Avaliação da ligação entre o CD22 e scFvs com linkers curto e longo usados em terapias CAR-T cell anti-CD22

• Ano de defesa: 2025
• Autor(a) principal: Pereira, Liandra Éllen Coelho
• Orientador(a): Lourenzoni, Marcos Roberto
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Área do conhecimento CNPq: CNPQ::CIENCIAS BIOLOGICAS
• Link de acesso: http://repositorio.ufc.br/handle/riufc/80389

Immunotherapy has emerged as an innovative strategy in the biotechnology sector for treating patients with advanced-stage cancers. Acute Lymphocytic Leukemia is one of the most aggressive forms of hematological cancer, and Adoptive Cell Therapy based on CAR (Chimeric Antigen Receptor) has stood out as a promising approach to achieving remission of this neoplasm. Among the CAR variants, studies based on the m971 antibody, which recognizes the CD22 protein present in B cells, have gained relevance. The CAR structure includes several domains such as the single-chain variable fragment (scFv), which is responsible for antigen recognition. This fragment is composed of the VH and VL domains of an antibody, connected by a linker. The linker length directly influences the flexibility, functionality, binding affinity, and stability of the antibody. In this context, the objective of this study was to produce scFvs with short- and long-linkers, derived from the m971 antibody, using a bacterial system for their expression and purification, as well as to evaluate their binding to CD22. The conformational stability and oligomerization state of the scFvs were investigated, along with the kinetics of interaction with the antigen. To achieve this, protocols for scFv expression in Escherichia coli and purification were standardized. The conformational stability and oligomerization state were assessed through different biophysical approaches. The binding of scFvs to CD22 was analyzed by flow cytometry using Raji B cell lines and biolayer interferometry assays to quantify interaction kinetics. The results indicated that the scFv with a long-linker had a higher purification yield than the scFv with a short-linker. Conformational stability analysis revealed differences in the structural profiles of the scFvs. Size-exclusion chromatography demonstrated that both scFvs form monomers, with secondary dimer formation observed for the scFv with a short-linker. Besides, the presence of the SUMO protein at the N-terminal region of the scFvs affected antigen binding, requiring its removal before the assays. Both scFvs bound to CD22; however, the scFv with a short-linker exhibited greater binding capacity at lower concentrations. The obtained affinity constants confirmed that the scFv with a short-linker has a stronger binding to CD22, corroborating the flow cytometry results. These findings contribute to a better understanding of the structural and functional properties of these scFvs, providing robust scientific foundations for the development of improved scFvs or other types of binding fragments.