(M1230-02-07) Development and Validation of a High-Sensitivity PK Assay for CLN-978 Supporting a First in Human Study in Systemic Lupus Erythematosus Using Quanterix SIMOA Technology

Purpose: CLN-978 is a novel, highly potent CD19 single chain variable fragment (scFv) x CD3 scFv bispecific T cell engager (Fig. 1) engineered to target CD19-expressing cells in development for patients with autoimmune diseases including systemic lupus erythematosus (SLE). Previously, CLN-978 was administered in a first-in-human study in patients with relapsed or refractory B-cell non-Hodgkin lymphoma (B-NHL). The corresponding PK assay lower limit of quantification (LLOQ), 2.5 ng/mL, was sufficient for drug quantification in which a 30 µg dose was administered. As the starting dose for Phase 1 dose escalation in SLE is lower (10 µg) a more sensitive PK assay with a sub-ng/mL LLOQ is required. We proposed that new reagents when tested with technology optimized for sensitivity, we’d achieve our targeted LLOQ. Newly sourced reagents were evaluated using Immuno PCR (IPCR). Single molecule array (SIMOA) was evaluated to improve the sensitivity of the original assay and to assess this technology for performance consistent with clinical bioanalytical PK method regulatory requirements.

Methods: Five F(ab')₂s and three mAbs targeting either the CD3 or CD19 scFvs were sourced using phage display and murine hybridoma technologies respectively. The CD3 and the CD19 extracellular domains (ECDs), human CD3 epsilon & CD3 delta heterodimer (CD3 HD) and human CD19 Fc chimera (CD19 Fc) originally used for the B-NHL method, were sourced commercially. Newly sourced reagents were tested with IPCR as this platform supports simultaneous evaluation of multiple reagent combinations in a single run. Samples, minimum required dilution (MRD) 1:4, were plated on washed, blocked, capture-reagent coated plates. Incubation and washing were followed by detection using reagents labeled with standard biotin-NHS ester chemistry, streptavidin conjugated with a proprietary DNA tag, and real-time (q) PCR. The best performing reagent pair was further evaluated using SIMOA. Assay development utilized EDC activated paramagnetic beads coupled with the CD3 scFv specific capture mAb 1 using standard published SIMOA protocols. Biotin coupling of the CD19 scFv specific detection mAb 3, was achieved using standard biotin-NHS ester chemistry. Calibrators and QCs MRD 1:50, and mAb 1 coupled paramagnetic beads were mixed 1:1 with Helper Beads in Super Block and loaded onto the HD-X Analyzer with biotinylated mAb 3, streptavidin beta galactosidase (SBG) and the resorufin β-D-galactopyranoside (RGP) substrate. Beads were arrayed against 216,000 femtoliter-sized wells, sealed with oil and imaged. Fluorescence directly proportional to the number of enzymes captured per bead, was analyzed and the average number of enzymes/bead (AEB) imported to Watson LIMS for data regression using a 4PL method with weighting 1/Y2.

Results: Newly sourced F(ab')₂s and mAbs, were tested in every combination as capture and detect to optimize the assay’s dynamic range, delta Ct signal – noise (S-N) using IPCR. Figure 2 shows S-N for top performing combinations compared with the original assay reagents, CD3 HD and CD19 Fc capture and detect respectively. Red S-N indicates the dynamic range provided by these original reagents: 2.07 at 2.5 ng/mL, and the best performing mAb 1 and mAb 3 reagent pair S-N: 2.49 at 0.156 ng/mL, a 16-fold improvement. As mAbs 1 and 3 provided the best dynamic range, these reagents were selected for assay development using SIMOA. Affinity alone did not predict the reagent pair providing the best dynamic range as accessibility and specificity also likely played a key role. Note the CD19 scFv /CD19 ECD KD: 0.074nM, versus the better performing CD19 scFv /mAb 3 KD: 1.04 nM (Figs. 1 and 2). The CLN-978 PK assay quantitative range was established with an 8-point calibration curve 8 – 0.0625 ng/mL and one anchor point below the LLOQ, dilutional linearity (DL), accuracy and precision (A&P), and selectivity in both normal and SLE sera during validation (Fig. 3 A&P and Selectivity). DL was evaluated from 30.625 - 0.0617 ng/mL.

Conclusion: We report the successful development and validation of a clinical PK assay, LLOQ 0.0625 ng/mL, for the quantification of CLN-978 in patients with SLE using SIMOA technology. The sensitivity achieved using SIMOA is 40-fold greater than the original assay’s LLOQ of 2.5 ng/mL thereby enabling the quantification of CLN-978 at the starting dose of 10 µg in a Phase 1 dose escalation study in SLE. The assay performance and validation data presented here are in accordance with clinical regulatory expectations supporting the use of SIMOA in providing high sensitivity PK methods for clinical studies.

Acknowledgements: CLN-978 was administered in a first-in-human study in patients with relapsed or refractory B-cell non-Hodgkin lymphoma (B-NHL; NCT05879744).
CLN-978 is in development for patients with autoimmune diseases including systemic lupus erythematosus (SLE; NCT06613360).
F(ab')₂s and mAbs were sourced from Bio-Rad and Twist Bioscience respectively.
The CD3 and CD19 extracellular domains were sourced from AcroBiosystems (CDD-H52W1) and R&D Systems (9269-CD) respectively.