Inhibrx : Poster Presentation

PABE: A Novel Enhanced Drug Tolerant assay format to detect Anti-Drug Antibodies in the presence of high concentrations of rhAAT-Fc

Varun Ramani1, Carson Veldstra1, Carolina Bellizzi1, Jason Zour1, Buket Onel2 1Inhibrx, Inc.; 2 BioAgilytix

M1130-06-39

PURPOSE

RESULT (S)

Polyethylene glycol (PEG) Precipitation, Acid dissociation (AD),

Bridging Electrochemiluminescent (ECL) or PABE

Detection of anti-drug antibodies (ADA) plays a key role for safety and efficacy assessment of biotherapeutics. Biotherapeutic drugs can be administered at high doses, potentially resulting in circulating therapeutic drug concentrations within mg/mL range. Therefore, developing robust ADA assays with high drug tolerance is critical.

The therapeutic drug INBRX-101 is a recombinant human Alpha-1 Antitrypsin (AAT)-Fc fusion protein being investigated as an augmentation therapy in patients with Alpha-1 Antitrypsin Deficiency (AATD). In clinical studies, ADA samples taken at steady state trough are expected to have ≥ 1.5 mg/mL of circulating INBRX-101 in addition to endogenous AAT. Therefore, development of an ADA method which could meet the needs of high drug tolerance for this program was pursued.

PABE (Polyethylene glycol (PEG) Precipitation, Acid dissociation (AD), Bridging Electrochemiluminescent (ECL)) is a novel Anti-Drug Antibody assay format that was developed at Inhibrx, Inc. and validated at BioAgilytix Labs, Durham, NC.

This assay detects ADA (both endogenous counterpart or AAT-bound ADA, and INBRX-101 bound ADA) in human serum samples. Using a combination of a bridging Master Mix format with a pre-treatment step involving PEG precipitation and acid dissociation resulted in a robust assay with a drastically improved drug tolerance (≥ 5mg/mL INBRX-101)

We validated the ADA assay following FDA guidelines from 2014 and 2019 for immunogenicity assessment. During validation, we tested drug tolerance using normal human serum, which typically contains over 1.0 mg/mL of endogenous AAT. We examined drug tolerance at three surrogate positive control (PC) concentrations (2000 ng/mL, 500 ng/mL, and 100 ng/mL). These PC levels were assessed with and without varying concentrations of INBRX-101, which contains two AAT molecules, ranging from 5000 µg/mL to 0 µg/mL.

All PC levels assessed in the presence of varying concentrations of INBRX-101 screened and confirmed positive. Thus, they were tolerant up to 5 mg/mL of INBRX-101.

The PC-spiked pooled normal human serum contains endogenous levels of AAT. Therefore, established INBRX-101 drug tolerance is in combination with normal human levels of endogenous AAT.

The target acceptance criteria for validation experiments were met, including intra- and inter-assay precision at the sensitivity level of the assay, cut points, sensitivity, selectivity, free drug and endogenous AAT tolerance, prozone (Hook) effect, stability, and

DRUG TOLERANCE RESULTS

and sensitivity (≤ 100 ng/mL).

Unlike the PandA5 assay format, PABE is simplified in its design and execution and does not require addition of excess drug material to samples. Additionally, it uses a bridging assay format instead of a sequential format to detect ADA. It will be used to evaluate INBRX-101 ADA and domain specificity in the ElevAATephase 2 registrational clinical trial sponsored by Inhibrx, Inc.

METHOD

1. PEG treatment (5% final concentration of PEG-8000) precipitates

immune complexes

• Dilution of controls and samples at a 1:1 ratio with 10% PEG-8000

solution and followed by overnight incubation to precipitate immune

complexes.

• Centrifugation and washing steps in 5% PEG 8000 solution

2. Acid Dissociation

• Dissociation of the resulting precipitated immune complexes in a

weak acid solution.

3. Bridging ECL with dual-labeled drug to capture and detect ADA

• Neutralization and Formation of bridged Drug:Antibody:Drug

complexes.

• Capturing samples and controls on a blocked, streptavidin coated

robustness.

CONCLUSION (S)

Best of PandA5

PABE

Best of Bridging Assays

1.	PABE format was effective in overcoming ultra-high
	concentrations of INBRX-101 in addition to endogenous AAT.
2.	Operational ease for high throughput, low non-specific
	background, improved sensitivity, and superior drug tolerance
	achieved.
3.	Possibly eliminates target interferences & enables efficient
	detection of immunoglobulin isotypes.
4.	ADA is seemingly effectively isolated & detected using this
	format regardless of how much therapeutic drug and its
	endogenous counterpart may be contained in the study
	samples.

• 100 ng/mL of PC detected in the presence of 5 mg/mL of INBRX-101 in addition to endogenous counterpart drug in NHS.
• Provided almost complete elimination of Drug Interference

REFERENCES

1. Creighton, W. D., Lambert, P. H., & Miescher, P. A. (1973). Detection of antibodies and soluble antigen- antibody complexes by precipitation with Polyethylene Glycol. The Journal of Immunology, 111(4), 1219-1227.https://doi.org/10.4049/jimmunol.111.4.1219
2. Ohlson, S., & Zetterstrand, K. (1985). Detection of circulating immune complexes by PEG precipitation combined with Elisa. Journal of Immunological Methods, 77(1), 87-93.https://doi.org/10.1016/0022-1759(85)90186-3
3. Moxness, M., Tatarewicz, S., Weeraratne, D., Murakami, N., Wullner, D., Mytych, D., Jawa, V., Koren, E., & Swanson, S. J. (2005).Immunogenicity testing by electrochemiluminescent detection for antibodies directed against therapeutic human monoclonal antibodies. Clinical Chemistry, 51(10), 1983- 1985.https://doi.org/10.1373/clinchem.2005.053272
4. Zoghbi, J., Xu, Y., Grabert, R., Theobald, V., & Richards, S. (2015). A breakthrough novel method to resolve the drug and target interference problem in immunogenicity assays. Journal of Immunological Methods, 426, 62-69.https://doi.org/10.1016/j.jim.2015.08.002

MSD plate.

• Followed by washing steps and addition of the MSD Gold Read

Buffer.

• Detection of the complexes using an MSD S600 plate reader.

4. The presence of ADA is determined by comparing the signal in the

samples or controls to a statistically derived threshold/assay cut point.

5. PABE is a novel assay format poised to find valuable

applications in the realm of ADA assays, particularly in

scenarios where elevated quantities of the therapeutic fusion

protein coexist alongside their native endogenous

counterparts.

SCP (S/N) = 1.19; CCP (% Inh.) = 22.8%

Drug Concentration (mg/mL)		5	2	1	0.1	0
	2000	S/N	6.96	8.89	8.01	9.55	9.70
	% Inh	83	86.5	85.3	88	86.8
PC Concentration (ng/mL)	500	S/N	2.46	2.64	2.88	2.81	3.22
% Inh	58.8	61.6	65	64.7	68.5
	100	S/N	1.30	1.32	1.43	1.42	1.44
	% Inh	27.1	26.1	32.2	32.5	33.5

CONTACT INFORMATION: Varun Ramani, [e-mail]

DISCLOSURES

V.Ramani, C.Veldstra, C.Bellizzi, and J.Zour are employees and shareholders of Inhibrx, Inc.

B. Onel is an employee of BioAgilytix Labs.

ACKNOWLEDGEMENTS

This study was funded by Inhibrx, Inc.