-Author name in bold denotes the presenting author
-Asterisk * with author name denotes a Non-ASH member
Clinically Relevant Abstract denotes an abstract that is clinically relevant.

PhD Trainee denotes that this is a recommended PHD Trainee Session.

Ticketed Session denotes that this is a ticketed session.

975 Structural and Functional Analysis of the Clonotypic B Cell Receptor Immunoglobulin in Splenic Marginal Zone Lymphoma: Ontogenetic and Therapeutic Implications

Program: Oral and Poster Abstracts
Type: Oral
Session: 622. Lymphomas: Translational - Non-Genetic: New Approaches and Models for Improving Lymphoma Therapies
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Non-Hodgkin lymphoma, Lymphomas, Translational Research, Elderly, B Cell lymphoma, Indolent lymphoma, Diseases, Immunology, Lymphoid Malignancies, Biological Processes, Molecular biology, Technology and Procedures, Study Population, Human
Monday, December 9, 2024: 5:00 PM

Anastasia Iatrou1,2*, Marco Patrone3*, Ioannis Sarrigeorgiou4*, Nikolaos Pechlivanis2*, David Graham Oscier5, Alexandra Traverse-Glehen, MD, PhD6*, Raphael Sandaltzopoulos1*, Niki Stavroyianni7*, Andreas Agathangelidis8*, Georgios Petrakis9*, Triantafyllia Koletsa9*, Peggy Lymberi4*, Paolo Ghia10,11, Anastasia Chatzidimitriou2,12*, Massimo Degano3* and Kostas Stamatopoulos2,12*

1Department of Molecular Biology and Genetics, Democritus University of Thrace, Alexandroupolis, Greece
2Institute of Applied Biosciences, Centre for Research and Technology Hellas, Thessaloniki, Greece
3Università Vita-Salute San Raffaele and Biocrystallography Group, Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy
4Immunology Laboratory, Immunology Department, Hellenic Pasteur Institute, Athens, Greece
5Department of Haematology, Royal Bournemouth Hospital, Bournemouth, United Kingdom
6Service de Pathologie Sud, Hospices Civils de Lyon, Pierre-Bénite, France
7Department of Hematology - BMT Unit, G. Papanicolaou Hospital, Thessaloniki, Greece
8School of Biology, National and Kapodistrian University of Athens, Athens, Greece
9Pathology Department, Medical School, Aristotle University of Thessaloniki, Thessaloniki, Greece
10Division of Experimental Oncology, B cell neoplasia, IRCCS Ospedale San Raffaele, Milan, Italy
11Medical School, Università Vita-Salute San Raffaele, Milano, Italy
12Department of Molecular Medicine and Surgery, Karolinska Institute, Stockholm, Sweden

Immunogenetic evidence implicates the B cell receptor immunoglobulin (BcR IG) in the natural history of splenic marginal zone lymphoma (SMZL). Indeed, SMZL carries distinct features of somatic hypermutation (SHM) amongst cases utilizing particular IGHV genes in their BcR IG. Moreover, the BcR IG gene repertoire in SMZL is restricted, whereby ~30% of cases utilize the IGHV1-2*04 gene and allele, notable for carrying a tryptophan (W) residue at position VH FR3-75 instead of the arginine (R) residue that is encoded by all remaining IGHV1-2 gene alleles and almost all other human IGHV genes and alleles. IGHV1-2*04 cases display heterogeneous CDR3 features and light chains, as well as pronounced intraclonal diversification within the heavy chain gene rearrangements. Overall, this constellation of features argues for heavy chain dominance in this major immunogenetic subgroup of SMZL. Against that, scant information exists regarding the functional and structural properties of the clonotypic BcR IG in SMZL, while the relevance of the IGHV1-2*04 overuse remains an enigma. In order to address this knowledge gap, we performed immunological, biochemical and crystallographic studies of the clonotypic BcR IG from SMZL cases that we expressed as recombinant monoclonal antibodies (rmAbs). ELISA against molecules that are common antigenic targets of naturally-occurring autoantibodies and disease-occurring/related pathological autoantibodies, namely DNA, actin, myosin, β-amyloid, TNP, thyroglobulin, IgG F(ab)’2 fragments, and trinitrophenyl (TNP), revealed that SMZL rmAbs (n=42, of which 14 utilized the IGHV1-2*04) were poly/autoreactive, regardless of the expressed IGHV gene and their SHM status. Of note, comparison with CLL rmAbs (n=35), known for their poly/autoreactivity, revealed significantly (p<0.05) stronger binding of the SMZL rmAbs to all tested autoantigens on autoantigen protein microarrays. Autoreactivity was corroborated by flow cytometry showing that 38/42 SMZL rmAbs bound viable MEC1, HEK293 and HS-5 cells; and, by immunohistochemistry using the SMZL rmAbs as primary Abs, which documented binding to human tissues (appendix, tonsil, lymph nodes and kidney), albeit with distinct profiles depending on the expressed IGHV gene. Next, in order to assess the functional relevance of the W residue in IGHV1-2*04 BcR IG, we used PCR-based site-directed mutagenesis for modifying W75 to R (IGHV1-2*04 W75R, n=10), documenting significant (p<0.05) differences in antigen reactivity profile between the authentic IGHV1-2*04 rmAbs vs the respective IGHV1-2*04 W75R rmAbs. Further, in order to test the hypothesis of heavy chain dominance, we coupled the IGHV1-2*04 heavy chains with random light chains and compared their antigen reactivity profile versus the authentic rmAbs, observing no differences. Finally, we crystallized two different IGHV1-2*04 SMZL BcR Fabs and determined their structures to high resolution. In all crystals obtained, regardless of pH and precipitating agent, pairs of Fab molecules were found to interact homotypically through a symmetrical stacking cation-π interaction mediated by the allele-specific W75 residue and side chain of R95 in the IGHV1-2*04 heavy chain, burying an extensive surface of ~650 Å2. In silico analysis with FOLDX highlighted the high energetic contribution of these residues in promoting the formation of the homodimers in the crystals. In conclusion, we document pronounced poly/autoreactivity in SMZL suggesting that SMZL likely derives from a progenitor B-cell population with highly restricted BcR IG structures and autoreactive potential. We also provide experimental support to the hypothesis of heavy chain dominance in the IGHV1-2*04 subgroup which raises the intriguing possibility that the IGHV1-2*04 heavy chain may represent a target for immunotherapeutic interventions in a sizeable fraction of SMZL.

Disclosures: Ghia: Astra Zeneca: Consultancy, Research Funding; Beigene: Consultancy; Abbvie: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Johnson & Johnson: Consultancy, Research Funding; Galapagos: Consultancy; Loxo@Lilly: Consultancy; MSD: Consultancy; Roche: Consultancy. Stamatopoulos: Bristol Myers Squibb: Honoraria; Roche: Research Funding; Novartis: Research Funding; AstraZeneca: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Lilly: Honoraria.

*signifies non-member of ASH