DESC:FIELD OF THE INVENTION
The present invention relates to methods and means relating to an in vitro cell-based assay. More particularly, the invention relates to a method for determining inhibition of cell adhesion by an antibody preparation based on binding to a specific binding partner, by means of a cell based assay.
BACKGROUND OF THE INVENTION
Biologic medicines are complex drugs and are prone to heterogeneity. Subtle differences in protein structure can lead to massive shifts in structure and function of the molecule. For this reason, regulatory bodies mandate extensive structural and functional characterization of biologic drugs for their review towards marketing approval. Such characterization forms an integral part of drug assessment studies.
Functional characterization is performed to assess potency and activity of the drug. This includes assessment of target and receptor binding as well as bioactivity such as complement binding, cell mediated toxicity, cytotoxicity, inhibition of cell adhesion, apoptosis etc. Functional evaluation is tailored based on the mechanism of action of a given drug. For example, functional studies of vedolizumab, a monoclonal antibody targeting a4ß7 integrin complex involves assessment of its ability to bind to the integrin complex, or conversely, an assessment of the extent to which it can inhibit binding of the complex to MAdCAM-1 (mucosal addressin cell adhesion molecule-1), one of its natural ligands. This can be a direct read-out of the potency of the drug. Some of the major challenges faced in arriving at a relevant assay of choice include choice of cell line, ease of assay methodology, qualification parameters, reduction in variability of output and mode of detection.
It is an object of the present invention to develop an efficient, time-saving cell-based assay with high sensitivity and less variability, for the functional assessment of vedolizumab.
SUMMARY OF THE INVENTION
Accordingly, the present invention discloses a sensitive cell adhesion inhibition assay to estimate the relative potency of an anti- a4ß7 antibody sample to inhibit MAdCAM-1-induced adhesion of cells. The method comprises incubating a pre-complex preparation comprising the antibody and a4ß7-expressing cells with MAdCAM-1 and later, detecting unbound cells spectrophotometrically. The method is rapid, saves time and is adapted for high throughput measurements.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Representative dose response curves (DRC) following assay optimization at varying concentrations of vedolizumab (250 ng/mL to 3.125 ng/mL), in unbound (non-adherant) cell populations
Figure 2: Representative dose response curves (DRC) following assay optimization at 125 ng/mL to 5 ng/mL (a) & 500 ng/mL to 5 ng/mL (b) of vedolizumab in unbound (supernatant) cell populations
Figure 3: Representative dose response curves (DRC) of unbound (supernatant) cell populations detected using Alamar Blue reagent (a) and CellTitre-Glo® luminescent reagent (b).
Figure 4: Representative dose response curves (DRC) comparing measurement from bound (a) and unbound (supernatant) (b) cells
DETAILED DESCRIPTION OF THE INVENTION
During inflammation, a4ß7 integrin expressed on the surface of a discrete subset of memory T-lymphocytes binds to MAdCAM-1 on gut endothelia, leading to lymphocyte trafficking into the gastrointestinal (GI) mucosa and gut associated lymphoid tissue (GALT). Vedolizumab (the active ingredient of Entivyo®) is a humanized IgG1 monoclonal antibody that targets and binds to a4ß7 integrin, thereby inhibiting T cell migration across the inflamed GI tissue into the gut. In other words, vedolizumab blocks the interaction of a4ß7 integrin with mucosal addressin cell adhesion molecule-1 (MAdCAM-1).
The present invention pertains to a sensitive cell-based assay to estimate the relative potency of an anti- a4ß7 antibody against a4ß7 integrin by measuring inhibition of MAdCAM-1-induced cell adhesion.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice and testing of the present invention, the preferred methods and materials are described herein.
In an embodiment, the invention discloses a method to estimate the relative potency of anti-a4ß7 antibody in a sample to inhibit MAdCAM-1-induced cell adhesion wherein the method comprises:
a) serially diluting the sample and a reference standard solution of the anti-a4ß7 antibody;
b) seeding a plurality of wells with non-adherent cells expressing a4ß7 on their surface in culture medium containing MnCl2;
c) incubating the wells of step b) with either the serially diluted samples or the serially diluted reference standard solution in medium containing MnCl2 thereby generating pre-complexes of cells with antibodies;
d) coating a plurality of solid substrates with MAdCAM-1 protein;
e) incubating the coated solid substrates with a pre-complex solution of step c) to allow binding of cells to MAdCAM-1, generating supernatant with unbound cells;
f) aspirating the supernatant containing unbound cells;
g) measuring viability of the unbound cells spectrophotometrically; and
h) comparing the viability of cells incubated with pre-complex with sample and pre-complex with standards, thereby measuring the relative potency;
wherein the incubation of step c) & e) are carried out for at least 60 minutes; and
wherein the method can measure relative potency of the anti-a4ß7 antibody sample at a concentration as high as 500 ng/mL and as low as 5 ng/mL in the sample.
In yet another embodiment, MAdCAM-1 is coated onto the solid substrate for at least overnight at a concentration of about 3 µg/mL.
In yet another embodiment, the spectrophotometric measurement employs reagents that facilitate measurement of preferably luminescence as a read-out of cell viability.
In yet another embodiment, the cells expressing a4ß7 are preferably RPMI 8866.
In a further embodiment, the concentration of MnCl2 in the culture medium is about 1 mM.
In yet another embodiment, the anti-a4ß7 antibody is vedolizumab.
Definitions:
The term ‘pre-complex’ as used herein refers to an association formed between one or more cells in the medium with one or more antibodies, as a result of binding of functionally active anti-a4ß7 antibody to the a4ß7 integrin receptor on the non-adherent cells upon incubation.
The term ‘potency’ as used herein refers to the measure of the biological activity using a suitable quantitative biological assay (also called potency assay), based on the attribute of the product which is linked to the relevant biological properties. The biological activity measured in present method is inhibition of MAdCAM-1-induced cell adhesion of otherwise non-adherant cells expressing a4ß7 integrin on the cell surface.
The term ‘reference standard’ refers to a currently or previously marketed recombinant protein product, also described as the ‘originator product or ‘branded product’ serving as a comparator in the studies.
The term ‘relative potency’ as used herein refers to the ability of a test sample, of unknown potency, to produce the desired response compared to a reference sample, when tested under the same conditions. Accordingly, present invention discloses measurement of relative potency of unknown sample containing anti-a4ß7 antibody compared to a reference sample of anti-a4ß7 antibody to inhibit MAdCAM-1-induced cell adhesion of otherwise non-adherant cells expressing a4ß7 integrin on the cell surface.
The term ‘serially diluting’ as used herein refers to stepwise dilution of a substance in solution.
Abbreviations:
a4ß7 - alpha4beta7 integrin
ATCC - American type culture collection
CO2 - carbondioxide
CV - coefficient of variation
DMEM- Dulbecco’s modified eagle medium
DRC - dose response curve
ECACC- European Collection of Authenticated Cell Cultures
FBS - fetal bovine serum
GALT - gut associated lymphoid tissue
MAdCAM-1- mucosal addressin cell adhesion molecule-1
MnCl2 - Manganese chloride
mM - millimolar
PBS - phosphate buffer saline
RFU - relative fluorescence unit
EXAMPLES
Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of this invention. The invention will now be described in greater detail by reference to the following non-limiting examples. The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.
Example 1: Selection of cell line
Two different cell lines (RPMI 8866 and HuT 78) were evaluated for development of assay measuring vedolizumab-induced inhibition of cell adhesion. Both cell lines express a4ß7 integrin. RPMI 8866 cell line was sourced from ECACC (Catalogue No.: 95041316) and maintained in RPMI 1640 medium with 10% FBS and necessary supplements. HuT 78 cell line was sourced from ATCC (Catalogue No.: TIB-161TM) and maintained in DMEM with 10% FBS and necessary supplements. Cells at varying cell densities per well were incubated with MAdCAM-1 at 370C for different periods of time. Cell viability was measured at the end of incubation period with mean relative fluorescence unit (RFU) (n=3) as the read-out. Results revealed superiority of RPMI 8866 cells when compared to HuT 78 cells in terms of comparability of RFU and amenability for performance of the assay (Table 1). Further, it was observed that HuT 78 cells displayed a higher tendency of forming clumps throughout the assay optimization process, hindering assay performance, thereby rendering the cell line less suitable.
Table 1: Selection of cell line
Cell seeding density
(NX106 cells/well) Incubation time (Cells + MAdCAM-1)
2 hours 15 hours
Mean RLU (n=3)
RPMI 8866 HuT 78 RPMI 8866 HuT 78
0.500 8065 6495 10809 9523
0.250 5252 4100 12948 9513
0.125 3133 1675 13143 5373
0.063 1035 1045 9601 3393
0.031 740 587 10809 1694
Example 2: Coating of MAdCAM-1 to substrate
96 well flat bottom high binding plate was coated with MAdCAM-1 solution (3 µg/mL) diluted in 1xPBS with Ca2+ and Mg2+ salts. Plate was sealed and incubated at 2-80C overnight or for upto 48 hours. At the end of incubation period, plate was washed twice with 1X PBS containing Ca2+ and Mg2+ salts. Plate was blocked with 5%BSA for 60±30 minutes at 370C.
Example 3: Optimization of vedolizumab dose response curve
Various concentration ranges of vedolizumab were considered for dose response curve optimization. For this, 0.4x106 cells per well (100 µL) of RPMI 8866 cells were seeded in microtiter plate, to which, various concentrations of vedolizumab were added (40 µL) and incubated at 370C at 5% CO2 for 60-90 mins to form ‘pre-complexes’ of the cells with vedolizumab. At the end of incubation period, 100 µL of the said pre-complex solution from each well was added to corresponding well in MAdCAM-1 coated plates and incubated at 370C at 5% CO2 for 60-75 mins. After completion of incubation 100 µL supernatant (unbound cells) was aspirated and transferred to another 96 well plate. To the aspirated cells, CellTitre-Glo® reagent (100 µL) was added and incubated for 30 mins in the dark at 250C. Luminescence was read using a spectrophotometer with 1000ms integration time. Table 2 shows dose response curve (DRC) when assay is performed in the concentration range of 250 ng/mL to 3.125 ng/mL, and Table 3 shows the same at 125 ng/mL to 5 ng/mL range. (also represented in Figure 1 & Figure 2) A statistically acceptable DRC with dose point distribution on sigmoidal curve with clear upper and lower asymptote saturation was observed in the concentration range of 125 ng/mL to 5 ng/mL and 500 ng/mL to 5ng/mL. Based on this data, vedolizumab dose points between 500 ng/mL and 5ng/mL was finalized for the cell adhesion assay so as to minimize day to day assay variations.
Table 2: Vedolizumab dose response curve in the concentration range of 250 ng/mL to 3.125 ng/mL
Vedolizumab Conc.
ng/mL Unbound (supernatant) Cells
Mean RLU (n=3)
Run 1 Run 2
250 5661 5406
125 5251 5256
62.5 5047 5662
50 5147 5592
25 4397 5272
12.5 1862 2709
6.25 1543 1959
3.125 1568 1712
Cells+MAdCAM 1468 1719
Cell Control 6141 5400
Table 3: Vedolizumab dose response curve in the concentration range of 125 ng/mL to 5 ng/mL
Vedolizumab Conc.
ng/mL Unbound (supernatant) Cells
Mean RLU (n=3)
Run 1 Run 2
125.000 367416 369933
75.000 386162 400435
50.000 377315 403713
40.000 372672 397712
32.000 349533 372650
25.600 265161 233374
20.480 135761 98288
16.384 76247 51969
10 31498 26391
5 33373 28316
MAdCAM+Cells 148949 229902
Cell Control 367045 379836
Example 4: Optimization of Mncl2 concentration in assay medium
To evaluate the influence of Mncl2 on cell adhesion ability with integrin, different concentrations (1mM, 2mM and 4mM) of MnCl2 in the assay medium were evaluated. Assay procedure as described in Example 3 was followed. As alterations in cell morphology was observed upon use of 4mM MnCl2, the same was not selected for further optimization studies. As evident from data in Table 4, DRC obtained upon using 1mM MnCl2 displayed a higher fold ratio (signal to noise ratio), and hence was selected for the assay.
Table 4: Dose response curve at 1mM & 2mM MnCl2 in unbound cells (in supernatant)
Unbound Cells (in supernatant)
Vedolizumab Concentration (ng/mL) 2mM Mncl2 1mM Mncl2
Mean RLU (n=3) CV% Mean RLU (n=3) CV%
125.000 191027 7 250253 3
75.000 201229 6 259953 1
50.000 182227 3 249880 3
40.000 166390 4 238092 1
32.000 138640 3 238337 4
25.600 108153 8 207170 5
20.480 64921 3 135389 2
16.384 37796 13 73885 6
10.000 13435 15 12751 88
5.000 5277 36 2707 51
MAdCAM+Cells 3319 66 1859 51
Cells + Assay Medium 262694 3 266584 3
Fold Ratio 79 143
Example 5: Formation of ‘pre-complex’ and transfer to MAdCAM-1 coated plate
Vedolizumab sample was serially diluted in pre-warmed assay medium. RPMI 8866 cells were seeded at a density of about 0.4 X106 cells per well (160µL) in 96 well U bottom plates. 40µL of serially diluted vedolizumab sample was added to each culture well in medium containing MnCl2 to allow binding of functionally active vedolizumab to a4ß7 integrin receptors on RPMI 8866 cells (hereinafter referred to as ‘pre-complex’) of functionally active vedolizumab and non-adherant RPMI 8866 cells. Plate was incubated at 370C, 5% CO2 for at least 60 minutes.
At the end of incubation, the preparation containing pre-complex was mixed for at least 10 times by pipette mixing. Separately, the blocking reagent in plate of Example 2 was discarded and 100 µL of the preparation containing pre-complex was transferred to the blocked plate. The plate was incubated at 370C, 5% CO2 for at least 60 minutes.
Example 6: Detection of viable cells
After completion of incubation of pre-complex preparation with MAdCAM-1 coated plate, 100µL of cell suspension was aspirated and transferred to 96 well white opaque plate carefully, without forming any foams/bubbles. 100µL of CellTitre-Glo® reagent (30 minutes in dark) or Alamar Blue (20% of the volume of medium in well, 4hrs incubation) was added to the wells and plate was incubated at 250C. Luminescence (CellTitre-Glo®) or fluorescence (Alamar Blue) was read using a multimode spectrophotometer with 1000ms as the integration time (top read). (Figure 3)
The relative potency of the samples with respect to the reference samples was calculated using the formula:
Percent relative potency (%) = Relative potency X 100
Measurement using CellTitre-Glo® reagent displayed higher signal to noise ratio compared to the same using Alamar blue for adherent cells as well as unbound (supernatant) cells. Also by using CellTitre-Glo® reagent, overall assay incubation period was reduced by ~4 Hrs. In addition, considering CV (%) between the replicates and dose point distribution on 4-PL curve, method was finalized with quantification of unbound (supernatant) cells for reporting results.
Example 7: Comparison of measurement of bound vs. unbound cells as a read-out of relative potency
The relative potency of vedolizumab samples was evaluated by measuring the binding of vedolizumab to a4ß7 integrin expressed on RPMI 8866 cells and inhibition of binding of cells to recombinant human MAdCAM-1. Briefly, RPMI 8866 cells were pre-incubated for at least 60 mins with various concentrations of vedolizumab to form pre-complexes. The said pre-complex-containing solution was then added to MAdCAM-1 coated plates. After incubation period of 60 mins, the unbound (supernatant) or bound cells were measured by luminescence detection reagent. The observed relative luminescence unit is directly proportional to the binding of vedolizumab to RPMI 8866 cells and inhibition of cell adhesion. Vedolizumab dose response curves were analysed by 4-parameter logistic fit using Softmax Pro software.
Compared to bound (adherent) cells, data from measurement of unbound cells was found to show statistically superior DRC points on the sigmoidal curve, which were comparatively more consistent and with minimum variation among triplicates. (Figure 4). Hence, measurement of unbound cells was chosen for cell-viability read-out in the assay.
The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments and examples are therefore to be considered in all respects illustrative rather than limiting the invention described herein.
,CLAIMS:CLAIMS
1. A method to estimate the relative potency of anti-a4ß7 antibody in a sample to inhibit MAdCAM-1-induced cell adhesion wherein the method comprises:
a) serially diluting the sample and a reference standard solution of the anti-a4ß7 antibody;
b) seeding a plurality of wells with non-adherent cells expressing a4ß7 on their surface in culture medium containing MnCl2;
c) incubating the wells of step b) with either the serially diluted samples or the serially diluted reference standard solution in medium containing MnCl2 thereby generating pre-complexes of cells with antibodies;
d) coating a plurality of solid substrates with MAdCAM-1 protein;
e) incubating the coated solid substrates with a pre-complex solution of step c) to allow binding of cells to MAdCAM-1, generating supernatant with unbound cells;
f) aspirating the supernatant containing unbound cells;
g) measuring viability of the unbound cells spectrophotometrically; and
h) comparing the viability of cells incubated with pre-complex with sample and pre-complex with standards, thereby measuring the relative potency;
wherein the incubation of step c) & e) are carried out for at least 60 minutes; and
wherein the method can measure relative potency of the anti-a4ß7 antibody sample at a concentration as high as 500 ng/mL and as low as 5 ng/mL in the sample.
2. The method as claimed in claim 1 wherein MAdCAM-1 is coated onto the solid substrate for at least overnight at a concentration of about 3 µg/mL.
3. The method as claimed in claim 1 wherein the spectrophotometric measurement employs reagents that facilitate measurement of preferably luminescence as a read-out of cell viability.
4. The method as claimed in claim 1wherein the cells expressing a4ß7 are preferably RPMI 8866.
5. The method as claimed in claim 1 wherein concentration of MnCl2 in the culture medium is about 1 mM.
6. The method as claimed in claim 1 wherein the anti-a4ß7 antibody is vedolizumab.