Isolation and Characterization of Monoclonal Antibodies for the Treatment of Lymphoid Malignancies

Chronic lymphocytic leukemia (CLL) and T cell acute lymphoblastic leukemia (T ALL) are part of lymphoid malignancies, a heterogeneous group of hematologic cancers derived from lymphoid progenitor cells or mature B, T, or natural killer (NK) lymphocytes. Despite major advances in the study of their pathogenic mechanisms and the ongoing research efforts for the introduction of more targeted therapeutic approaches, both CLL and T ALL continue to present significant clinical challenges. The need to develop innovative therapeutic approaches and to identify novel druggable targets is particularly evident in aggressive disease subsets and in relapsed or refractory patients. In recent years, monoclonal antibody-based therapeutics – thanks to their high specificity, safety, reproducibility and versatility – have contributed greatly to advances in targeted oncology. Their demonstrated activity across multiple hematologic malignancies has highlighted the translational opportunities offered by targeted antibody development. This doctoral project addressed two complementary objectives, focusing on antibody-based strategies against relevant targets in lymphoid malignancies. Project A was carried out at Fondazione Toscana Life Sciences (TLS) in the HARD Lab group, under the supervision of Dr. Cristina Tinti and Dr. Piero Pileri, whereas Project B was performed during a research stay at Gulbenkian Institute for Molecular Medicine (GIMM) in the João Barata’s laboratory, under the supervision of Dr. João Barata and Dr. Rita Fragoso, as part of an ongoing research program of the host group. Project A focused on the isolation and production of monoclonal antibodies (mAbs) targeting CEMIP2, a recently characterized protein, proposed here as a novel potential target in CLL. mAbs were generated from antigen-specific antibody-secreting cells (ASCs) isolated with a refined high-throughput single-cell screening using the CellCelector platform. This new method overcomes limitations of prior manual screening by providing the expected advantages in terms of cells isolated in a single session and time required. Five antibodies (mAb#2, #21, #34, #13 and #12) showed strong binding to CEMIP2 on the CLL cell line MEC 1. Their antigen specificity was verified by testing them on CEMIP2 knockout cells. These antibodies represent a set of research tools for investigating the function of CEMIP2 in chronic lymphocytic leukemia, thus supporting future preclinical studies. Project B focused on the in vitro functional characterization of an anti-interleukin-7 receptor alpha (IL-7Rα) monoclonal antibody (FJB45) in T-ALL cell lines and patient-derived xenograft (PDX) cells. Antibody treatment substantially reduced cell viability and proliferation and caused marked inhibition of downstream STAT5 phosphorylation in cells expressing wild-type IL-7R. In contrast, the treatment with the antibody didn’t cause a decrease in cell viability and proliferation in PDX cells expressing a mutated version of IL-7R, which leads to constitutive signaling and characterizes almost 10% of T-ALL patients. These results showed that the antibody efficacy in inhibiting cell viability and proliferation is dependent on its modulation of downstream signaling. Furthermore, the antibody demonstrated cell killing ability through antibody-dependent cellular cytotoxicity (ADCC) and showed efficient internalization into target cells, supporting its potential suitability for antibody–drug conjugate development. Overall, this work integrates antibody discovery and functional characterization exploring antibody-based approaches against biologically relevant targets in CLL and T-ALL and providing a foundation for further preclinical and therapeutic development.