Synergistic T cell signaling by 41BB and CD28 is optimally achieved by membrane proximal positioning within parallel chimeric antigen receptors.

Second generation (2G) chimeric antigen receptors (CARs) contain a CD28 or 41BB co-stimulatory endodomain and elicit remarkable efficacy in hematological malignancies. Third generation (3G) CARs extend this linear blueprint by fusing both co-stimulatory units in series. However, clinical impact has been muted despite compelling evidence that co-signaling by CD28 and 41BB can powerfully amplify natural immune responses. We postulate that effective dual co-stimulation requires juxta-membrane positioning of endodomain components within separate synthetic receptors. Consequently, we designed parallel (p)CARs in which a 2G (CD28+CD3ζ) CAR is co-expressed with a 41BB-containing chimeric co-stimulatory receptor. We demonstrate that the pCAR platform optimally harnesses synergistic and tumor-dependent co-stimulation to resist T cell exhaustion and senescence, sustaining proliferation, cytokine release, cytokine signaling, and metabolic fitness upon repeated stimulation. When engineered using targeting moieties of diverse composition, affinity, and specificity, pCAR T cells consistently elicit superior anti-tumor activity compared with T cells that express traditional linear CARs.

ALIX Regulates Tumor-Mediated Immunosuppression by Controlling EGFR Activity and PD-L1 Presentation.

The immunosuppressive transmembrane protein PD-L1 was shown to traffic via the multivesicular body (MVB) and to be released on exosomes. A high-content siRNA screen identified the endosomal sorting complexes required for transport (ESCRT)-associated protein ALIX as a regulator of both EGFR activity and PD-L1 surface presentation in basal-like breast cancer (BLBC) cells. ALIX depletion results in prolonged and enhanced stimulation-induced EGFR activity as well as defective PD-L1 trafficking through the MVB, reduced exosomal secretion, and its redistribution to the cell surface. Increased surface PD-L1 expression confers an EGFR-dependent immunosuppressive phenotype on ALIX-depleted cells. An inverse association between ALIX and PD-L1 expression was observed in human breast cancer tissues, while an immunocompetent mouse model of breast cancer revealed that ALIX-deficient tumors are larger and show an increased immunosuppressive environment. Our data suggest that ALIX modulates immunosuppression through regulation of PD-L1 and EGFR and may, therefore, present a diagnostic and therapeutic target for BLBC.

c-Met PET Imaging Detects Early-Stage Locoregional Recurrence of Basal-Like Breast Cancer.

UNLABELLED: Locoregional recurrence of breast cancer poses significant clinical problems because of frequent inoperability once the chest wall is involved. Early detection of recurrence by molecular imaging agents against therapeutically targetable receptors, such as c-Met, would be of potential benefit. The aim of this study was to assess (18)F-AH113804, a peptide-based molecular imaging agent with high affinity for human c-Met, for the detection of early-stage locoregional recurrence in a human basal-like breast cancer model, HCC1954. METHODS: HCC1954 tumor-bearing xenograft models were established, and (18)F-AH113804 was administered. Distribution of radioactivity was determined via PET at 60 min after radiotracer injection. PET and CT images were acquired 10 d after tumor inoculation, to establish baseline distribution and uptake, and then on selected days after surgical tumor resection. CT images and caliper were used to determine the tumor volume. Radiotracer uptake was assessed by (18)F-AH113804 PET imaging. c-Met expression was assessed by immunofluorescence imaging of tumor samples and correlated with (18)F-AH113804 PET imaging results. RESULTS: Baseline uptake of (18)F-AH113804, determined in tumor-bearing animals after 10 d, was approximately 2-fold higher in the tumor than in muscle tissue or the contralateral mammary fat pad. The tumor growth rate, determined from CT images, was comparable between the animals with recurrent tumors, with detection of tumors of low volume (<10 mm(3)) only possible by day 20 after tumor resection. (18)F-AH113804 PET detected local tumor recurrence as early as 6 d after surgery in the recurrent tumor-bearing animals and exhibited significantly higher (18)F-AH113804 uptake (in comparison to mammary fatty tissue), with a target-to-background (muscle) ratio of approximately 3:1 (P < 0.01). The c-Met expression of individual resected tumor samples, determined by immunofluorescence, correlated with the respective (18)F-AH113804 imaging signals (r = 0.82, P < 0.05). CONCLUSION: (18)F-AH113804 PET provides a new diagnostic tool for the detection of c-Met-expressing primary tumor and has potential utility for the detection of locoregional recurrence from an early stage.

The Breast Cancer-Associated Glycoforms of MUC1, MUC1-Tn and sialyl-Tn, Are Expressed in COSMC Wild-Type Cells and Bind the C-Type Lectin MGL.

Aberrant glycosylation occurs in the majority of human cancers and changes in mucin-type O-glycosylation are key events that play a role in the induction of invasion and metastases. These changes generate novel cancer-specific glyco-antigens that can interact with cells of the immune system through carbohydrate binding lectins. Two glyco-epitopes that are found expressed by many carcinomas are Tn (GalNAc-Ser/Thr) and STn (NeuAcα2,6GalNAc-Ser/Thr). These glycans can be carried on many mucin-type glycoproteins including MUC1. We show that the majority of breast cancers carry Tn within the same cell and in close proximity to extended glycan T (Galß1,3GalNAc) the addition of Gal to the GalNAc being catalysed by the T synthase. The presence of active T synthase suggests that loss of the private chaperone for T synthase, COSMC, does not explain the expression of Tn and STn in breast cancer cells. We show that MUC1 carrying both Tn or STn can bind to the C-type lectin MGL and using atomic force microscopy show that they bind to MGL with a similar dead adhesion force. Tumour associated STn is associated with poor prognosis and resistance to chemotherapy in breast carcinomas, inhibition of DC maturation, DC apoptosis and inhibition of NK activity. As engagement of MGL in the absence of TLR triggering may lead to anergy, the binding of MUC1-STn to MGL may be in part responsible for some of the characteristics of STn expressing tumours.