Allogeneic CD20-targeted γδ T cells exhibit innate and adaptive antitumor activities in preclinical B-cell lymphoma models.

OBJECTIVES: Autologous chimeric antigen receptor (CAR) αß T-cell therapies have demonstrated remarkable antitumor efficacy in patients with haematological malignancies; however, not all eligible cancer patients receive clinical benefit. Emerging strategies to improve patient access and clinical responses include using premanufactured products from healthy donors and alternative cytotoxic effectors possessing intrinsic tumoricidal activity as sources of CAR cell therapies. γδ T cells, which combine innate and adaptive mechanisms to recognise and kill malignant cells, are an attractive candidate platform for allogeneic CAR T-cell therapy. Here, we evaluated the manufacturability and functionality of allogeneic peripheral blood-derived CAR+ Vδ1 γδ T cells expressing a second-generation CAR targeting the B-cell-restricted CD20 antigen. METHODS: Donor-derived Vδ1 γδ T cells from peripheral blood were ex vivo-activated, expanded and engineered to express a novel anti-CD20 CAR. In vitro and in vivo assays were used to evaluate CAR-dependent and CAR-independent antitumor activities of CD20 CAR+ Vδ1 γδ T cells against B-cell tumors. RESULTS: Anti-CD20 CAR+ Vδ1 γδ T cells exhibited innate and adaptive antitumor activities, such as in vitro tumor cell killing and proinflammatory cytokine production, in addition to in vivo tumor growth inhibition of B-cell lymphoma xenografts in immunodeficient mice. Furthermore, CD20 CAR+ Vδ1 γδ T cells did not induce xenogeneic graft-versus-host disease in immunodeficient mice. CONCLUSION: These preclinical data support the clinical evaluation of ADI-001, an allogeneic CD20 CAR+ Vδ1 γδ T cell, and a phase 1 study has been initiated in patients with B-cell malignancies (NCT04735471).

Arv1 regulates PM and ER membrane structure and homeostasis but is dispensable for intracellular sterol transport.

The pan-eukaryotic endoplasmic reticulum (ER) membrane protein Arv1 has been suggested to play a role in intracellular sterol transport. We tested this proposal by comparing sterol traffic in wild-type and Arv1-deficient Saccharomyces cerevisiae. We used fluorescence microscopy to track the retrograde movement of exogenously supplied dehydroergosterol (DHE) from the plasma membrane (PM) to the ER and lipid droplets and high performance liquid chromatography to quantify, in parallel, the transport-coupled formation of DHE esters. Metabolic labeling and subcellular fractionation were used to assay anterograde transport of ergosterol from the ER to the PM. We report that sterol transport between the ER and PM is unaffected by Arv1 deficiency. Instead, our results indicate differences in ER morphology and the organization of the PM lipid bilayer between wild-type and arv1Δ cells suggesting a distinct role for Arv1 in membrane homeostasis. In arv1Δ cells, specific defects affecting single C-terminal transmembrane domain proteins suggest that Arv1 might regulate membrane insertion of tail-anchored proteins involved in membrane homoeostasis.

Vesicle trafficking from a lipid perspective: Lipid regulation of exocytosis in Saccharomyces cerevisiae.

The protein cargo transported by specific types of vesicles largely defines the different secretory trafficking pathways operating within cells. However, mole per mole the most abundant cargo contained within transport vesicles is not protein, but lipid. Taking a "lipid-centric" point-of-view, we examine the importance of lipid signaling, membrane lipid organization and lipid metabolism for vesicle transport during exocytosis in budding yeast. In fact, the essential requirement for some exocytosis regulatory proteins can be bypassed by making simple manipulations of the lipids involved. During polarized exocytosis the sequential steps required to generate post-Golgi vesicles and target them to the plasma membrane (PM) involves the interplay of several types of lipids that are coordinately linked through PI4P metabolism and signaling. In turn, PI4P levels are regulated by PI4P kinases, the Sac1p PI4P phosphatase and the yeast Osh proteins, which are homologs of mammalian oxysterol-binding protein (OSBP). Together these regulators integrate the transitional steps required for vesicle maturation directly through changes in lipid composition and organization.