Uncovering the Tumor Antigen Landscape in Patients Undergoing Dendritic Cell/Myeloma Fusion Vaccine with Lenalidomide Maintenance Treatment

Background: Identifying novel antigenic targets for immune-based therapy in Multiple Myeloma (MM) is a critical area of investigation. The BMT CTN 1401 (NCT02728102), a national randomized trial evaluating dendritic cell (DC)/MM fusion cells following autologous transplant in patients with newly diagnosed MM, offers a unique source of bone marrow aspirates that can be interrogated for novel antigen discovery studies. In the present analysis, we characterize the personalized and shared tumor-associated (TAA) and tumor-specific (TSA) antigens derived from MM in the bone marrow and patterns of associated T cell clonotypes at baseline (time of enrollment).

Methods: We developed an in silico-experimental framework that integrates tumor bulk RNAseq, Whole Exome Sequencing (WES), and T Cell Receptor (TCR) profiling data. We established translation probabilities and assessment of tumor specificity by analyzing >6,500,000 translated regions from raw public Ribosome profiling data and >1,000,000 transcribed regions from healthy control samples across 29 tissues. We developed an AI/ML classifier to define TAAs and TSAs, ensuring each antigenic peptide is highly expressed in tumor samples, translated, presented, and has high immunogenic potential. We applied our model to bone marrow aspirate samples collected at time of study enrollment pre-transplant from 16 patients to identify private TAAs and TSAs, as well as antigens shared across patients. Plasma-specific antigen expression was further explored using single-cell immunoprofiling data from bone marrow tumor samples in a subset of 10 patients from this cohort.

Results: We demonstrated a high occurrence of tumor-specific antigens and neoantigens in MM cells at the time of enrollment. Notably, 50% of the identified neoantigens exhibited shared antigenic mutations in more than 2 patients. Nineteen of these neoantigens were further prioritized and identified as highly immunogenic and highly expressed in MM malignant plasma cells from the matched single-cell experiments. Four of these neoantigens, typically expressed in immune-privileged tissues, including cancer-testis antigens (such as MAGEC1 and TPTE) and brain-specific genes (such as UCHL1), were found to be highly expressed in MM tumor cells. Notably, UCHL1 showed a median 80-fold higher expression in MM bone marrow samples compared to public healthy controls. We observed a substantial enrichment of highly shared tumor antigenic peptides (n=33) among up to 10 patients, originating from regions not typically highly expressed or translated, such as retroelements and downstream/upstream open reading frames (ORFs). A significant association was observed between TSA and TAA abundance at baseline and the extent of α/β clonotypic expansion of tumor-infiltrating T cells, as identified from bulk and single-cell immunoprofiling data. MHC I Dextramer technology with 10x Genomics is being employed to further validate the identified antigens and characterize antigen-specific expanded T cell clones in the studied MM patients post-vaccine.

Conclusion: These findings highlight the role of the baseline tumor antigenic landscape in the cohort of patients subsequently randomized to undergo DC/MM vaccination or maintenance therapy alone. We identified personal and shared tumor antigens, with a minority observed in most patients studied. We will interrogate the expanded T cell clonotypes following vaccination to better understand the dominant epitopes being recognized and their association with clinical outcomes.

Acknowledgments: Support for this study was provided by grants #U10HL069294 and #U24HL138660 to the Blood and Marrow Transplant Clinical Trials Network from the National Heart, Lung, and Blood Institute and the National Cancer Institute along with contributions by Celgene Corporation and the Multiple Myeloma Research Foundation Fellowship Program. The content is solely the responsibility of the authors and does not necessarily represent the official views of the above-mentioned parties.