DESC:FIELD OF INVENTION
The present invention relates to a method for determining specific binding affinity of an antibody (Ab) to human immunoglobulin receptors, specifically to human Fc gamma receptors (Fc?R). In particular, the present invention relates to methods and means for use in an Ab-Fc?R binding assay for determining the binding affinity of a pharmaceutical composition comprising said antibody.
BACKGROUND OF INVENTION
Immunoglobulins are highly attractive therapeutic agents for treatment of various diseases. Immunoglobulins contain Fab (antigen-binding) and Fc (crystallizable) regions. The former binds to the target antigens while the latter binds to Fc receptors present on various cells including monocytes, macrophages, and natural killer cells.
There are different types of Fc receptors (abbreviated FcR), which are classified based on the type of antibody they recognize. These include Fc-gamma receptors (Fc?R) that bind IgG, Fc-alpha receptors (FcaR) that bind IgA and Fc-epsilon receptors (FceR) that bind IgE.
Human Fc gamma receptors are divided into three families such as Fc?RI (CD64), Fc?RII (CD32) and Fc?RIII (CD16) and further, divided into six isoforms such as Fc?RI, Fc?RIIa, Fc?RIIb, Fc?RIIc, Fc?RIIIa and Fc?RIIIb. These receptors differ in characteristics such as their cellular distribution, binding affinities, and/or their effector functions.
Binding of Fc portion of an antibody to Fc gamma receptors on cells lead to different effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), cell dependent cytotoxicity (CDC), phagocytosis, endocytosis and cytokine release. Further, such resulting effector functions are dependent on the type of Fc gamma receptors to which Fc portion of an antibody is bound. Such binding is also influenced by various factors, including glycosylation at Asn 297 position of the antibody. Further, assessing the interaction between Fc portion of an antibody and Fc? receptor using binding assays can potentially be used as a surrogate for evaluating their interaction activities.
Hence, it is one of the recommendations in regulatory guidance to test antibodies for their binding to the Fc binding proteins including FcRn, C1q and Fc-gamma receptors such as CD16, CD32, and CD64.
Furthermore, Fc?RI have higher affinity towards Fc portion of IgGs compared to other Fc?RII and Fc?RIII receptors. Of the Fc?R isoforms Fc?RIIa, Fc?RIIb and Fc?RIIIb have weaker affinity towards Fc portion of an immunoglobulin and hence known as weak affinity receptors. Food and Drug Administration (FDA) recommends to report rate of association and dissociation between test monoclonal antibody and Fc?R in terms of association constant (Ka), dissociation constant (Kd) respectively and equilibration constant KD.
Measurement of these kinetic constants between Fc of a monoclonal antibody and Fc?R may be difficult using traditional methods such as ELISA or flow cytometry. ELISA measures only apparent affinity constants which may not represent an actual affinity constant and measurement of kinetic parameters via flow cytometry are laborious. Also, there exists an additional challenge in using these traditional methods for measuring kinetic parameters for low affinity receptors such as Fc?RIIa, Fc?RIIb and Fc?RIIIb.
Surface Plasmon Resonance (SPR) is a preferred method for measurement of binding activity because of its ability to perform measurements in real time and also exhibit increased sensitivity while measuring kinetic parameters. However, data obtained from SPR should be available in a format to fit into steady state kinetics to calculate the said kinetic constants such as Ka and Kd.
However, one another challenge is the use of SPR based method to capture Ka, kd and KD in case of low affinity receptors, as steps involved in the SPR method may interfere in the measurement of kinetic constants. To apply the steady state model, the saturation in binding needs to be achieved between low affinity Fc?R and monoclonal antibody. To achieve such saturation levels, increased amount of low affinity receptors are required. However, due to cost factor and availability of such low affinity receptor, it may not be easy to perform affinity binding assay for different lots of monoclonal antibodies.
Karlsson et al., showed that with proper experimental design, the kinetics measured with SPR are the same as those of solution based methods. (Karlsson, R. and Falt, A. Experimental design for kinetic analysis of protein-protein interactions with surface plasmon resonance biosensors. Journal of Immunological Methods 200: 121-133; (1997).) Hence, it is necessary to have an optimized SPR method to achieve maximum immobilization/capturing of the binding partners (in this case, either -Fc containing antibody or Fc gamma Receptor) on the surface of the sensor chip to measure the binding activity of the same,
Hence, it is an objective of the present invention to develop an optimized assay format to measure kinetic constants such as Ka and Kd wherein the said assay format provides effective immobilization/capturing of the binding partners that does not interfere with the binding activity of weak affinity Fc gamma receptors such as Fc?RIIa, Fc?RIIb and Fc?RIIIb to the Fc portion of an antibody in the assay.
Therefore, the present invention provides an optimized SPR method, where in the obtained data can be fitted into steady state kinetics to measure kinetic constant without use of increased amount of Fc?R. Additionally the present invention provides a single cycle kinetics assay for measuring binding affinities such as Ka, Kd and KD between weak affinity Fc gamma receptors and Fc portion of a therapeutic antibody.
SUMMARY OF THE INVENTION
The present invention discloses an optimized SPR method to measure kinetic constants such as association constant (Ka), dissociation constant (Kd), and equilibrium constant (KD) between Fc portion of an antibody and low affinity Fc gamma receptors, wherein the method employs protein-L to capture said antibody. The method is optimized, by selecting suitable pH and concentration of immobilization buffer for effective immobilization of protein-L. Further the use of protein-L is devoid of cross reactivity with Fc gamma receptors.
Furthermore, the said SPR based method is performed at a temperature between 4 and 12º C to control rate of reaction thereby measuring kinetic constants such as Ka, Kd and KD.
Further, the method as disclosed in the present invention relates to a single cycle kinetic assay for measuring binding affinity between Fc portion of an antibody and Fc gamma receptors wherein a sample containing varying concentrations of Fc gamma receptors is used for binding and wherein the assay format does not require regeneration of said Fc Gamma Receptors.
The optimized method of the present invention is capable to detect binding affinity between Fc of an antibody and weak affinity Fc?R in terms of equilibrium dissociation constant (KD) in the range of micro molar.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Illustrates an adjusted sensorgram of Fc?RIIa prepared in various pH containing acetate buffer and is represented in terms of Response units (RU) versus time.
Figure 2: Illustrates 1:1 binding fitted sensorgram of single cycle kinetics wherein various concentration of Fc?RIIa passed on to X-Mab captured by immobilized protein-L.
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. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.
The present invention discloses a method for measuring binding affinity between weak affinity Fc?R and Fc portion of an antibody wherein the method employs immobilization of protein L on to a surface for capturing therapeutic antibody in a sample.
In one embodiment, the invention discloses a method for determining affinity between Fc of said antibody in a sample and Fc?R comprising steps of:
a) Immobilization of protein L present in a suitable buffer on to a sensor surface
b) Capturing of said antibody in a sample using immobilized protein L of step (a)
c) Adding Fc?R to the captured therapeutic antibody and
d) Measuring association constant (ka), dissociation constant (kd) and equilibrium constant (KD) between said antibody and Fc?R,
wherein the said method is performed at about 4ºC -12ºC.
In another embodiment of the invention, the said SPR based method is performed at lower temperature to measure the rate of association and dissociation that in turn helps in measuring association and dissociation constant.
In another embodiment the present invention discloses a sensitive surface plasmon resonance method to measure affinity between Fc portion of an antibody and Fc?R, wherein the said method employs use of protein L to capture the antibody.
In yet another embodiment of the invention, protein L is immobilized on to a gold sensor surface, which is covalently attached to carboxymethylated dextran. Commercially available carboxymethylated dextran coated chips such as, but not restricted to CM3, CM4 and CM5, are used.
In yet another embodiment of the invention, immobilization of protein L on CM5 surface involves amine chemistry, wherein the amino groups of protein L attaches to carboxyl groups present on the CM5 chip surface.
In one embodiment, the sample is any antibody which exhibits Fc binding to Fc gamma receptors, more particularly therapeutic antibody such as monoclonal antibodies or the like. Additionally, the said therapeutic antibody is a chimeric or humanized or human antibody.
In another embodiment of the invention, the therapeutic antibody contains kappa light chain which is captured by said immobilized protein L upon passing on the surface.
In yet another embodiment of the invention, the binding between Fc?R passed on to the therapeutic captured antibody is measured in terms of resonance units (RU).
The method as disclosed in the present invention provides optimized conditions including but not limited to concentration and pH of the buffer to achieve maximum immobilization of protein L on the surface of the sensor chip.
In one embodiment of the invention, the buffer employed during immobilization of protein L is an acidic buffer. The method as disclosed in the present invention, wherein the acidic buffer is acetate buffer comprising pH in the range of 4.0 to 5.0.
In yet another embodiment, the method provides capturing of therapeutic antibody via binding to protein L, immobilized to the surface of the sensor chip. The immobilized protein L captures the therapeutic antibody, wherein said protein L has affinity towards Kappa light chain of the antibody. Due to protein L binding to the light chain, the capturing may not impact or result in any conformational changes to the binding sites of Fc?R in the therapeutic antibody.
The Fc? Receptors as disclosed in the present invention, comprises Fc?RI, Fc?RII and Fc?RIII receptors and more specifically low affinity receptors such as Fc?RIIa, Fc?RIIb, Fc?RIIIa and/or Fc?RIIIb.
In yet another embodiment, the weak affinity Fc?R is passed on the surface of said therapeutic antibody captured on immobilized protein L, at a predetermined flow rate that allows effective binding of Fc?R to Fc portion of the therapeutic antibody.
The present invention provides an SPR based method of performing single cycle kinetics assay between Fc of immunoglobulin and Fc?R that is devoid of any regeneration steps between multiple doses of Fc?R, comprising steps of:
a). Immobilization of protein L present in a suitable buffer on to a sensor surface
b). Capturing of said antibody in a sample using immobilized protein L of step (a)
c). Adding varying concentrations of Fc?R on the captured therapeutic antibody and
d) Measuring the binding affinity between said therapeutic antibody and said Fc?R.
The present invention discloses an optimized SPR method to measure kinetic constants such as Ka, Kd and KD, wherein the said method employs use of protein-L to capture one of the binding partners. The said optimization comprises, selecting appropriate pH and concentration of buffer, and performing at lower temperature to control rate of reaction such as association and dissociation between Fc and low affinity Fc gamma receptor. The SPR based method, as disclosed in the present invention is performed without using higher amount of the receptor to calculate the said kinetic constants by fitting the said data values in steady state kinetics.
Definitions
The term used herein equilibrium dissociation constant (KD) is calculated from the four kinetic constants of two state kinetic binding reaction KD= Kd/Ka.
The term used here in association constant (Ka) is kinetic constant which depicts the rate of formation of complex when analyte passed through the ligand immobilized surface.
The term used here in dissociation constant (Kd) is kinetic constant which depicts the rate of dissociation of analyte from the ligand-analyte complex from the surface.
X-Mab as used here in the invention is an immunoglobulin G (Ig G).
“Resonance unit” or “Response unit” or “RU” used herein refers to unit of measurement of change in the actual angle shift in reflected light in SPR.
Examples
The present invention discloses an optimized SPR method for measuring binding affinity between Fc of an antibody present in a sample and low affinity Fc gamma receptors such as Fc?RIIa, wherein the method employs immobilization of protein L to capture therapeutic antibody followed by passing a sample comprising Fc?RIIa (analyte).
Various parameters were optimized in said SPR method to achieve maximum sensitivity. Those parameters include selection of a suitable concentration and pH of buffer for maximum immobilization of protein L on CM5 surface, and use of CM5 sensor chip comprising four flow cells.
Once, suitable buffer was selected for maximum immobilization of protein L on surface, cross reactivity of Fc gamma receptors was performed.
Further, temperature for performing the assay was optimized to measure association and dissociation constants during binding interaction between Mab and low affinity receptor such as Fc gamma R II.
Example 1: Selection of suitable pH of an acidic buffer for immobilization of protein L
To select suitable buffer for maximum immobilization of protein L on CM5 sensor chip, 20 µg/mL of protein L was prepared in 10 mM acetate buffer containing various pH such as 4.0, 4.5, 5.0 and 5.5. Protein L present in various pH containing buffers were used for immobilization using amine chemistry as per standard protocol available/provided by vendor (Biacore).
After immobilization resonance units were measured by passing light through the surface to know maximum immobilization of protein L. Binding resonance units presented in below Table 1 are also represented in Figure 1.
Protein L (20µg/ml) in various buffers Binding RU’s
10 mM Acetate pH 5.5 -1947
10 mM Acetate pH 5.0 2538
10 mM Acetate pH 4.5 5501
10 mM Acetate pH 4.0 6599

Table 1: Binding RU’s of protein L in various pH containing acetate buffer.
Form the above results, it is evident that maximum RU’s were observed with protein L (20µg/ml) present in 10 mM Acetate buffer containing pH 4.0.
Example 2: Cross reactivity of Fc gamma receptors with Protein L
To check whether Fc?RIIa low affinity receptors has any cross reactivity with protein L, 0.5 µM of Fc?RIIa was prepared and diluted in HBS-EP+ running buffer (composition: 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v Surfactant polysorbate 20) and was allowed to pass through flow cell 2 over the protein L immobilized surface at a flow rate of 20 µL/min.
After the regeneration step, 7.5 nM of X-Mab, a therapeutic antibody diluted in HBS-EP+ buffer was allowed to pass through flow cell 2 over protein L immobilized surface at a flow rate of 20 µL/min as a positive control.
Resonance units (RU) of both Fc?RIIa and X-Mab were measured and results are represented in the below Table 2.
Sample Concentration Flow Cell Binding RU’s
Fc?RIIa 0.5 µM 2 5
X-Mab 7.5 nM 2 511
Table 2: Binding RU’s of Fc?RIIa and X-mAb to immobilized protein L.
From the results as shown in Table 2, it is evident that there is no nonspecific binding of Fc?RIIa to Protein L, whereas the positive control binding confirms the activity of immobilized protein L.
Example 3: SPR based method for measurement of binding affinity
To measure binding affinity between X-Mab and Fc gamma RIIa in a single cycle kinetics assay, 30 nM concentration of X-Mab was passed through flow cell 2 over protein L immobilized surface at a flow rate of 30 µL/min with contact time of 15s and stability period of 60s.
To perform single cycle kinetics, various concentrations of Fc?RIIa receptor which were diluted with HBS-EP+ (400nM to 25 nM(2-fold serial dilution) and were passed over flow cell 2 as well as flow cell 1 at a flow rate of 30uL/min with contact time of 60s and dissociation time of 60s. The Fc?RIIa receptor passed over flow cell 1 acts as a negative control. After running all the concentrations in an increasing order, regeneration was done with 10 mM NaOH at a flow rate of 30uL/min with contact time of 15s and stability period of 240s. The flow cells are washed with HBS-EP+ at a flow rate of 30 µL/min with contact time of 350s and stability period of 350s. All the steps in this assay was performed at 4ºC.
Result of the assay is calculated in terms of RU, which were further fitted in Single state kinetic model to calculate kinetic association (Ka), dissociation constants (Kd) and equilibrium dissociation constant (KD) and are represented in Table 3 and the representative sensorgram is represented Figure 2.
Sample ka (1/Ms) kd (1/s) KD
(M) Rmax (RU) Chi² (RU²)
X-Mab 0.47 x 1006 0.309 6.66 x 10-07 63 0.101
Table 3: Kinetic rate constants of binding of FcgRIIa and X-mAb.
Example 4: Assessment of repeatability of the SPR method
To assess, repeatability of the disclosed method, five independent kinetic cycles were conducted in one experiment as described in example 3. Results of the said method is mentioned in below table 4.
Cycle ka (1/Ms) kd (1/s) KD (M) Rmax (RU) Chi² (RU²)
Cycle 1 0.36 x 1006 0.2979 8.26 x 10-07 104 0.098
Cycle 2 0.35 x 1006 0.2773 7.84 x 10-07 119 0.132
Cycle 3 0.38 x 1006 0.2705 7.21 x 10-07 116 0.129
Cycle 4 0.37 x 1006 0.2732 7.38 x 10-07 119 0.121
Cycle 5 0.37 x 1006 0.2665 7.28 x 10-07 119 0.164

Dated this 14h day of April 2016 Signature: __________________
V. R. Srinivas, Ph.D.
Dr. Reddy’s Laboratories Limited
,CLAIMS:1. A method for determining the affinity between Fc of an antibody in a sample and Fc?R comprising steps of:
a) immobilizing protein L in a suitable buffer onto a sensor surface
b) capturing of the antibody in the sample using immobilized protein L of step (a)
c) adding Fc?R to the captured therapeutic antibody and
measuring association constant (ka) and/or dissociation constant (kd) and/or equilibrium constant (KD) between said antibody and Fc?R.
2. A surface plasmon resonance based method of performing single cycle kinetics assay between Fc of an antibody and Fc?R, that is devoid of any regeneration steps between multiple doses of Fc?R, comprising steps of:
a. immobilization of protein L present in a suitable buffer onto a sensor surface
b. capturing of said antibody in a sample using immobilized protein L of step (a)
c. adding Fc?R to the captured antibody and
measuring association constant (ka) and/or dissociation constant (kd) and/or equilibrium constant (KD) between said therapeutic antibody and said Fc?R.
3. The method according to claim 1 or claim 2 wherein, the said method is performed at about 4°C to about 12°C.
4. The method according to claim 1 or claim 2 wherein, the said antibody is therapeutic antibody.
5. The method according to claims 4 wherein, the said therapeutic antibody is chimeric or human or humanized antibody.
6. The method according to claims 1 or claim 2 wherein, the said buffer pH is about 4.0 to about 5.0.
7. The method according to claim 1 or claim 2 wherein, the said sensor surface is a gold sensor surface, which is covalently attached to carboxymethylated dextran.
8. The method according to claim 1 or claim 2 wherein, the said Fc?R is a low affinity receptor.