Drug-regulated CD33-targeted CAR T cells control AML using clinically optimized rapamycin dosing.

Chimeric antigen receptor (CAR) designs that incorporate pharmacologic control are desirable; however, designs suitable for clinical translation are needed. We designed a fully human, rapamycin-regulated drug product for targeting CD33+ tumors called dimerizaing agent-regulated immunoreceptor complex (DARIC33). T cell products demonstrated target-specific and rapamycin-dependent cytokine release, transcriptional responses, cytotoxicity, and in vivo antileukemic activity in the presence of as little as 1 nM rapamycin. Rapamycin withdrawal paused DARIC33-stimulated T cell effector functions, which were restored following reexposure to rapamycin, demonstrating reversible effector function control. While rapamycin-regulated DARIC33 T cells were highly sensitive to target antigen, CD34+ stem cell colony-forming capacity was not impacted. We benchmarked DARIC33 potency relative to CD19 CAR T cells to estimate a T cell dose for clinical testing. In addition, we integrated in vitro and preclinical in vivo drug concentration thresholds for off-on state transitions, as well as murine and human rapamycin pharmacokinetics, to estimate a clinically applicable rapamycin dosing schedule. A phase I DARIC33 trial has been initiated (PLAT-08, NCT05105152), with initial evidence of rapamycin-regulated T cell activation and antitumor impact. Our findings provide evidence that the DARIC platform exhibits sensitive regulation and potency needed for clinical application to other important immunotherapy targets.

Comparable dimerization found in wildtype and familial Alzheimer's disease amyloid precursor protein mutants.

Alzheimer's disease (AD) is a progressive and fatal neurodegenerative disorder marked by memory impairment and cognitive deficits. A major component of AD pathology is the accumulation of amyloid plaques in the brain, which are comprised of amyloid beta (Aß) peptides derived from the amyloidogenic processing of the amyloid precursor protein (AßPP) by ß- and γ-secretases. In a subset of patients, inheritance of mutations in the AßPP gene is responsible for altering Aß production, leading to early onset disease. Interestingly, many of these familial mutations lie within the transmembrane domain of the protein near the GxxxG and GxxxA dimerization motifs that are important for transmembrane interactions. As AßPP dimerization has been linked to changes in Aß production, it is of interest to know whether familial AßPP mutations affect full-length APP dimerization. Using bimolecular fluorescence complementation (BiFC), blue native gel electrophoresis, and live cell chemical cross-linking, we found that familial Alzheimer's disease (FAD) mutations do not affect full-length AßPP dimerization in transfected HEK293 and COS7 cells. It follows that changes in AßPP dimerization are not necessary for altered Aß production, and in FAD mutations, changes in Aß levels are more likely a result of alternative proteolytic processing.

Lowering of amyloid beta peptide production with a small molecule inhibitor of amyloid-ß precursor protein dimerization.

The amyloid ß precursor protein (APP) is a single-pass transmembrane glycoprotein that is ubiquitously expressed in many cell types, including neurons. Amyloidogenic processing of APP by ß- and γ-secretases leads to the production of amyloid-ß (Aß) peptides that can oligomerize and aggregate into amyloid plaques, a characteristic hallmark of Alzheimer's disease (AD) brains. Multiple reports suggest that dimerization of APP may play a role in Aß production; however, it is not yet clear whether APP dimers increase or decrease Aß and the mechanism is not fully understood. To better understand the relationship between APP dimerization and production of Aß, a high throughput screen for small molecule modulators of APP dimerization was conducted using APP-Firefly luciferase enzyme complementation to detect APP dimerization. Selected modulators identified from a compound library of 77,440 compounds were tested for their effects on Aß generation. Two molecules that inhibited APP dimerization produced a reduction in Aß levels as measured by ELISA. The inhibitors did not change sAPPα or γ-CTF levels, but lowered sAPPß levels, suggesting that blocking the dimerization is preventing the cleavage by ß-secretase in the amyloidogenic processing of APP. To our knowledge, this is the first High Throughput Screen (HTS) effort to identify small molecule modulators of APP dimerization. Inhibition of APP dimerization has previously been suggested as a therapeutic target in AD. The findings reported here further support that modulation of APP dimerization may be a viable means of reducing the production of Aß.