DESC:The present invention relates to a method for detecting anti-drug antibodies in a biological sample, generated against biological therapeutics with minimal interference from a soluble target.
BACKGROUND
Monoclonal antibodies are an increasingly promising class of biological therapeutics for the targeted therapy of several diseases including cancer and autoimmune disorders. Administration of protein-based drugs however run the risk of immunogenicity in the recipient with possibly adverse consequences. One such response is that of the generation of anti-drug antibodies (ADA). Due to the heterogeneity in biological response to a given drug, differences in terms titre, type and affinities of ADAs are generated, because of which, immunogenicity testing for ADAs needs to be developed based on factors including the type of drug and disease-specific factors.
Assays for the detection and quantification of ADA mostly utilize their specific binding properties to the drug itself. However, the said detection is complex due to the presence of interference factors in the sample. Soluble drug target is an example of an interference factor, the presence of which could give a false read-out. Interference ultimately leads to either underestimation of the levels of ADA present in the biological sample or detection of false positives, affecting the performance and sensitivity of the assay. Hence high emphasis is required on mitigating such interferences while optimizing a given immunogenicity testing format.

Tocilizumab, an anti-IL-6R antibody, binds to both soluble and membrane bound IL-6R with the same affinity. In healthy volunteers, reported serum IL-6R levels are 25-75 ng/mL, which upon administration of single dose of tocilizumab can increase up to approximately 300 ng/mL. This high level of circulating serum IL-6R can pose a challenge in ADA assay, also referred to as target interference.
The objective of present invention is to develop a specific immunogenicity assay to measure anti-drug antibodies elicited against anti-IL6R antibody, with minimal interference from free IL-6R present in the sample.
SUMMARY OF THE INVENTION
Accordingly, present invention discloses a method for measuring antibodies (“ADA”) against an IL-6 receptor antagonist (“Anti IL-6R”) in a sample wherein the sample has or is suspected to have interference from free IL-6R in the sample. The method employs an assay format that mitigates interference from free IL-6R in the sample substantially.
DETAILED DESCRIPTION
Present invention discloses a method for measuring antibodies (“ADA”) against IL-6 receptor antagonist (“Anti IL-6R”) in a sample wherein the sample has or is suspected to have interference from free IL-6R.
Present invention discloses a method for measuring antibodies (“ADA”) against IL-6 receptor antagonist (“Anti IL-6R”) in a sample by means of electrochemiluminescence.
In an embodiment, present invention discloses a method for measuring anti-drug antibody (“ADA”) elicited against an IL-6 receptor antagonist (“Anti IL-6R”) in a sample, wherein the method comprises:
a) diluting the sample to upto 80 fold in an acidic solution;
b) incubating the diluted sample of step a) to facilitate acid dissociation of protein complexes in the sample;
c) further incubating the acid dissociated samples of step b) with a solution comprising IL-6, biotinylated Anti IL-6R and ruthenylated Anti IL-6R, thereby allowing binding of ADA to the biotinylated Anti IL-6R and the ruthenylated Anti IL-6R;
d) contacting the solution of step c) to a solid support coated with streptavidin, thereby allowing binding of the complex of step c) to the solid support; and
e) detecting the bound complex of step d), by electrochemiluminescence detection;
wherein, the method mitigates interference by free IL-6R substantially.
In an embodiment, the method mitigates interference by free IL-6R upto a concentration of 350 ng/mL in the solution.
In another embodiment, the IL-6 is recombinant IL-6.
In an embodiment, the IL-6 receptor antagonist is tocilizumab.
In another embodiment, the acidic solution comprises acetic acid.
The invention describes a method for measuring anti-drug antibodies generated against an IL-6 receptor antagonist in a sample, wherein the sample dilution factor, assay diluent buffer, contact antibodies and wash program are selected to mitigate non-specific interferences from the sample, leading to enhanced sensitivity and substantially high target tolerance.
As can be obvious to a person skilled in the art, the labelled Anti-IL-6R pair of step c) (viz., biotinylated Anti IL-6R and ruthenylated Anti IL-6R) can be replaced with any commercially available appropriate labelled anti-IL-6R pair for capture and detection purposes and shall further be detected using appropriate substrate for detection.
Definitions

The term “drug” as used herein refers to an IL-6 receptor antagonist.
The term “free IL-6R” refers to IL-6R present in the sample that is not membrane-bound.
The term “negative control” or “NC” refers to pooled normal human serum from healthy volunteers.
The term “positive control” of “PC” refers to pooled normal human serum that has been spiked with monoclonal or polyclonal anti-IL-6R antibody.
The term “substantially” in the context of mitigating interference by free IL-6R as used herein refers to the fact that the method mitigates said interference to upto free IL-6R concentration of 350 ng/mL in the sample.
The term ‘sample’ as used herein the invention refers to blood sample or serum sample isolated from the blood of healthy volunteers or patients who had been administered with IL-6 receptor antagonist. The said sample has or is suspected to have non-specific interferences.
The term “Anti-drug antibody” or “ADA” refers to an antibody that is elicited/raised/formed/produced against the drug in animals including humans.
Certain specific aspects and embodiments of the invention are more fully described by reference to the following examples. However, these examples should not be construed as limiting the scope of the invention in any manner.
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.
During development of the method for detecting anti-IL-6 receptor antagonist in a sample, various parameters were optimized to achieve acceptable sensitivity and interference mitigation. One such critical parameter was the optimal dilution of the test sample such that the signal-to-noise ratio is enhanced substantially. Other parameters optimized are optimal combination of biotinylated (capture) and ruthenylated (detection) reagents, among others.
Examples of present invention describe methodology relating to anti-IL-6R antibodies, but not limited to tocilizumab.
Example 1: Optimization of minimal required dilution (MRD)
Negative control (NC) sampled were obtained by pooling normal human serum from healthy volunteers. Positive control (PC) samples were obtained by spiking pooled normal human serum (or NC) with monoclonal or polyclonal anti-IL-6R antibody.
To titrate and establish the optimal combination of biotinylated (capture) and ruthenylated (detection) reagents, positive and negative control samples were analyzed under different conditions. Biotinylated-tocilizumab (BT-TC) and ruthenylated-tocilizumab (ST-TC) were prepared using multiple concentration combinations. Once the optimal concentrations for BT-TC and ST-TC were chosen, the MRD was tested at various fold dilutions. The condition that met the best signal-to-noise (S/N) criteria, which was 1:80 dilution, was used for further analysis. (Table1)
Concentration MRD 1:20 MRD 1:40 MRD 1:80
No. anti TCZ (ng/mL) TCZ (µg/mL) IL-6R (ng/mL) Mean %CV S/N Mean %CV S/N Mean Value %CV S/N
1 500 0 0 5377.0 0.7 28.1 2619.5 2.3 14.3 1402.0 1.7 7.8
2 50 0 0 723.5 2.4 3.8 440.5 0.5 2.4 308.0 0.5 1.7
3 500 250 0 732.0 1.9 3.8 801.5 3.4 4.4 710.0 2.6 4.0
4 500 100 0 1714.0 4.7 9.0 1409.5 0.5 7.7 1013.5 1.9 5.7
5 500 50 750 2670.0 1.6 13.9 1862.5 1.3 10.2 1170.5 0.7 6.5
6 500 10 0 4808.5 4.3 25.1 2488.5 1.2 13.6 1305.0 0.5 7.3
7 500 5 0 5083.0 1.1 26.5 2451.5 2.9 13.4 1370.5 2.3 7.7
8 500 0 750 5086.0 5.2 26.6 2508.0 1.1 13.7 1358.5 3.9 7.6
9 50 0 750 871.5 1.7 4.6 503.0 1.7 2.7 1358.5 3.9 7.6
10 500 10 750 3128.5 0.5 16.3 1740.0 0.7 9.5 962.0 3.5 5.4
NEGATIVE CONTROL
11 0 0 0 202.0 0.7 1.1 185.0 2.3 1.0 192.0 1.5 1.1
12 0 0 50 214.0 2.6 1.1 195.0 2.2 1.1 188.5 0.4 1.1
13 0 0 100 216.0 2.6 1.1 200.5 0.4 1.1 189.5 1.1 1.1
14 0 0 350 282.0 0.0 1.5 227.5 0.3 1.2 202.5 0.3 1.1
15 0 0 750 376.5 1.3 2.0 265.0 3.7 1.4 224.5 0.3 1.3
16 0 0 0 191.5 1.1 1.0 183.0 0.0 1.0 179.0 4.0 1.0
Table 1
Example 2: Evaluation of mitigation of interference by free IL-6R
Tocilizumab binds to both soluble and membrane bound IL-6R with the same affinity. In healthy volunteers, reported serum IL-6R levels are 25-75 ng/mL, which upon administration of single dose of tocilizumab can increase up to approximately 300 ng/mL, this high level of circulating serum IL-6R can pose a challenge in ADA assay, also referred to as target interference.
Target interference was initially evaluated by comparing unspiked assay controls to assay controls spiked with either 100 ng/mL, 350 ng/mL or 750 ng/mL of IL-6R. For assessing mitigation of target interference, samples were diluted 80 fold in acetic acid solution and incubated under shaking conditions to facilitate acid dissociation of protein complexes in the sample. The acid dissociated samples were further incubated with a master mix solution comprising IL-6, biotinylated Anti IL-6R (capture reagent) and ruthenylated Anti IL-6R (detection reagent), thereby allowing binding of ADA to the biotinylated Anti IL-6R and the ruthenylated Anti IL-6R. Following this step, the solution containing the aforementioned solution containing bound complex was added to a polypropylene plate coated with streptavidin and incubated under shaking conditions. Ruthenylated Anti IL-6R was measured by electrochemiluminescence. Employing the method, target interference was found to be mitigated to upto a concentration of at least 350 ng/mL of free IL-6R. (Table 2)

Concentration Assay
Sample anti-TCZ (ng/mL) Drug TCZ (µg/mL) IL-6R (ng/mL) Mean Value %CV S/N
PCH+IL6R350 500 0 350 2818.500 1.7 33.9
PCH+IL6R500 500 0 500 2972.500 1.5 35.8
PCL+IL6R350 25 0 350 227.000 3.1 2.7
PCL+IL6R500 25 0 500 230.000 1.2 2.8
DPC+IL6R350 500 10 350 2693.500 0.2 32.4
DPC+IL6R500 500 10 500 2687.000 1.1 32.3
NC+IL6R10 0 0 10 82.500 0.9 1.0
NC+IL6R50 0 0 50 83.500 5.9 1.0
NC+IL6R100 0 0 100 87.000 4.9 1.0
NC+IL6R250 0 0 250 90.500 5.5 1.1
NC+IL6R350 0 0 350 92.000 6.1 1.1
Table 2

,CLAIMS:CLAIMS
We claim:
1. A method for measuring anti-drug antibody (“ADA”) elicited against an IL-6 receptor antagonist (“Anti IL-6R”) in a sample, wherein the method comprises:
a) diluting the sample to upto 80 fold in an acidic solution;
b) incubating the diluted sample of step a) to facilitate acid dissociation of protein complexes in the sample;
c) further incubating the acid dissociated samples of step b) with a solution comprising IL-6, biotinylated Anti IL-6R and ruthenylated Anti IL-6R, thereby allowing binding of ADA to the biotinylated Anti IL-6R and the ruthenylated Anti IL-6R;
d) contacting the solution of step c) to a solid support coated with streptavidin, thereby allowing binding of the complex of step c) to the solid support; and
e) detecting the bound complex of step d), by electrochemiluminescence;
wherein, the method mitigates interference by free IL-6R substantially.
2. The method as claimed in claim 1 wherein, the IL-6 is recombinant IL-6.
3. The method as claimed in claim 1 wherein, the IL-6 receptor antagonist is tocilizumab.
4. The method as claimed in claim 1 wherein, the acidic solution comprises acetic acid.