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CD19-specific KIR-CAR/Dap12 shows antigen-specific cytotoxicity in vitro.

SS1-KIRS2 does not affect endogenous TCR expression.

Figure legends for supplemental data.

SS1-KIRS2/DAP12 T cells mediate robust anti-tumor activity in vivo.

Chimeric antigen receptors (CAR) bearing an antigen-binding domain linked in cis to the cytoplasmic domains of CD3ζ and costimulatory receptors have provided a potent method for engineering T-cell cytotoxicity toward B-cell leukemia and lymphoma. However, resistance to immunotherapy due to loss of T-cell effector function remains a significant barrier, especially in solid malignancies. We describe an alternative chimeric immunoreceptor design in which we have fused a single-chain variable fragment for antigen recognition to the transmembrane and cytoplasmic domains of KIR2DS2, a stimulatory killer immunoglobulin-like receptor (KIR). We show that this simple, KIR-based CAR (KIR-CAR) triggers robust antigen-specific proliferation and effector function in vitro when introduced into human T cells with DAP12, an immunotyrosine-based activation motifs-containing adaptor. T cells modified to express a KIR-CAR and DAP12 exhibit superior antitumor activity compared with standard first- and second-generation CD3ζ-based CARs in a xenograft model of mesothelioma highly resistant to immunotherapy. The enhanced antitumor activity is associated with improved retention of chimeric immunoreceptor expression and improved effector function of isolated tumor-infiltrating lymphocytes. These results support the exploration of KIR-CARs for adoptive T-cell immunotherapy, particularly in immunotherapy-resistant solid tumors. Cancer Immunol Res; 3(7); 815–26. ©2015 AACR.

Cytotoxicity induced by SS1-KIRS2, but not receptor expression at the cell surface, depends upon DAP12 co-expression.

Mesothelin-specific CD3ζ CARs and SS1-KIRS2/Dap12 show comparable antigen-specific in vitro cytotoxicity toward EM-meso cells.

FAP-specific KIR-CAR/Dap12 T cells induce bone marrow hypocellularity and loss of body weight.

Early diagnosis of lung cancer followed by surgery presently is the most effective treatment for non–small cell lung cancer (NSCLC). An accurate, minimally invasive test that could detect early disease would permit timely intervention and potentially reduce mortality. Recent studies have shown that the peripheral blood can carry information related to the presence of disease, including prognostic information and information on therapeutic response. We have analyzed gene expression in peripheral blood mononuclear cell samples including 137 patients with NSCLC tumors and 91 patient controls with nonmalignant lung conditions, including histologically diagnosed benign nodules. Subjects were primarily smokers and former smokers. We have identified a 29-gene signature that separates these two patient classes with 86% accuracy (91% sensitivity, 80% specificity). Accuracy in an independent validation set, including samples from a new location, was 78% (sensitivity of 76% and specificity of 82%). An analysis of this NSCLC gene signature in 18 NSCLCs taken presurgery, with matched samples from 2 to 5 months postsurgery, showed that in 78% of cases, the signature was reduced postsurgery and disappeared entirely in 33%. Our results show the feasibility of using peripheral blood gene expression signatures to identify early-stage NSCLC in at-risk populations. [Cancer Res 2009;69(24):9202–10]

Supplementary Methods, Tables 1-10 from Gene Expression Profiles in Peripheral Blood Mononuclear Cells Can Distinguish Patients with Non–Small Cell Lung Cancer from Patients with Nonmalignant Lung Disease

Soft-tissue sarcomas (STS) are a group of lethal mesenchymal tumors that are a major cause of cancer related morbidity for those under the age of 20.PI3K inhibitors have been found to be effective against subset of STS but are associated with significant toxicities limiting their use.We have thought to test a selective fibroblast activation protein (FAP) targeted delivery of PI3K/AKT/mTOR inhibitor omipalisib to STS in pre-clinical setting as FAP is overwhelmingly expressed agressive soft tissue malignancies. Experimental Design: Patient derived fibrosarcomas (aSMA-,FAP+,pAKT+) were isolated by flow cytometry.FAP(-)HT1080 fibrosarcoma cell lines were used as a negative control whereas methylcoanthrene induced mouse fibrosarcoma transfected with human FAP cDNA were used as positive control.Cell expression of FAP,AKT,mTOR was compared using SDS Page,IHC,and flow cytometry.FAP ligand coupled to omipalisib(FAPL-PI3Ki)was then evaluated in-vitro and in small animal models. Results: Cell lines with known FAP expression demonstrated significant uptake of FAPL conjugated NIR fluorochrome.Wild type cells(hFAP-) showed no ligand binding both in flow cytometry and fluorescent microscopy(p Citation Format: Feredun Azari, Gregory T. Kennedy, Ashley Chang, Bilal Nadeem, Azra Din, Isvita Marfatia, Steven Albelda, Philip Low, Madduri Srinivasarao, Ramesh Mukkamala, Neil T. Sullivan, Evgeniy Eruslanov, Sunil Singhal. Selective delivery of fibroblast activation protein conjugated dual phosphoinositide 3-kinase–AKT Kinase-mTOR inhibitor associated with decreased tumor proliferation and on-target toxicity in high-grade soft-tissue sarcomas [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A016.

Objective: Cancer-associated fibroblasts (CAFs) are a key component of the tumor microenvironment and affect neoplastic progression by stromal remodeling, paracrine signaling, potentiating cancer cell proliferation, and immune system evasion. As such, they are a potential target for optimizing therapeutic and diagnostic strategies. The purpose of our study was to evaluate if Fibroblast Activation Protein (FAP) can be a suitable target for molecular imaging guided cancer localization during surgery because FAP is overwhelmingly expressed by CAFs. Methods: Patient derived lung adenocarcinoma FAP+/alpha SMA+ myofibroblasts (CAFs) were isolated by flow cytometry and cultured according to institutional best practices. Normal fibroblasts from 3T3 cell line were used as negative control whereas normal lung fibroblasts transfected with human FAP cDNA were used as positive control. Cell expression of FAP was compared using SDS Page analysis and flow cytometry. FAP ligand coupled to NIR S0456 fluorochrome (FAP-S0456) was then evaluated with in-vitro dose escalation, cell-toxicity study, fluorescence parameters, and immunofluorescence microscopy. Results: Patient derived lung cancer (LC) CAFs had lower FAP-S0456 dye binding expression compared to transfected fibroblasts (TF), while normal fibroblasts (FAP-, aSMA-) had no FAP-S0456 dye binding detected on FACS direct antibody analysis (MFI 1033 vs 244, p Conclusion: FAP transmembrane receptor is highly expressed in cancer associated fibroblasts and can serve as a potential target for FAP ligand conjugated near-infrared tracers. This study demonstrates proof of principle of utilizing FAP-S0456 in CAF in-vitro models and paves a path for pre-clinical diagnostic and therapeutic validation for surgical applications. Citation Format: Feredun Azari, Gregory Kennedy, Ashley Chang, Bilal Nadeem, Neil Sullivan, Emanuel Encarnado, Steven Albelda, Evgeniy Eruslanov, Sunil Singhal. Fibroblast activation protein transmembrane receptor targeted near-infrared fluorochrome specifically identifies patient derived lung cancer associated fibroblasts in pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4142.

Abstract Complement-mediated pulmonary edema results from increases in lung capillary hydraulic conductivity (Lp), possibly by receptor-mediated mechanisms. We considered the Lp effects of vitronectin and the vitronectin-containing complement complex SC5b-9, which ligate the integrin α v β 3 . Vitronectin, SC5b-9, and SC5b-9–enriched zymosan-activated serum all rapidly increased Lp, as determined by the split-drop technique in single lung capillaries of rat lung. The Lp increases were inhibited by a monospecific (LM609) and a polyclonal (R838) antibody against the α v β 3 integrin but not by an irrelevant monoclonal antibody isotype matched with LM609, by a monoclonal antibody against the α v β 5 integrin, or by preimmune rabbit serum. Vitronectin monomers failed to increase Lp. The tyrosine kinase blockers genistein and methyl 2,5-dihydroxycinnamate caused significant concentration-dependent inhibitions of Lp increases due to vitronectin and zymosan-activated serum. By contrast, the protein kinase C blocker calphostin C had no major effect. We conclude that (1) multivalent ligation of the luminally located α v β 3 integrin of lung capillary endothelium increases transcapillary liquid flux, and (2) the dominant signal transduction pathway for this effect occurs through tyrosine kinase activation.

The expression and function of integrin subunits was examined by immunohistochemical staining of normal and malignant tissues and by producing specific changes in avian beta subunit cDNA that were subsequently expressed in mammalian cells. Most tissues express only a restricted number of integrins. These include primarily those thought to function as collagen/laminin receptors. With the exception of metastatic melanomas, tumors show a general down regulation of integrins. Structure/function studies of the beta subunit show that the cytoplasmic domain is required for inclusion in adhesion plaques and for promotion of adhesive functions; that the transmembrane domain is required for subunit association, but not proper alpha subunit selection; and that the amino terminal one third of the subunit must remain intact for subunit selection and ligand binding to occur.

Neutrophils are important innate immune cells involved in microbial clearance at sites of infection. However, the roles of neutrophils in cancer development are not clear. We hypothesized that neutrophils might mediate antitumor effects at the initial tumor formation stage. To test this hypothesis, we injected 2x106 mouse lung Lewis cancer cells into the flanks of C57BL/6 and Cxcr2-/- mice, the latter lack the receptor for ELR+ CXC neutrophil chemokines. We observed increased tumor growth in Cxcr2-/- mice as compared with controls within 3 weeks of injection. FACS analysis indicated fewer neutrophils and more IL-17-producing cells in the tumors of Cxcr2-/- mice than those of C57BL/6 mice. We further tested the roles of neutrophils using anti-Ly6G antibody depletion at the time of tumor cell injection. We demonstrated that tumors grew faster in the neutrophil depleted group, while the percentages of IL-17-producing cells in the tumors and the levels of G-CSF in the plasma were also increased in the absence of neutrophils. To test whether neutrophils have a direct cytotoxic effect on LLC cells, we performed in vitro cell killing assays. We demonstrated neutrophils isolated from both circulation and bone marrow exerted cytotoxic effects. In conclusion, by using Cxcr2-/- and neutrophil antibody-depleted C57BL/6 mice, we have demonstrated that initial tumor growth of LLC cells is enhanced in association with inaccessibility of neutrophils and activation of the IL-17/G-CSF axis.

Foxp3+ Treg cells limit autoimmunity and maintain immune homeostasis but also restrict immune responses to cancer. Thus, Tregs are a promising target for modulation of host anti-tumor immune responses. We have shown that targeting of Foxp3+ Tregs using histone/protein deacetylase inhibitors (HDACi) promotes Foxp3 acetylation and increases Treg production and suppression function. By contrast, there are no studies concerning the role of histone/protein acetyltransferases (HAT) targeting in Tregs. HATs are classified into 3 main families; GNAT, MYST and p300/CBP. Here, we found that conditional deletion of p300 in Tregs caused lymphoproliferation, weight loss, arthritis and dermatitis in mice. Likewise, deletion of p300 in Tregs, or its pharmacologic inhibition, decreased Foxp3+ Treg production and impaired Treg-mediated suppression of homeostatic proliferation, and blocked Treg-dependent donor-specific allograft tolerance. In contrast with these data, p300 targeting decreased TC1 lung tumor volume in immunocompetent C57BL/6 mice, but had no direct effect on TC1 tumor cell proliferation in vitro, or on the growth of TC1 implanted tumors in RAG1-/- immunodeficient mice. We conclude that in real world tumor models using immunocompetent mice, p300 targeting impaired Treg function without overt impairing effector T cell responses or inducing autoimmunity. We thereby identify p300 as a critical target for modulation of Foxp3+ Treg function in the context of anticancer therapy.