This invention relates to methods for determining MHC class II binding activity of preparations of lymphocyte activation gene-3 (LAG-3) protein, or fragments, derivatives, or analogues thereof, and to probes and kits for use in the methods.
LAG-3 protein is a CD4 homolog type I membrane protein with four extracellular immunoglobulin superfamily domains. Similar to CD4, LAG-3 oligomerizes at the surfaces of T cells and binds to MHC class II molecules on antigen-presenting cells (APCs) but with significantly higher affinity than CD4. LAG-3 is expressed on activated CD4+ and CD8+ T lymphocytes where it associates with the CD3/T cell receptor complex at the cell surface and negatively regulates signal transduction. As a consequence, it negatively regulates T cell proliferation, function, and homeostasis. LAG-3 is upregulated on exhausted T cells compared with effector or memory T cells. LAG-3 is also upregulated on tumor infiltrating lymphocytes (TILs), and blockade of LAG-3 using anti-LAG-3 antibody can enhance anti-tumour T cell responses.
IMP321 is a recombinant soluble LAG-3Ig fusion protein that binds to MHC class II with high avidity. It is a first-in-class immunopotentiator targeting MHC class II-positive antigen-presenting cells (APCs) (Fougeray et al.: A soluble LAG-3 protein as an immunopotentiator for therapeutic vaccines: Preclinical evaluation of IMP321. Vaccine 2006, 24:5426-5433; Brignone et al.: IMP321 (sLAG-3) safety and T cell response potentiation using an influenza vaccine as a model antigen: A single-blind phase I study. Vaccine 2007, 25:4641-4650; Brignone et al.: IMP321 (sLAG-3), an immunopotentiator for T cell responses against a HBsAg antigen in healthy adults: a single blind randomised controlled phase I study. J Immune Based Ther Vaccines 2007, 5:5; Brignone et al.: A soluble form of lymphocyte activation gene-3 (IMP321) induces activation of a large range of human effector cytotoxic cells. J Immunol 2007, 179:4202-4211). IMP321 has been tested in previously-treated advanced renal cell carcinoma patients known to be immunosuppressed and shown to induce an increase in the percentage of circulating activated CD8 T cells and of long-lived effector-memory CD8 T cells in all patients treated by repeated injections over 3 months, without any detectable toxicity (Brignone et al.: A phase I pharmacokinetic and biological correlative study of IMP321, a novel MHC class II agonist in patients with advanced renal cell carcinoma. Clin Cancer Res 2009, 15:6225-6231). A concentration of only a few ng/mL IMP321 has been shown to be active in vitro on APC, showing the great potency of IMP321 as an agonist of the immune system (Brignone, et al., 2009, supra).
In a study in metastatic breast carcinoma (MBC) patients, Brignone et al. (First-line chemoimmunotherapy in metastatic breast carcinoma: combination of paclitaxel and IMP321 (LAG-3Ig) enhances immune responses and antitumor activity. Journal of Translational Medicine 2010, 8:71) demonstrated that IMP321 expanded and activated for several months both the primary target cells (MHC class II-positive monocytes/dendritic cells) to which IMP321 binds, and the secondary target cells (NK/CD8+ effector memory T cells) which are activated subsequently. By pooling results from all 30 patients and comparing tumor regression with an appropriate historical control group, they saw a doubling of the objective response rate which suggests that IMP321 is a potent agonist of effective anti-cancer cellular immune responses in this clinical setting.
WO 99/04810 describes use of LAG-3 protein, or fragments or derivatives thereof, as an adjuvant for vaccination, and in cancer treatment. Use of LAG-3 protein, or fragments or derivatives thereof, for the treatment of cancer and infectious disease is described in WO 2009/044273.
In view of the medical uses of LAG-3, and fragments or derivatives thereof, there is a need to provide preparations of such compounds that comply with good manufacturing practices (GMP). Such practices are required in order to conform to the guidelines recommended by agencies that control authorization and licensing for manufacture and sale of active pharmaceutical products. These guidelines provide minimum requirements that a pharmaceutical manufacturer must meet to assure that the products are of high quality and do not pose any risk to the consumer or public. As part of the quality control procedure in GMP-grade manufacture of proteins, it is necessary to determine whether preparations of such compounds retain a high level of bioactivity.
We have found, however, that several conventional methods for determining protein-protein interactions are not suitable for determining specific binding of the LAG-3 derivative IMP321 to MHC class II molecules expressed on the surface of immune cells. In particular, fluorescence-activated cell sorting (FACS) was not suitable for distinguishing IMP321 preparations with differing abilities to bind to MHC class II-expressing cells. No upper plateaus were observed at increasing concentrations of IMP321 for the binding curves obtained using FACS. This prevents calculation of the relative potencies of different preparations, which requires converged plateaus (parallelism).
We have also found that IMP321 binds non-specifically to plates used for MesoScale Discovery (MSD) electrochemiluminescent (ECL) assays, and Enzyme-Linked Immunosorbent Assays (ELISAs). Whilst non-specific binding of IMP321 to plates used for ELISA and MSD assays was dramatically reduced by use of casein as a blocking reagent, this lowered the absolute signal in the MSD assay. No upper plateaus were observed for binding curves obtained using assays in which cells expressing MHC class II molecules were immobilised to the MSD plates. A different ELISA technique was also tested, in which cells expressing MHC class II molecules were transferred to another plate after binding of IMP321, in order to minimise the effect of non-specific binding of IMP321 to the plates. However, the well-to-well signal variation was found to be unacceptable. In view of this, it was concluded that neither MSD ECL assays nor ELISA assays could be used to determine specific binding of IMP321 to the immobilised cells in a quality control assay to test GMP-grade product.
There is a need, therefore, to provide a method for determining MHC class II binding activity of preparations of LAG-3 protein, or fragments, derivatives, or analogues thereof, which is suitable for use as a quality control assay in GMP-grade production of such compounds.
According to the invention, there is provided 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).
The term “bio-layer interferometry (BLI)” is used herein to refer to a fibre-optic assay based on phase-shift interferometry, for example as described in U.S. Pat. No. 5,804,453 (Chen). Developments to the BLI technique, including developments aimed at enhancing the sensitivity and accuracy of analyte detection, are described in WO 2005/047854 and WO 2006/138294 of ForteBio, Inc.
U.S. Pat. No. 5,804,453 describes a probe, method, and system for detecting analyte binding to a fibre-optic end surface. Analyte detection is based on a change in the thickness at the end surface of the optical fibre resulting from the binding of analyte molecules to the surface, with greater amount of analyte producing a greater thickness-related change in the interference signal. The change in interference signal is due to a phase shift between light reflected from the end of the fibre and from the binding layer carried on the fibre end, as illustrated particularly in FIGS. 7a and 7b of U.S. Pat. No. 5,804,453.
The probe described in U.S. Pat. No. 5,804,453 includes a fibre optic section having a proximal end tip and a distal end tip and a reagent layer disposed on the distal end tip. The reagent layer reacts (or bonds) with the substance (analyte) being detected. The fibre optic section has a first index of refraction and the reagent layer has a second index of refraction. When any of the substance bonds to the reagent layer, a resulting layer including the reagent layer and the substance is formed. The resulting layer can be treated as having a homogeneous index of refraction.
The method permits the concentration of a substance in a sample solution to be determined using the fibre optic probe. The method includes steps of (i) immersing the distal end of the fibre optic probe into the sample solution, (ii) optically coupling a light source with the proximal end of the fibre optic probe, (iii) detecting at least a first light beam reflected from an interface between the distal end surface of the fibre optic section and the reagent layer, and a second light beam reflected from an interface between the reagent layer and the sample solution, reflected from the distal end of the fibre optic probe, (iv) detecting an interference pattern formed by the first and second light beams at a first time, (v) detecting an interference pattern formed by the first and second light beams at a second time, and (vi) determining whether the substance is present in the sample solution based on whether a shift occurs in the interference patterns. The concentration of the substance may be determined based on a shift in the interference patterns and based on a differential between the first and second times.
The system for detecting the concentration of a substance in a sample solution has a light source for providing a light beam, a fibre optic probe, a detector, a fibre optic coupler, a fibre optic connector, and a processor. The fibre optic coupler includes a first fibre optic section having a proximal end for receiving an incident light beam, a second fibre optic section having a proximal end for delivering the reflected interference light beam to the detector, and a third fibre optic section having a distal end for connecting to the fibre optic probe. The fibre optic probe includes a proximal end for connecting to the fibre optic coupler, and a distal end tip with a reagent layer disposed thereon. The fibre optic probe produces at least a first reflected beam and a second reflected beam from the incident light beam. The detector detects an interference pattern formed by the first and second reflected beams. The coupler optically couples the light source with the fibre optic probe and optically couples the fibre optic probe with the detector. The processor determines a phase associated with an interference pattern detected by the detector at a first time, determines a phase associated with an interference pattern detected by the detector at a second time, and determines the concentration of the substance based on a shift in the phases associated with the interference patterns detected by the detector at the first and second times.
We have appreciated that the BLI technique can be used to determine the MHC class II binding activity of preparations of LAG-3 protein, or fragments, derivatives, or analogues thereof, and that such methods are particularly useful as a quality control assay in GMP-grade production of such compounds.
In particular embodiments, methods of the invention comprise determining binding of the LAG-3 protein, fragment, derivative, or analogue, to MHC class II molecules present on MHC class II-expressing cells. In such embodiments, the LAG-3 protein, fragment, derivative, or analogue may be immobilised to a reagent layer of a BLI probe, and the MHC class II-expressing cells are in solution.
The probe, method, and system described in U.S. Pat. No. 5,804,453 may be used in accordance with the present invention for determining the MHC class II binding activity of a preparation of LAG-3 protein, or a fragment, derivative, or analogue thereof, as exemplified below by binding of the recombinant LAG-3 protein derivative IMP321 to MHC class II-expressing Raji cells.
Referring to Figure a below, a biosensor probe 100 includes an optical fibre 102, and a reagent layer 104, comprising a blocking reagent (e.g. BSA) and IMP321, at a distal tip of the optical fibre 102. The blocking reagent and IMP321 may be bound to the tip of the optical fibre 102 by soaking the tip in a solution having a predetermined concentration of IMP321, or the blocking reagent, for a predetermined period.
An incident light beam 110 is sent through the optical fibre 102 toward its distal end. At the interface 106 defined between the optical fibre 102, which has a first index of refraction, and the reagent layer 104, which has a second index of refraction, a first portion 112 of the incident light beam 110 will be reflected, while a second portion 114 of the incident light beam 110 will continue through the reagent layer 104. Typically, the blocking reagent and IMP321 will be small relative to the wavelength of the incident light beam 110, from an optical perspective, so the blocking reagent and the IMP321 can be treated as forming a single reagent layer 104. At an interface 108 defined at the exposed surface of the reagent layer 104, of the second portion 114 of the incident beam 110, a first portion 116 will be reflected, while a second portion 118 will pass into the adjacent medium. Of the first portion 116 of the second portion 114 of the incident beam 110, a first portion 160 will be transmitted back through the optical fibre 102, while a second portion (not shown) will be reflected at the interface 106 back into the reagent layer 104.
At a proximal end of the optical fibre 102, the reflected beams 112 and 160 are detected and analysed. At any given point along the optical fibre 102, including its proximal end, the reflected beams 112 and 160 will exhibit a phase difference. Based on this phase difference, the thickness S1 of the reagent layer 104 can be determined.
Referring to
The total thickness S2 of this combined layer will be greater than the thickness S1 of the reagent layer 104 alone. Thus, similar to the probe 100 of
At a second interface 128 between the combined layer and the sample solution 134, a second portion 124 of the second portion 120 of the incident beam 110 is reflected, while a third portion 122 of the second portion 120 of the incident beam 110 continues through the sample solution 134. Of the second portion 124 of the second portion 120 of the incident beam 110, a first portion 126 continues back through the optical fibre 102, while a second portion (not shown) is reflected back into the combined layer at the interface 106.
At a proximal end of the optical fibre 102, the reflected beams 112 and 126 are detected and analysed. At any given point along the optical fibre 102, including its proximal end, the reflected beams 112 and 126 will exhibit a phase difference. Based on this phase difference, the thickness S2 of the combined layer can be determined.
By determining the difference between the thickness S2 of the combined layer and the thickness S1 of the reagent layer 104, the thickness of the cell layer 132 can be determined. The thickness S2 of the combined layer is determined (or “sampled”) at discrete points in time. In this way, the rate of increase of the difference between the thickness S2 of the combined layer and the thickness S1 of the reagent layer 104 (i.e., the rate of increase in thickness of the cell layer 132) can be determined. Based on this rate, the rate of binding of the immobilised IMP321 to MHC class II molecules on the Raji cells can be determined within a very short incubation period.
The diameter of Raji cells is approximately 5-7 μM, 1000 times the wavelength of light, so might be expected to affect the results obtained. However, the signal readout is around 1-2 nM, indicating that light is reflected near the surface of the cells. We have found that the signal change is repeatable, correlated with cell binding, and that the binding rate change is within the measurement range, so can be used to determine binding of Raji cells to IMP321 immobilised at the tip of the optical fibre.
The MHC class II binding activity of the preparation may be determined as the rate of binding of the LAG-3 protein, fragment, derivative, or analogue to the MHC class II molecules.
We have found that the binding rate obtained using the BLI assay depends on the density of MHC class II-expressing cells in the solution, whereas the binding rate is low and relatively flat when the density of non-MHC class II-expressing cells is increased. A higher rate, as well as a higher upper plateau of the binding curve, are obtained if the MHC class II-expressing cells are present at a density of at least 4E6/mL, preferably at least 6E6/mL or 8E6/mL.
We have found that the specificity of the BLI assay is improved when the reagent layer of the BLI probe has been pre-treated with a blocking reagent to minimise non-specific binding of the MHC class II-expressing cells to the reagent layer. Any suitable blocking reagent can be used, for example blocking reagents comprising inert protein such as albumin, for example bovine serum albumin (BSA).
The MHC class II-expressing cells may be immune cells expressing MHC class II molecules. Suitable examples include antigen-presenting cells, or cells of cell lines derived from immune cells. In particular embodiments, the MHC class II-expressing cells are B cells or cells of a B cell line, for example Raji cells.
We have found that MHC class II-expressing cells used for methods of the invention may be thawed, ready-to-use cells obtained from a frozen stock solution. Use of such cells eliminates the requirement to culture cells immediately before a method of the invention is carried out, can help to ensure reliability and reproducibility of results obtained by methods of the invention, and can also allow results obtained at different times to be compared.
Methods of the invention may comprise 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, for example as described in Example 6 below.
Methods of the invention may further comprise determining MHC class II 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 II 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.
The MHC class II binding activity of the reference sample, at a predetermined concentration, may be set as 100% and diluted to various desired concentrations, for example to allow qualification or validation of measurements of MHC class II binding activity of a preparation comprising LAG-3 protein, or a fragment, derivative or analogue thereof, made using a method of the invention.
In some embodiments, 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. Suitable treatments include, for example, deglycosylation (for example by treatment with a PNGase), storage at 37° C. for at least 12 days, oxidation (for example by treatment with 1% or 0.1% hydrogen peroxide), treatment with acid or alkali, or exposure to light for at least 5 days.
Example 6 below describes in detail a BLI assay for determining the MHC class II binding activity of immobilised IMP321 to Raji cells in solution.
There is also provided according to the invention 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.
There is further provided 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 a reagent layer to which the LAG-3 protein, or fragment, derivative, or analogue thereof, is immobilised, and MHC class II-expressing cells.
In some embodiments, the reagent layer of the BLI probe has been pre-treated with a blocking reagent to minimise non-specific binding of the MHC class II-expressing cells to the reagent layer. Any suitable blocking reagent may be used, for example a blocking reagent comprising inert protein such as albumin, for example bovine serum albumin (BSA).
In some embodiments the MHC class II-expressing cells are frozen cells.
In some embodiments the MHC class II-expressing cells are Raji cells.
The MHC class II-expressing cells may be present at a density of at least 1E6/mL, preferably at least 4E6/mL, or 8E6/mL.
A kit of the invention may further include a reference sample, for example as described above, comprising LAG-3 protein, or a fragment, derivative, or analogue thereof. Preferably the MHC class II binding activity of the reference sample is known (for example as determined by a CCL4 release assay, described below).
Probes and kits of the invention may be used in methods of the invention.
The LAG-3 protein may be an isolated natural or recombinant LAG-3 protein. The LAG-3 protein may comprise an amino sequence of LAG-3 protein from any suitable species, such as a primate or murine LAG-3 protein, but preferably a human LAG-3 protein. The amino acid sequence of human and murine LAG-3 protein is provided in FIG. 1 of Huard et al (Proc. Natl. Acad. Sci. USA, 11: 5744-5749, 1997). The sequence of human LAG-3 protein is repeated in
Derivatives of LAG-3 protein include soluble fragments, variants, or mutants of LAG-3 protein that are able to bind MHC class II molecules. Several derivatives of LAG-3 protein are known that are able to bind to MHC class II molecules. Many examples of such derivatives are described in Huard et al (Proc. Natl. Acad. Sci. USA, 11: 5744-5749, 1997). This document describes characterization of the MHC class II binding site on LAG-3 protein. Methods for making mutants of LAG-3 are described, as well as a quantitative cellular adhesion assay for determining the ability of LAG-3 mutants to bind class II-positive Daudi cells. Binding of several different mutants of LAG-3 to MHC class II molecules was determined. Some mutations were able to reduce class II binding, while other mutations increased the affinity of LAG-3 for class II molecules. Many of the residues essential for binding MHC class II proteins are clustered at the base of a large 30 amino acid extra-loop structure in the LAG-3 D1 domain. The amino acid sequence of the extra-loop structure of the D1 domain of human LAG-3 protein is GPPAAAPGHPLAPGPHPAAPSSWGPRPRRY (SEQ ID NO: 2), the underlined sequence in
The LAG-3 protein derivative may comprise the 30 amino acid extra-loop sequence of the human LAG-3 D1 domain, or a variant of such sequence with one or more conservative amino acid substitutions. The variant may comprise amino acid sequence that has at least 70%, 80%, 90%, or 95% amino acid identity with the 30 amino acid extra-loop sequence of the human LAG-3 D1 domain.
The derivative of LAG-3 protein may comprise an amino acid sequence of domain D1, and optionally domain D2, of LAG-3 protein, preferably human LAG-3 protein.
The derivative of LAG-3 protein may comprise an amino acid sequence that has at least 70%, 80%, 90%, or 95% amino acid identity with domain D1, or with domain D1 and D2, of LAG-3 protein, preferably human LAG-3 protein.
The derivative of LAG-3 protein may comprise an amino acid sequence of domains D1, D2, D3, and optionally D4, of LAG-3 protein, preferably human LAG-3 protein.
The derivative of LAG-3 protein may comprise an amino acid sequence that has at least 70%, 80%, 90%, or 95% amino acid identity with domain D1, D2, and D3, or with domain D1, D2, D3, and D4, of LAG-3 protein, preferably human LAG-3.
Sequence identity between amino acid sequences can be determined by comparing an alignment of the sequences. When an equivalent position in the compared sequences is occupied by the same amino acid, then the molecules are identical at that position. Scoring an alignment as a percentage of identity is a function of the number of identical amino acids at positions shared by the compared sequences. When comparing sequences, optimal alignments may require gaps to be introduced into one or more of the sequences to take into consideration possible insertions and deletions in the sequences. Sequence comparison methods may employ gap penalties so that, for the same number of identical molecules in sequences being compared, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. Calculation of maximum percent identity involves the production of an optimal alignment, taking into consideration gap penalties.
Suitable computer programs for carrying out sequence comparisons are widely available in the commercial and public sector. Examples include MatGat (Campanella et al., 2003, BMC Bioinformatics 4: 29; program available from http://bitincka.com/ledion/matgat), Gap (Needleman & Wunsch, 1970. J. Mol. Biol. 48: 443-453), FASTA (Altschul et al., 1990, J. Mol. Biol. 215: 403-410; program available from http://www.ebi.ac.uk/fasta), Clustal W 2.0 and X 2.0 (Larkin et al., 2007, Bioinformatics 23: 2947-2948; program available from http://www.ebi.ac.uk/tools/clustalw2) and EMBOSS Pairwise Alignment Algorithms (Needleman & Wunsch, 1970, supra; Kruskal, 1983, In: Time warps, string edits and macromolecules: the theory and practice of sequence comparison, Sankoff & Kruskal (eds), pp 1-44, Addison Wesley; programs available from http://www.ebi.ac.uk/tools/emboss/align). All programs may be run using default parameters.
For example, sequence comparisons may be undertaken using the “needle” method of the EMBOSS Pairwise Alignment Algorithms, which determines an optimum alignment (including gaps) of two sequences when considered over their entire length and provides a percentage identity score. Default parameters for amino acid sequence comparisons (“Protein Molecule” option) may be Gap Extend penalty: 0.5, Gap Open penalty: 10.0, Matrix: Blosum 62.
The sequence comparison may be performed over the full length of the reference sequence.
The LAG-3 protein derivative may be fused to Immunoglobulin Fc amino acid sequence, preferably human IgG1 Fc amino acid sequence, optionally by a linker amino acid sequence.
The ability of a derivative of LAG-3 protein to bind to MHC class II molecules may be determined using a quantitative cellular adhesion assay as described in Huard et al (supra). The affinity of a derivative of LAG-3 protein for MHC class II molecules may be at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the affinity of human LAG-3 protein for class II molecules. Preferably the affinity of a derivative of LAG-3 protein for MHC class II molecules is at least 50% of the affinity of human LAG-3 protein for class II molecules.
Embodiments of the invention are described below, by way of example only, with reference to the following drawings in which:
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.
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.
The binding curves obtained are shown in
This example describes evaluation of a Meso Scale Discovery (MSD) assay to determine binding of IMP321 to Raji cells.
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.
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.
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.
The MSD assay used is shown schematically in
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.
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.
The results obtained for samples containing IMP321 with and without Raji cells are shown in
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.
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.
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.
Briefly, microplates were blocked with blocking reagent at 25° C. for 2 hours. Samples and rituxan control were diluted with dilution buffer to 2 μg/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).
The results are shown in
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.
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.
The results are shown in
The results show that Casein was the best blocking reagent to reduce non-specific binding of IMP321 to ELISA plates.
Evaluation of Use of Meso Scale Discovery (MSD) Assay, with Casein Blocking Buffer, to Determine Binding of IMP321 to Raji Cells
This example describes evaluation of an MSD assay to determine binding of IMP321 to Raji cells at different seeding densities using casein blocking buffer.
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.
The results are shown in
Binding of IMP321 to Raji cells was compared with binding of IMP321 to HLA-DRdim 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
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.
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.
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:
The results are shown in the table below.
The results show dose-dependent IMP321 binding to plate-immobilised Raji cells.
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:
The results are shown in the table below:
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.
It was concluded that a direct cell-based ELISA cannot be used to demonstrate specific binding of IMP321 to plate-immobilised Raji cells.
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.
The results are shown in the table below:
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.
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.
1) Raji cells: ATCC/CCL-86
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
NOTE: 1) Use reverse pipetting to ensure accuracy.
1) Hydrate the biosensors in PBS for at least 10 min
2) Prepare the assay plate. In a black polypropylene microplate, transfer 200 μL 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:
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.
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
8) Calculate relative binding potency of the sample using EC50 ratio of the Reference Standard and the Sample.
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.
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:
Absolute value (Assay 1 Result−Assay 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.
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.
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
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
It was concluded from these results that the BLI assay is able to determine specific binding of immobilised IMP321 to Raji cells in solution.
Correlation of IMP321 Binding Activity Measured by BLI Assay with Known Binding Potency
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.
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).
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
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.
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.
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.
It was concluded that the bioactivity of the different IMP321 samples correlated with the bioactivity as determined by CCL4 release assay.
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
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:
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.
Number | Date | Country | Kind |
---|---|---|---|
201611180971.4 | Dec 2016 | CN | national |
Filing Document | Filing Date | Country | Kind |
---|---|---|---|
PCT/CN2017/116889 | 12/18/2017 | WO | 00 |