Monitoring disease status and treatment performance in multiple myeloma

Introduction

Multiple Myeloma (MM) is a common blood cancer caused by malignant plasma cells that overproduce a monoclonal immunoglobulin (M-protein). The M-protein is a personalized biomarker. Since current M-protein quantification methods lack in sensitivity, we have developed a LC-MS/MS method to quantify clonotypic peptides from the variable region of the M-protein (MS-MRD). Therapeutic monoclonal antibodies (t-MAbs) play an important role in MM treatment but often lead to a depletion of total immunoglobulin (Ig) levels which requires intravenous IgG administration. Monitoring t-MAb and polyclonal Ig levels could provide important information for therapy administration. Here, we demonstrate the monitoring of M-proteins, t-MAbs, and Ig constant regions in one LC-MS/MS run providing a holistic view of therapy response kinetics.

Methods

Clonotypic peptides for M-proteins and t-MAbs were selected based on linearity and sensitivity in serial dilutions. Ig constant regions of all classes and subclasses were monitored using (sub)class-unique peptides. With a multi-enzyme de novo protein sequencing approach, we obtained the M-protein sequences (M-inSight, Sebia). Patient sera collected between 2013 and 2024 from four MM patients who have received multiple treatment lines were analyzed with MS-MRD. Sera were analyzed using PASEF DDA on a timsTOF pro - PaSER platform (Bruker). Subsequently, patient-specific libraries and dia-PASEF methods were generated for sensitive disease and therapy monitoring. Peak areas of the selected clonotypic, t-MAb, and Ig constant region peptides were filtered from raw dia-PASEF data files and further processed for quantification.

Results

Clonotypic peptides were identified in all four patients and serial dilutions demonstrated an assay sensitivity of 1 mg/L, which is 1,000-fold more sensitive compared to routine M-protein evaluation. MS-MRD analyses in patient 1 and 2 showed the lowest M-protein concentrations since diagnosis in 2013 and 2016 with teclistamab (a novel anti-CD3 and -BCMA bispecific antibody) given under compassionate use as the 8th and 6th line of therapy respectively. Monitoring all administered t-mAbs was feasible in all patients and the measured concentrations corresponded to the administration dates. A 1,000-fold lower concentration of teclistamab compared to daratumumab and isatuximab (anti-CD38 t-MAbs) was observed, corresponding to the significantly different dosing of these drugs. MS-MRD revealed rapid and deep depletion of all Ig classes which is a side-effect of teclistamab, followed by a selective increase in IgG due to intravenous IgG administration to counter this side-effect.

Conclusion

This case series demonstrated the robustness of the M-protein as personalized biomarker during follow-up periods of up to 11 years. Quantification of the M-protein, t-mAbs, and all Ig (sub)classes within a single mass spectrometry run enables dynamic monitoring of novel therapeutics and therapy response.