Preparation and preliminary bioevaluation studies of ⁶⁸Ga‐NOTA‐rituximab fragments as radioimmunoscintigraphic agents for non‐Hodgkin lymphoma

Abstract

Rituximab is used for the treatment of non‐Hodgkin lymphoma (NHL). This study focuses on development of 68Ga‐labeled rituximab fragments, (68Ga‐NOTA‐F (ab′)‐rituximab and 68Ga‐NOTA‐F (ab′)2‐rituximab, as PET‐imaging agents for NHL. Rituximab was digested with immobilized pepsin and papain to yield F (ab′)2 and Fab fragments respectively that were characterized by size exclusion HPLC (SE‐HPLC) and SDS‐PAGE. They were conjugated with p‐SCN‐Bn‐NOTA, labeled with 68Ga and characterized by SE‐HPLC. Intact rituximab was labeled with gallium‐68 for comparison. Specificity of 68Ga‐labeled immunoconjugates was ascertained by immunoreactivity and cell binding studies in Raji cells, while biodistribution studies were performed in normal Swiss mice. Gradient SDS‐PAGE under nonreducing condition showed molecular weights of F (ab′)2‐rituximab and F (ab′)‐rituximab as approximately 100 and 40 Kd, respectively. Radiochemical purity (RCP) of 68Ga‐NOTA‐F (ab′)2‐rituximab and 68Ga‐NOTA‐F (ab′)‐rituximab were 98.2 ± 0.5% and 98.8 ± 0.2% respectively with retention times of 17.1 ± 0.1 min and 19.3 ± 0.1 min in SE‐HPLC. 68Ga‐labeled rituximab fragments were stable in saline and serum up to 2‐hour post preparation and exhibited specificity to CD20 antigen. Immunoreactivity of 68Ga‐labeled immunoconjugates was greater than 80%. Clearance of the fragmented radioimmunoconjugates was predominantly through renal route. Preliminary results from this study demonstrate the potential of 68Ga‐ NOTA‐F (ab′)2‐rituximab and 68Ga‐NOTA‐F (ab′)‐rituximab as PET imaging agents for NHL.

Noninvasive Evaluation of CD20 Expression Using 64Cu-Labeled F(ab′)2 Fragments of Obinutuzumab in Lymphoma

“…Moreover, both 64 Cu-NOTA-F(ab9) 2 -obinutuzumab in CCL-155 tumors and 64 Cu-NOTA-F(ab9) 2 -IgG in Ramos tumors showed significantly lower tumor contrast at 48 h after injection (respectively: T/B 5 1.5 6 0.5 and 1.1 6 0.3; T/M 5 7.9 6 4.0 and 4.6 6 2.4). Although 68 Ga-labeled F(ab) and F(ab9) 2 for rituximab have previously been developed, they were evaluated at only the cellular level (21). Therefore, our study confirmed that 64 Cu-labeled F(ab9) 2 -obinutuzumab is promising for evaluating the differential CD20 expression of lymphoma noninvasively.…”

“…Although antibody fragments can significantly enhance blood clearance and tumor contrast, these small tracers typically undergo rapid renal filtration and elimination (16). Radiolabeled agents of similar or smaller size would also have high renal uptake (21). These lead to high kidney accumulation and may induce nephrotoxicity.…”

Invasive ductal carcinoma and small lymphocytic lymphoma/chronic lymphocytic leukemia manifesting as a collision breast tumor: A case report and literature review

“…For collision breast tumors of BC and NHL, the use of some specially formulated contrast agents may be helpful for differentiating. The potential of 68Ga-NOTA-F (ab′)2-rituximab and 68Ga-NOTA-F (ab′)-rituximab as PET imaging agents for NHL has been reported [ 19 ].…”

Does the Number of Bifunctional Chelators Conjugated to a mAb Affect the Biological Activity of Its Radio-Labeled Counterpart? Discussion Using the Example of mAb against CD-20 Labeled with 90Y or 177Lu

“…The most popular procedure for determining the number of DOTA molecules conjugated to the antibody is the colorimetric method measuring the absorbance of a solution containing complexes of metal ions (such as Cu 2+ , Pb 2+ , Y 3+ , or Tb 3+ ) and Arsenazo­(III). It measures the number of mAb-bound metal chelator molecules available for metal binding. ,− Those methods bring some benefits like simplicity and relatively fast determination. However, the main limitation is the low sensitivity in the micromolar range, so the accuracy of the DOTA/mAb ratio is very low.…”

“…A few literature reports cover the use of rats in research on radiolabeled Rituximab, ,− but the overwhelming majority of the reports relate to the murine model, Table . The latter model encompasses both healthy ,− ,,, and immunodeficient mouse strain, that is, NOD/SCID and Nude. ,,, Transplantable xenograft models were subcutaneously grafted with RAJI, DAUDI, or RAMOS cells from existing cell lines, not from the primary tumor origin (patient-derived). The advantage of this approach was undoubtedly rapid result, relative simplicity, high yield, usefulness as first step investigation, determining in vivo proof-of-concept from in vitro findings like immunoreactivity and examining the efficacy of radiotherapeutics on human tumor cells.…”