DESC:METHOD FOR DETECTION OF ANTI-DRUG ANTIBODY
FIELD OF THE INVENTION
The present invention relates to a bioanalytical method for detecting anti-drug antibodies. Particularly, the invention relates to detection of neutralizing anti-drug antibodies in a sample.
BACKGROUND OF THE INVENTION
Biologics are structurally complex proteins developed by genetic engineering; purified and formulated with processes optimized based upon the nature of protein. A major goal in the development of a biologic is to ensure its safety, purity, potency and efficacy. As immunogenicity studies provide information about the safety profile of a given drug, regulatory bodies recommend, head-on-head immunogenicity assessment, among others.
Immunogenicity refers to propensity of drug to generate immune response to itself or to induce immunologically related non-clinical or adverse clinical effect. Among assessment of the several different outcomes of an immunogenic response in the body, that of neutralizing anti-drug antibodies (hereinafter referred to as “NAB”) is indispensable as generation of NABs can lead to potential neutralization of effectiveness of the drug.
IL-6 is a cytokine that regulates immune and inflammatory responses and its overproduction plays a role in autoimmune disorders like rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA). Tocilizumab is a recombinant, humanized, monoclonal antibody raised against IL-6 receptor (IL-6R) that competitively inhibits the binding of IL-6 to its receptor. Tocilizumab is capable of binding to both membrane-bound and soluble IL-6R and thereby inhibits further signal transduction, suppressing excess production of IL-6. Considering the safety concerns associated with the presence of neutralizing anti-drug antibodies, it is important to design a sensitive assay that is able to detect NAB generated against anti-IL6R in the body with superior sensitivity, target tolerance and drug tolerance, among others.
SUMMARY OF THE INVENTION
Accordingly, the present invention discloses a method for detecting neutralizing anti-drug antibodies against anti-IL-6R antibodies present in a sample. In an embodiment, the invention discloses a highly sensitive, selective and tolerant method for detecting and quantifying neutralizing anti-drug antibodies (NAB) against an anti-IL6R antibody in a sample, the method comprising:
a) incubating the sample with an acidic buffer to dissociate immune complexes present therein;
b) contacting the treated sample of step a) with a biotin-conjugated anti-IL6R antibody and a streptavidin-coated substrate overnight to allow affinity-based complex formation of components present therein;
c) eluting NAB from the complex formed at step b) with a buffer at low pH;
d) incubating the dissociated NAB of step c) in a solution containing anti-IL6R antibodies that are conjugated to a detectable label;
e) contacting the solution of step d) with IL-6R immobilized to a substrate;
f) detecting the intensity of the label on the substrate of step e) by colorimetry, luminometry or fluorimetry;
wherein the method is interspersed with intermittent wash steps;
wherein assay signal is inversely proportional to the neutralizing activity of the sample; and
wherein the method can detect as low as 195.313 ng/mL monoclonal NAB and 390.625 ng/mL polyclonal NAB in the sample;
wherein the method is able to tolerate as high as 500 ng/mL, 10 µg/mL and 350 ng/mL of IL-6, anti-IL6R antibody and IL6R in the sample respectively.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Dose response curve of sulfo-tag conjugated tocilizumab for determination of EC 50
Figure 2: Dose response curve of sulfo-tag conjugated tocilizumab at varying coating concentrations of IL-6R
Figure 3: Representative dose response curve of sulfo-tag conjugated tocilizumab for determination of EC50
DETAILED DESCRIPTION OF THE INVENTION
Definitions
The term “assay selectivity” as used herein is the ability of the assay to detect the neutralizing anti-drug antibodies (NAB) specific to the drug in the presence of other potentially interfering substances in the sample.
The term “assay sensitivity” as used herein refers to the lowest antibody concentration at which neutralizing activity can be detected.
The term “dissociation positive control” or “DPC” as used herein refers to pooled normal human serum sample spiked with anti-tocilizumab neutralizing antibody to a concentration of about 500.0 ng/mL and with tocilizumab to about 10.0 µg/mL
The term “drug tolerance” as used herein refers to the maximal amount of free drug i.e., anti-IL6R antibody in present invention, present in the sample that still results in detectable neutralizing anti-drug antibody signal.
The term “high positive control” or “HPC” as used herein refers to pooled normal human serum sample spiked with anti-tocilizumab neutralizing antibody to a concentration of about 1000.0 ng/mL.
The term “low positive control” or “LPC” as used herein refers to pooled normal human serum sample spiked with anti-tocilizumab neutralizing antibody to a concentration of about 500.0 ng/mL.
The term “negative quality control” or “NQC” used herein refers to pooled serum obtained from normal healthy humans.
The term “positive quality control” or “PC” used herein refers to serum sample spiked with monoclonal anti-tocilizumab antibody and/or polyclonal anti-tocilizumab antibody.
The term “signal to noise ratio” or “S/N ratio” as used herein refers to mean relative light units (RLU) of sample or control /Mean RLU of NQC or blank.
The term “target tolerance” as used herein refers to the maximal amount of the drug’s target i.e., IL6R and IL6 present in the sample that still results in detectable neutralizing anti-drug antibody signal.
The term “% CV” as used herein refers to standard deviation/Mean RLU values between replicates.
The term “% Neutralization” as used herein refers to the output of {(mean signal value of NQC - mean signal value of sample) / (mean signal value of NQC) }*100
The present invention discloses a method for detecting neutralizing anti-drug antibodies against anti-IL-6R antibodies present in a sample.
In an embodiment, the invention discloses a highly sensitive, selective and tolerant method for detecting and quantifying neutralizing anti-drug antibodies (NAB) against an anti-IL6R antibody in a sample, the method comprising:
g) incubating the sample with an acidic buffer to dissociate immune complexes present therein;
h) contacting the treated sample of step a) with a biotin-conjugated anti-IL6R antibody and a streptavidin-coated substrate overnight to allow affinity- based complex formation of components present therein;
i) eluting NAB from the complex formed at step b) with a buffer at low pH;
j) incubating the dissociated NAB of step c) in a solution containing anti-IL6R antibodies that are conjugated to a detectable label;
k) contacting the solution of step d) with IL-6R immobilized to a substrate;
l) detecting the intensity of the label on the substrate of step e) by colorimetry, luminometry or fluorimetry;
wherein the method is interspersed with intermittent wash steps;
wherein assay signal is inversely proportional to the neutralizing activity of the sample; and
wherein the method can detect as low as 195.313 ng/mL monoclonal NAB and 390.625 ng/mL polyclonal NAB in the sample;
wherein the method is able to tolerate as high as 500 ng/mL, 10 µg/mL and 350 ng/mL of IL-6, anti-IL6R antibody and IL6R in the sample respectively.
In an embodiment, the buffer at low pH is at a pH of about 2.
In an embodiment, the first-labelled anti-IL6R antibody is biotin-labelled anti-IL6R antibody.
In another embodiment, the neutralizing anti-drug antibodies (NAB) is generated against monoclonal and/or polyclonal anti-IL6R antibody.
In another embodiment, the monoclonal anti-IL6R antibody is tocilizumab.
Abbreviations:
ADA: anti-drug antibodies
BCA: bicinchoninic acid
BT-TC: biotin-conjugated tocilizumab
CV: coefficient of variation
DPC: dissociation positive control
EC50: half maximal effective concentration
HPC: high positive control
IL-6: interleukin 6
IL-6R: interleukin-6 receptor
LPC: low positive control
MSD: Meso Scale Discovery
NAB: neutralizing anti-drug antibodies
NQC: negative quality control
OD: optical density
PBS: phosphate buffered saline
PBST: phosphate buffered saline with a low concentration of detergent solution
PNHS: pooled normal human serum
PQC: positive quality control
S/N ratio: signal to noise ratio
ST-TC: sulfo-tag conjugated tocilizumab
TC: tocilizumab
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: Biotin-conjugation of Tocilizumab
Optimization of biotinylation procedure was aimed to generate a labeled TC such that its functional activity is not affected significantly. Tocilizumab was diluted to 2.0 mg/mL and buffer exchanged to 1X PBS. The conjugation reaction was set up by incubating antibody solution after addition of Biotin at RT for 2 hours and finally buffer exchanged to formulation buffer using a desalting column. The concentration of biotin-conjugated Tocilizumab (BT-TC) was estimated by BCA protein estimation. After conjugation, BT-TC was evaluated for its functional activity. For this, potency of conjugated Tocilizumab was evaluated against unconjugated Tocilizumab via IL-6 induced growth-inhibition assay.
Table 1: Relative potency (%) of Biotin-conjugated Tocilizumab
Sample Run-1 Run-2 Run-3 Average %CV
BT-TC 73.9 85.3 89.6 83 10

Example 2: Sulfo-tag conjugation of Tocilizumab
Optimization of sulfo-tag labeling procedure was aimed to generate a labeled Tocilizumab such that its functional activity was not affected significantly. Tocilizumab was diluted to 2.0 mg/mL and buffer exchanged to 1X PBS. The conjugation reaction was set up by incubating antibody solution after addition of Sulfo-label solution at RT for 2 hours and finally buffer exchanged to formulation buffer using a desalting column. The concentration of Sulfo-tag conjugated Tocilizumab (ST-TC) was estimated by BCA protein estimation. After conjugation, ST-TC was evaluated for its functional activity. For this potency of conjugated Tocilizumab was evaluated against unconjugated Tocilizumab via IL-6 induced growth-inhibition assay.
Table 2: Relative potency (%) of Sulfo-labelled Tocilizumab
Sample Run-1 Run-2 Run-3 Average %CV
ST-TC 106.7 104.7 85.6 99 12

Example 3: Optimization of coating concentration of IL-6R
Microplate wells were coated with 50 µL/well of IL-6R (0.750, 0.500, 0.250 and 1.000 µg/mL ) diluted to required concentration in 1X PBS and incubated overnight at 2-8°C. After wash program, plates were blocked with 250 µL/well of 5% MSD Blocker-A and incubated at 25°C for at least 1 hour. Plates were washed. 50 µL/well of ST-TC from 5142.857 ng/mL to 0.029 ng/mL diluted in assay diluent (5% MSD Blocker A) was added to the plate and incubated for 1 hour with 300 rpm at 25°C. Following next wash, 150 µL/well of MSD Gold Read buffer was added and plate was read at 620 nm. A four parameter logistic graph was plotted by using concentration of ST-TC on X-axis and mean signal value on Y-axis. The dose response curves are presented in Figure 2. The coating concentration at which a sigmoid curve for was obtained was fixed for the assay. Accordingly, the coating concentration of 0.750 µg/mL was selected for the assay.
Example 4: Optimization of dose response curve for sulfo-tag conjugated tocilizumab to determine EC-50 concentration
Microplate wells were coated with 50 µL/well of IL-6R diluted to 0.750 µg/mL concentration in 1X PBS and incubated overnight at 2-8°C. After wash program, the plates were blocked with 250 µL/well of 5% MSD Blocker-A and incubated at 25°C for at least 1 hour. After a subsequent wash, 50 µL/well of ST-TC at concentrations ranging from 6000 ng/mL to 0.080 ng/mL diluted in assay diluent (5% MSD Blocker A in PBS) was added to the plate and incubated for 1 hour at 25°C. 150 µL/well of MSD Gold Read buffer was added and plate was read at 620 nm. An eleven point dose response curve was generated using ST-TC in the aforementioned concentration range to select the EC 50 concentration of ST-TC. For this, a four parameter logistic graph was plotted by using concentration of ST-TC on X-axis and mean signal value on Y-axis. The representative dose response curve is presented in Figure 3. The ST-TC concentration of 100 ng/mL was chosen for the assay.
Example 5: Optimization of BT-TC concentration to increase drug tolerance and target tolerance of assay
In normal human volunteers, the expected level of IL-6R is observed to be between about 25.0 and about 75.0 ng/mL. In patients with rheumatoid arthritis, these levels are observed to be elevated compared to healthy individuals. Also, after administration of Tocilizumab, mean serum concentration of IL-6R increases in a dose dependent manner to about 350.0 ng/mL around day 14 when the ADA sampling begins. This can lead to interference in the assay signal due to the target (i.e, IL-6R in the present invention). The interference of IL-6R in the assay also depends on the assay format and hence it is important to test the interference that may arise from the presence of IL-6R in the current assay and plan for mitigation if required. The assay format should be target tolerant and not allow IL-6R induced interference which may give rise to false positives. In addition, it was expected that the present method should be able to tolerate tocilizumab concentration of at least about 10.0 µg/ml which is the reported Cmax of tocilizumab (SC) in normal healthy volunteers, so that minimal or no drug interference is observed in the assay.
To enhance the drug and target tolerance of the assay, affinity capture and elution step was introduced in the present method. BT-TC was added to the sample after dilution. The sample was then transferred to the Streptavidin coated plate for overnight incubation wherein BT-TC bound to NAB then binds to the streptavidin coated on the plate. Anti tocilizumab Neutralizing Antibodies were then eluted by dissociation with acid. Control samples (HPC, LPC and NQC) as well as samples spiked with anti-IL-6R antibody and IL-6R were used for the evaluation. Different concentrations of BT-TC were evaluated. At the BT-TC concentration range between 25.0 µg/mL and 75.0 µg/mL the optimal percentage neutralization at HPC, LPC and spiked samples was obtained. Hence, BT-TC concentration of 50.0 µg/mL was selected for the assay.
Example 6: Estimation of IL-6 tolerance of the assay
Interference in NAB assay may arise from the circulating IL-6 in the serum sample. The circulating IL-6 can bind to the IL-6R coated onto the plate and thus generate false positive results. Hence, it is essential for the method to be tolerant of IL-6. The serum concentration of IL-6 is known to be about 10.0 pg/mL in normal healthy volunteers and with administration of Tocilizumab, the serum concentration of IL-6 is observed to increase to about 30.0 pg/mL. Hence the assay was evaluated for IL-6 tolerance. For this, control samples (HPC, LPC, DPC and NQC) were spiked with different concentrations of IL-6 ( 10.0, 100.0, 500.0 and 1000.0 ng/mL). DPC sample constituted LPC (500.000 ng/mL) + drug (10.000 µg/mL). As reported in Table 3, the method is able to tolerate IL-6 concentration of 500.0 ng/mL.

Table 3: IL-6 tolerance of the assay

Sample Mean signal % CV % neutralization S/N
HPC-1000(1) 1373.500 20 96 0.04
LPC-500(1) 13807.500 5 59 0.41
HPC-1000(2) 278.000 7 99 0.01
LPC-500(2) 15913.000 1 53 0.47
DPC-1 22911.000 2 32 0.68
DPC-2 18847.000 18 44 0.56
NQC-1 7557.500 73 78 0.22
NQC-2 34907.000 2 -4 1.04
NQC-3 32507.500 1 4 0.96
Mean NQC 33707.250
HPC+ IL-6 (1000.000 ng/mL ) 1095.000 2 97 0.03
HPC + IL-6 (500.000 ng/mL ) 1110.000 5 97 0.03
HPC + IL-6 (100.000 ng/mL) 1139.000 0 97 0.03
HPC + IL-6 (10.000 ng/mL) 616.500 7 98 0.02
LPC+ IL-6 (1000.000 ng/mL ) 11416.000 2 66 0.34
LPC + IL-6 (500.000 ng/mL ) 13427.000 3 60 0.40
LPC + IL-6 (100.000 ng/mL) 12082.000 1 64 0.36
LPC + IL-6 (10.000 ng/mL) 12153.500 0 64 0.36
DPC+ IL-6 (1000.000 ng/mL ) 22598.500 1 33 0.67
DPC + IL-6 (500.000 ng/mL ) 23836.500 2 29 0.71
DPC + IL-6 (100.000 ng/mL) 23287.500 4 31 0.69
DPC + IL-6 (10.000 ng/mL) 10921.500 15 68 0.32
NQC+ IL-6 (1000.000 ng/mL ) 27217.500 2 19 0.81
NQC + IL-6 (500.000 ng/mL ) 30653.000 4 9 0.91
NQC + IL-6 (100.000 ng/mL) 33969.500 3 -1 1.01
NQC + IL-6 (10.000 ng/mL) 34627.500 1 -3 1.03

Example 7: Assay procedure

Samples for detecting NAB were diluted in 0.2M Glycine (pH 2.3) and incubated at 25°C for 15-20 minutes. 10µL/ well of BT-TC diluted to a concentration of 50.0 µg/mL was added to all the samples and further incubated at 25°C for about 5 minutes. About 85 µL of the sample in 10 µL of neutralizing buffer (1M Tris-HCl, pH 9.5) was added to streptavidin-coated plate and incubated at room temperature, overnight. Following this, plates were washed as per wash program and samples were further subjected to acid dissociation by adding 85 µL of (0.2M Glycine, pH 2.3) and incubated at 25°C for about 15 minutes. The acid dissociated sample was neutralized by incubation of 50 µL of the sample with 70µL (100ng/ml) ST-TC and 20µL of 1M Tris-HCl at 25°C.
Separately, microplate wells were coated with IL-6R by adding 0.75µg/mL IL-6R solution to the plate and incubated at 2-8°C overnight. Plate was washed and blocked with 150 µL/well of blocking reagent (5% MSD Blocker A) and incubated at 25°C for 1 hour. 25 µL/well of the aforementioned neutralized sample was added to the blocked MSD Plate and incubated at 25°C, 1 hour in dark. After washing, 150 µL/well of MSD gold read buffer was added to the plate and the plate was read at 620nm.
(All wash programs unless otherwise specified comprise washing with 250 µL /well of 0.05% PBST.)
Example 8: Evaluation of assay sensitivity with monoclonal Anti-Tocilizumab Antibody as positive control and polyclonal Anti-Tocilizumab antibody as positive control
Commercially available Monoclonal and polyclonal anti-TC Neutralizing antibody was used as positive control for demonstrating the assay sensitivity. Three 12 point dose response curves starting from 50000.0 ng/mL to 24.414 ng/mL with a two-fold serial dilution at each step were generated for establishing the assay sensitivity for both monoclonal and polyclonal antibody respectively. As shown in Table 4, the assay sensitivity is 195.313 ng/mL for monoclonal anti-tocilizumab antibody and 390.625 ng/mL with polyclonal anti-tocilizumab antibody.

Table4: Sensitivity of the method
Monoclonal Anti-Tocilizumab Antibody
Sensitivity curve-1 Sensitivity curve-2 Sensitivity curve-3
Sample conc (ng/mL). Mean signal % neutralization S/N ratio Mean % neutralization S/N ratio Mean signal % neutralization S/N ratio
50000.000 76.000 99 0.007 85.500 99 0.006 81.000 99 0.006
25000.000 75.500 99 0.007 90.000 99 0.006 79.000 99 0.006
12500.000 71.000 99 0.006 85.500 99 0.006 79.000 99 0.006
6250.000 72.500 99 0.006 98.000 99 0.007 82.000 99 0.006
3125.000 82.500 99 0.007 116.000 99 0.008 92.500 99 0.007
1562.500 126.500 99 0.011 253.500 98 0.018 136.000 99 0.011
781.250 1692.500 85 0.150 4793.000 66 0.339 2756.000 78 0.218
390.625 6445.000 43 0.572 9560.000 32 0.677 7313.500 42 0.579
195.313 8677.500 23 0.770 11839.000 16 0.838 9680.000 23 0.766
97.656 10696.000 5 0.950 13453.500 5 0.952 10928.500 13 0.865
48.828 11588.500 -3 1.029 14431.000 -2 1.022 11781.500 7 0.933
24.414 11973.000 -6 1.063 13840.000 2 0.980 11540.500 9 0.914
Mean NQC 11263.000 14126.000 12630.000
Polyclonal Anti-Tocilizumab Antibody
Sensitivity curve-1 Sensitivity curve-2 Sensitivity curve-3
Sample conc (ng/ml). Mean signal % neutralization S/N ratio Mean % neutralization S/N ratio Mean signal % neutralization S/N ratio
50000.000 82.000 99 0.007 86.000 99 0.006 84.000 99 0.007
25000.000 84.000 99 0.007 85.000 99 0.006 87.500 99 0.007
12500.000 78.500 99 0.007 96.500 99 0.007 98.000 99 0.008
6250.000 96.500 99 0.009 214.000 98 0.015 209.000 98 0.017
3125.000 213.500 98 0.019 2109.000 85 0.149 1289.000 90 0.102
1562.500 2162.000 81 0.192 7323.500 48 0.518 6360.000 50 0.504
781.250 6671.500 41 0.592 11343.000 20 0.803 9751.000 23 0.772
390.625 8551.500 24 0.759 12783.500 10 0.905 10951.000 13 0.867
195.313 9852.500 13 0.875 13670.500 3 0.968 11955.000 5 0.947
97.656 11005.000 2 0.977 14387.000 -2 1.018 12887.500 -2 1.020
48.828 11462.000 -2 1.018 14402.500 -2 1.020 12377.000 2 0.980
24.414 11796.500 -5 1.047 14788.000 -5 1.047 13149.500 -4 1.041
Mean NQC 11263.000 14126.000 12630.000

Example 9: Evaluation of assay selectivity
Commercially available Monoclonal anti-TC Neutralizing antibody was used for evaluation of assay selectivity. Five normal human serum samples were spiked with NAB at HPC, LPC and DPC (500 ng/mL + drug 10.0 µg/mL) levels. DPC samples were prepared with both DRL-Tocilizumab and Actemra® (Reference product). Un-spiked individual samples were also evaluated. A pool of five individual samples was also prepared and evaluated at all levels. As shown in Table 5, the method is selective.
Table 5: Selectivity of the method
Sample Mean signal % CV % neutralization S/N ratio
Individual sample-1 (S1)
S1-NC 12085.000 1 0 0.998
S1-HPC 796.500 2 93 0.066
S1-LPC 5647.500 3 53 0.466
S1-DPC-DRL 9990.000 5 18 0.825
S1-DPC-Actemra 10184.000 3 16 0.841
Individual sample-2 (S2)
S2-NC 12665.000 1 -5 1.046
S2-HPC 888.000 0 93 0.073
S2-LPC 7545.500 1 38 0.623
S2-DPC-DRL 10134.500 2 16 0.837
S2-DPC-Actemra 10621.000 3 12 0.877
Individual sample-3 (S3)
S3-NC 11946.500 1 1 0.986
S3-HPC 590.000 3 95 0.049
S3-LPC 5324.000 3 56 0.440
S3-DPC-DRL 9890.500 1 18 0.816
S3-DPC-Actemra 9078.000 2 25 0.749
Individual sample-4 (S4)
S4-NC 11710.500 6 3 0.967
S4-HPC 975.500 6 92 0.081
S4-LPC 6191.000 4 49 0.511
S4-DPC-DRL 9746.000 2 20 0.805
S4-DPC-Actemra 10370.000 4 14 0.856
Individual sample-5 (S5)
S5-NC 12987.000 1 -7 1.072
S5-HPC 1160.000 1 90 0.096
S5-LPC 6407.000 2 47 0.529
S5-DPC-DRL 10811.500 2 11 0.893
S5-DPC-Actemra 10943.000 2 10 0.903
Pooled Individual sample-1-5 (pool)
pool-NC 13068.000 3 -8 1.079
pool-HPC 736.000 3 94 0.061
pool-LPC 6106.000 1 50 0.504
pool-DPC-DRL 10486.000 1 13 0.866
pool-DPC-Actemra 10425.500 2 14 0.861
,CLAIMS:We claim:
1. A method for detecting and quantifying neutralizing anti-drug antibodies (NAB) against an anti-IL6R antibody in a sample, wherein the method comprises:
a) incubating the sample with an acidic buffer to dissociate immune complexes present therein;
b) contacting the treated sample of step a) with a biotin-conjugated anti-IL6R antibody and a streptavidin-coated substrate to allow affinity-based complex formation of components present therein;
c) eluting NAB from the complex formed at step b) with a buffer at low pH;
d) incubating the dissociated NAB of step c) in a solution containing anti-IL6R antibodies that are conjugated to a detectable label;
e) contacting the solution of step d) with IL-6R immobilized to a substrate;
f) detecting the intensity of the label on the substrate of step e) by colorimetry, luminometry or fluorimetry;
wherein the method can detect as low as 195.313 ng/mL NAB; and
wherein the method can tolerate as high as 500 ng/mL IL-6, 10 µg/mL anti-IL6R antibody and 350 ng/mL IL6R.
2. The method as claimed in claim 1 wherein, pH of the acidic buffer is about 2.
3. The method as claimed in claim 1 wherein, the neutralizing anti-drug antibodies (NAB) are generated against monoclonal and/or polyclonal anti-IL6R antibody.
4. The method as claimed in claim 1 wherein, the monoclonal anti-IL6R antibody is tocilizumab.