Background

[PubMed]

CD3 antigen is a protein complex that is expressed on the T cell surface and is involved in the signal transduction of T cell activation. More than 95% of the circulating T cells in blood and activated T cells in inflamed tissues express CD3 antigen (1-3). Blocking CD3 antigen with monoclonal antibodies (mAbs) has been shown to be effective in suppressing the immunoreaction in inflammatory and graft-versus-host disease conditions. OKT3 is a mouse anti-human CD3 mAb that has been widely used to prevent and treat acute allograft rejection in clinical transplantation (4-6). However, the clinical utility of OKT3 therapy is limited by inducing a neutralizing anti-mouse antibody response and a cytokine release syndrome (7, 8). The syndrome has been thought to be derived from the enormous release of cytokines, particularly TNF-α from T cells in response to the antibody, and to be attributed to the cross-linking of T cells bearing CD3 molecules and Fc receptor (FcR)-bearing immune effector cells that bind to the Fc portion of the antibodies. The cross-linking activates both the T cells and the FcR-bearing cells, leading to the massive release of cytokines (9, 10).

To reduce potential side effects and immunogenicity, Cole et al. generated a humanized version of the OKT3 mAb visilizumab (HuM291) (11). Visilizumab is characterized by decreased binding with FcRs due to the introduced mutations within the IgG₂ Fc at amino acid residues 234 and 237 (Val to Ala), and by decreased ability to activate human complements because of the human IgG₂ M3 isotype. Compared with OKT3 mAb, visilizumab has been shown to be much less mitogenic to T cells and to induce less toxicity from cytokine release in vivo, while retaining the ability to modulate the CD3 complex and inhibit the mixed lymphocyte reaction. The potential therapeutic effects of visilizumab have been examined in several immune system–related disorders, including ulcerative colitis, Crohn's disease, renal allograft rejection, and acute graft-versus-host disease (8, 12, 13)., However, visilizumab induces a rapid and severe lymphopenia after infusion in humans (12). The reason for this transient lymphopenia is unknown because the path of migration from blood after visilizumab infusion has not been clarified. Malviya et al. radiolabeled visilizumab with ^99mTc and checked its in vitro and in vivo specificity for CD3-positive cells (2). The design of this study was based on their hypothesis that the use of a radiolabeled antibody against CD3 antigen might assist in the selective detection of resting and activated T cells and thus serve as a diagnostic tool for imaging T cell traffic and lymphocytic infiltration of tissues and organs affected in autoimmune diseases. Ultimately, the use of a radiolabeled antibody would provide a rationale for treatment with anti-CD3 mAb and would serve as a tool for therapy follow-up.

Synthesis

[PubMed]

To label visilizumab and the control antibody, Malviya et al. tested both direct and indirect labeling methods (2). The control mAb was a humanized IgG₂ antibody without FcR and without CD3 binding. The direct labeling with ^99mTc was achieved with the 2-mercapthoethanol method. The indirect labeling was completed first by conjugating the heterobifunctional linker succinimidyl-6-hydrazinonicotinate hydrochloride (SHNH) with the mAb to obtain a SHNH-mAb conjugate, and then by coupling the SHNH-mAb complex with ^99mTc to obtain the desired product, ^99mTc-SHNH–mAb. With the direct method, a high labeling efficiency (~99%) with a high specific activity (≥2,600 MBq/mg (70.3 mCi/mg)) was achieved for the control mAb but not for visilizumab. The directly labeled control mAb was then used in all experiments. With the indirect method, a high labeling efficiency (≥90%) with a high specific activity (10,360–11,100 MBq/mg (280–300 mCi/mg) was obtained for visilizumab but not for the control mAb (labeling efficiency: <40%; specific activity: <4,440 MBq/mg (<120 mCi/mg)). An average of 4–10 hydrazino groups were conjugated to each molecule of visilizumab. The indirect labeling method was then used to label visilizumab, and the labeled product ^99mTc-SHNH–visilizumab was used in all experiments. The reason for the differences of labeling efficiency and specific activity between the two antibodies was not given. Both ^99mTc-SHNH–visilizumab and the ^99mTc-labeled control mAb were stable in human serum for up to 24 h at 37°C (87% for visilizumab and 89% for control Ab). The cysteine challenge assay also demonstrated high stability of the radiolabeled mAbs up to a 500:1 ratio between cysteine and the radiolabeled mAb. Sodium dodecyl sulphate polyacrylamide gel electrophoresis confirmed that visilizumab (in molecular weight) was not modified by the labeling procedure, as shown by a single band at 150 kDa.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Malviya et al. first analyzed the CD3 expression on human peripheral blood mononuclear cells (hPBMCs), human lymphoma cells (HuT78), T cell–derived human acute lymphoblastic leukemia cells (CEM), and immortalized T lymphocytes (Jurkat cell line) with fluorescence-activated cell sorter analysis after staining cells with a fluorescein isothiocyanate–conjugated anti-CD3 antibody (2). The investigators observed a mean fluorescence intensity of 300 for hPBMCs (80% positive cells), 38 for CEM (50% positive cells), 18 for HuT78 (>95% positive cells), and 30 for Jurkat cells (60% positive cells), indicating different CD3 expression levels among the cell lines. hPBMCs and HuT78 were then used for experiments. Specific binding saturation on HuT78 cells was obtained at ^99mTc-SHNH–visilizumab concentrations of more than 5 × 10^-10 mol, and ^99mTc-SHNH–visilizumab binding was displaced by a 100-fold molar excess of unlabeled antibody, indicating that ^99mTc-SHNH–visilizumab retained its specific binding activity with CD3 antigens expressed on HuT78 cells. The immunoreactive fraction of ^99mTc-SHNH–visilizumab on hPBMCs in two experiments was 68% and 75%, respectively.

Animal Studies

Human Studies

[PubMed]

No references are currently available.

References

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Malviya G., D'Alessandria C., Bonanno E., Vexler V., Massari R., Trotta C., Scopinaro F., Dierckx R., Signore A. Radiolabeled humanized anti-CD3 monoclonal antibody visilizumab for imaging human T-lymphocytes. J Nucl Med. 2009;50(10):1683–91. [PubMed: 19759100]

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Cole M.S., Stellrecht K.E., Shi J.D., Homola M., Hsu D.H., Anasetti C., Vasquez M., Tso J.Y. HuM291, a humanized anti-CD3 antibody, is immunosuppressive to T cells while exhibiting reduced mitogenicity in vitro. Transplantation. 1999;68(4):563–71. [PubMed: 10480417]

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