Synthesis and Preliminary Assessment of ^99m tc-HYNIC-Fab´s (Rituximab) for Non-Hodgkin Lymphoma Diagnosis

Introduction: Non-Hodgkin lymphoma (NHL) is the most common hematologic malignancy; more than 90% are B cell lymphomas and express CD-20 antigen. Rituximab® (RTX) is an IgG1κ chimeric anti-CD20 mAb that binds specifically to the CD20-antigen on B lymphocytes. Our group previously reported that ^99mTc-RTX represents a promising molecular imaging agent for NHL [1]. When used for tumor imaging, intact IgG exhibits high liver uptake. Antibody fragments (Fab´s) are quickly eliminated from blood and normal tissues (except kidneys), achieving high tumor/blood and tumor/normal tissue ratios with renal clearance. The development of radiolabeled Fab´s directed against specific targets may become a new strategy for NHL staging and surveillance.

Objective: To radiolabel Fab´s (RTX) with ^99mTc and to perform its chemical and biological evaluation.

Methodology: We performed antibody fragmentation with papain and, once purified, fragments were identified by MaldiTOF/TOF and derivatized with Suc-HYNIC as a bifunctional coupling agent. A mixture of Tricine/SnCl₂.2H₂O was added to Fab´s (RTX)-HYNIC and radiolabeled with ^99mTcO₄^-. Radiochemical purity was determined by HPLC. The in-vitro radiochemical stability of the radiolabeled Fab´s were analyzed in saline and serum up to 4 h. In-vitrobinding and competition assays were performed using Ramos and Raji cell lines up to 90 min. Biodistribution studies were evaluated in normal Balb/c mice and in Raji tumor-bearing Nude mice at 0.5 and 1 h.

Results: Radiochemical purity of radiolabeled Fab´s were ≥90%. The in-vitro radiochemical stability studies showed that the radioconjugate was stable and no significant transchelation was detected. In-vitro binding and competition assays confirm that after its derivatization and radiolabeling, Fab´s (RTX) retained its specificity of binding to CD-20 antigen. This results confirm that Fab´s (RTX) affinity for CD20+ NHL cells remained unaffected after its derivatization. In-vivobiodistribution studies show that radiolabeled Fab´s has renal uptake with neglectable uptake in other organs, indicating that the primary route of clearance is renal. Lymph-node/muscle ratios of 4.00 and 2.55 at 0.5 and 1 h post injection, respectively.

Conclusions: Fab´s (RTX) were easily and rapidly labeled demonstrating good stability and radiochemical purity. Based on lymph-node uptake and lymph-node/muscle ratios, ^99mTc-HYNIC-Fab´s (RTX) may be useful for tumor molecular imaging agent for NHL.