DESC:INTRODUCTION
The present invention relates to a method for measuring anti-drug antibodies in a biological sample, generated against a biologic drug with minimal interference from the soluble drug.
BACKGROUND
One of the main factors of assessment during development of large molecule drugs is that of assessment of immunogenic response elicited by the drug in the body. Such assessment and monitoring are important components of pre-clinical and clinical studies to understand drug exposure, safety and efficacy. Factors that influence drug immunogenicity are molecule size, structure, purity, formulation composition, dosage etc. in addition to health status of the patient and possible drug co-administration.
Immunogenicity response is characterized by elicitation of anti-drug antibodies (ADA) in the body which can result in responses ranging from no adverse events to deleterious events in the subject of administration. Due to the heterogeneity in biological response to a given drug, there can be differences in the titer, type and affinities of ADAs generated. Also, neutralizing anti-drug antibodies (NAB) are a subset of ADAs which, by binding to the protein drug negate the binding ability of the latter to its target, thereby impacting drug efficacy. In addition to this, elicited ADAs could also cross-bind and neutralize activity of one or more other endogenous protein(s). Therefore, it is essential to assess and monitor immunogenicity during drug development process.
Assays for the detection and quantification of ADAs 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 such as the drug itself, or its soluble target, which can result in a false read-out. Also, in the case of monoclonal antibody drugs, assessment of ADAs elicited becomes challenging due to structural similarity between drug and the analyte (ADA); as well as high drug concentration in the sample. Thus, interference ultimately leads to either underestimation of the levels of ADA present in the sample or detection of false positives, affecting the performance and sensitivity of the assay. Hence high emphasis is required on understanding and mitigating such interferences while optimizing a given immunogenicity testing format.
Nivolumab is a targeted biologic drug that acts as immune checkpoint inhibitor by binding to PD-1, thereby preventing interaction with its ligand PDL1/PDL2. (Physiological binding of PD-1 with PDL1/PDL2 otherwise results in suppression of antigen-stimulated lymphocyte proliferation, migration and cytokine production and attenuation of effector T cell response, resulting in immunological tolerance.) Nivolumab has received regulatory approval for several indications including melanoma, non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), classical Hodgkin’s lymphoma (cHL) and urothelial carcinoma, among others.
The objective of present invention is to develop a specific immunogenicity assay to measure anti-drug antibodies elicited against anti-PD-1 antibody (drug), with minimal interference from free drug present in the sample.
SUMMARY OF THE INVENTION
Accordingly, present invention discloses a simple and robust method for measuring antibodies (“ADA”) elicited against anti-PD-1 antibody (“drug”) in a sample by affinity capture elution of the ADA, wherein the sample has or is suspected to have interference from free drug in the sample. The method employs an assay format that mitigates interference from free drug in the sample substantially. Drug tolerance of the disclosed method to detect as low as 100 ng/mL of ADA was found to be as high as 500 µg/mL of nivolumab in the sample.
DETAILED DESCRIPTION OF THE INVENTION
Present invention discloses a method for measuring anti-drug antibodies (“ADA”) elicited against anti-PD-1 antibody or antigen binding fragment thereof (“drug”) in a sample wherein the sample has or is suspected to have interference from free anti-PD-1 antibody or antigen binding fragment thereof.
Drug tolerance of the disclosed method to detect as low as 100 ng/mL of ADA was found to be as high as 500 µg/mL of anti-PD-1 antibody or antigen binding fragment thereof in the sample.
In an embodiment, the present invention discloses a method for measuring anti-drug antibodies (“ADA”) in a sample elicited against anti-PD-1 antibody or antigen binding fragment thereof (“drug”) wherein the method comprises:
a) incubating the sample with an acidic buffer containing glycine to dissociate immune complexes;
b) capturing ADAs in the acid-dissociated sample of step a) on to a plate containing an alkaline Tris-buffer coated with the drug;
c) incubating the plate with an acidic buffer to dissociate ADA captured on the plate in step b);
d) eluting the ADA from the plate with the acidic buffer;
e) incubating the eluted ADA onto a second plate containing the alkaline Tris-buffer so as to coat the ADA onto the second plate;
f) incubating the coated ADAs of step e) with labelled drug; and
g) detecting the labelled drug using standard protocols of enzyme linked immunosorbent assay;
wherein the method can detect as low as 100 ng/mL of ADA in the sample in the presence of about 500 µg/mL to about 2000 µg/mL of drug in the sample.

In another embodiment, the labelled drug is detected by means of electrochemiluminescence, luminescence or fluorescence.
In an embodiment, the anti-PD-1 antibody is nivolumab, pembrolizumab, dostarlimab or cemiplimab.
The invention describes a method for measuring anti-drug antibodies generated against anti-PD-1 antibody or antigen binding fragment (“drug”) thereof in a sample wherein factors comprising sample dilution, pH of buffer, concentration of assay reagents and wash program are selected so as to reduce non-specific interferences from the sample, leading to enhanced sensitivity and substantially high drug tolerance.
As can be obvious to a person skilled in the art, the labelled drug of step f) can be any of the commercially available appropriate label 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 anti-PD1 antibody or antigen-binding fragment thereof.
The term ‘sample’ or matrix’ as used herein the invention refers to blood sample or serum sample obtained from a blood repository.
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.
Abbreviations:
PD-1 - Programmed Cell Death Protein 1
PD-L1 - Programmed Death ligand 1
MRD – Minimum Required Dilution
POD – Peroxidase
TMB – 3,3’,5,5’-tetramethylbenzidine
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-PD-1 antibody or antigen binding fragment (“drug”) in a sample, various parameters were optimized to achieve acceptable sensitivity and interference mitigation. Examples of present invention describe methodology relating to anti-PD-1 antibody or antigen binding fragment (“drug”) in a sample, including but not limited to nivolumab.
Example 1: Assessment of anti-nivolumab antibodies
An Affinity, Capture and Elution (ACE) based immunoassay was developed for the detection of anti-Nivolumab antibodies in a sample. For this, the sample was first subject to acid dissociation step by treatment with Glycine-HCl (pH~2.3) which results in dissociation of the drug and anti-drug antibodies complex. Acid dissociated ADAs are then captured on to a plate coated with Nivolumab. The captured ADAs are further eluted using Glycine-HCl buffer and coated onto a fresh plate. After this, primary antibody (Digoxigenylated Nivolumab) and secondary antibody (anti-digoxigenin POD) is added. Following this, TMB substrate is added which results in the development of color that is measured at 450 nm. At assay MRD of 1:40, the preliminary assay sensitivity is 1.221 ng/mL of ADA in the absence of free drug and 100 ng/mL of ADA in presence of up to 500 µg/mL of free drug. Similar steps were followed for detection of anti-Pembrolizumab antibodies in a sample. Except in this procedure eluted ADAs were detected by two different types of mechanism, one of which was digoxigenylated Pembrolizumab with anti-digoxigenin POD, and another one was biotinylated Pembrolizumab with streptavidin-Poly HRP. At assay MRD of 1:50 and with digoxigenylated-Pembrolizumab based detection mechanism, the screening assay sensitivity is 3.906 ng/mL of ADA in the absence of free drug and 100 ng/mL of ADA in presence of up to 2000 µg/mL of free drug.
Example 2: Drug tolerance
Free drug present in the sample could interfere with ADA detection by competing with the capture and detection reagents for binding with ADA, thereby impacting assay sensitivity. Drug tolerance is defined as the maximal amount of free drug present in the sample that can still result in a detectable ADA signal. For this, different combinations of normal pooled plasma (NPP) samples were spiking with different concentrations of nivolumab (100 µg/mL to 500 µg/mL) and commercially available anti-nivolumab antibodies (100 ng/mL to 1000 ng/mL) (Biorad, catalog no. HCA 300). The said concentrations are indicated in Table 1.
SAMPLE
(Anti-NU antibody+NU) Sample ID Mean OD %CV S/N
1000 ng/mL+500 µg/mL drug C01 0.936 4 20
1000 ng/mL+250 µg/mL drug C02 1.265 0 27
1000 ng/mL+100 µg/mL drug C03 1.792 2 39
1000 ng/mL+ No drug C04 2.710 2 59
500 ng/mL+500 µg/mL drug C05 0.493 3 11
500 ng/mL+250 µg/mL drug C06 0.679 3 15
500 ng/mL+100 µg/mL drug C07 1.037 7 23
500 ng/mL+ No drug C08 1.489 3 32
250 ng/mL+500 µg/mL drug C09 0.258 4 6
250 ng/mL+250 µg/mL drug C10 0.361 4 8
250 ng/mL+100 µg/mL drug C11 0.543 4 12
250 ng/mL+ No drug C12 0.872 1 19
100 ng/mL+500 µg/mL drug C13 0.136 0 3
100 ng/mL+250 µg/mL drug C14 0.166 5 4
100 ng/mL+100 µg/mL drug C15 0.212 3 5
100 ng/mL+ No drug C16 0.422 1 9
500 µg/mL drug C17 0.050 17 1
250 µg/mL drug C18 0.045 1 1
100 µg/mL drug C19 0.051 17 1
*NU - Nivolumab
Table 1
According to the acceptance criteria, if mean optical density (OD) of sample is greater than or equal to the mean OD at PSCP (plate specific cut point), the assay tolerance at that drug level is said to be achieved. PSCP is calculated by multiplying the average OD value of negative quality control (NQC) with screening normalization factor (0.046*1.02 = 0.047). Negative quality control (NQC) refers to unspiked NPP.
It was found that all the samples with test ADA concentrations ranging from 100 ng/mL to 1000 ng/mL gave OD value greater than PSCP at nivolumab concentration as high as 500 µg/mL. It was concluded that the assay can tolerate up to 500 µg/mL drug at ADA concentration as low as 100 ng/mL.
Example 3: Drug tolerance of other anti-PD1 antibody
Commercially available anti-Pembrolizumab positive control (Biorad; Cat# HCA298) was treated with different drug concentration ranging from 10µg/mL to 2000µg/mL and was tested at HPC, MPC and LPC level. The said concentrations are indicated in Table 2. The drug controls were kept without any ADA spiked for each concentration. Assay tolerance to the drug is defined as the highest drug concentration at which the signal produced by the positive control exceeds the screening cut-point. The screening cut-point was calculated by multiplying the mean NC of the plate with screening cut-point factor 1.295.
For positive control HCA298, the calculated screening cut-point was 0.094. The positive control has shown response, higher than the plate specific SCP across all the drug concentrations at HPC, MPC, LPC level. It was concluded that the assay can tolerate up to 2000µg/mL drug at 100ng/mL of anti-drug antibody concentration.
SAMPLE
(Anti-PB antibody+PB) Sample ID Mean OD %CV S/N
1000 ng/mL+2000 µg/mL drug HPC-D-2000 0.543 1 7
1000 ng/mL+1500 µg/mL drug HPC-D-1500 0.431 0 6
1000 ng/mL+1000 µg/mL drug HPC-D-1000 0.610 1 8
1000 ng/mL+500 µg/mL drug HPC-D-500 0.953 1 13
1000 ng/mL+250 µg/mL drug HPC-D-250 1.199 1 16
1000 ng/mL+100 µg/mL drug HPC-D-100 1.866 0 25
1000 ng/mL+50 µg/mL drug HPC-D-50 1.863 0 25
1000 ng/mL+10 µg/mL drug HPC-D-10 2.442 2 33
1000 ng/mL+ No drug HPC-D-0 2.656 5 35
500 ng/mL+2000 µg/mL drug MPC-D-2000 0.408 1 5
500 ng/mL+1500 µg/mL drug MPC-D-1500 0.268 4 4
500 ng/mL+1000 µg/mL drug MPC-D-1000 0.467 2 6
500 ng/mL+500 µg/mL drug MPC-D-500 0.715 0 10
500 ng/mL+250 µg/mL drug MPC-D-250 0.674 2 9
500 ng/mL+100 µg/mL drug MPC-D-100 0.967 1 13
500 ng/mL+50 µg/mL drug MPC-D-50 1.233 4 16
500 ng/mL+10 µg/mL drug MPC-D-10 1.545 2 21
500 ng/mL+ No drug MPC-D-0 1.616 1 22
100 ng/mL+2000 µg/mL drug LPC-D-2000 0.101 1 1
100 ng/mL+1500 µg/mL drug LPC-D-1500 0.119 0 2
100 ng/mL+1000 µg/mL drug LPC-D-1000 0.129 1 2
100 ng/mL+500 µg/mL drug LPC-D-500 0.184 1 2
100 ng/mL+250 µg/mL drug LPC-D-250 0.202 2 3
100 ng/mL+100 µg/mL drug LPC-D-100 0.309 4 4
100 ng/mL+50 µg/mL drug LPC-D-50 0.344 1 5
100 ng/mL+10 µg/mL drug LPC-D-10 0.395 2 5
100 ng/mL+ No drug LPC-D-0 0.465 6 6
*PB - Pembrolizumab
Table 2
Example 4: Sensitivity of method with Anti-Nivolumab antibody
Sensitivity can be defined as the lowest ADA concentration at which a signal greater than or equal to the cut point of the assay is obtained. In other words, sensitivity of the assay should be sufficient to be able to detect low levels of ADA.
For this, commercially available monoclonal Anti-Nivolumab antibody (Bio-Rad; Catalogue number: HCA 300; Conc.:500 µg/mL) was used as positive control. Normal pooled plasma was spiked with the Anti-Nivolumab antibody with concentrations ranging from 0.076 ng/mL to 2500 ng/mL in duplicate (Plate 1 & Plate 2) as depicted in Table 2. A dose response curve was plotted at said concentration range. Sensitivity of the assay was found to be 1.221 ng/mL.
Screening assay sensitivity samples
Plate-1 Plate-2
Sample Concentration ng/mL Mean Value CV% S/N Mean Value CV% S/N
S01 2500.000 3.286 1 68 3.307 2 66
S02 1250.000 2.972 0 62 2.915 5 58
S03 625.000 1.929 3 40 1.776 8 36
S04 312.500 1.086 5 23 1.041 10 21
S05 156.250 0.544 2 11 0.576 2 12
S06 78.125 0.307 1 6 0.306 1 6
S07 39.063 0.171 1 4 0.184 2 4
S08 19.531 0.113 0 2 0.114 5 2
S09 9.766 0.078 1 2 0.078 6 2
S10 4.883 0.063 1 1 0.062 6 1
S11 2.441 0.054 1 1 0.054 1 1
S12 1.221 0.053 3 1 0.052 5 1
S13 0.610 0.047 6 1 0.049 1 1
S14 0.305 0.048 3 1 0.048 3 1
S15 0.152 0.047 1 1 0.046 1 1
S16 0.076 0.046 1 1 0.045 0 1
Table 3
Example 5: Sensitivity of method with other anti-PD1 antibody
Sensitivity was assessed by testing serial dilutions of a positive control antibody (Biorad; Cat# HCA298) of known concentration ranging from 2000ng/mL to 0.061ng/mL, using pooled matrix from treatment-naïve subjects. The dilution series was twofold, and total of sixteen dilutions were tested to evaluate the sensitivity of both screening and confirmatory assay. Sensitivity with PC HCA298 was evaluated by two different analysts in two separate runs.
With PC HCA298, assay sensitivity estimated from two runs for screening assay is 3.906ng/mL of ADA and for confirmatory assay is 0.977ng/mL of ADA.
Screening assay sensitivity samples
Plate-1 Plate-2
Sample Concentration ng/mL Mean Value CV% S/N Mean Value CV% S/N
S01 2000.000 3.338 2 68 3.212 0 35
S02 1000.000 3.242 0 62 3.062 1 33
S03 500.000 2.767 2 40 2.444 0 27
S04 250.000 1.822 2 23 1.607 4 18
S05 125.000 0.978 2 11 0.827 0 9
S06 62.500 0.541 2 6 0.528 3 6
S07 31.250 0.345 6 4 0.298 10 3
S08 15.625 0.205 9 2 0.213 4 2
S09 7.813 0.141 3 2 0.148 3 2
S10 3.906 0.108 1 1 0.118 3 1
S11 1.953 0.049 3 1 0.108 8 1
S12 0.977 0.110 3 1 0.094 1 1
S13 0.488 0.081 3 1 0.089 0 1
S14 0.244 0.076 1 1 0.087 2 1
S15 0.122 0.075 3 1 0.081 1 1
S16 0.061 0.073 0 1 0.084 0 1
Table 4
Example 6: Selectivity of method
Selectivity is the ability of the assay to detect the ADA specific to the drug of interest in the presence of other potentially interfering substances in the matrix. The positive and negative controls are prepared in the matrix and inhibition of signal in presence of the drug without any interference from matrix components would indicate a selective response.
5 sample replicates (S1 to S5) were used for evaluating selectivity of method. Drug at 1000 ng/mL (High positive control; HPC) and 100 ng/mL (Low positive control; LPC) levels were spiked in the individual test matrices (S1 to S5), and were run along with unspiked controls. The sampling and resultant OD is depicted in Table 3. HPC and LPC of all 5 individual test matrices showed OD values above the PSCP and % inhibition more than CCP. Hence the method is highly sensitive.
Matrix ID OD Without Drug OD With Drug % Inhibition Outcome Sample types
S1 0.055 0.056 -1.82 Positive Negative control
S2 0.049 0.049 0.00 Negative
S3 0.052 0.050 3.85 Negative
S4 0.052 0.051 1.92 Negative
S5 0.051 0.051 0.00 Negative
S1 2.489 0.078 96.87 Positive HPC (1000 ng/mL)
S2 2.323 0.068 97.07 Positive
S3 2.498 0.074 97.04 Positive
S4 2.431 0.081 96.67 Positive
S5 2.516 0.073 97.10 Positive
S1 0.350 0.057 83.71 Positive LPC (100 ng/mL)
S2 0.348 0.052 85.06 Positive
S3 0.360 0.053 85.28 Positive
S4 0.367 0.058 84.20 Positive
S5 0.368 0.052 85.87 Positive
Table 5
Example 7: Prozone effect
Prozone or hook effect is a false-negative signal that can be obtained at very high concentrations of the analyte (ADA). Due to an excess amount of ADA in the sample, availability of capture and detection reagents may be insufficient to bind all of the ADA, and the assay hence can give an erroneous low response or signal.
To determine impact of prozone effect in the method disclosed, test matrices with 16 different dilutions of ADA (1.221 ng/mL to 40,000 ng/mL) were run in duplicates (Run 1 & Run 2) using method described in Example 1. The sample concentrations and results are depicted in Table 4 & Figure 1. It was found that the signal-to-noise (S/N) ratios and OD values are observed to be saturated starting from 2500 ng/mL up to the highest concentration evaluated of 40,000 ng/mL and no %CV failures were observed. Thus, it is evident that no prozone or hook effect was observed up to 40,000 ng/mL of ADA concentration.
Samples Concentration (ng/mL) Run-1 Run-2 % CV between S/N of Run-1 and Run-2
Mean-OD Value S/N Replicate %CV Mean-OD Value S/N Replicate %CV
S1 40000.000 3.331 65 1 3.277 71 1 6
S2 20000.000 3.350 66 1 3.337 73 2 7
S3 10000.000 3.345 66 1 3.335 72 1 7
S4 5000.000 3.499 69 1 3.412 74 1 6
S5 2500.000 3.364 66 1 3.233 70 1 4
S6 1250.000 2.886 57 4 2.491 54 4 3
S7 625.000 1.818 36 7 1.430 31 6 10
S8 312.500 0.951 19 2 0.822 18 1 3
S9 156.250 0.532 10 10 0.436 9 5 7
S10 78.125 0.294 6 11 0.223 5 2 12
S11 39.063 0.163 3 2 0.136 3 1 6
S12 19.531 0.099 2 2 0.090 2 1 0
S13 9.766 0.070 1 0 0.067 1 4 4
S14 4.883 0.056 1 2 0.052 1 3 3
S15 2.441 0.051 1 3 0.048 1 3 3
S16 1.221 0.048 1 5 0.047 1 5 5
Table 6
,CLAIMS:CLAIMS
We claim:
1. A method for measuring anti-drug antibodies (“ADA”) elicited against anti-PD-1 antibody or antigen binding fragment thereof (“drug”) in a sample wherein the method comprises:
a) dissociating the drug and anti-drug antibodies complex in the sample by incubating the sample with an acidic buffer containing glycine;
b) capturing ADAs in the acid-dissociated sample of step a) on to a plate containing an alkaline Tris-buffer coated with the drug;
c) incubating the plate with an acidic buffer to dissociate ADA captured on the plate in step b);
d) eluting the ADA from the plate with the acidic buffer;
e) incubating the eluted ADA onto a second plate containing the alkaline Tris-buffer so as to coat the ADA onto the second plate;
f) incubating the coated ADAs of step e) with labelled drug; and
g) detecting the labelled drug using enzyme linked immunosorbent assay;

2. The method as claimed in claim 1 wherein, the anti-PD-1 antibody is nivolumab, pembrolizumab, dostarlimab or cemiplimab.

3. The method as claimed in claim 1 wherein, the sensitivity of the method is up to 3.906ng/ml of ADA

4. The method as claimed in claim 1 wherein the said method can detect as low as 100 ng/mL of ADA in the sample in the presence of about 500 µg/mL to about 2000 µg/mL of drug in the sample.