Arben Nace

Rational development and characterization of humanized anti-EGFR variant III chimeric antigen receptor T cells for glioblastoma.

A chimeric antigen receptor redirects T cells to treat glioblastoma. CAR T cells drive glioblastoma therapy Immunotherapy with chimeric antigen receptor (CAR) T cells can successfully treat B cell malignancies, but expansion into solid tumors has been limited by the lack of availability of tumor-specific antigens. Now, Johnson et al. target CAR T cells to a variant III mutation of the epidermal growth factor receptor (EGFRvIII), which is thought to be enriched in glioblastoma stem cells. They found that a low-affinity single-chain variable fragment was specific for EGFRvIII over wild-type EGFR and that CAR T cells transduced with this fragment were able to target antigen-expressing cells in vitro and in vivo in multiple mouse xenograft models of human glioblastoma. These cells are currently being moved into the clinic in a phase 1 clinical trial. Chimeric antigen receptors (CARs) are synthetic molecules designed to redirect T cells to specific antigens. CAR-modified T cells can mediate long-term durable remissions in B cell malignancies, but expanding this platform to solid tumors requires the discovery of surface targets with limited expression in normal tissues. The variant III mutation of the epidermal growth factor receptor (EGFRvIII) results from an in-frame deletion of a portion of the extracellular domain, creating a neoepitope. We chose a vector backbone encoding a second-generation CAR based on efficacy of a murine scFv–based CAR in a xenograft model of glioblastoma. Next, we generated a panel of humanized scFvs and tested their specificity and function as soluble proteins and in the form of CAR-transduced T cells; a low-affinity scFv was selected on the basis of its specificity for EGFRvIII over wild-type EGFR. The lead candidate scFv was tested in vitro for its ability to direct CAR-transduced T cells to specifically lyse, proliferate, and secrete cytokines in response to antigen-bearing targets. We further evaluated the specificity of the lead CAR candidate in vitro against EGFR-expressing keratinocytes and in vivo in a model of mice grafted with normal human skin. EGFRvIII-directed CAR T cells were also able to control tumor growth in xenogeneic subcutaneous and orthotopic models of human EGFRvIII+ glioblastoma. On the basis of these results, we have designed a phase 1 clinical study of CAR T cells transduced with humanized scFv directed to EGFRvIII in patients with either residual or recurrent glioblastoma (NCT02209376).

Fgf9 from dermal γδ T cells induces hair follicle neogenesis after wounding

Understanding molecular mechanisms for regeneration of hair follicles provides new opportunities for developing treatments for hair loss and other skin disorders. Here we show that fibroblast growth factor 9 (Fgf9), initially secreted by γδ T cells, modulates hair follicle regeneration after wounding the skin of adult mice. Reducing Fgf9 expression decreases this wound-induced hair neogenesis (WIHN). Conversely, overexpression of Fgf9 results in a two- to threefold increase in the number of neogenic hair follicles. We found that Fgf9 from γδ T cells triggers Wnt expression and subsequent Wnt activation in wound fibroblasts. Through a unique feedback mechanism, activated fibroblasts then express Fgf9, thus amplifying Wnt activity throughout the wound dermis during a crucial phase of skin regeneration. Notably, humans lack a robust population of resident dermal γδ T cells, potentially explaining their inability to regenerate hair after wounding. These findings highlight the essential relationship between the immune system and tissue regeneration. The importance of Fgf9 in hair follicle regeneration suggests that it could be used therapeutically in humans.

Reengineering chimeric antigen receptor T cells for targeted therapy of autoimmune disease

Engineering T cells to treat autoimmunity Autoimmune diseases such as lupus and rheumatoid arthritis lack therapies that specifically target only the disease-causing cells. Inspired by the clinical success of using chimeric antigen receptor T cells to treat certain types of cancers, Ellebrecht et al. asked whether a similar approach might also work against antibody-driven autoimmune diseases. They engineered T cells to express chimeric receptors consisting of the disease-causing autoantigen desmoglein 3 fused to signaling domains that activate T cells. When given to diseased mice, the engineered T cells targeted and killed B cells that express antibodies targeting desmoglein 3, hinting that such a strategy may be an effective way to treat antibody-driven autoimmune diseases. Science, this issue p. 179 A proof-of-principle study indicates that engineered T cells may be an effective, targeted therapy for autoimmunity. Ideally, therapy for autoimmune diseases should eliminate pathogenic autoimmune cells while sparing protective immunity, but feasible strategies for such an approach have been elusive. Here, we show that in the antibody-mediated autoimmune disease pemphigus vulgaris (PV), autoantigen-based chimeric immunoreceptors can direct T cells to kill autoreactive B lymphocytes through the specificity of the B cell receptor (BCR). We engineered human T cells to express a chimeric autoantibody receptor (CAAR), consisting of the PV autoantigen, desmoglein (Dsg) 3, fused to CD137-CD3ζ signaling domains. Dsg3 CAAR-T cells exhibit specific cytotoxicity against cells expressing anti-Dsg3 BCRs in vitro and expand, persist, and specifically eliminate Dsg3-specific B cells in vivo. CAAR-T cells may provide an effective and universal strategy for specific targeting of autoreactive B cells in antibody-mediated autoimmune disease.

Regeneration of fat cells from myofibroblasts during wound healing

Hair follicles: Secret to prevent scars? Although some animals easily regenerate limbs and heal broken flesh, mammals are generally not so gifted. Wounding can leave scars, which are characterized by a lack of hair follicles and cutaneous fat. Plikus et al. now show that hair follicles in both mice and humans can convert myofibroblasts, the predominant dermal cell in a wound, into adipocytes (see the Perspective by Chan and Longaker). The hair follicles activated the bone morphogenetic protein (BMP) signaling pathway and adipocyte transcription factors in the myofibroblast. Thus, it may be possible to reduce scar formation after wounding by adding BMP. Science, this issue p. 748; see also p. 693 Hair follicles convert wound myofibroblasts to adipocytes through the bone morphogenetic protein signaling pathway, revealing a target to decrease scarring. Although regeneration through the reprogramming of one cell lineage to another occurs in fish and amphibians, it has not been observed in mammals. We discovered in the mouse that during wound healing, adipocytes regenerate from myofibroblasts, a cell type thought to be differentiated and nonadipogenic. Myofibroblast reprogramming required neogenic hair follicles, which triggered bone morphogenetic protein (BMP) signaling and then activation of adipocyte transcription factors expressed during development. Overexpression of the BMP antagonist Noggin in hair follicles or deletion of the BMP receptor in myofibroblasts prevented adipocyte formation. Adipocytes formed from human keloid fibroblasts either when treated with BMP or when placed with human hair follicles in vitro. Thus, we identify the myofibroblast as a plastic cell type that may be manipulated to treat scars in humans.

Generation of folliculogenic human epithelial stem cells from induced pluripotent stem cells

Epithelial stem cells (EpSCs) in the hair follicle bulge are required for hair follicle growth and cycling. The isolation and propagation of human EpSCs for tissue engineering purposes remains a challenge. Here we develop a strategy to differentiate human iPSCs (hiPSCs) into CD200+/ITGA6+ EpSCs that can reconstitute the epithelial components of the hair follicle and interfollicular epidermis. The hiPSC-derived CD200+/ITGA6+ cells show a similar gene expression signature as EpSCs directly isolated from human hair follicles. Human iPSC-derived CD200+/ITGA6+ cells are capable of generating all hair follicle lineages including the hair shaft, and the inner and outer root sheaths in skin reconstitution assays. The regenerated hair follicles possess a KRT15+ stem cell population and produce hair shafts expressing hair specific keratins. These results suggest an approach for generating large numbers of human EpSCs for tissue engineering and new treatments for hair loss, wound healing and other degenerative skin disorders.

Direct conversion of mouse and human fibroblasts to functional melanocytes by defined factors

Direct reprogramming provides a fundamentally new approach for the generation of patient-specific cells. Here, by screening a pool of candidate transcription factors, we identify that a combination of three factors, MITF, SOX10 and PAX3, directly converts mouse and human fibroblasts to functional melanocytes. Induced melanocytes (iMels) activate melanocyte-specific networks, express components of pigment production and delivery system, and produce melanosomes. Human iMels properly integrate into the dermal-epidermal junction, and produce and deliver melanin pigment to surrounding keratinocytes in a 3D organotypic skin reconstruct. Human iMels generate pigmented epidermis and hair follicles in skin reconstitution assays in vivo. The generation of iMels has important implications for studies of melanocyte lineage commitment, pigmentation disorders and cell replacement therapies.

Pre-clinical validation of a humanized anti-EGFR variant III chimeric antigen receptor and phase I trial of CART-EGFRvIII in glioblastoma

Chimeric antigen receptors are synthetic molecules designed to re-direct T cells to specific surface antigens; CAR-modified T cells can mediate long-term durable remissions in B cell malignancies, but expanding this platform to solid tumors requires the discovery of novel surface targets with limited expression. The variant III mutation of the epidermal growth factor receptor (EGFR variant III) is the most common variant of the EGF receptor observed in human tumors, and results from an in-frame deletion of a portion of the extracellular domain. In glioblastoma, the EGFRvIII mutation is oncogenic, portends a poor prognosis, and is thought to be enriched in glioblastoma stem cells. However, because the neoepitope of EGFR variant III is based on a small peptide sequence, an antibody or single-chain variable fragment (scFv) directed to this epitope must be rigorously tested to confirm lack of cross-reactivity to the ubiquitously expressed normal EGFR. Having selected a candidate murine scFv directed to EGFRvIII and a vector backbone encoding a second generation CAR, we generated a panel of humanized scFvs and tested their specificity and function as soluble proteins and in the form of CAR-transduced T cells. The lead candidate scFv was tested in vitro for its ability to direct CAR-transduced T cells to kill antigen-bearing targets effectively, and proliferate and secrete cytokines specifically in response to antigen. We further evaluated the specificity of the lead candidate CAR by comparing it to a cetuximab-based CAR which does not discriminate between EGFR and EGFR variant III; the two CARs, along with negative controls, were tested in vitro against primary cells derived from a panel of normal tissues, and in vivo in immunodeficient mice grafted with normal human skin, which naturally expresses EGFR. CAR-T cells were also able to control tumor growth in xenogeneic subcutaneous and orthotopic models of human EGFR variant III+ glioblastoma. We have designed a Phase I clinical study of CAR T cells transduced with humanized scFv directed to EGFR variant III in patients with glioblastoma.