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Purpose <p>CD4<sup>+</sup> T cells (T helper and T reg) play an important role in the immune system and are influential in autoimmune diseases (e.g., rheumatoid arthritis, inflammatory bowel disease) and cancer (antitumor immunity). Non-invasive, whole-body anti-CD4 immunoPET can provide dynamic and spatial information (localization, proliferation, and migration) on CD4<sup>+</sup> T cells. The cys-diabody format enables site-specific radiolabeling and rapid renal clearance, which results in high-contrast images at early time points.</p> Procedures <p>In this work, an anti-CD4 cys-diabody based on the hybridoma GK1.5 was reengineered by CDR-grafting (GK1.5 FR cDb) for higher expression in mammalian cell lines. An N-glycosylation motif in the variable light chain domain framework was removed by site-directed mutagenesis, resulting in GK1.5 N80D cDb. To investigate the impact of the variable domain glycan on the <i>in vivo</i> biodistribution and pharmacokinetics, both cys-diabodies were site-specifically conjugated with deferoxamine-maleimide and radiolabeled by chelation of zirconium-89. Serial immunoPET/CT imaging was used for non-invasive, whole-body assessment of specific targeting, biodistribution, and differential clearance of the two novel anti-CD4 cys-diabodies.</p> Results <p>The anti-CD4 cys diabody was successfully re-engineered by CDR-grafting&#xa0;(GK1.5 FR cDb) and aglycosylation (GK1.5 N80D cDb), resulting in a higher expression yield (~ tenfold increase) without impacting antigen specificity or affinity. Both cys-diabody variants were successfully <sup>89</sup>Zr-radiolabeled with similar specific activity and radiochemical purity. ImmunoPET imaging of <sup>89</sup>Zr-GK1.5 FR cDb and <sup>89</sup>Zr-GK1.5 N80D cDb in immunocompetent mice showed CD4 antigen-specific lymphoid tissue uptake in vivo. <sup>89</sup>Zr-GK1.5 FR cDb exhibited rapid hepatic clearance, resulting in significantly reduced uptake in lymph nodes and the spleen. Removal of the N-glycosylation motif in <sup>89</sup>Zr-GK1.5 N80D cDb restored diabody-typical biodistribution (renal clearance), resulting in higher target tissue uptake.</p> Conclusion <p>The novel reengineered anti-CD4 GK1.5 N80D cDb overcomes the previous production yield bottleneck and provides same-day <sup>89</sup>Zr-immunoPET imaging for non-invasive, whole-body visualization of murine CD4<sup>+</sup> T cells.</p> Graphical Abstract <p></p>

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Reengineered Anti-CD4 Cys-diabody Variants for 89Zr-immunoPET of CD4+ T Cells in Immunocompetent Mice

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年8月7日

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  • Felix B. Salazar,
  • Richard Tavaré,
  • Arya Ökten,
  • Maciej Kujawski,
  • Anna M. Wu,
  • Kirstin A. Zettlitz

摘要

Purpose

CD4+ T cells (T helper and T reg) play an important role in the immune system and are influential in autoimmune diseases (e.g., rheumatoid arthritis, inflammatory bowel disease) and cancer (antitumor immunity). Non-invasive, whole-body anti-CD4 immunoPET can provide dynamic and spatial information (localization, proliferation, and migration) on CD4+ T cells. The cys-diabody format enables site-specific radiolabeling and rapid renal clearance, which results in high-contrast images at early time points.

Procedures

In this work, an anti-CD4 cys-diabody based on the hybridoma GK1.5 was reengineered by CDR-grafting (GK1.5 FR cDb) for higher expression in mammalian cell lines. An N-glycosylation motif in the variable light chain domain framework was removed by site-directed mutagenesis, resulting in GK1.5 N80D cDb. To investigate the impact of the variable domain glycan on the in vivo biodistribution and pharmacokinetics, both cys-diabodies were site-specifically conjugated with deferoxamine-maleimide and radiolabeled by chelation of zirconium-89. Serial immunoPET/CT imaging was used for non-invasive, whole-body assessment of specific targeting, biodistribution, and differential clearance of the two novel anti-CD4 cys-diabodies.

Results

The anti-CD4 cys diabody was successfully re-engineered by CDR-grafting (GK1.5 FR cDb) and aglycosylation (GK1.5 N80D cDb), resulting in a higher expression yield (~ tenfold increase) without impacting antigen specificity or affinity. Both cys-diabody variants were successfully 89Zr-radiolabeled with similar specific activity and radiochemical purity. ImmunoPET imaging of 89Zr-GK1.5 FR cDb and 89Zr-GK1.5 N80D cDb in immunocompetent mice showed CD4 antigen-specific lymphoid tissue uptake in vivo. 89Zr-GK1.5 FR cDb exhibited rapid hepatic clearance, resulting in significantly reduced uptake in lymph nodes and the spleen. Removal of the N-glycosylation motif in 89Zr-GK1.5 N80D cDb restored diabody-typical biodistribution (renal clearance), resulting in higher target tissue uptake.

Conclusion

The novel reengineered anti-CD4 GK1.5 N80D cDb overcomes the previous production yield bottleneck and provides same-day 89Zr-immunoPET imaging for non-invasive, whole-body visualization of murine CD4+ T cells.

Graphical Abstract