Antitumor activity of AZD0754, a dnTGFßRII-armored, STEAP2-targeted CAR-T cell therapy, in prostate cancer.

Prostate cancer is generally considered an immunologically "cold" tumor type that is insensitive to immunotherapy. Targeting surface antigens on tumors through cellular therapy can induce a potent antitumor immune response to "heat up" the tumor microenvironment. However, many antigens expressed on prostate tumor cells are also found on normal tissues, potentially causing on-target, off-tumor toxicities and a suboptimal therapeutic index. Our studies revealed that six-transmembrane epithelial antigen of prostate-2 (STEAP2) was a prevalent prostate cancer antigen that displayed high, homogeneous cell surface expression across all stages of disease with limited distal normal tissue expression, making it ideal for therapeutic targeting. A multifaceted lead generation approach enabled development of an armored STEAP2 chimeric antigen receptor T cell (CAR-T) therapeutic candidate, AZD0754. This CAR-T product was armored with a dominant-negative TGF-ß type II receptor, bolstering its activity in the TGF-ß-rich immunosuppressive environment of prostate cancer. AZD0754 demonstrated potent and specific cytotoxicity against antigen-expressing cells in vitro despite TGF-ß-rich conditions. Further, AZD0754 enforced robust, dose-dependent in vivo efficacy in STEAP2-expressing cancer cell line-derived and patient-derived xenograft mouse models, and exhibited encouraging preclinical safety. Together, these data underscore the therapeutic tractability of STEAP2 in prostate cancer as well as build confidence in the specificity, potency, and tolerability of this potentially first-in-class CAR-T therapy.

Opposing functions of circadian protein DBP and atypical E2F family E2F8 in anti-tumor Th9 cell differentiation.

Interleukin-9 (IL-9)-producing CD4+ T helper cells (Th9) have been implicated in allergy/asthma and anti-tumor immunity, yet molecular insights on their differentiation from activated T cells, driven by IL-4 and transforming growth factor-beta (TGF-ß), is still lacking. Here we show opposing functions of two transcription factors, D-binding protein (DBP) and E2F8, in controlling Th9 differentiation. Specifically, TGF-ß and IL-4 signaling induces phosphorylation of the serine 213 site in the linker region of the Smad3 (pSmad3L-Ser213) via phosphorylated p38, which is necessary and sufficient for Il9 gene transcription. We identify DBP and E2F8 as an activator and repressor, respectively, for Il9 transcription by pSmad3L-Ser213. Notably, Th9 cells with siRNA-mediated knockdown for Dbp or E2f8 promote and suppress tumor growth, respectively, in mouse tumor models. Importantly, DBP and E2F8 also exhibit opposing functions in regulating human TH9 differentiation in vitro. Thus, our data uncover a molecular mechanism of Smad3 linker region-mediated, opposing functions of DBP and E2F8 in Th9 differentiation.

Treg deficiency-mediated TH 1 response causes human premature ovarian insufficiency through apoptosis and steroidogenesis dysfunction of granulosa cells.

Immune dysregulation has long been proposed as a component of premature ovarian insufficiency (POI), but the underlying mediators and mechanisms remain largely unknown. Here we showed that patients with POI had augmented T helper 1 (TH 1) responses and regulatory T (Treg ) cell deficiency in both the periphery and the ovary compared to the control women. The increased ratio of TH 1:Treg cells was strongly correlated with the severity of POI. In mouse models of POI, the increased infiltration of TH 1 cells in the ovary resulted in follicle atresia and ovarian insufficiency, which could be prevented and reversed by Treg cells. Importantly, interferon (IFN) -γ and tumor necrosis factor (TNF) -α cooperatively promoted the apoptosis of granulosa cells and suppressed their steroidogenesis by modulating CTGF and CYP19A1. We have thus revealed a previously unrecognized Treg cell deficiency-mediated TH 1 response in the pathogenesis of POI, which should have implications for therapeutic interventions in patients with POI.

Combination of apoptotic T cell induction and self-peptide administration for therapy of experimental autoimmune encephalomyelitis.

BACKGROUND: Clinical trials on multiple sclerosis with repeated injections of monoclonal antibodies depleting CD4+ T cells have not resulted in much success as a disease therapy. Here, we developed an immunotherapy for EAE in mice by combining a transient depletion of T cells together with the administration of neuron derived peptides. METHODS: EAE was induced in SJL and C57BL/6 mice, by proteolipid protein peptide PLP139-151 (pPLP) and myelin-oligodendrocyte glycoprotein MOG35-55 (pMOG) peptides, respectively. Anti-CD4 and anti-CD8 antibody were injected intraperitoneally before or after peptide immunization. EAE scores were evaluated and histology data from brain and spinal cord were analyzed. Splenocytes were isolated and CD4+, CD4+CD25- and CD4+CD25+ T cells were purified and cultured in the presence of either specific peptides or anti-CD3 antibody and proliferation of T cells as well as cytokines in supernatant were assessed. FINDINGS: This experimental treatment exhibited therapeutic effects on mice with established EAE in pPLP-susceptible SJL mice and pMOG-susceptible C57BL/6 mice. Mechanistically, we revealed that antibody-induced apoptotic T cells triggered macrophages to produce TGFß, and together with administered auto-antigenic peptides, generated antigen-specific Foxp3+ regulatory T cells (Treg cells) in vivo. INTERPRETATION: We successfully developed a specific immunotherapy to EAE by generating autoantigen-specific Treg cells. These findings have overcome the drawbacks of long and repeated depletion of CD4+ T cells, but also obtained long-term immune tolerance, which should have clinical implications for the development of a new effective therapy for multiple sclerosis. FUND: This research was supported by the Intramural Research Program of the NIH, NIDCR.

Extracellular Vesicles from Apoptotic Cells Promote TGFß Production in Macrophages and Suppress Experimental Colitis.

The clearance of apoptotic cells is an essential process to maintain homeostasis of immune system, which is regulated by immunoregulatory cytokines such as TGFß. We show here that Extracellular Vesicles (EVs) were highly released from apoptotic cells, and contributed to macrophage production of TGFß in vitro and in vivo. We further elucidated mechanistically that phosphatidylserine in EVs was a key triggering-factor, and transcription factor FOXO3 was a critical mediator for apoptotic EV-induced TGFß in macrophages. Importantly, we found that macrophages pre-exposed to EVs exhibited an anti-inflammatory phenotype. More strikingly, administration of EVs in vivo promotes Tregs differentiation and suppresses Th1 cell response, and ameliorates experimental colitis. Thus, apoptotic-EV-based treatment might be a promising therapeutic approach for human autoimmune disease.

Manipulating regulatory T cells: a promising strategy to treat autoimmunity.

CD4(+)CD25(+)Foxp3(+)regulatory T cells (Treg cells) are extremely important in maintaining immune tolerance. Manipulation of Treg cells, especially autoantigen-specific Treg cells is a promising approach for treatments of autoimmune disease since Treg cells may provide the advantage of antigen specificity without overall immune suppression. However, the clinical application of Treg cells has long been limited due to low numbers of Treg cells and the difficulty in identifying their antigen specificity. In this review, we summarize studies that demonstrate regression of autoimmune diseases using Treg cells as therapeutics. We also discuss approaches to generate polyclonal and autoantigen-specific Treg cells in vitro and in vivo. We also discuss our recent study that describes a novel approach of generating autoantigen-specific Treg cells in vivo and restoring immune tolerance by two steps apoptosis-antigen therapy.

The DNA-binding inhibitor Id3 regulates IL-9 production in CD4(+) T cells.

The molecular mechanisms by which signaling via transforming growth factor-ß (TGF-ß) and interleukin 4 (IL-4) control the differentiation of CD4(+) IL-9-producing helper T cells (TH9 cells) remain incompletely understood. We found here that the DNA-binding inhibitor Id3 regulated TH9 differentiation, as deletion of Id3 increased IL-9 production from CD4(+) T cells. Mechanistically, TGF-ß1 and IL-4 downregulated Id3 expression, and this process required the kinase TAK1. A reduction in Id3 expression enhanced binding of the transcription factors E2A and GATA-3 to the Il9 promoter region, which promoted Il9 transcription. Notably, Id3-mediated control of TH9 differentiation regulated anti-tumor immunity in an experimental melanoma-bearing model in vivo and also in human CD4(+) T cells in vitro. Thus, our study reveals a previously unrecognized TAK1-Id3-E2A-GATA-3 pathway that regulates TH9 differentiation.