# Differential substrate degradation by super-degron: EGFP in wild-type mouse cells, PD-1 requires CRBN humanization

## TL;DR

A new method using a super-degron tag and thalidomide analogs can degrade specific proteins in mouse cells, but some proteins require humanized components for effective degradation.

## Contribution

The super-degron tag combined with iberdomide enables protein degradation in wild-type mouse cells, with PD-1 degradation requiring CRBN humanization.

## Key findings

- SD-tagged EGFP was degraded in wild-type mouse cells using iberdomide or mezigdomide.
- SD-tagged PD-1 required human-type CRBNI391V for degradation in mouse cells.
- Iberdomide suppressed tumor growth faster than anti-PD-1 antibodies by degrading PD-1 in vivo.

## Abstract

Protein knockdown using a zinc finger degron tag and thalidomide analogs was previously considered ineffective in mouse cells. However, using EGFP as an indicator, we found that a super-degron tag (SD) enables degradation in mouse cells when combined with iberdomide or mezigdomide. While SD-tagged EGFP was degraded in wild-type mouse cells, SD-tagged PD-1 required human-type CEREBLON (CRBNI391V) for degradation. In mice with CRBNI391V, endogenous PD-1 tagged with SD was efficiently degraded in T cells both in vitro and in vivo. Compared with anti-PD-1 antibody treatment, the degradation of PD-1 led to more rapid activation of CD8+ T cells. Moreover, pomalidomide that crosses the brain-blood barrier reduced PD-1 expression in the brain. These results suggest that SD and thalidomide analogs can be used for in vitro and in vivo protein knockdown in mice, although some conditional settings are required.

•Iberdomide induced degradation of SD-tagged EGFP in mouse, rat, and human cells•CRBN humanization enhanced degradation efficiency by 10-100 fold in mouse cells•Unlike EGFP, SD-tagged PD-1 was not degraded in wild-type mouse cells•In vivo, iberdomide suppressed MC-38 tumor growth faster than an antibody by degrading SD-tagged PD-1

Iberdomide induced degradation of SD-tagged EGFP in mouse, rat, and human cells

CRBN humanization enhanced degradation efficiency by 10-100 fold in mouse cells

Unlike EGFP, SD-tagged PD-1 was not degraded in wild-type mouse cells

In vivo, iberdomide suppressed MC-38 tumor growth faster than an antibody by degrading SD-tagged PD-1

Natural sciences; Biological sciences; Immunology