(W1230-02-06) Strategies for the Development and Characterization of Sensitive Anti-oligonucleotide Antibodies for Application in Anti-drug Antibody Assays

Purpose: Oligonucleotide (ON) therapeutics are an evolving class of drugs, which are short single-stranded synthetic DNAs or RNAs that can modulate RNA to address a wide range of targets for many indications. As for other modalities, there is a continual need to develop critical reagents for immunogenicity assays to support preclinical and clinical development studies. Due to their relatively small size (~7-15 kDa) and chemical modifications in their structure, employed to improve stability and PK properties^1-2, there can be considerable challenges to develop surrogate positive controls for these molecules (i.e., lack of robust immune responses in animals). These positive controls, sensitive to the target drug, are critical reagents for anti-drug-antibody (ADA) assays, serving as reference antibodies for evaluation of immunogenicity in study samples. For a stereopure antisense ON, ASO-1, traditional rabbit polyclonal antibody generation failed due to the low immunogenic properties of this ASO. Pivoting to recombinant antibody generation via a phage display platform containing a library of 40+ billion functional human antibody genes increase the likelihood of identifying sensitive antibodies. This strategy enabled the development of an ADA assay with high sensitivity and good drug tolerance.

Methods: Antibodies were generated either via inoculation of rabbits at Lampire Biological Labs (ASO-1) or through BioRad’s HuCAL platform (ASO-1, ASO-2, ASO-3). At Lampire, 3 rabbits were immunized with an initial round of inoculation of 4 injections of ASO-1-KLH over 3 months. After a 7-week resting period, a second round of immunizations were performed with 2 injections of ASO-1-BSA over 1.5 months to boost the anti-ASO-1-specific immune response. Rabbit serum was harvested, and total IgG was isolated using a protein A column. Further affinity purification was attempted. At BioRad, the Human Combinatorial Antibody Libraries (HuCAL) presented on phage was incubated with the immobilized target ASO-1. ASO-2 and ASO-3 were included as additional closely related antigens to increase selection pressure for highly specific antibodies. Antibody phages with affinity toward ASO-1 are eluted and further enriched, after which the phagemid DNA encoding the antibodies were sequenced. After isolation, monovalent Fab fragments were expressed for primary screening on target ASO-1 and counter-screening on control ASO-2 and ASO-3. Hits were sequenced, expressed, and conjugated with human IgG1 Fc to make synthetic IgG for evaluating affinity ranking via Octet and expected ADA assay performance. Two clones of (1) high ADA assay performance and (2) high or medium affinity were selected for humanization, which thereafter were used to evaluate ADA assay performance. The ADA assay used to detect anti-ASO-1 antibodies was a direct ELISA employing an immobilizer amino microtiter plate coated with 5′- and 3′-free amine ASO-1. The plate was washed and antibody positive controls (PCs), negative controls (NCs), and samples were added, incubated for 2 hours at 37°C. After washing, horseradish peroxidase (HRP)‑conjugated protein A/G was added and incubated for approximately 1 hour at 37°C. After another wash step, tetramethylbenzidine (TMB) substrate solution was added. TMB reacts with the HRP conjugate, producing a colorimetric signal proportional to the amount of anti-ASO antibodies bound to the plate. The plate was then incubated at room temperature (RT) for approximately 15 minutes, protected from light. After the TMB incubation, acidic stop solution was added to the plate, and it was read on a spectrophotometer at 450 nm.

Results: A total of 19 primary-screen positive clones with identified through BioRad’s phage-display library. Of these, 15 were selected for affinity measurement and confirmation of ADA assay performance. A K_D range of 1–126 nM was determined for these clones, two of which were in the single nanomolar range. Signal-to-noise ratio (SNR) ADA performance data in PBS and human serum was used to select two clones for humanization; similar rank-order performance was found between free amine and biotin-based immobilization strategies (Figure 1). While nearly all clones demonstrated high specificity for ASO-1 over ASO-2 and ASO-3, there was a trend suggesting that nucleobase sequence may drive antibody specificity more than backbone and 2′ modifications (Figure 2). Humanized recombinant antibodies yielded an ADA assay sensitivity of approximately 50 ng/mL (2-fold SNR) in both human and non-human primate (NHP) serum, when evaluated as an equimolar control (Figure 3). This sensitivity was a 1000-fold improvement over the polyclonal rabbit antibodies. Excellent drug tolerance was also demonstrated in both human ( >1000 μg/mL) and NHP ( >300 μg/mL) ADA assays (Figure 3).

Conclusion: Consideration of alternative strategies for the development and characterization of critical reagents for ADA assays is critical, as advances are made in evolving molecules that lack a strong immunogenic response³. The strategy presented here highlights methodologies that can meet this need, yielding recombinant antibody surrogate PCs capable of exceeding current industry-accepted standards for detection of anti-drug antibodies⁴. Furthermore, evaluation of the closely related ASOs (either sequence, ASO-2, or chemical modification, ASO-3) provided evidence suggesting nucleobase sequence may be more important than backbone and 2′ modifications in driving specificity.

References: 1. Bano N, Ehlinger C, Yang TY, Swanson M, Allen S. Considerations in the Immunogenicity Assessment Strategy for Oligonucleotide Therapeutics (ONTs). AAPS J. 2022 Aug 26;24(5):93. doi: 10.1208/s12248-022-00741-x. PMID: 36028587.
2. US Department of Health and Human Services, US FDA, Center for Drug Evaluation and Research Guidance for Industry, Clinical Pharmacology Considerations for the Development of Oligonucleotide Therapeutics. Silver Spring, MD, USA (2022).
3. Henry SP, Arfvidsson C, Arrington J, Canadi J, Crowe D, Gupta S, Lohmann S, Massonnet B, Mytych D, Rogers T, Rogers H, Stebbins C, Stovold C, Verthelyi D, Vigil A, Xuan C, Xu Y, Yu R, Klem T. Assessment of the Immunogenicity Potential for Oligonucleotide-Based Drugs. Nucleic Acid Ther. 2022 Oct;32(5):369-377. doi: 10.1089/nat.2021.0112. Epub 2022 Sep 29. PMID: 36178478.
4. US Department of Health and Human Services, US FDA, Center for Drug Evaluation and Research, Center for Biologics Evaluation and Research, Guidance for Industry, Immunogenicity Testing of Therapeutic Protein Products —Developing and Validating Assays for Anti-Drug Antibody Detection. Rockville, MD, USA (2019).

Acknowledgements: The authors thank Amy Donner for editorial assistance and Stephany Standley, Frank Favaloro, Stearne Briem, Stephen Friend, Amber Lindsey, Richard Looby, Leah McCarthy, Andrew McGlynn, Ashwini Ranade, Jeff Rossi, Jason Dufresne, and Snehlata Tripathi for their support in the synthesis and formulation of all oligonucleotides and their conjugates. Disclosure: Authors are employees of Wave Life Sciences, and this work was funded by Wave Life Sciences.