DESC:FIELD OF INVENTION
The present invention relates to methods and means for determining the relative binding activity of a sample therapeutic antibody based on the affinity of said antibody for a specific binding partner thereof. In particular, this invention relates to methods and means for use in a Mab-FcRn binding assay for characterization of a pharmaceutical composition comprising said therapeutic antibody.
BACKGROUND OF INVENTION
Biomolecules suspected to influence the pharmacology of recombinant protein therapeutics are selected to assess their binding interactions. These in vitro pharmacological model may then be used in several applications, such as optimizing new batches of therapeutic Recombinant monoclonal antibodies, developing bio-similar or bio-better drug candidates.
Therapeutic Recombinant humanized monoclonal antibodies (MAbs) achieve their effects either directly by inducing apoptosis or indirectly by inducing antibody-dependent, cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). The mechanism of action generally involves an antibody’s Fab (antigen-binding) and Fc (crystallizable) regions. The former binds to antigens; the latter bind to Fc receptors found on monocytes, macrophages, and natural killer cells.
The EMA draft guidance recommends testing antibodies such as for their binding to the Fc receptor FcRn. The neonatal, intracellular FcRn receptor is responsible for transport of IgG across the placenta. FcRn binds to IgG and albumin at low pH but not at high pH. This receptor is responsible for “salvage” of internalized IgG or albumin and therefore endows these proteins with a long half-life. Structurally, the FcRn molecule is similar to Class I MHC and consists of a specific heavy chain combined with ß-2 microglobulin.
By virtue of this pH-specific interaction with IgG-Fc, FcRn mediates IgG homeostasis in human adults by maintaining serum IgG levels, and also transfers maternal IgGs from mother to fetus via the placenta. The Fc-FcRn interaction is also critical for keeping IgG-based therapeutic molecules in circulation thereby enhancing their serum half-life. A homogeneous cell-based flow cytometric FcRn binding assay was established to characterize the Fc-FcRn interaction of therapeutic IgG-based molecules. It is a competition-based assay, wherein the IgG-Fc containing test molecule competes with a fixed concentration of fluorescently-labeled IgG-Fc moiety in solution for binding to the cell-expressed FcRn. The cell-bound fluorescence is read on a flow cytometer. Response of the test sample is analyzed relative to the standard sample and the results are reported as % relative binding. (J Immunol Methods. 2013 Apr 30;390(1-2):81-91. doi: 10.1016/j.jim.2013.01.011. Epub 2013 Feb 4).
However, it has been recently reported (Protein Aggregation and Potency, Cromwell and Gazzano-Santoro 2006) that in cell based flow cytometry method, the percentage of protein aggregation in the FcRn binding assay correlates with % relative binding affinity of the therapeutic antibody to FcRn receptor. Percentage increase of aggregate in the assay proportionally increases the relative potency of the therapeutic antibody. It has been observed that for a 1.5% aggregate difference, the therapeutic antibody is hyperpotent by 40% (Figure 2 & Figure 3). Furthermore, it is reported that methionine oxidation at the Fc binding site of a monoclonal antibody decreases the potential binding affinity to the FcRn receptor (Gaza-Bulseco et al. Effect of methionine oxidation of a recombinant monoclonal antibody on the binding affinity to protein A and protein G. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 870 (2008) 55–62.). The effect of differential aggregates mask the actual Fc changes wherein, the Mab-FcRn binding assay shows acceptable binding for a sample with higher percentage of aggregation and is oxidized. This effect limited the utility of the assay for accurate determination of binding affinity and detecting other changes in the antibody, such as oxidation, that are known to affect receptor binding
Interference of aggregate in the FcRn-Mab binding, to assay relative binding activity is a significant challenge in the development of protein therapeutics. Therefore there is a need to understand the correlation between relative binding activity and aggregation, and formulate assay methodologies that accurately characterize the protein therapeutics.
The present invention provides methods and means for determining the relative binding activity of a sample therapeutic antibody (X-Mab) to the FcRn receptor. The principle object of the present invention is to provide a Mab-FcRn binding assay, where in the measure of % relative binding of the therapeutic antibody (X-Mab) is not influenced by protein aggregation, while being sensitive to molecular level changes such as oxidation.
SUMMARY OF THE INVENTION
In one aspect the present invention provides methods and means for assaying a sample therapeutic antibody thereof and to perform a binding assay for said therapeutic antibody, wherein said competitive binding assay is performed between labelled Mab-F(ab’)2 immune complex and unlabelled therapeutic (X-Mab)-F(ab’)2 immune complex to the FcRn receptors.
In another aspect the invention comprises a FcRn-Mab binding assay, comprising
(a) administering simultaneously a first unlabelled X-Mab-F(ab’)2 immune complex and, a second labelled Mab-F(ab’)2 immune complex for specific binding to FcRn receptor expressing cells, under conditions suitable for competitive binding between said unlabelled and said labelled immune complex
(b) allowing binding of the immune complex to the FcRn receptor expressing cells
(c) measuring the binding of labelled Mab-F(ab’)2 immune complex to said FcRn receptor,
(d) repeating steps (a) to (c) for varying ratios of labelled to unlabelled immune complex
(e) comparing the specific binding measured in step (c) to a reference standard therapeutic antibody (R-Mab) binding to an FcRn receptor and
(f) using information obtained by the comparison to assess the relative binding activity of the therapeutic antibody (X-Mab) with the reference standard therapeutic antibody (R-Mab).
The Mab-FcRn binding assay of the present invention, measures % relative binding of the therapeutic antibody (X-Mab) is not influenced by protein aggregation, while being sensitive to molecular level changes in the Fc region such as oxidation. The said assay can be used as an analytical tool for comparing the binding of different Immunoglobulin G-Fc containing molecules to FcRn. Additional objects, features and advantages of the invention will be apparent from the description herein.
BRIEF DESCRIPTION OF DRAWINGS
Figure1: Represents dose response curve of Therapeutic-X-Mab binding to FcRn.
Figure 2: Represents dose response curves of Therapeutic –X-Mab differing in % aggregate.
Figure 3: Represents Correlation between Percent aggregates with Relative Binding in FcRn Binding Assay.
Figure 4: Represents dose response curves of Therapeutic X-Mab control verses Oxidized (O-Mab) binding to FcRn
Figure 5: Represents dose response curves of X-Mab control verses 5% aggregate sample binding to FcRn performed via Mab-FcRn binding assay of the present invention.
LIST OF ABBREVIATIONS
FcRn - Neonatal Fc receptor
F(ab')2 - variable domain of an antibody
Fc - Constant domain of antibody
X-Mab – Test therapeutic monoclonal antibody for which the relative binding activity is assayed
R-Mab – The reference standard antibody against which the binding activity of the test therapeutic antibody is assayed
O-Mab – Oxidised sample of test therapeutic antibody
FACS - Fluorescence-activated cell sorting
DETAILED DESCRIPTION OF THE INVENTION
Unless defined otherwise, 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 or testing of the present invention, the preferred methods and materials are now described.
The present invention pertains to methods and means for measuring the relative binding activity of a therapeutic antibody (X-Mab) to a specific binding receptor such as an FcRn receptor to evaluate specificity of said therapeutic antibody (X-Mab) to a reference standard antibody (R-Mab).
In a broad aspect of this invention, methods to determine structural and functional integrity of a therapeutic antibody are disclosed, wherein an antigen independent immune complex with said therapeutic antibody (X-Mab) are provided for use in the Mab-FcRn binding assay. The antigen independent immune complex for use in the present invention are obtained by treating the first mixture of therapeutic antibody (X-Mab) with F(ab’)2 of anti-human kappa antibody.
The F(ab')2 fragment antibodies are generated by pepsin digestion of whole IgG antibodies to remove most of the Fc region while leaving intact some of the hinge region. The F(ab’)2 of anti-human kappa antibody are incubated with the therapeutic antibody (X-Mab) to obtain X-Mab- F(ab’)2 immune complexs as described in the example section of this invention disclosure. The F(ab’)2 of anti-human kappa antibody used in the present invention is procured from commercially available sources (Cappel™ - MP BIOMEDICALS Goat F(AB')2 Fragment to Human Kappa Chain (Bound)).
Use of F(ab')2 fragments of anti-human kappa antibody avoids unspecific binding to FcRn receptors on host cells. Immune complex formed with said F(ab')2 fragment does not hinder the Fc molecular binding of the therapeutic antibody with the FcRn Receptor.
In a typical Mab-FcRn assay, the therapeutic X-Mab competes with the labelled Mab added to the assay to bind to the FcRn receptor. The binding of the therapeutic X-Mab is evaluated by acquiring the measure of labelled Mab binding with the FcRn receptor as in Figure1.
The Mab-FcRn assay of the present invention is a competitive assay, wherein the immune complexes of therapeutic antibody (X-Mab) is allowed to compete with immune complexes of labeled Mab to bind to the FcRn receptors.
The labelling of Mab as known in the art is a detectable label and is selected such that conjugation or addition of the label to the Mab is detectable with changes in binding as the ratio of labelled to unlabelled Mab. The result is used to effectively map specificity and, structural and functional integrity of the therapeutic X-Mab.
Additionally a second mixture of labelled Mab is incubated with F(ab’)2 of anti-human kappa antibody to form Mab-F(ab’)2 Immune complex for use in the present invention as described in the examples section.
The immune complex of the labelled Mab and unlabeled therapeutic X-Mab used in the Mab-FcRn assay are used to eliminate the interference of aggregation in cell based flow cytometry method for assessing FcRn binding affinity.
In another embodiment the present invention comprises a FcRn-Mab binding assay, comprising
(g) administering simultaneously a first unlabelled X-Mab -F(ab’)2 immune complex and, a second labelled Mab-F(ab’)2 immune complex for specific binding to FcRn receptor expressing cells, under conditions suitable for competitive binding between said unlabelled and said labelled immune complex
(h) allowing binding of the immune complex to the FcRn receptor expressing cells
(i) measuring the binding of labelled Mab- F(ab’)2 immune complex to said FcRn receptor,
(j) repeating steps (a) to (c) for varying ratios of labelled to unlabelled immune complex
(k) comparing the specific binding measured in step (c) to a reference standard therapeutic antibody binding to an FcRn receptor and
(l) using information obtained by the comparison to assess the relative binding activity of the therapeutic antibody (X-Mab) with the reference standard therapeutic antibody (R-Mab).
FcRn receptors expressing cells for use in the present invention may for instance be produced by expression in host cells, in any way known in the art. They may also be present on the surface of a cell, such as a eukaryotic cell, for instance a yeast cell or a mammalian cell, transfected with nucleic acid enabling expression of an FcRn receptor. The term "host cell" (or recombinant "host cell"), as used herein, is intended to refer to a cell into which a recombinant expression vector has been introduced. Recombinant host cells include, for example, transfectomas, such as CHO cells, NS/0 cells, and lymphocytic cells or the like.
The competitive binding of the labelled and unlabelled Mab-F(ab’)2 immune complex in the Mab-FcRn assay is measured by flow cytometer which acquires the measure of labelled bound Mab-F(ab’)2 immune complex to the FcRn receptors. The flow cytometer acquired measure is further used to plot a Logit curve to calibrate the % binding activity of the therapeutic antibody (X-Mab) with the reference standard antibody (R-Mab).
The relative binding activity of a therapeutic antibody (X-Mab) is a measure of the binding activity in a Mab-FcRn binding assay relative to the activity of a reference standard antibody (R-Mab) for which therapeutic efficacy is assessed. Use of a method according to the invention thus enables determining the binding activity with an FcRn receptor without the interference of aggregation in a cell-based assay. Furthermore, the use of a method and means according to the present invention enables characterization of the therapeutic antibody (X-Mab) by simply measuring the binding activity of the immune complexed binding of the therapeutic antibody (X-Mab) to the FcRn receptor.
The Mab-FcRn binding assay is unbiased to aggregations and demonstrated sensitivity to differences in antibody oxidation. A third mixture of therapeutic antibody (Mab) was oxidized by treatment with H2O2 to allow for complete Fc oxidation. The binding affinity of the oxidized therapeutic antibody (O-Mab) was measured in accordance with the Mab-FcRn assay of the present invention to evaluate changes to FcRn binding as a function of levels of oxidized therapeutic antibody (O-Mab). The assay exhibited a dose-dependent response to the level of sample oxidation which is sensitive to the oxidation levels in Fc, without a bias towards the aggregate content.
In another embodiment the present invention comprises a FcRn-Mab binding assay, wherein
(a) administering simultaneously a first therapeutic unlabelled Mab-F(ab’)2 immune complex and, a second of labelled Mab-F(ab’)2 immune complex for specific binding to FcRn receptor expressing cells, under conditions suitable for competitive binding between said unlabelled and said labelled mAb immune complexes.
(b) allowing simultaneous binding of therapeutic unlabelled oxidized O-Mab-F(ab’)2 immune complex and, labelled Mab-F(ab’)2 immune complex for specific binding to FcRn receptor expressing cells, under conditions suitable for competitive binding between said unlabelled and said labelled immune complexes
(c) allowing independent competitive binding of the immune complexs of (a) and (b) to the respective FcRn receptor expressing cells
(d) measuring the binding of labelled Mab- F(ab’)2 immune complexs to said FcRn receptor of (a) and (b),
(e) comparing the specific binding measured in step (d) to a reference standard therapeutic antibody (R-Mab) binding to an FcRn receptor and
(f) using information obtained by the comparison to assess % relative binding activity of the therapeutic antibody (X-Mab), Oxidised sample of therapeutic antibody (O-Mab) with the reference standard antibody (R-Mab) as represented in Figure 4.
The Mab-FcRn assay of the present invention is useful for more accurately assessing FcRn binding for therapeutic antibodies (Mab’s) via cell based flow cytometry FcRn binding assay
The instant invention further provides techniques to identifying desired therapeutic antibodies and provides methods to determine the binding activity of said therapeutic antibodies with reference standard antibodies without the high accuracy.
The invention is more fully understood by reference to the following examples. These examples should not, however, be construed as limiting the scope of the invention
EXAMPLES
A. Labeling of Mab
The Monoclonal Antibody (Mab) to be labelled was buffer exchanged with PBS using 50 kDa amicon filters. Commercially available label DyLight™488 (Make: Thermo Scientific Pierce, Cat #46402 - DyLight™ 488 NHS-Ester, 1mg) was used to label the Mab. 140 µg (14 µl of 10 mg/ml) of DyLight™488 was added to 1 ml of (2 mg/ml) Mab in PBS. The mixture was incubated for 1 h in the absence of light at room temperature. Unlabelled fluorophore was removed and the labelled Mab was buffer exchanged. The labelled Mab sample was stored at -20°C in smaller aliquots at 2 mg/ml stock concentration.
B. Formation of Immune Complex

Immune complexes are formed by treating the unlabelled therapeutic antibody (X-Mab) and DyLight 488 Mab with F(ab’)2 of anti-human kappa antibody. The F(ab’)2 fragment was commercially obtained from (Cappel™ - MP BIOMEDICALS Goat F(AB')2 Fragment to Human Kappa Chain (Bound)). To the first mixture of therapeutic antibody X-Mab was added with goat F(ab’)2 to human kappa at 1:4 W/W and the second mixture of 1µg/ml Dylight 488 Mab was added with goat F(ab’)2 to human kappa at 1:4 W/W. The first and second mixture were independently incubated at 37 °C for 1 h at 300 rpm (dilutions in PBS at pH 6).
C. Culturing of HEK293 cells
HEK 293 cells stably expressing FcRn receptors (FcRn + ß2m) on cell surface were obtained from LONZA. MCBs (Master Cell Banks) were prepared for this cells using the freezing medium (growth medium + 5% DMSO). The growth medium used for propagating HEK293-FcRn- ß2m stable cells was DMEM with 10% FBS, 1% penicillin-streptomycin mixture, 0.6mg/ml Neomycin G418 and 0.5µg/ml Puromycin. The FcRn and ß2m expression on HEK293 cells across different passages was confirmed by western blot analysis. The cell passage up to X+10 and cell age 48 h and 72 h were used for assay development. The cells are maintained at 70-80 % confluency for use in the assay.
D. FcRn binding assay
HEK293 cells which are around 70-80% confluent is washed with PBS twice. To this was added 5 ml of 0.025 % trypsin EDTA in T175 flask and incubated at 37 °C for one minute. The flask was gently tapped to dislodge the cells and added with 15 ml of growth medium. Cells were spin at 200g for 5 minutes to take out the supernatant, and added with 30ml of PBS pH 6.0. The cells suspension was spin again at 200 g for 5 minutes and added with 10 ml of PBS pH 6.0. The cell count was noted. The cells were diluted with PBS pH6.0. The number of cells per tube was 0.2 x106cells/ml.
Dilutions of X-Mab-F(ab’)2 immune complex (4X) were prepared in PBS pH6.0 in a deep well block and aliquot 250 µl of all X-Mab-F(ab’)2 immune complex into fresh deep well block. In the DyLight control wells 250 µl of PBS was added. Equal volume of diluted DyLight 488- Mab-F(ab’)2 immune complex was added in all wells. The deep well block was mixed at 600 rpm for 30 seconds in shaker. 200 µl of sample was added to U bottom tubes containing 200 µl of cells to make the final volume in the tubes at 400 µl. The tubes were incubated for 3.5 h– 4 h at 25 °C. After incubation samples were acquired in flow cytometer at FL1.
E. Data acquisition and analysis
The flow cytometer, BD FACS Calibur’s fluorescence 1 (FL1) detector, equipped with a 530/30 band pass filter was used to analyze light exited at 493 nm and emitted at 518 nm by the stained cells. Density plots were set up with SSC signal on the Y-axis (logarithmic scale) and FSC signal on the X-axis (linear scale). Live cells gated into R1 were used for assessing fluorescent signaling events on histograms set with counts/events on the Y-axis (linear scale) and FL1-height (FL1-H) on the X-axis (logarithmic scale). The geomean value was obtained from histogram statistics for each concentration of X-Mab-F(ab’)2 immune complex and was noted in Table 1. The parametric logistic graphs of concentration vs. geomean values was plotted as in Figure 5.
Table 1: % relative potency and & aggregates of X-Mab (Therapeutic Antibody) and R-Mab (Reference Standard antibody) binding to FcRn

Sample % aggregate % relative potency
R-Mab 2.6 100
X-Mab 5 101
,CLAIMS:1. A method of performing cell based monoclonal antibody (Mab)-FcRn binding assay comprising
(a) administering simultaneously a first unlabelled X-Mab-F(ab’)2 immune complex and, a second labelled Mab-F(ab’)2 immune complex for specific binding to FcRn receptor expressing cells, under conditions suitable for competitive binding between said unlabelled and said labelled immune complex
(b) allowing binding of the both immune complexes in step (a) to the FcRn receptor expressing cells
(c) measuring the binding of labelled Mab-F(ab’)2 immune complex to said FcRn receptor,
(d) repeating steps (a) to (c) for varying ratios of labelled to unlabelled immune complex,
wherein the said method is not influenced by amount of aggregate content present in said sample.
2. A method according to claim 1, wherein said immune complexes are antigen independent immune complexes.
3. A method according to claim 2, wherein said immune complexes are obtained by treating therapeutic monoclonal antibody (X-Mab) with F(ab’)2 of anti human kappa antibody.
4. A method according to claim 2, wherein said therapeutic monoclonal antibody is labeled or unlabelled antibody.
5. A method according to claim 2, antigen independent complex normalizes the influence of aggregate content present in the sample while measuring binding affinity between X-Mab and FcRn receptor.
6. A method of performing cell based monoclonal antibody (Mab)-FcRn binding assay comprising
(a) administering simultaneously a first unlabelled X-Mab-F(ab’)2 immune complex and, a second labelled Mab-F(ab’)2 immune complex for specific binding to FcRn receptor expressing cells, under conditions suitable for competitive binding between said unlabelled and said labelled immune complex
(b) allowing binding of both the immune complex in step (a) to the FcRn receptor expressing cells
(c) measuring the binding of labelled Mab-F(ab’)2 immune complex to said FcRn receptor,
(d) repeating steps (a) to (c) for varying ratios of labelled to unlabelled immune complex
(e) comparing the specific binding measured in step (c) to the specific binding of a reference standard therapeutic antibody (R-Mab) to FcRn receptor and
(f) using information obtained by the comparison to assess the relative binding activity of the therapeutic antibody (X-Mab) with the reference standard therapeutic antibody (R-Mab)
wherein, the said method is not influenced by amount of aggregate content present in said sample.
7. A method of performing Mab-FcRn assay, wherein the assay is not influenced by aggregate content of a monoclonal antibody in a sample, and sensitive to detect Fc oxidation, comprising the binding of antigen independent immune complex of said Mab to FcRn.