METHOD OF DETECTING NEUTRALIZING ANTI-DRUG ANTIBODIES AGAINST BIO-THERAPEUTICS
INTRODUCTION
The present invention relates to a method for detecting neutralizing anti-drug antibodies in a biological sample, generated against biological therapeutics with minimal interference.
BACKGROUND
Monoclonal antibodies are an increasingly promising class of biological therapeutics for the targeted therapy of several diseases including cancer. Administration of these drugs is, however, often associated with immunological responses in the recipient with possibly adverse consequences. One such response is that of the generation of anti-drug antibodies, which could have neutralizing or non-neutralizing effects on the action of the drug.
Neutralizing antibodies (hereinafter referred to as "NAB") bind to the administered therapeutic antibody thereby neutralizing the activity of the antibody. This obstructs the normal function of the administered antibody, thereby impacting its bioavailability and efficacy. Therefore, screening and detection of NAB is important and mandated by regulatory agencies when assessing safety and immunogenicity of a therapeutic drug.
NAB detection could be cell-based or non-cell-based. Competitive non-cell based assays, which are relatively easier to perform, while being sensitive and reproducible, commonly utilize the NAB target itself for capture and detection. The said detection is however hindered by the presence of interference factors in the. Interference ultimately results either in underestimation of the levels of NAB or detection of false positives, thereby affecting the performance and sensitivity of the assay. Hence high emphasis is

required on mitigating such interferences while optimizing a given immunogenicity testing format.
HER2 is a protein overexpressed in approximately 20-30% of early stage breast cancers. HER2 targeting therapeutics that bind to the extracellular domain of HER2 (for e.g., trastuzumab, pertuzumab and trastuzumab emtansine) help control the proliferative nature in these types of cancers to a great extent. Administration of HER2 targeting therapeutics may generate anti-drug antibodies (that includes neutralizing antibodies), the detection of which is hindered by the presence of interference factors. Potential interference factors include: shed HER2-ECD, the circulating drug itself and other more non-specific interference. Shed HER2-ECD, which is the proteolytically cleaved extracellular domain of HER2/neu, is found at varying levels in the serum of HER2 overexpressing breast cancer patients. During immunogenicity testing, shed HER2-ECD could bind to the capture drug and give rise to false positive results. In addition, the circulating drug itself can bind to circulating NAB to form immune complexes, giving rise to false negative results. It is therefore essential to mitigate the effects of these interference factors while developing a suitable immunogenicity testing assay.
Hence the primary objective of the invention is to develop an immunogenicity assay format that can overcome interference to an acceptable extent during detection and characterization of neutralizing anti-drug antibodies in a biological sample.
SUMMARY OF THE INVENTION
The present invention discloses a method for detecting of neutralizing anti-drug antibodies generated against HER2 targeting drugs, wherein the method mitigates interference due to shed HER2-ECD and free drug, substantially. The said method comprises a pre-treatment step of the biological samples, which involves sequestration of shed HER2-ECD using anti-HER2-antibody, followed by separation of the said sequestered complex by magnetic separation. The pre-treatment step further comprises acid dissociation of the samples. The resultant acid dissociated sample is subject to

detection of neutralizing anti-drug antibodies. By the method described herein, interference is mitigated substantially.
Specifically, the results show that said method allows detection of neutralizing anti-trastuzumab antibodies in the presence of free drug as high as 100 ug/ml in the serum sample, pointing to high drug tolerance of the assay.
BRIEF DESCRIPTION OF THE DRAWING
Figure 1: Comparison of assay response in the presence and absence of target depletion by anti-HER2 antibody in the method
DETAILED DESCRIPTION
The present invention discloses a method for detecting neutralizing anti-drug antibodies (NAB) generated against HER2 targeting therapeutics with minimal interference.
Detection of NAB in biological samples obtained from HER2 overexpressing patients is challenging due to the presence of interference factors. A significant source of interference is the proteolytically cleaved extracellular domain of HER2 (shed HER2-ECD) which is found at levels as high as approximately 2ug/ml in metastatic breast cancer (MBC) patients. During NAB testing, as the HER2 targeting drug itself is used for detection, shed HER2-ECD circulating in the serum/plasma can also compete with NAB for binding with drug, giving rise to false output.
Further, the circulating HER2 targeting drug itself is present at varying levels in the serum/plasma of the drug administered patients. This can bind with the free NAB, forming circulating immune complexes in the serum/plasma. These complexes mask the NAB from detection, thereby generating a false negative output.
Therefore it is essential to mitigate the above mentioned interferences (in addition to non-specific interference from the serum) prior to detection of NAB.

The present invention discloses a method wherein optimal matrix dilution, target depletion and acid dissociation steps are employed in combination in a pre-treatment step to achieve a read-out with minimal interference.
Specifically, with respect to anti-trastuzumab neutralizing antibodies, the assay sensitivity is as high as 540.5 ng/ml. Further, the assay shows free drug tolerance up to 200ug/mL of drug in the presence of NAB as high as 1000 ng/mL and free drug tolerance up to lOOug/mL of drug with NAB at 500ng/mL. Also, the target tolerance is 1000 ng/ml of shed HER2-ECD. The method detects NAB at high concentrations of shed HER2-ECD with minimal false read-out from interference factors, demonstrating its utility in highly progressed diseased conditions also.
Various embodiments are disclosed herein as methods that describe the invention in detail.
In an embodiment, the claimed invention discloses a method for detecting neutralizing anti-drug antibodies (NAB) in a biological sample generated against a bio-therapeutic, comprising a pre-treatment step that comprises:
a) depleting soluble target by ligand based capture and separation; and
b) incubating the sample in acidic buffer;
wherein, the said method mitigates drug and soluble target interference substantially.
In another embodiment, the said soluble target is shed HER2-ECD.
In yet another embodiment, the drug is trastuzumab, pertuzumab or trastuzumab emtansine.
In yet another preferred embodiment, the depletion of soluble target is achieved by ligand-based capture and magnetic separation.
In an embodiment the acidic buffer contains glycine-HCI.

In another embodiment, the method of detecting NAB utilizes cell based or non-cell based assays.
In a further embodiment, the non-cell based assay for detecting NAB utilizes competitive ligand binding assay.
In another embodiment, the said competitive ligand binding assay involves uses a labelled drug.
In yet another embodiment, the said label is biotin.
The resultant sample containing free and/or bound biotinylated drug is added to plates with the immobilized target, subjecting the sample to competitive ligand binding. After washing, bound drug is detected by enzyme-substrate colorimetric reaction wherein Streptavidin coupled to horse radish peroxidase (HRP) and 3,3\5,5'-Tetramethylbenzidine (TMB) as a substrate to HRP. However, the bound drug shall be detected using an appropriate substrate for performing enzyme-linked immunosorbent assay.
Specifically, the method disclosed detects neutralizing anti-trastuzumab antibody as high as 540.5 ng/ml.
Definitions
The term 'Biological sample' as used herein refers to blood, plasma or serum sample
The term "shed HER2-ECD" refers to the soluble proteolytically cleaved extracellular domain of the transmembrane-spanning protein HER2/neu that is overexpressed in HER2-positive cancers.
The term "HER2 targeting therapeutic" refers to a drug that targets the extracellular domain of HER2/neu for mediating its therapeutic activity.

The term used here in "Neutralizing antibody" refers to an antibody that is raised/formed/produced against the drug in animals including humans that bind to the drug and neutralize its activity.
"Positive control (PC)" as used here in the invention, is a sample (serum obtained from healthy volunteers) spiked with anti-trastuzumab antibodies to mimic the physiological condition of trastuzumab treated patient's sample.
"Negative quality control (NQC)" is an unspiked sample (pooled serum/plasma obtained from healthy volunteers) in which no anti-trastuzumab antibodies have been added.
"Sensitivity" of the assay is defined as the lowest concentration of positive control antibody preparation which consistently provides positive signal in the assay.
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
During the development of the immunogenicity assay for the detection of anti-drug antibodies (ADA), specifically neutralizing antibodies (NAB), various parameters were optimized. One such parameter was that of mitigation of serum induced interference of the samples. The resultant samples were subject to detection of NAB.
The Examples described below detail the method for performing the said immunogenicity testing taking the specific example of detecting anti-trastuzumab antibodies as the NAB.

Example 1: Sample preparation
Pooled serum samples from drug na'i've (not treated with trastuzumab) were spiked with varying concentrations of human anti-trastuzumab antibodies (Product code: HCA177, AbD Serotec®) for use as Positive Control (PC). Unspiked pooled serum of healthy individuals was treated as Negative Quality Control (NQC) in the study. In addition, PC samples spiked with trastuzumab were also used to mimic the physiological conditions of trastuzumab treated (drug administered) patients and to determine the drug tolerance of the assay (Dissociation positive control, DPC).
To study the shed HER2-ECD induced interference in the assay, recombinant human HER2-ECD levels were spiked in the samples to not more than 1000 ng/ml.
Wherever applicable, immune complexes were allowed to form by incubating the respective samples at 37°C for 1 hour with end to end mixing. For acid dissociation, samples were diltuted with acidic buffer and incubated at 25°C, 500 rpm for 15-30 mins.
Example 2: Sample pre-treatment for the removal of interference
Serum sample was incubated with streptavidin dyna beads and biotinylated anti-HER2 antibody at 37°C for 60 min under shaking conditions. Post incubation, the beads were separated out by applying magnet. The bead-free supernatant is subject to acid dissociation in 200mM Glycine-HCI at 25°C for 8-10 min under shaking conditions.
Example 3: Capture and elution of neutralizing antibodies using labelled trastuzumab
The acid dissociated samples from Example 2 were incubated with trastuzumab coupled with magnetic beads at 37°C for 1-2 hrs, to capture the anti-trastuzumab antibodies. Post-incubation, the anti-trastuzumab were eluted from the magnetic beads and incubated with biotinylated Trastuzumab in neutralization buffer. The above complex was added to immobilized rHUHER2-ECD and blocked with blocking buffer. The anti-

trastuzumab neutralizing antibody will bind to biotinylated trastuzumab and inhibit its binding to HER2-ECD. The amount of biotin is detected with streptavidin HRP and chromogenic substrate TMB. The colour development is stopped by adding stop solution. Absorbance (OD) at 450 nm was measured.
WE CLAIM:
I. A method for detecting neutralizing anti-drug antibodies (NAB) in a biological sample, comprising a pre-treatment step comprising:
c) depleting soluble target by ligand based capture and separation; and
d) incubating the sample in acidic buffer;
wherein, the said method mitigates drug and soluble target interference substantially.
2. A method according to claim 1, wherein the said soluble target is shed HER2-ECD.
3. A method according to claim 1, wherein the drug is trastuzumab, pertuzumab or trastuzumab emtansine.
4. A method according to claim 1, wherein the acidic buffer contains glycine-HCI.
5. A method according to claim 1 wherein the method of detecting NAB utilizes cell based or non-cell based assays.