Near-infrared fluorescence imaging of non-Hodgkin's lymphoma CD20 expression using Cy7-conjugated obinutuzumab

Obinutuzumab is the first fully humanized and glycoengineered monoclonal antibody (mAb) directly targeting CD20 antigen, which is expressed on B cell lymphocytes and the majority of non-Hodgkin's lymphoma (NHL). This study aims to design a diagnostic molecular probe, Cy7-Obinutuzumab (Cy7-Obi), in which Cy7 is a near-infrared fluorescent dye. This probe is used to noninvasively image CD20 antigen expressed in NHL cells.

Cy7-Obi probe was synthesized through nucleophilic substitution reaction between NHS-Cy7 and obinutuzumab. After purification, the conjugate was fully characterized by a series of methods. The immunoreactivity and molecular specificity of the probe were confirmed using flow cytometry and in vitro microscopy on Raji (CD20-positive) cells. For in vivo imaging, Cy7-Obi probe (1 nmol) was injected intravenously in severe combined immunodeficiency (SCID) mice bearing Raji tumors which overexpress CD20 (n = 3) and was imaged with near-infrared fluorescence (NIRF) at 6, 9, 12, 24, 60, and 96 h post-probe injection. For pre-block, obinutuzumab (3.25 mg) was injected intravenously in tumor-bearing mice 6 h before the administration of Cy7-Obi probe.

The synthesized Cy7-Obi probe in this paper mimics obinutuzumab in both structure and function. Flow cytometry analysis of the probe and obinutuzumab on Raji cells showed minor difference in binding affinity/specificity with CD20. The probe showed significant fluorescence signal when it was examined on Raji cells using in vitro microscopy. The fluorescence signal can be blocked by pretreatment with obinutuzumab. The probe Cy7-Obi also showed high tumor uptake when it was examined by in vivo optical imaging on Raji tumor-bearing mice. The tumor uptake can be blocked by pretreatment with obinutuzumab (n = 3, p < 0.05). The in vivo imaging results were also confirmed by ex vivo imaging of dissected organs. Finally, the probe Cy7-Obi has shown excellent tumor targeting and specificity through immunofluorescence analysis.

We have shown that humanized Cy7-Obi probe can be used for NIRF imaging successfully. The probe may be an effective and noninvasive diagnostic molecular probe capable of tracking CD20 overexpression in NHL.