Abstract 2721: FF01: a Novel Humanized Heavy Chain Antibody Targeting Endogenous Retroviruses with Anti-Lymphoma Activity

Background: Human endogenous retroviruses (HERVs) are transposable elements (TE) derived from retrovirus infections that occurred millions of years ago. Despite constituting 8% of our genome, their biological role still needs to be explored. HERV-K (HML-2), a subgroup of HERVs, represents the most recent retroviruses integrated into the human germline DNA. Members of the HML-2 subgroup retain complete ORF with coding potential and have garnered the most attention in disease-related studies. HERVs exhibit physiological expression in embryonic stem cells but are heavily methylated and remain almost silent in normal adult tissues. Although the mechanisms underlying HERV-K contribution to tumor cell growth and survival remain poorly understood, aberrant expression of HERVs has been demonstrated in various human tumor tissues and lymphoma. These findings led us to explore HERV-K as a potential therapeutic target in B cell lymphomas .Methods: RNA data were analyzed with Telescope. Validations were performed by qPCR, Western blot, flow cytometry, and ELISA. Env structure was obtained with AlphaFold and peptides were purchased from GeneScript. Antibody-dependent cellular cytotoxicity (ADCC) was performed with the ADCC Reporter Bioassay (Promega). Internalization was assessed using the Incucyte Fabfluor-pH Antibody labeling reagents. Results: We assessed the TE expression in RNA-seq datasets of diffuse large B cell lymphoma (DLBCL) and B cells from healthy individuals. An MDS plot showed that TEs could separate neoplastic from healthy samples, demonstrating that tumoral and normal specimens have distinct TE and HERV expression profiles. Focusing on tumor samples, DLBCLs were divided into four clusters with different prognoses. Similar results were obtained in a 47 B-cell lymphoma cell line panel. Focusing on the HML-2 family in lymphoma patients, we identified many of them upregulated in tumors. We validated RNA and protein expression of the HML-2 envelope (env) in seventeen lymphoma cell lines, and in two of them, we also proved the protein surface expression. Env-specific siRNA reduced env expression determined a decrease in cell proliferation, suggesting a contribution to lymphoma cell growth. In silico examination of the env identified four linear regions with potential for antibody binding, and peptides covering them were synthesized. We screened 150 lymphoma patients' serum samples for IgG antibodies binding to the peptides, and all peptides showed positive hits. We used a phage display screen to obtain one nanobody, FF01, binding one peptide. FF01 induced ADCC in the cell line with high surface env expression and not in a negative one. FF01 was internalized in an expressing cell, proving the principle for using it as a component of an antibody-drug conjugate. Conclusions: Our data demonstrate that HERVs are in DLBCL patients and propose FF01 as a new candidate to target HERV Env+ lymphomas. Citation Format: Filippo Spriano, Luciano Cascione, Jacopo Sgrignani, Nikolai Bendik, Sara Napoli, Giulio Sartori, Eleonora Cannas, Tao Gongo, Alberto J. Arribas, Davide Rossi, Davide F. Robbiani, Andrea Cavalli, Francesco Bertoni. FF01: a novel humanized heavy chain antibody targeting endogenous retroviruses with anti-lymphoma activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2721.