It is a summary of an article published by Cell Press Publishers to genetically target E3 ligases to enhance CAR T therapy

1. https://www.cell.com/cell-chemical-biology/abstract/S2451-9456(23)00386-0 1

2. About me
• Shafi Ullah from Pakistan
• Ph.D. in Chemical Biology,
Shanghai Jiao Tong University,
School of Pharmacy
• Advisor: Professor Zhōu Hǔchén (周虎臣)
• Postdoctoral Researcher in Professor
Zhāng Míngmíng’s (张明明) group
2/16

3. Author affiliation
3/16

4. Max Jan MD, PhD
4/16

5. Ubiquitin proteosome system
Hijack
E3-ligase system
via PROTACs
5/16
Ubiquitin proteosome system is the
physiological protein degradation
pathway for misfolded/damaged proteins

6. PROTACs-Target protein degraders
6/16
Targeted protein degradation PROTAC is a suitable strategy for undruggable proteins such as
GPCRs, Kinases and Ion channels that can not effectively be targeted by traditional drugs.
PROTACs does not need to bind to the druggable pocket.
Many of the proteins that suppress cell therapies remain undruggable, and the genes that encode
them are often not amenable to genetic knockdown or knockout.

7. Theme of the study
(transgene-based targeted protein degradation)
1. Inducible protein-protein interactions to control endogenous protein abundance are an
alternative approach to rewire cellular circuitry and target suppressive pathways in cellular
immunotherapies by exploiting ubiquitin proteasome system.
2. Inspired by PROTAC technology, this study reported the development of all-human
sequence E3-recruiting fusion proteins, termed synthetic substrate receptors (SySRs) to
induce conditional, multi-specific degradation of sets of proteins that limit CAR T cell
potency.
3. The group developed SySRs targeting the multiple SMAD transcription factors
(SMAD2/3) that enforce T cell-suppressive TGFβ signaling. SMAD degradation
enhanced CAR T cell proliferation and anti-tumor function.
Glycine-Serine
Linker SMAD2/3 binding
domains
Activated E3 ligase will degrade SMAD2/3 via Ubiquitin-Proteasome System
SMAD2
SMAD3
E3 ligase-binding
domains (SySRs)
E3
ligase
Ub
7/16

8. Reprogramming E3 ligase specificity against GFP using synthetic
substrate receptors (SySRs) for platform optimization (Figure 1)
SySRs were successfully designed to degrade GFP in multiple cell lines without affecting normal cell
function of CRL5 E3 ligase.
Note: SOCS2 is one of multiple SOCS box containing proteins that bind to the CRL5 E3 ligase and act
as substrate receptors (SySRs) to direct CRL5 to target proteins for degradation. 8/16

9. Designing targeted transgenes against SARA-SMAD2/3 (Figure 2)
SMAD2 and SMAD3 transcription factors are recruited to the cell surface and TGFβ-family receptors
by the protein SARA. Hence, they created a SARA-SOCS2 fusion protein and this putative SMAD
degrader induced the depletion of SMAD2-GFP and SMAD3-GFP fusion proteins in Jurkat cells. 9/16

10. Designing drug-inducible transgenes against GFP & SMAD2/3
(Figure S2)
Designing Lenalidomide-inducible transgens: Zinc-finger domain whose association with the
CRL4CRBN E3 ubiquitin ligase is conditional upon the addition of the molecular glue anti-cancer drug
lenalidomide. Yet its effect was partial. 10/16

11. Designing synthetic circuit-inducible transgenes against GFP & SMAD2/3
(Figure 2)
Designing a synNotch circuit in which a CD19 sensor induced the expression of a GFP degrader
transcriptional response element as well as that of endogenous SMAD2/SMAD3.
11/16

12. Designing SMAD2/3 degrader CAR T cells (Figure 3)
The SMAD degrader CAR T cells had
reduced CAR surface abundance with
mult-cistronic transgene however,
SMAD degrader CAR T cells had
higher cytolysis ability and
proliferated more than conventional
CAR T cells in the presence of TGFβ.
12/16

13. SMAD2/3 degrader CAR T cells inhibits transcriptional response of CAR T
cells to TGFβ (Figure 5)
The gene expression profiling of 780
genes identified 24 genes in
conventional CAR T cells that were
upregulated. Similarly they identified
24 genes in conventional CAR T cells
that were downregulated with TGFβ.
The expression of TGFβ-responsive
genes was also influenced in SMAD
degrader CAR T cells.
13/16

14. SMAD2/3 degrader CAR T cells enhanced in vivo CAR T cell early
expansion and anti-tumor potency in murine xenograft model (Figure 6)
The SMAD degrader CAR T cells depleted tumor cells in all mice groups and
proliferated in relatively higher number in peripheral blood, yet comparable in
bone marrow and spleen.
14/16

15. Summary
A multi-specific SMAD degrader transgene reduced CAR T cell responsiveness to TGFβ
by genetic retargeting of E3 ligase and enhanced anti-tumor potency.
15/16

16. Make it a great day