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

## TL;DR

Researchers improved an anti-CD4 cys-diabody for better production and imaging of CD4+ T cells in mice using immunoPET.

## Contribution

A reengineered anti-CD4 cys-diabody variant with improved expression yield and renal clearance for immunoPET imaging.

## Key findings

- The GK1.5 N80D cDb variant showed higher expression yield and typical renal clearance.
- 89Zr-labeled cys-diabodies enabled specific imaging of CD4+ T cells in immunocompetent mice.
- Removing the N-glycosylation motif improved target tissue uptake and imaging contrast.

## Abstract

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.

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.

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.

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.

The online version contains supplementary material available at 10.1007/s11307-025-02043-y.