The same signal, different fates, the root of the difference between CD28 CAR-T and 4-1BB CAR-T

Synthetic receptors that mimic natural T cell signals are being used in immunotherapy for cancer, autoimmunity, and infection.

Chimeric antigen receptors (CARs), chimeric co-stimulatory receptors, and engineered T cell receptors (TCRs) are used to transfer genes into T cells to specifically redirect T cells, initiate effector T cell activation, and promote T cell proliferation And effect function.

The most successful application is CARs, which consist of extracellular antigen-specific single-chain variable immunoglobulin fragments (scFv) fused to the intracellular activation signal domain of T cells.

CARs exhibit good anti-tumor effects, but they also cause life-threatening cytokine storms and neurotoxicity, which are all related to activated intracellular domain signals.

The basic structure of CAR and the costimulatory domain (Document 1) After the TCR is activated, the ITAM on the CD3 𝜀, 𝛾 chain connected to the TCR is phosphorylated.

Combining signals from costimulatory molecules and cytokines, changes the T cell transcription program, induces proliferation, promotes cytotoxicity, and stimulates the release of cytokines.

The difference CARs between CD28 and 4-1BB CAR-T combine CD3 with the costimulatory molecule CD28 or 4-1BB.

Both CD28/CD3CAR-T and 4-1BB/CD3CAR-T have achieved good results, but there is still a big difference in the cellular behavior of the two.

CD28/CD3 CAR-T began to proliferate vigorously 7 days after infusion, but it rarely persists for more than 60 days.

4-1BB/CD3CAR-T reaches its peak 7-14 days after infusion, and can last for several months thereafter.

4-1BB/CD3 CARs have greater mitochondrial mass, and the cells show more memory phenotypes, and can retain effector functions for a long time under chronic antigen stimulation.

The internal mechanism of the difference between CD28 and 4-1BB CAR-T CD28/CD3 and 4-1BB/CD3CART cell phenotype, function and survival time difference, which mechanism determines? CAR signal pathway and function (Reference 2) through CD28/ The activation signals of CD3 CAR and 4-1BB/CD3CAR will produce almost the same protein phosphorylation events (PLC-γ1, ZAP-70, CAR-CD3 pTyr142, etc.
phosphorylation), suggesting that they use the same signal pathway.

Phosphorylation of signal pathway (Reference 3) Although the two use the same signal pathway, mass spectrometry quantitative results show that the phosphorylation level caused by CD28/CD3 is significantly higher than that of 4-1BB/CD3.

Mass spectrometry quantitative phosphorylation kinetics (Document 3) Under the condition of the same activation marker (CD69) level, CD28/CD3 CAR-T will produce more effector molecules (GZMB, TNF, IFN-G, IL-2, etc.
), This is consistent with the stronger performance of its effect function.

The level of CAR-T effector molecules (document 3) In the case of the same activation marker (CD69) level, CD28/CD3 CAR-T produces a higher level of cell exhaustion markers (immune checkpoint molecules: PD-1, Lag- 3.
Tim-3, etc.
), which is consistent with the longer duration of 4-1BB and longer effect function.

CAR-T depletion marker level (Reference 3) Xiaobian summarized CD28 and 4-1BB are the two most commonly used costimulatory factors for CAR-T.

More and more studies have shown that the CAR-T cell phenotype, effect level and survival time produced by the two are significantly different.

Many top cell therapy research centers in the world have done a lot of research to find the source of the differences between the two.

This article was completed by Fred Hutchinson Cancer Research Center and published in the Science sub-Journal Sci.
Signal.

The research results show that CD28 and 4-1BB use the same signal pathway, but the phosphorylation level caused by CD28 activation is significantly higher than that of 4-1BB, and therefore produces a higher level of effector molecules, so CD28CAR-T has a stronger cell effect function .

But at the same time, CD28 CAR-T also expresses higher levels of depletion markers, which may also explain why its survival time is shorter.

References 1.
Ling Wu et al, Signaling from T cell receptors (TCRs) and chimeric antigen receptors (CARs) on T cells, Cellular & Molecular Immunology, https://doi.
org/10.
1038/s41423-020-0470-32 .
Nico M.
Sievers et al,CARs: Beyond T Cells and T Cell-Derived Signaling Domains,Int.
J.
Mol.
Sci.
2020, 21, 35253.
Alexander I.
Salter et al, Phosphoproteomic analysis of chimeric antigen receptor signaling reveals Kinetic and quantitative differences that affect cell function,Sci.
Signal.
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