibroblast-like cell line cloned from strain L. The results are shown in FIG. 8. The results show that non-specific binding of IMP321 to MSD plates was significantly reduced in the presence of casein blocker. However, the specific binding signal was low, and no upper plateau of the IMP321 dose-binding curve was observed.

[0118] It was concluded that the MSD assay using casein blocking buffer cannot be used to demonstrate specific binding of IMP321 to plate-immobilised Raji cells.

EXAMPLE 5

Evaluation of Use of ELISA Assays to Determine Binding of IMP321 to Raji Cells

[0119] This example describes an evaluation of the ability of cell-based direct ELISA and cell-based transfer ELISA to determine binding of IMP321 to Raji cells.

[0120] Direct ELISA (similar to the assay described in Example 3) was carried out in the presence of different blocking reagents (5% BSA, 10% FBS, 0.5% Casein, or 3% gelatin) with different amounts of plate-immobilised Raji cells (10,000, 5,000, or 2,500 cells), and different concentrations of IMP321 or IMP321 treated with Peptide-N-Glycosidase F (PNGase F, an amidase that cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins). The conditions used for the direct ELISA assay are summarised in the tables below:

TABLE-US-00004 Culture plate wells Conditions 1 A-G 5% BSA, PNGase IMP321, 10,000 cells 2 A-G 10% FBS, PNGase IMP321, 10,000 cells 3 A-G 5% BSA, PNGase IMP321, 5,000 cells 4 A-G 10% FBS, PNGase IMP321, 5,000 cells 5 A-G 5% BSA, PNGase IMP321, 2,500 cells 6 A-G 10% FBS, PNGase IMP321, 2,500 cells 7 A-G 0.5% Casein, IMP321, 10,000 cells 8 A-G 3% gelatin, IMP321, 10,000 cells 9 A-G 0.5% Casein, IMP321, 5,000 cells 10 A-G 3% gelatin, IMP321, 5,000 cells 11 A-G 0.5% Casein, IMP321, 2,500 cells 12 A-G 3% gelatin, IMP321, 2,500 cells H 1-12 No blocking reagent (NSB)

TABLE-US-00005 Culture plate wells IMP321 concentration (ng/ml) A 1-12 1000 B 1-12 500 C 1-12 250 D 1-12 125 E 1-12 62.5 F 1-12 31.25 G 1-12 15.625 H 1-12 0

[0121] The results are shown in the table below.

TABLE-US-00006 1 2 3 4 5 6 7 8 9 10 11 12 A 1.771 1.345 1.812 2.254 1.834 2.394 0.160 1.970 0.136 2.306 0.112 2.596 B 1.298 1.346 1.357 1.632 1.320 1.796 0.188 1.554 0.206 1.881 0.180 2.565 C 0.910 0.762 1.090 0.939 0.893 1.287 0.241 1.392 0.255 2.096 0.231 1.947 D 0.395 0.377 0.476 0.443 0.415 1.124 0.176 0.688 0.197 0.823 0.163 0.770 E 0.283 0.290 0.303 0.280 0.297 0.329 0.142 0.417 0.131 0.464 0.135 0.399 F 0.310 0.279 0.297 0.304 0.304 0.294 0.180 0.389 0.143 0.359 0.149 0.303 G 0.366 0.333 0.274 0.327 0.281 0.270 0.185 0.361 0.207 0.316 0.168 0.295 H 0.342 0.286 0.350 0.360 0.370 0.318 0.190 0.337 0.200 0.305 0.197 0.276

[0122] The results show dose-dependent IMP321 binding to plate-immobilised Raji cells.

[0123] To check whether IMP321 binds non-specifically to the ELISA plates, a direct ELISA was carried out in the absence of Raji cells, under the conditions summarised in the table below:

TABLE-US-00007 Culture plate wells Conditions 1 A-G 5% BSA, PNGase IMP321 2 A-G 10% FBS, PNGase IMP321 3 A-G 0.5% Casein, IMP321 4 A-G 3% gelatin, IMP321 H 1-4 No blocking reaegent (NSB)

[0124] The results are shown in the table below:

TABLE-US-00008 1 2 3 4 A 1.898 2.625 1.056 2.587 B 1.808 2.607 1.960 2.527 C 1.626 2.443 2.326 2.400 D 1.381 1.526 1.101 1.710 E 0.857 0.896 0.342 0.856 F 0.388 0.419 0.193 0.375 G 0.300 0.302 0.128 0.245 H 0.210 0.129 0.143 0.104

[0125] The results show strong non-specific binding of IMP321 to the ELISA plate in the absence of plate-immobilised Raji cells. Neither casein nor gelatin blocking reagents, nor PNGase treatment of IMP321, removed the non-specific binding.

[0126] It was concluded that a direct cell-based ELISA cannot be used to demonstrate specific binding of IMP321 to plate-immobilised Raji cells.

[0127] A transfer cell ELISA was carried out to determine binding of different concentrations of IMP321, or IMP321 treated with PNGase, to immobilised Raji cells. Raji cells were transferred to another plate after binding to IMP321 or treated IMP321. The conditions used for the assay are summarised in the tables below.

TABLE-US-00009 Culture plate wells Conditions B 1-12 Raji cells and WT IMP321 C 1-12 Raji cells and treated IMP321 D 1-12 Raji cells and treated IMP321 F 1-12 No cells and WT IMP321 G 1-12 No cells and treated IMP321 H 1-12 No cells and treated IMP321

TABLE-US-00010 WT or treated IMP321 concentration Culture plate wells (ng/ml) 1 B-D, F-H 1000 2 B-D, F-H 500 3 B-D, F-H 250 4 B-D, F-H 125 5 B-D, F-H 62.5 6 B-D, F-H 31.25 7 B-D, F-H 15.63 8 B-D, F-H 7.813 9 B-D, F-H 3.906 10 B-D, F-H 1.953 11 B-D, F-H 0.977 12 B-D, F-H 0

[0128] The results are shown in the table below:

TABLE-US-00011 1 2 3 4 5 6 7 8 9 10 11 12 A B 2.031 1.815 0.956 0.475 0.223 0.159 0.098 0.053 0.034 0.026 0.018 0.009 C 0.878 0.642 0.276 0.166 0.082 0.042 0.035 0.020 0.012 0.011 0.010 0.010 D 1.129 0.594 0.361 0.206 0.076 0.049 0.029 0.020 0.012 0.010 0.010 0.011 E F 0.011 0.010 0.015 0.008 0.007 0.007 0.006 0.007 0.007 0.006 0.007 0.008 G 0.011 0.010 0.008 0.007 0.007 0.008 0.007 0.007 0.007 0.008 0.008 0.008 H 0.013 0.011 0.009 0.009 0.009 0.008 0.008 0.009 0.008 0.008 0.006 0.009

[0129] The results show that the well-to-well signal variation is not acceptable for a quality control method. The method is also labour-intensive. It was concluded that a cell-based transfer ELISA cannot be used to demonstrate specific binding of IMP321 to plate-immobilised Raji cells.

EXAMPLE 6

A Cell-Based Assay to Measure the Binding Activity of a Preparation of the LAG-3 Protein Derivative IMP321 Using Bio-Layer Interferometry (BLI)

[0130] IMP321 is a soluble recombinant derivative of LAG-3 protein with high affinity to MHC class II molecules. This example describes a cell-based assay to measure the binding activity of IMP321 to MHC class II-expressing Raji cells using BLI. The assay is simple and quick, and allows comparison between reference standards and samples.

[0131] FIG. 9 shows schematically, on the left, a BLI probe with a protein A-conjugated sensor and IMP321 immobilised to the distal tip of the optical fibre of the sensor, with the tip of the sensor immersed in a sample solution containing Raji cells. The basic steps of the method are set out on the right of the figure. The assay is described in more detail below.

Materials:

[0132] 1) Raji cells: ATCC/CCL-86

2) RPMI 1640: Invitrogen/22400-089

3) HI-FBS: Invitrogen/10100147

4) DPBS: Hyclone/SH30028.01B

5) BSA: Sigma/A3032

6) IMP321 Reference Material

7) Raji Cell Growth Medium: RPMI 1640, 10% HI-FBS

8) Binding Assay Diluent: DPBS, 0.5% BSA

9) Protein A Tray (ForteBio-18-5010)

[0133] 10) 96-flat-bottom-well black plate (Greiner-655209)
11) Single- and multi-channel pipettes: Sartorius and Eppendorf/various
12) Cell counter: Roche/Cedex HiRes and Beckman/ViCell
13) Bio-Layer Interferometer: Fortebio/Octet Red with software version 7.0 or later

Methods:

1. Preparation of Ready-to-Use Raji Cells

[0134] 1) Remove N vial(s) of Raji cells from the liquid nitrogen freezer and quickly thaw in a 37 C. water bath. [0135] 2) Aseptically transfer the vial contents to a sterile centrifuge tube containing approximately N9 mL of Raji Cell Growth Media. Mix well by gently pipetting. [0136] 3) Centrifuge the cells 5 min at 300g. Resuspend cells in Binding Assay Diluent and count them with a cell counter or a hemacytometer. [0137] 4) Add the volume of cell stock suspension to a sufficient volume of Binding Assay Diluent to adjust cell densities to 4.0E6-8.0E6 cells per mL and keep on ice for use.

2. Preparation of IMP321 Reference Standard, Control and Samples

[0138] NOTE: 1) Use reverse pipetting to ensure accuracy. [0139] 2) Vortex gently to avoid or minimize creating foam and bubbles [0140] 1) Reference standard preparation: [0141] 1.1) Thaw a vial of IMP321 Reference Material as needed. Store at 2-8 C. Expiration is 7 days from date of thaw [0142] 1.2) Dilute IMP321 Reference Material to approximately 1.0 mg/mL in Formulation Buffer. Prepare fresh and use fresh. Determine the protein concentration spectrophotometrically using Formulation Buffer as a blank. [0143] 1.3) Based on measured protein concentration, dilute RM to prepare standard curve to the appropriate concentrations as described below. Mix dilutions by votexing.

TABLE-US-00012 Volume of Volume of Assay Tube IMP321 concentration IMP321 Dilution Diluent A ~30 mg/mL B ~1.0 mg/mL 40 L of A 1160 L C 62.5 g/mL 40 L of B XXX mL D 12.5 g/mL 400 L of C 1600 L E 3125 ng/mL 400 L of D 1200 L F 1562.5 ng/mL 200 L of D 1400 L G 781.25 ng/mL 100 L of D 1500 L H 390.625 ng/mL 50 L of D 1550 L I 78.125 ng/mL 400 L of H 1600 L J 0 1000 L [0144] 1.4) Use dilutions C-J for the standard curve. Additional concentrations may be used if needed, to include the linear portion of the curve and the upper and lower plateaus. [0145] 2) Preparation of Control [0146] 2.1) The control is an independent dilution of Reference Material from Tube C prepared in Step 1.3 above. Further dilute as described in the Table above. Mix dilutions by votexing. [0147] 2.2) Use dilutions C-J for the Control. [0148] 3) Preparation of Samples [0149] 3.1) Based on protein concentration, dilute IMP321 Samples to approximately 1.0 mg/mL in Assay Diluent. Prepare fresh and use fresh. [0150] 3.2) Further dilute to prepare standard curve to the appropriate concentrations as described in the Table above. Mix dilutions by votexing. [0151] 3.3) Use dilutions C-J for the Samples. Additional concentrations may be used if needed, to include the linear portion of the curve and the upper and lower plateaus.

3. Detection Steps in the Octet System

[0152] 1) Hydrate the biosensors in PBS for at least 10 min
2) Prepare the assay plate. In a black polypropylene microplate, transfer 200 L per well of PBS, Assay Diluent, titrations of IMP321 in AD, or Raji cells respectively into the appropriate wells according to the Sample Plate Map below:

TABLE-US-00013 Sample Plate Map 1 2 3 4 5 6 7 8 9 10 11 12 A B L B L B S B E E E E E B B L B L B S B E E E E E C B L B L B S B E E E E E D B L B L B S B E E E E E E B L B L B S B E E E E E F B L B L B S B E E E E E G B L B L B S B E E E E E H B L B B B S B E E E E E 1 2 3 4 5 6 7 DPBS DA DA Sample DA Cell DA B = Buffer S = Sample L = Loading E = Empty
3) Set up a kinetic assay with the parameter settings described below.
4) Enter location and file name for saving the data.
5) Click GO to run the assay.

TABLE-US-00014 Assay Step Data Sample Step Number Name Column Step Type Assay Time(s) 1 Equilibration 1 Custom 60 2 Loading 2 Loading 120 3 Baseline 3 Baseline 60 4 Loading2 4 Loading 500 5 Baseline 5 Baseline 60 6 Association 6 Association 500 7 Dissociation 7 Dissociation 120

4. Analyze Data

[0153] 1) In the Octet Data Analysis software, load the data folder to be analyzed.
2) In the Processing tab, select Association step. Then click on the quantitate the Selected Step.
3) Input Concentration information accordingly.
4) In the Results tab, select R equilibrium (Req) as the binding rate equation. This equation will fit the binding curve generated during the experiment and calculate a response at equilibrium as the output signal.
5) Click on Calculate Binding Rate. Results will be displayed automatically in the table.
6) Click the Save Report button to generate a MS Excel report file.
7) Use SoftMax Pro, a 4-parameter logistic curve-fitting program, to generate a standard curve or sample curve by Binding rate (nm) against the IMP321 concentration expressed ug/mL. An example is shown in FIG. 10.
8) Calculate relative binding potency of the sample using EC50 ratio of the Reference Standard and the Sample.

5. System Suitability and Assay Acceptance Criteria.

[0154] An assay is valid if it meets ALL following criteria:

1) Ready to use Raji cell viability>=60%
2) Relative activity of the control is within 80%-120%
3) Signal to Background ratio of the control (Parameter D/Parameter A)>=2.
4) Parallelism (comparability): slope ratio with the Standard is between 0.8 and 1.4.
5) If the result for the assay control does not meet the criteria listed above, the assay is considered invalid.

6. Reportable Value:

[0155] 1) For a clinical sample, the reportable value for a sample is defined as the mean of two or three valid and independent assay results as detailed below: [0156] % Difference is calculated as follows:

Absolute value (Assay 1 ResultAssay 2 Result)/Mean value (Assay 1 Result,Assay 2 Result)100%

2) If the % Difference of the two assay results<=20%, report mean results of the two assays.
3) If the % Difference of the two assay results>20%, perform 1 additional valid assay.
4) If the CV of the three sample assay results<=25%, report mean results of the three assays.
5) If the CV of the three sample assay results>25%, there is no reportable value. Initiate a discrepancy with a re-test plan.
6) If the reportable value for a sample does not meet specifications listed in the COA, initiate a discrepancy with a retest plan.

7. Retest Plan

[0157] Perform the retest of a sample as follows:

1) Retest the sample with three valid and independent assays
2) If the CV of the three sample assay results<=25%, report mean results of the three assays.
3) If the CV of the three sample assay results>25%, there is no reportable value.
4) If the retest result is out of specification (OOS) listed in the COA, the conclusion is fail.

EXAMPLE 7

Determination of Specific Binding of Immobilised IMP321 to Raji Cells in Solution in a BLI Assay

[0158] A BLI assay as described in Example 6 was used to determine binding of immobilised IMP321 to different concentrations of Raji cells in solution (8E6/mL, 4E6/mL, 2E6/mL, 1E6/ml). Jurket cells were used as a negative control. The association and dissociation curves obtained are shown in FIG. 11(a). FIG. 11(b) shows a graph of the binding signal obtained for the different Raji cell concentrations. The results show that the binding signal was dependent on the concentration of Raji cells, i.e. the higher the concentration of Raji cells, the higher the binding rate and upper plateau obtained. No specific binding of Jurket cells was observed in the same assay.

[0159] A further BLI assay was performed as described in Example 6, but to compare binding of immobilised IMP321 to Raji cells with binding of immobilised Humira or Avastin. The association and dissociation curves obtained are shown in FIG. 12(a). FIG. 12(b) shows a graph of the binding signal obtained for the different immobilised proteins. The results show that IMP321, but not Humira or Avastin, binds to Raji cells.

[0160] It was concluded from these results that the BLI assay is able to determine specific binding of immobilised IMP321 to Raji cells in solution.

EXAMPLE 8

[0161] Correlation of IMP321 Binding Activity Measured by BLI Assay with Known Binding Potency

[0162] Samples of IMP321 diluted from reference standard with different levels of Raji cell binding potency were used in a BLI assay to determine whether the binding activity measured by the assay correlated with the known binding potency of the samples. The results are shown in the table below. FIG. 13 shows a plot of the percentage binding potency, measured by BLI assay, versus their expected potency;

TABLE-US-00015 Potency determined by Sample binding potency BLI assay Percentage recovery 50% 55% 110% 75% 80% 107% 100% 98% 98% 125% 135% 108% 150% 150% 100%

[0163] The results show a good correlation between the binding potency measured by BLI assay, and the expected binding potency. Mean recoveries of each sample were from 90% to 110%, with good parallelism of binding curves (i.e. acceptable slope ratio and converged plateaus).

EXAMPLE 9

Use of Frozen Cells in a BLI Assay to Determine MHC Class II Binding Activity

[0164] A BLI assay as described in Example 6 was carried out to compare binding of immobilised IMP321 to Raji cells in solution obtained from culture or from a frozen stock solution. A plot of the binding signal obtained for binding of different concentrations of immobilised IMP321 to cultured Raji cells in solution is shown in FIG. 14(a). A plot of the binding signal obtained for binding of different concentrations of immobilised IMP321 to previously frozen Raji cells in solution is shown in FIG. 14(b).

[0165] The results show that the frozen Raji cells behave very similarly to the cultured Raji cells, and so the frozen stock solution can be used in place of a fresh culture solution, thereby providing improved assay robustness and transferability.

EXAMPLE 10

In-Process Sample Testing

[0166] BLI assays as described in Example 6 were carried out to determine the MHC class II binding activity of various different preparations of IMP321, and to compare the bioactivity of the preparations as determined by CCL4 release assay.

[0167] THP-1 is a human single nuclear leukaemia cell line. When induced with LAG-3 protein, or stressed samples, THP-1 cells secrete cytokine CCL4 which can be quantified with a CCL4 ELISA kit. The level of CCL4 release can be used to measure the bioactivity of a preparation of LAG-3 protein, or a fragment, derivative, or analogue thereof.

TABLE-US-00016 Bioactivity Bioactivity IMP321 Sample (CCL4 release) (binding) SD140817K01 102% 92% 20140801-T0 101% 89% 20140802-T0 102% 91% 20140801-T0-PC 98% 102% 20140802-T0-PC 97% 91% 20140801-D-25-5D 104% 93% 20140802-D-25-5D 96% 87% 20140803-T0 110% 86% 20140804-T0 104% 100%

[0168] It was concluded that the bioactivity of the different IMP321 samples correlated with the bioactivity as determined by CCL4 release assay.

EXAMPLE 11

BLI Assay Testing of Stressed IMP321 Samples and Correlation to a Cell-Based CCL4 Release Assay

[0169] BLI assays as described in Example 6 were used to determine MHC class II binding activity of IMP321 samples that have been exposed to different treatments (deglycosylation by treatment with PNGase, storage at 37 C., oxidation by treatment with 1% or 0.1% hydrogen peroxide, treatment with acid at pH 3.0, 3.6, or 3.1, treatment with alkali at pH 9.2, 9.75, or exposure to light). The results are shown in FIGS. 15-24.

[0170] FIG. 15(a) shows a plot of the downstream CCL4 release obtained by cell-based assay for binding of different concentrations of immobilised IMP321, or deglycosylated IMP321, to Raji cells;

[0171] FIG. 15(b) shows a plot of the binding signal obtained by BLI assay for binding of different concentrations of immobilised IMP321, or deglycosylated IMP321, to Raji cells;

[0172] FIG. 16 shows plots of the signal for binding of different concentrations of immobilised IMP321, or IMP321 stored inappropriately (at 37 C. for 12 days) to Raji cells. The results shown in FIG. 16(a) were obtained by cell-based assay measuring CCL4 release, and the results shown in FIG. 16(b) were obtained by BLI assay;

[0173] FIG. 17 shows plots of the signal for binding of different concentrations of immobilised IMP321, or IMP321 stored inappropriately (at 37 C. for 1 month) to Raji cells. The results shown in FIG. 17(a) were obtained by cell-based assay measuring CCL4 release, and the results shown in FIG. 17(b) were obtained by BLI assay;

[0174] FIG. 18 shows plots of the signal obtained by cell-based assay measuring CCL4 release (FIG. 18a), or by BLI assay (FIG. 18b), for binding of different concentrations of immobilised IMP321 untreated, or oxidised IMP321 (with 1% hydrogen peroxide), to Raji cells;

[0175] FIG. 19 shows plots of the signal obtained by cell-based assay measuring CCL4 release (FIG. 19a), or by BLI assay (FIG. 19b), for binding of different concentrations of immobilised IMP321 untreated, or oxidised IMP321 (with 0.1% hydrogen peroxide), to Raji cells;

[0176] FIG. 20 shows plots of the signal obtained by cell-based assay measuring CCL4 release (FIG. 20a), or by BLI assay (FIG. 20b), for binding of different concentrations of immobilised IMP321 untreated, or acid-treated (at pH 3.0), to Raji cells;

[0177] FIG. 21 shows plots of the signal obtained by cell-based assay measuring CCL4 release (FIG. 21a), or by BLI assay (FIG. 21b), for binding of different concentrations of immobilised IMP321 untreated, or acid-treated (at pH 3.1, or pH 3.6), to Raji cells;

[0178] FIG. 22 shows plots of the signal obtained by cell-based assay measuring CCL4 release (FIG. 22a), or by BLI assay (FIG. 22b), for binding of different concentrations of immobilised IMP321 untreated, or base-treated (at pH 9.2 or pH 9.75), to Raji cells;

[0179] FIG. 23 shows plots of the signal obtained by cell-based assay measuring CCL4 release (FIG. 23a), or by BLI assay (FIG. 23b), for binding of different concentrations of immobilised IMP321 untreated, or light-exposed (at 25 C. for 5 days), to Raji cells; and

[0180] FIG. 24 shows plots of the signal obtained by cell-based assay measuring CCL4 release (FIG. 24a), or by BLI assay (FIG. 24b), for binding of different concentrations of immobilised IMP321 untreated, or light-exposed (at 25 C. for 10 days).

[0181] The bioactivity (as determined by CCL4 release of the different IMP321 samples, compared with their MHC class II binding activity (determined by a method as described in Example 6) is shown in the table below:

TABLE-US-00017 FIG. Bioactivity Bioactivity No. Sample (CCL4 release) (binding) 15 IMP321 PNGase treated None NRR 16 IMP321 stored at 37 C. (12 D = NRR NRR 12 days) 17 IMP321 stored at 37 C. (1 M = None None 1 month) 18 IMP321 stored at 37 C. (1 D = 84% 77% 1 day) Control IMP321 Oxidation, 1% H.sub.2O.sub.2 10% None at 37 C. (1 D = 1 day) 19 IMP321 stored at 37 C. (1 D = 84% 85% 1 day) Control IMP321 Oxidation, 0.1% 21% NRR H.sub.2O.sub.2 at 37 C. (1 D = 1 day) 20 IMP321, pH 7.0 at RT (1 D = 87% 126% 1 day) IMP321, Acid pH 3.0 at RT 7% None (1 D = 1 day) 21 IMP321, pH 7.0 at RT (1 D = NA 95% 1 day) IMP321, Acid pH 3.6 at RT 29% NRR (1 D = 1 day) IMP321, Acid pH 3.1 at RT 15% NRR (1 D = 1 day) 22 IMP321, pH 7.0 at 37 C. (1 D = 79% 94% 1 day) IMP321, Alkali pH 9.2 at 17% NRR 37 C. (1 D = 1 day) IMP321, Alkali pH 9.75 at None None 37 C. (1 D = 1 day) 23 IMP321, Dark, 5 D = 5 days 100% 100% IMP321, Light, 5 D = 5 days 87% 74% 24 IMP321, Dark, 10 D = 10 days 100% 100% IMP321, Light, 10 D = 10 73% 75% days

[0182] The results show a good correlation between the bioactivity of each treated IMP321 sample, as determined by CCL4 release, and its MHC class II binding activity, as determined by BLI assay according to the invention. It was concluded that determination of MHC class II binding activity by BLI assay can be used to determine the bioactivity of IMP321 preparations.