T-cell engineered with a fully humanized B-cell maturation antigen-specific T-cell antigen coupler receptor effectively target multiple myeloma.

B-cell maturation antigen (BCMA) is a clinically validated target for multiple myeloma. T-cell engineered with chimeric antigen receptors (CARs) directed against BCMA have demonstrated robust therapeutic activity in clinical trials, but toxicities remain a significant concern for a subset of patients, supporting continued investigation of other engineered T-cell platforms that may offer equal efficacy with an improved toxicity profile. The authors recently described a BCMA-specific, T-cell-centric synthetic antigen receptor, the T-cell antigen coupler (TAC) receptor, that can be used to engineer T-cell with robust anti-myeloma activity. Here the authors describe the creation of a fully humanized BCMA-specific TAC receptor. Single-chain variable fragments (scFvs) were developed from BCMA-specific F(ab)s that were identified in a fully human phage display library. Twenty-four configurations of the F(ab)s were evaluated in a medium-throughput screening using primary T-cell, and a single F(ab), TRAC 3625, emerged as the most robust following in vitro and in vivo evaluation. An optimized BCMA-specific TAC receptor was developed through iterations of the BCMA-TAC design that evaluated a next-generation TAC scaffold sequence, different domains connecting the TAC to the 3625 scFv and different orientations of the TRAC 3625 heavy and light variable regions.

The mammalian-membrane two-hybrid assay (MaMTH) for probing membrane-protein interactions in human cells.

Cell signaling, one of key processes in both normal cellular function and disease, is coordinated by numerous interactions between membrane proteins that change in response to stimuli. We present a split ubiquitin-based method for detection of integral membrane protein-protein interactions (PPIs) in human cells, termed mammalian-membrane two-hybrid assay (MaMTH). We show that this technology detects stimulus (hormone or agonist)-dependent and phosphorylation-dependent PPIs. MaMTH can detect changes in PPIs conferred by mutations such as those in oncogenic ErbB receptor variants or by treatment with drugs such as the tyrosine kinase inhibitor erlotinib. Using MaMTH as a screening assay, we identified CRKII as an interactor of oncogenic EGFR(L858R) and showed that CRKII promotes persistent activation of aberrant signaling in non-small cell lung cancer cells. MaMTH is a powerful tool for investigating the dynamic interactomes of human integral membrane proteins.

Bypass of glycan-dependent glycoprotein delivery to ERAD by up-regulated EDEM1.

Trimming of mannose residues from the N-linked oligosaccharide precursor is a stringent requirement for glycoprotein endoplasmic reticulum (ER)-associated degradation (ERAD). In this paper, we show that, surprisingly, overexpression of ER degradation-enhancing α-mannosidase-like protein 1 (EDEM1) or its up-regulation by IRE1, as occurs in the unfolded protein response, overrides this requirement and renders unnecessary the expression of ER mannosidase I. An EDEM1 deletion mutant lacking most of the carbohydrate-recognition domain also accelerated ERAD, delivering the substrate to XTP3-B and OS9. EDEM1 overexpression also accelerated the degradation of a mutant nonglycosylated substrate. Upon proteasomal inhibition, EDEM1 concentrated together with the ERAD substrate in the pericentriolar ER-derived quality control compartment (ERQC), where ER mannosidase I and ERAD machinery components are localized, including, as we show here, OS9. We suggest that a nascent glycoprotein can normally dissociate from EDEM1 and be rescued from ERAD by reentering calnexin-refolding cycles, a condition terminated by mannose trimming. At high EDEM1 levels, glycoprotein release is prevented and glycan interactions are no longer required, canceling the otherwise mandatory ERAD timing by mannose trimming and accelerating the targeting to degradation.

Mannose trimming is required for delivery of a glycoprotein from EDEM1 to XTP3-B and to late endoplasmic reticulum-associated degradation steps.

Although the trimming of α1,2-mannose residues from precursor N-linked oligosaccharides is an essential step in the delivery of misfolded glycoproteins to endoplasmic reticulum (ER)-associated degradation (ERAD), the exact role of this trimming is unclear. EDEM1 was initially suggested to bind N-glycans after mannose trimming, a role presently ascribed to the lectins OS9 and XTP3-B, because of their in vitro affinities for trimmed oligosaccharides. We have shown before that ER mannosidase I (ERManI) is required for the trimming and concentrates together with the ERAD substrate and ERAD machinery in the pericentriolar ER-derived quality control compartment (ERQC). Inhibition of mannose trimming prevents substrate accumulation in the ERQC. Here, we show that the mannosidase inhibitor kifunensine or ERManI knockdown do not affect binding of an ERAD substrate glycoprotein to EDEM1. In contrast, substrate association with XTP3-B and with the E3 ubiquitin ligases HRD1 and SCF(Fbs2) was inhibited. Consistently, whereas the ERAD substrate partially colocalized upon proteasomal inhibition with EDEM1, HRD1, and Fbs2 at the ERQC, colocalization was repressed by mannosidase inhibition in the case of the E3 ligases but not for EDEM1. Interestingly, association and colocalization of the substrate with Derlin-1 was independent of mannose trimming. The HRD1 adaptor protein SEL1L had been suggested to play a role in N-glycan-dependent substrate delivery to OS9 and XTP3-B. However, substrate association with XTP3-B was still dependent on mannose trimming upon SEL1L knockdown. Our results suggest that mannose trimming enables delivery of a substrate glycoprotein from EDEM1 to late ERAD steps through association with XTP3-B.