Identifying highly active anti-CCR4 CAR T cells for the treatment of T-cell lymphoma.

A challenge when targeting T-cell lymphoma with chimeric antigen receptor (CAR) T-cell therapy is that target antigens are often shared between T cells and tumor cells, resulting in fratricide between CAR T cells and on-target cytotoxicity on normal T cells. CC chemokine receptor 4 (CCR4) is highly expressed in many mature T-cell malignancies, such as adult T-cell leukemia/lymphoma (ATLL) and cutaneous T-cell lymphoma (CTCL), and has a unique expression profile in normal T cells. CCR4 is predominantly expressed by type-2 and type-17 helper T cells (Th2 and Th17) and regulatory T cells (Treg), but it is rarely expressed by other T helper (Th) subsets and CD8+ cells. Although fratricide in CAR T cells is generally thought to be detrimental to anticancer functions, in this study, we demonstrated that anti-CCR4 CAR T cells specifically depleted Th2 and Tregs, while sparing CD8+ and Th1 T cells. Moreover, fratricide increased the percentage of CAR+ T cells in the final product. CCR4-CAR T cells were characterized by high transduction efficiency, robust T-cell expansion, and rapid fratricidal depletion of CCR4-positive T cells during CAR transduction and expansion. Furthermore, mogamulizumab-based CCR4-CAR T cells induced superior antitumor efficacy and long-term remission in mice engrafted with human T-cell lymphoma cells. In summary, CCR4-depleted anti-CCR4 CAR T cells are enriched in Th1 and CD8+ T cells and exhibit high antitumor efficacy against CCR4-expressing T-cell malignancies.

Monitoring Therapeutic Response to Anti-FAP CAR T Cells Using [18F]AlF-FAPI-74.

PURPOSE: Despite the success of chimeric antigen receptor (CAR) T-cell therapy against hematologic malignancies, successful targeting of solid tumors with CAR T cells has been limited by a lack of durable responses and reports of toxicities. Our understanding of the limited therapeutic efficacy in solid tumors could be improved with quantitative tools that allow characterization of CAR T-targeted antigens in tumors and accurate monitoring of response. EXPERIMENTAL DESIGN: We used a radiolabeled FAP inhibitor (FAPI) [18F]AlF-FAPI-74 probe to complement ongoing efforts to develop and optimize FAP CAR T cells. The selectivity of the radiotracer for FAP was characterized in vitro, and its ability to monitor changes in FAP expression was evaluated using rodent models of lung cancer. RESULTS: [18F]AlF-FAPI-74 showed selective retention in FAP+ cells in vitro, with effective blocking of the uptake in presence of unlabeled FAPI. In vivo, [18F]AlF-FAPI-74 was able to detect FAP expression on tumor cells as well as FAP+ stromal cells in the tumor microenvironment with a high target-to-background ratio. We further demonstrated the utility of the tracer to monitor changes in FAP expression following FAP CAR T-cell therapy, and the PET imaging findings showed a robust correlation with ex vivo analyses. CONCLUSIONS: This noninvasive imaging approach to interrogate the tumor microenvironment represents an innovative pairing of a diagnostic PET probe with solid tumor CAR T-cell therapy and has the potential to serve as a predictive and pharmacodynamic response biomarker for FAP as well as other stroma-targeted therapies. A PET imaging approach targeting FAP expressed on activated fibroblasts of the tumor stroma has the potential to predict and monitor therapeutic response to FAP-targeted CAR T-cell therapy. See related commentary by Weber et al., p. 5241.

Potentiating adoptive cell therapy using synthetic IL-9 receptors.

Synthetic receptor signalling has the potential to endow adoptively transferred T cells with new functions that overcome major barriers in the treatment of solid tumours, including the need for conditioning chemotherapy1,2. Here we designed chimeric receptors that have an orthogonal IL-2 receptor extracellular domain (ECD) fused with the intracellular domain (ICD) of receptors for common γ-chain (γc) cytokines IL-4, IL-7, IL-9 and IL-21 such that the orthogonal IL-2 cytokine elicits the corresponding γc cytokine signal. Of these, T cells that signal through the chimeric orthogonal IL-2Rß-ECD-IL-9R-ICD (o9R) are distinguished by the concomitant activation of STAT1, STAT3 and STAT5 and assume characteristics of stem cell memory and effector T cells. Compared to o2R T cells, o9R T cells have superior anti-tumour efficacy in two recalcitrant syngeneic mouse solid tumour models of melanoma and pancreatic cancer and are effective even in the absence of conditioning lymphodepletion. Therefore, by repurposing IL-9R signalling using a chimeric orthogonal cytokine receptor, T cells gain new functions, and this results in improved anti-tumour activity for hard-to-treat solid tumours.

Enhancing CAR T cell persistence through ICOS and 4-1BB costimulation.

Successful tumor eradication by chimeric antigen receptor-expressing (CAR-expressing) T lymphocytes depends on CAR T cell persistence and effector function. We hypothesized that CD4+ and CD8+ T cells may exhibit distinct persistence and effector phenotypes, depending on the identity of specific intracellular signaling domains (ICDs) used to generate the CAR. First, we demonstrate that the ICOS ICD dramatically enhanced the in vivo persistence of CAR-expressing CD4+ T cells that, in turn, increased the persistence of CD8+ T cells expressing either CD28- or 4-1BB-based CARs. These data indicate that persistence of CD8+ T cells was highly dependent on a helper effect provided by the ICD used to redirect CD4+ T cells. Second, we discovered that combining ICOS and 4-1BB ICDs in a third-generation CAR displayed superior antitumor effects and increased persistence in vivo. Interestingly, we found that the membrane-proximal ICD displayed a dominant effect over the distal domain in third-generation CARs. The optimal antitumor and persistence benefits observed in third-generation ICOSBBz CAR T cells required the ICOS ICD to be positioned proximal to the cell membrane and linked to the ICOS transmembrane domain. Thus, CARs with ICOS and 4-1BB ICD demonstrate increased efficacy in solid tumor models over our current 4-1BB-based CAR and are promising therapeutics for clinical testing.

Engineered CAR T Cells Targeting the Cancer-Associated Tn-Glycoform of the Membrane Mucin MUC1 Control Adenocarcinoma.

Genetically modified T cells expressing chimeric antigen receptors (CARs) demonstrate robust responses against lineage restricted, non-essential targets in hematologic cancers. However, in solid tumors, the full potential of CAR T cell therapy is limited by the availability of cell surface antigens with sufficient cancer-specific expression. The majority of CAR targets have been normal self-antigens on dispensable hematopoietic tissues or overexpressed shared antigens. Here, we established that abnormal self-antigens can serve as targets for tumor rejection. We developed a CAR that recognized cancer-associated Tn glycoform of MUC1, a neoantigen expressed in a variety of cancers. Anti-Tn-MUC1 CAR T cells demonstrated target-specific cytotoxicity and successfully controlled tumor growth in xenograft models of T cell leukemia and pancreatic cancer. These findings demonstrate the therapeutic efficacy of CAR T cells directed against Tn-MUC1 and present aberrantly glycosylated antigens as a novel class of targets for tumor therapy with engineered T cells.

Structural determinants of human ζ-globin mRNA stability.

BACKGROUND: The normal accumulation of adult α and ß globins in definitive erythrocytes is critically dependent upon processes that ensure that the cognate mRNAs are maintained at high levels in transcriptionally silent, but translationally active progenitor cells. The impact of these post-transcriptional regulatory events on the expression of embryonic ζ globin is not known, as its encoding mRNA is not normally transcribed during adult erythropoiesis. Recently, though, ζ globin has been recognized as a potential therapeutic for α thalassemia and sickle-cell disease, raising practical questions about constitutive post-transcriptional processes that may enhance, or possibly prohibit, the expression of exogenous or derepresssed endogenous ζ-globin genes in definitive erythroid progenitors. METHODS: The present study assesses mRNA half-life in intact cells using a pulse-chase approach; identifies cis-acting determinants of ζ-globin mRNA stability using a saturation mutagenesis strategy; establishes critical 3'UTR secondary structures using an in vitro enzymatic mapping method; and identifies trans-acting effector factors using an affinity chromatographical procedure. RESULTS: We specify a tetranucleotide 3'UTR motif that is required for the high-level accumulation of ζ-globin transcripts in cultured cells, and formally demonstrate that it prolongs their cytoplasmic half-lives. Surprisingly, the ζ-globin mRNA stability motif does not function autonomously, predicting an activity that is subject to structural constraints that we subsequently specify. Additional studies demonstrate that the ζ-globin mRNA stability motif is targeted by AUF1, a ubiquitous RNA-binding protein that enhances the half-life of adult ß-globin mRNA, suggesting commonalities in post-transcriptional processes that regulate globin transcripts at all stages of mammalian development. CONCLUSIONS: These data demonstrate a mechanism for ζ-globin mRNA stability that exists in definitive erythropoiesis and is available for therapeutic manipulation in α thalassemia and sickle-cell disease.

The B-cell superantigen Finegoldia magna protein L causes pulmonary inflammation by a mechanism dependent on MyD88 but not B cells or immunoglobulins.

OBJECTIVE AND DESIGN: To determine whether Finegoldia magna protein L (PL) causes lung inflammation and, if so, whether the response is dependent on its immunoglobulin (Ig)-binding B-cell superantigenic property. MATERIAL: Pulmonary inflammatory reactions were analyzed at various time points after intratracheal administration of PL to various strains of mice. RESULTS: PL caused peribronchial and perivascular inflammation that peaked at 18-24 h. Polymorphonuclear cells (PMNs) began to accumulate in bronchoalveolar lavage fluid (BALF) of PL-challenged mice by 4 h and accounted for >90% of leukocytes by 18-24 h. Inflammation was marked by the appearance of MIP-2, KC, TNF-α, and IL-6 in the BALF with peak levels attained 4 h after PL administration. PL-induced pulmonary inflammation was associated with increased airway hyper-reactivity following inhalation of methacholine. The inflammatory reaction was unabated in mice lacking B cells and immunoglobulins. In contrast, PL-induced inflammation was abrogated in MyD88-deficient mice. PL-induced responses required alveolar macrophages. CONCLUSIONS: These results strongly suggest that PL-induced lung inflammation is dependent on an innate MyD88-dependent pathway rather than the Ig-binding properties of this microbial B cell superantigen. We propose that this pulmonary inflammatory reaction is caused by the interaction of PL with a Toll-like receptor expressed on alveolar macrophages.

Toll-like receptor 7-induced naive human B-cell differentiation and immunoglobulin production.

BACKGROUND: Toll-like receptors contribute to the establishment of adaptive immune responses. OBJECTIVE: The reported studies were conducted to examine the effects of the Toll-like receptor (TLR)-7 ligand, resiquimod, on human naive B-cell differentiation. METHODS: Naive human B cells were cultured with resiquimod in the presence or absence of IL-2 and IL-10. Secreted IgM and IgG were measured by ELISA, and IL-6, IL-10, and IFN-alpha were measured by a multiplex protein array. Cell proliferation was assessed by measuring [(3)H]thymidine uptake. mRNA for activation-induced cytidine deaminase and I(gamma 1)-C(mu) circle transcripts was measured by means of RT-PCR. RESULTS: Resiquimod induced the production of IgM and, to a lesser extent, IgG by naive human B cells in association with the secretion of IL-6 and IL-10, and a weak proliferative response. IL-2 and IL-10 synergized with resiquimod in markedly augmenting resiquimod-induced IgM and IgG production and proliferation. Resiquimod also stimulated production of IgG by B cells isolated from the blood of a patient with the X-linked hyper-IgM syndrome, with a greater response when these cells were costimulated with IL-2 and IL-10. The stimulated naive B cells from healthy volunteers displayed molecular evidence of immunoglobulin class-switch recombination-namely the appearance of activation-induced cytidine deaminase and I(gamma 1)-C(mu) circle transcripts. CONCLUSION: Perturbation of TLR-7 on naive human B cells can lead to the induction of immunoglobulin class switch and IgG production in the absence of B-cell receptor cross-linking and CD40-CD40L interaction. The results are relevant to vaccine development and mechanisms by which microbial infection may lead to autoimmunity.

The intrathymic pathogenesis of myasthenia gravis.

The thymus is considered to play an important role in the pathogenesis of Myasthenia gravis, an autoimmune disease characterized by antibody-mediated skeletal muscle weakness. However, its role is yet to be defined. The studies described herein summarize our efforts to determine how intrathymic expression of the neuromuscular type of acetylcholine (ACh) receptors is involved in the immunopathogenesis of this autoimmune disease. We review the work characterizing the expression of neuromuscular ACh receptors in the thymus and advance a new hypothesis that examines the intrathymic expression of this autoantigen in disease pathogenesis.

A new model linking intrathymic acetylcholine receptor expression and the pathogenesis of myasthenia gravis.

The thymus is thought to play an important role in the pathogenesis of myasthenia gravis (MG), an autoimmune disease characterized by skeletal muscle weakness. However, its role remains a mystery. The studies described represent our efforts to determine how intrathymic expression of the neuromuscular type of acetylcholine receptors (nAChRs) is involved in the immunopathogenesis of MG. We review our work characterizing the expression of the alpha subunit of nAChR (nAChRalpha) in the thymus and advance a new hypothesis that examines the intrathymic expression of this autoantigen in disease pathogenesis.

Intrathymic expression of neuromuscular acetylcholine receptors and the immunpathogenesis of myasthenia gravis.

The thymus has been considered to play an important role in the pathogenesis of myasthenia gravis (MG), an autoimmune disease characterized by skeletal muscle weakness. However, the pathogenic role of the thymus still remains a mystery. The neuromuscular type of acetylcholine receptor (AChR) was the first self-protein associated with a defined autoimmune disease that was found to be expressed by thymic stromal populations. The studies described herein represent our efforts to determine how this "promiscuous" autoantigen expression may be involved in the immunopathogenesis of MG. We review our work, characterizating the expression of the alpha subunit of AChR (AChRalpha) in the thymus, and advance a new hypothesis that examines the intrathymic expression of this autoantigen in disease pathogenesis.