EACR25-1028

Immunopeptidomics approach for the identification of neoantigens in multiple myeloma

G. Zuleger¹, N. de Andrade Krätzig², P. Giansanti³, S. Lutzenberger¹, A. Wagner⁴, D. Busch⁵, B. Meyer⁴, R. Rad², E. Bräunlein¹, A. Krackhard⁶

¹Technical University of Munich, School of Medicine and Health, IIIrd Medical Department, TUM University Hospital, Munich, Germany
²Technical University of Munich, School of Medicine and Health, IInd Medical Department, TUM University Hospital, Munich, Germany
³Technical University of Munich, Bavarian Biomolecular Mass Spectrometry Center, Freising, Germany
⁴Technical University of Munich, Department for Neurosurgery, TUM University Hospital, Munich, Germany
⁵Technical University of Munich, School of Medicine and Health, Institute for Medical Microbiology, Immunology and Hygiene, Munich, Germany
⁶Malteser Krankenhaus St. Franziskus-Hospital, Ist Medical Department, Flensburg, Germany

Introduction:

Multiple myeloma (MM) is a clonal plasma cell malignancy characterised by abnormal production of monoclonal proteins (M proteins). It accounts for approximately 10% of all haematological malignancies and remains incurable despite therapeutic advances. Immunotherapeutic strategies, including chimeric antigen receptor (CAR) T-cell therapy, monoclonal antibodies, antibody-drug conjugates and bispecific antibodies, have demonstrated the potential to harness the immune system to target MM cells. Adoptive T-cell receptor (TCR)-T cell therapy, which targets tumour-specific neoantigens presented via the human leukocyte antigen (HLA) complex, is not limited to extracellular proteins, thereby broadening the spectrum of targetable antigens. Early studies show promising results and a favourable safety profile.

Material and method:

We used an immunopeptidomics approach to identify neoantigens in MM. Whole exome sequencing (WES) and RNA sequencing (RNAseq) of MM cell lines, primary MM tissue from osteolytic lesions and autologous PBMCs (as healthy controls) were combined with immunoprecipitation of HLA peptide complexes (pHLA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of myeloma samples. Candidate neoantigens (NeoAgs) were rigorously validated based on MS spectra and a database search was performed to exclude single nucleotide polymorphisms (SNPs) and canonical RNA editing events. To assess immunogenicity, HLA-matched allogeneic and autologous PBMCs were stimulated in an accelerated co-cultured dendritic cell (acDC) assay followed by IFNγ ELISpot readout. Neoantigen-specific TCRs were analyzed by comparing the clonal frequencies of unstimulated and stimulated cells using single-cell RNA sequencing (scRNAseq).

Result and discussion:

A total of twelve neoantigens were identified in three MM cell lines and seven in eight primary tumour samples. In MM cell lines, neoantigens were derived equally from DNA and RNA variants, whereas in primary tumour tissue, most were derived from RNA variants. Four neoantigens from MM cell lines showed immunogenicity. Three were derived from somatic mutations in PRPF8, FBXL6 and IQGAP2, while the fourth immunogenic candidate arose from an RNA variant in an IgV pseudogene. One neoantigen from MM cell lines and three from primary tumour tissue were used in an scRNAseq approach to identify neoantigen-specific TCRs. The 20 clones with the highest fold change in clonal frequency are currently being tested for reactivity.

Conclusion:

Mass spectrometry-based immunopeptidomics allows the identification of neoantigens in multiple myeloma with potential for personalised TCR-based therapies. However, given the low mutational burden of MM, direct neoantigen identification remains challenging, highlighting the need to enhance MS sensitivity for improved detection.