Spotlight on the DMPK and Bioanalysis Strategy of Antibody Oligonucleotide Conjugates (AOCs)

Antibody oligonucleotide conjugates (AOCs) are a novel targeted therapeutic that combines oligonucleotides, such as small interfering RNAs (siRNAs) or anti-sense oligonucleotides (ASOs), with specific antibodies or antibody fragments like the Fc fragment which targets the transferrin receptor. This strategic combination enables AOCs to effectively target previously untreatable tissue and cell types compared with oligonucleotide drugs, presenting significant potential for advancing patient therapies. Conventional delivery systems for oligonucleotide drugs like lipid nanoparticles (LNPs) or GalNAc-based platforms often exhibit limited organ targeting, predominantly focusing on the liver which limits their applications. Effective targeting is crucial for ensuring drug efficacy and safety, further elevating the prominence of AOCs as therapeutic agents. Consequently, numerous biotech companies including  Avidity Biosciences, Tallac Therapeutics, Dyne Therapeutics, Denali Therapeutics, etc., are dedicating their research efforts to the development of AOCs. Markedly, AOC 1001 (Tfr1-targeted mAB for DM1 disease with anti-DMPK siRNA) was launched by Avidity Biosciences as the first AOC drug worldwide, which has entered the clinical phase^[1]. AOC 1044 (Tfr1-targeted mAB for DMD disease with anti-DMD PMO) has obtained FDA fast-track and orphan drug designation, which further underscores the vibrancy and potential of AOC-related therapies. This article will summarize the pharmacokinetics research strategies of AOCs, particularly highlighting the critical aspects of bioanalysis associated with AOC drugs and various detection platforms essential for the advancement of AOC therapeutics.

Pharmacokinetics study strategies of AOC drugs

There are diverse conjugation methods of oligonucleotides compared to typical small molecules in antibody-drug conjugates (ADCs). Currently, four primary conjugation strategies dominate the landscape involving ionic interactions, avidin/biotin mediated-affinity conjugation, direct conjugation, and base pairing mediated-hybridization (Figure 1)^[2]. The conjugation mode significantly influences the release profile of the oligonucleotide within cellular lysosomes, thereby impacting the efficacy of AOC drugs. Furthermore, each conjugation approach necessitates the development of suitable bioanalytical methods for pharmacokinetic (PK) studies tailored to the specific characteristics of the AOC.

Figure 1. General methods of oligonucleotide conjugation in AOCs: Ionic interactions, affinity conjugation, direct conjugation, and hybridization^[2].

The DMPK (Drug Metabolism and Pharmacokinetics) study of AOC drugs primarily focuses on the study of ADME (absorption, distribution, metabolism, and excretion) characteristics. The DMPK study of AOCs is divided into three main components: in vitro ADME, drug-drug interactions (DDI), and in vivo pharmacokinetics (PK), spanning three stages, including preclinical discovery, preclinical development, and clinical development. During the early stage, efforts are concentrated on screening lead molecules based on in vitro pharmacodynamics and stability assay. Several screened molecules undergo investigation during the preclinical phase, assessing both in vivo pharmacodynamics (PD) and PK studies to elucidate the dose-response relationship and tissue distribution of AOC drugs, thereby advancing the leading AOC candidates into the Investigational New Drug (IND) research stage. Within the DMPK study of AOCs, bioanalysis plays a crucial role in facilitating these complexes in vitro and in vivo studies. Consequently, the selection of appropriate bioanalytical methods is essential for the investigation of AOC drugs.

Table 1. Overview of strategies for AOC drug DMPK study at different stages

Bioanalysis and detection methods for AOC drugs

The bioanalysis of AOC drugs is relatively intricate, which is similar to ADC. Traditional single-platform approaches are often inadequate for comprehensive bioanalysis of AOCs. Hence, a multi-platform strategy is required. Typically, this involves the combination of ligand binding assay (LBA), LC/MS, and RT-qPCR platform to achieve effective bioanalysis of AOCs. In our summary displayed in Figure 2, we delineate the key bioanalytical components associated with AOCs in pharmacokinetic (PK) studies, highlighting the primary analytical platforms involved. These components include the analysis of total antibodies, conjugated antibodies, conjugated/free oligonucleotides, immunogenicity, biomarkers, etc. Subsequently, we will provide an in-depth discussion of each bioanalytical aspect and the associated internal validation processes.

Figure 2. Integrative bioanalysis methods and corresponding analysis platforms for AOC drugs

2.1 Bioanalysis of total antibodies

The bioanalysis of total antibodies to AOC is typically conducted on the LBA platform. In the process of developing the total antibody analytical method for AOC drugs, it is necessary to devise suitable analytical methods including specific or general methods that align with the structure characteristics of the antibody, the targeted molecule, and the specific phase of research. Appropriate analysis platforms, like ELISA and MSD, are determined by the requirements for sensitivity and sample throughput.

2.2 Bioanalysis of conjugated antibodies

The LBA platform is typically utilized as the primary method for analyzing conjugated antibodies. Generally, the binding of AOC-related oligonucleotides often occurs in a non-antibody affinity manner due to the low affinity and poor specificity of critical reagents for anti-oligo antibodies. For example, the conjugated antibody will be detected by Avidin-HRP when the siRNA-conjugated AOCs with biotin-labeled sense strands serve as a standard. Also, the biotin-labeled complementary strand of ASO can be employed as the detection reagents for the bioanalysis of ASO-conjugated AOC(Figure 3).

Figure 3. Schematic of the ELISA assay format for AOC conjugated antibody bioanalysis, A: siRNA-conjugated AOC and B: ASO-conjugated AOC.

It is noteworthy that there are certain inherent limitations in this analytical approach. For instance, the biotin-labeled AOC must be used to detect conjugated antibodies for the PK assay. While biotin is labeled on the sense strand of siRNA, this labeling may affect drug absorption, distribution, and metabolism. Furthermore, due to the limitations of the assay format, it is possible to overestimate the quantity of molecular species with a high oligo antibody ratio (OAR), leading to an inaccuracy in the quantification of conjugated antibodies.

For these reasons, the quantification of conjugated oligonucleotides and total antibodies is more frequently adopted to systematically characterize the ADME characteristics of AOC drugs in vivo during the AOC drug bioanalysis.

2.3 Bioanalysis of conjugated oligonucleotides

In the analysis of conjugated oligonucleotides, the general workflow begins with the use of protease to degrade the AOC, followed by the analysis of siRNA or ASO. WuXi AppTec DMPK is equipped with mature platforms such as RT-qPCR, hybridization ELISA/MSD, and LC/MS for oligonucleotide analysis (Figure 4). Different analysis platforms can be selected according to the required sensitivity as well as different needs such as the detection of metabolites.

Figure 4. Schematic of AOC-coupled oligonucleotide analysis

Generally, the hybridization MSD platform is recommended to analyze conjugated oligonucleotides due to its superior sensitivity, robustness, and wide range of quantitative capabilities. The marketed siRNA drug Inclisiran has been quantitatively analyzed in monkey plasma using hybridization MSD platform, revealing a linear range for the detection of the antisense strand between 3pmol/L to 600 pmol/L (1.5 pmol/L as the anchor point). It demonstrated that the hybridization MSD platform has excellent stability according to the inter-precision/accuracy of the quantification standard curve samples and control samples (Figure 5).

Figure 5. Standard curves and precision & accuracy evaluation for the analysis of marketed siRNA drug Inclisiran using hybridization MSD method

The Hybridization MSD platform also serves as the primary methodology for the analysis of free oligonucleotides in AOC bioanalysis. Detecting free oligonucleotides typically necessitates a more sensitive analytical platform due to the need for molar quantity for AOC-conjugated oligonucleotides and the rapid clearance characteristics of the oligonucleotide, particularly for AOC drugs with stable conjugation.

2.4 Immunogenicity evaluation

Universally, the oligonucleotides are less immunogenic. Similar to ADC, the immunogenicity evaluation of AOCs focused on their antibody fractions. Notably, to achieve a more comprehensive evaluation of total anti-drug antibodies (ADA) for AOCs, it is advisable to include the inhibition ratio of AOC and naked antibody in the methodological validation during the IND application. This may also help to clarify the specific regions of AOC targeted by the anti-drug antibody. The detailed procedures for the immunogenicity evaluation of AOCs are shown in Figure 6.

Figure 6. Schematic of ADA assessment process for AOC

2.5 Analysis of biomarkers

The preclinical biomarker analysis of AOC encompasses a comprehensive evaluation of oligonucleotide targets analysis (mRNA or expressed proteins), antibody target expression analysis, immune cell response analysis, cytokines profiling, and other markers evaluation (Figure 7). Biomarker analysis mainly focuses on the evaluation of the efficacy and safety of AOC. And pharmacodynamic assay involves the analysis of oligonucleotide targets, including quantifying target mRNA levels by the RT-qPCR platform and quantifying expressed proteins by the LBA platform. For prevalent siRNA targets, internal target-related analysis methods have been developed that can be used directly. The assessment of safety biomarkers primarily involves cytokine release and immunophenotyping. To meet standard operational requirements, a suite of in-house methods for commonly analyzed immune-related cytokines and immunophenotyping panels has been established. Additionally, customization of the analytical content to align with specific project needs can be offered, which ensures a tailored approach to biomarker evaluation.

Figure 7. Schematic diagram of AOC-related biomarker analysis, including the analysis of oligonucleotide target expression (mRNA or its expression protein), antibody ligand expression, immune cell response, cytokine, and other markers involved in the pathway.

Conclusion and discussion

AOC drugs have significantly broadened the therapeutic landscape for oligonucleotides, leading to increased enthusiasm for their development. Unlike traditional monoclonal antibodies and oligonucleotides, AOCs introduce additional complexity in the assessment of ADME effectiveness and safety, requiring tailored bioanalytical methods.

WuXi AppTec DMPK possesses extensive expertise in preclinical bioanalysis. Currently, dozens of AOC projects have been managed. The establishment and validation of preclinical bioanalytical methods to meet FDA/NMPA/TGA IND submission requirements have been executed. The integrated bioanalytical platform for AOCs in WuXi AppTec DMPK enables efficient evaluation of in vivo/vitro ADME characteristics, thereby enhancing the understanding of both efficacy and safety, and supporting more safety assessments and clinical studies in the future.

Authors: Maotian Zhou, Xue Zhang, Miaomiao Song, Liping Ma, Lili Xing

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