Checkpoint inhibitors, anti-PD1/anti-PD-L1 activities are channeled via CD28 co-stimulation

Two new back-to-back studies in Science showed that T cell recovery activity attributed to checkpoint inhibitors, anti-PD1/anti-PD-L1, is mediated via CD28 co-stimulation. One of the study focused on analysis of biochemical events leading to PD1 signaling in an in vitro reconstitution model and another study provided corroborating data using mouse model. 

Summary results from both studies suggest that anti-PD1/PD-L1 activity is lost when T cells lack co-stimulatory molecule CD28. However, readouts here are more complicated since PD-L1 itself could bind to CD28 ligand B7 molecule. Also, I noticed that by default T cells with inducible CD28 deficiency (CD28^f/f Cre^ERT2+) show less accumulation and/or survival (low cell density) compared to WT counterparts.

Another confusion has to do with the fact that at least in one of the clinical trials anti-CD28 antibodies produced severe cytokine release syndrome and program was discontinued. It could be that anti-PD1/PDL1 therapy activates only certain type of T cells while anti-CD28 could have targeted much larger T cell population.

David Usharauli

  

In vitro comparison of human CAR-T cells expressing either CD28 or 4-1BB costimulatory domains

Today journal Immunity published new [short] study from Carl June's lab describing some functional and metabolic differences between CAR-T cells expressing CD28 or 4-1BB costimulatory domains.

It is not a particularly cutting-edge study. But since its focus is human CAR-T cells such limited experiments are still expected. Though I will anticipate that soon publications of in vitro studies on human primary cells [in top journals] will require incorporation of specific gene targeting experiments using CRISPR-Cas9 technology. Such experiments are now technically feasible and will be more informative.

In short, for this study the authors compared proliferation/expansion and metabolic/energy source of two CAR constructs, one with CD28 and another with 4-1BB (aka CD137) signaling domains, as shown below. FMC63 is anti-CD19 scFv and SS1 is anti-mesothelin scFv.

Human primary T cells were electroporated with these two CAR constructs. Efficacy of CAR expression was 90%.

Then, these transduced CAR-T cells were stimulated with magnetic beads coated with a recombinant anti-CD19 or mesothelin-Fc (such stimulation allowed selective activation of CAR-T cells via CAR construct). It revealed that CAR construct with 4-1BB domain imparted superior long-term expansion functionality on CAR-T cells.

Metabolic analysis revealed that unlike CD28 construct, 4-1BB CAR-T cells showed enrichment for genes responsible for lipid oxidation, implying 4-1BB signaling activates metabolic pathway resembling that of naive and memory T cells (involving mitochondrial oxidation of free fatty acids).

Indeed, transmission electron microscopy data confirmed that 4-1BB CAR-T cells were enriched for mitochondria. 

In summary, this study provided supporting evidence that 4-1BB CAR construct endows T cells with superior expansion/persistence quality. Initial studies on this topic suggested that CD28 CAR construct displayed superior "effector" activity against tumor, while 4-1BB CAR construct showed longer survival [superior memory function]. Subject is not settled, but in discussion section, the authors do note that mixing both CAR constructs could provide better outcome compared to individual construct.

David Usharauli