Leonard Joseph Appleman

T cell anergy and costimulation

Summary:  T lymphocytes play a key role in immunity by distinguishing self from nonself peptide antigens and regulating both the cellular and humoral arms of the immune system. Acquired, antigen‐specific unresponsiveness is an important mechanism by which T cell responses to antigen are regulated in vivo. Clonal anergy is the term that describes T cell unresponsiveness at the cellular level. Anergic T cells do not proliferate or secrete interleukin (IL)‐2 in response to appropriate antigenic stimulation. However, anergic T cells express the IL‐2 receptor, and anergy can be broken by exogenous IL‐2. Anergy can be induced by submitogenic exposure to peptide antigen in the absence of a costimulatory signal provided by soluble cytokines or by interactions between costimulatory receptors on T cells and counter‐receptors on antigen‐presenting cells. The molecular events that mediate the induction and maintenance of T cell anergy are the focus of this review. The molecular consequences of CD28–B7 interaction are discussed as a model for the costimulatory signal that leads to T cell activation rather than the induction of anergy.

CD28 Costimulation Mediates Down-Regulation of p27kip1 and Cell Cycle Progression by Activation of the PI3K/PKB Signaling Pathway in Primary Human T Cells

CD28 provides a costimulatory signal that cooperates with the TCR/CD3 complex to induce T cell activation, cytokine production, and clonal expansion. We have recently shown that CD28 directly regulates progression of T lymphocytes through the cell cycle. Although a number of signaling pathways have been linked to the TCR/CD3 and to CD28, it is not known how these two receptors cooperate to induce cell cycle progression. Here, using cell-permeable pharmacologic inhibitors of phosphatidylinositol 3-hydroxykinase (PI3K) and mitogen-activated protein kinase kinase (MEK1/2), we show that cell cycle progression of primary T lymphocytes requires simultaneous activation of PI3K- and MEK1/2-dependent pathways. Decreased abundance of cyclin-dependent kinase inhibitor p27kip1, which requires simultaneous TCR/CD3 and CD28 ligation, was dependent upon both MEK and PI3K activity. Ligation of TCR/CD3, but not CD28 alone, resulted in activation of MEK targets extracellular signal-related kinase 1/2, whereas ligation of CD28 alone was sufficient for activation of PI3K target protein kinase B (PKB; c-Akt). CD28 ligation alone was also sufficient to mediate inactivating phosphorylation of PKB target glycogen synthase kinase-3 (GSK-3). Moreover, direct inactivation of GSK-3 by LiCl in the presence of anti-CD3, but not in the presence of anti-CD28, resulted in down-regulation of p27kip1, hyperphosphorylation of retinoblastoma tumor suppressor gene product, and cellular proliferation. Thus, inactivation of the PI3K-PKB target GSK-3 could substitute for CD28 but not for CD3 signals. These results show that the PI3K-PKB pathway links CD28 to cell cycle progression and suggest that p27kip1 integrates mitogenic MEK- and PI3K-dependent signals from TCR and CD28 in primary T lymphocytes.

CD28 costimulation mediates T cell expansion via IL-2-independent and IL-2-dependent regulation of cell cycle progression

In the presence of TCR ligation by Ag, CD28 pathway mediates the most potent costimulatory signal for T cell activation, cytokine secretion, and T cell expansion. Although CD28 costimulation promotes T cell expansion due to IL-2 secretion and subsequent signaling via the IL-2 receptor, recent studies indicate that the dramatic T cell expansion mediated through the unopposed CD28 stimulation in CTLA4-deficient mice is IL-2 independent. Therefore, we sought to dissect the effects of CD28 and IL-2 receptor pathways on cell cycle progression and determine the molecular mechanisms by which the CD28 pathway regulates T cell expansion. Here we show that CD28 costimulation directly regulates T cell cycle entry and progression through the G1 phase in an IL-2-independent manner resulting in activation of cyclin D2-associated cdk4/cdk6 and cyclin E-associated cdk2. Subsequent progression into the S phase is mediated via both IL-2-dependent and IL-2-independent mechanisms and, although in the absence of IL-2 the majority of T cells are arrested at the G1/S transition, a significant fraction of them progresses into the S phase. The key regulatory mechanism for the activation of cyclin-cdk complexes and cell cycle progression is the down-regulation of p27kip1 cdk inhibitor, which is mediated at the posttranscriptional level by its ubiquitin-dependent degradation in the proteasome pathway. Therefore, CD28 costimulation mediates T cell expansion in an IL-2-independent and IL-2 dependent manner and regulates cell cycle progression at two distinct points: at the early G1 phase and at the G1/S transition.

A Phase I Study of Foretinib, a Multi-Targeted Inhibitor of c-Met and Vascular Endothelial Growth Factor Receptor 2

Purpose: Foretinib is an oral multikinase inhibitor targeting Met, RON, Axl, and vascular endothelial growth factor receptor. We conducted a phase I, first-time-in-human, clinical trial using escalating doses of oral foretinib. The primary objectives are to identify a maximum tolerated dose and determine the safety profile of foretinib. Secondary objectives included evaluation of plasma pharmacokinetics, long-term safety after repeated administration, preliminary antitumor activity, and pharmacodynamic activity. Experimental Design: Patients had histologically confirmed metastatic or unresectable solid tumors for which no standard measures exist. All patients received foretinib orally for 5 consecutive days every 14 days. Dose escalation followed a conventional “3+3” design. Results: Forty patients were treated in eight dose cohorts. The maximum tolerated dose was defined as 3.6 mg/kg, with a maximum administered dose of 4.5 mg/kg. Dose-limiting toxicities included grade 3 elevations in aspartate aminotransferase and lipase. Additional non–dose-limiting adverse events included hypertension, fatigue, diarrhea, vomiting, proteinuria, and hematuria. Responses were observed in two patients with papillary renal cell cancer and one patient with medullary thyroid cancer. Stable disease was identified in 22 patients. Foretinib pharmacokinetics increased linearly with dose. Pharmacodynamic evaluation indicated inhibition of MET phosphorylation and decreased proliferation in select tumor biopsies at submaximal doses. Conclusions: The recommended dose of foretinib was determined to be 240 mg, given on the first 5 days of a 14-day cycle. This dose and schedule were identified as having acceptable safety and pharmacokinetics, and will be the dose used in subsequent phase II trials. Clin Cancer Res; 16(13); 3507–16. ©2010 AACR.

Neoadjuvant Dose-Dense Methotrexate, Vinblastine, Doxorubicin, and Cisplatin With Pegfilgrastim Support in Muscle-Invasive Urothelial Cancer: Pathologic, Radiologic, and Biomarker Correlates

PURPOSE In advanced urothelial cancer, treatment with dose-dense methotrexate, vinblastine, doxorubicin, and cisplatin (ddMVAC) results in a high response rate, less toxicity, and few dosing delays. We explored the efficacy and safety of neoadjuvant ddMVAC with pegfilgrastim support in muscle-invasive urothelial cancer (MIUC). PATIENTS AND METHODS Patients with cT2-cT4, N0-1, M0 MIUC were enrolled. Four cycles of ddMVAC were administered, followed by radical cystectomy. The primary end point was pathologic response (PaR) defined by pathologic downstaging to ≤ pT1N0M0. The study used Simons optimal two-stage design to evaluate null and alternative hypotheses of PaR rate of 35% versus 55%. Secondary end points included toxicity, disease-free survival (DFS), radiologic response (RaR), and biomarker correlates, including ERCC1. RESULTS Between December 2008 and April 2012, 39 patients (cT2N0, 33%; cT3N0, 18%; cT4N0, 3%; cT2-4N1, 43%; unspecified, 3%) were enrolled. Median follow-up was 2 years. Overall, 49% (80% CI, 38 to 61) achieved PaR of ≤ pT1N0M0, and we concluded this regimen was effective. High-grade (grade ≥ 3) toxicities were observed in 10% of patients, with no neutropenic fevers or treatment-related death. One-year DFS was 89% versus 67% for patients who achieved PaR compared with those who did not (hazard ratio [HR], 2.6; 95% CI, 0.8 to 8.1; P = .08) and 86% versus 62% for patients who achieved RaR compared with those who did not (HR, 4.1; 95% CI, 1.3 to 12.5; P = .009). We found no association between serum tumor markers or ERCC1 expression with response or survival. CONCLUSION In patients with MIUC, neoadjuvant ddMVAC was well tolerated and resulted in significant pathologic and radiologic downstaging.

The high-dose aldesleukin "select" trial: A trial to prospectively validate predictive models of response to treatment in patients with metastatic renal cell carcinoma

Purpose: High-dose aldesleukin (HD IL2) received FDA approval for the treatment of metastatic renal cell carcinoma (MRCC) in 1992, producing a 14% objective response rate (ORR) and durable remissions. Retrospective studies suggested that clinical and pathologic features could predict for benefit. The Cytokine Working Group conducted this prospective trial to validate proposed predictive markers of response to HD IL2. Experimental Design: Standard HD IL2 was administered to prospectively evaluate whether the ORR of patients with mRCC with “good” predictive pathologic features based on an “integrated selection” model [ISM (e.g., clear-cell histology subclassification and carbonic anhydrase-9 (CA-9) IHC staining] was significantly higher than the ORR of a historical, unselected population. Archived tumor was collected for pathologic analysis including tumor programmed death-ligand 1 (PD-L1) expression. Results: One hundred and twenty eligible patients were enrolled between June 11 and September 7; 70% were Memorial Sloan Kettering Cancer Center (New York, NY) intermediate risk, 96% had clear cell RCC, and 99% had prior nephrectomy. The independently assessed ORR was 25% (30/120, 95% CI, 17.5%–33.7%, P = 0.0014; 3 complete responses, 27 partial responses) and was higher than a historical ORR. Thirteen patients (11%) remained progression free at 3 years and the median overall survival was 42.8 months. ORR was not statistically different by ISM classification (“good-risk” 23% vs. “poor-risk” 30%; P = 0.39). ORR was positively associated with tumor PD-L1 expression (P = 0.01) by IHC. Conclusions: In this prospective, biomarker validation study, HD IL2 produced durable remissions and prolonged survival in both “good” and “poor-risk” patients. The proposed ISM was unable to improve the selection criteria. Novel markers (e.g., tumor PD L1 expression) appeared useful, but require independent validation. Clin Cancer Res; 21(3); 561–8. ©2014 AACR.

MET Signaling Pathway: A Rational Target for Cancer Therapy

In Journal of Clinical Oncology (JCO), two groups of investigators provide important evidence for the critical role of MET in malignant transformation. Graziano et al report a dose-proportional risk of recurrence and decreased survival associated with increasing MET gene copy number in stages II to III gastric adenocarcinoma. Lennerz et al show that MET amplification in gastroesophogeal carcinomas is uncommon, observed in only 2% of their patient cohort. However, an increase in MET gene copy number was associated with higher presenting tumor stage and grade as well as shorter median survival. These two reports add to a growing body of evidence that MET is a key driver of oncogenic transformation in a defined subset of cancers. This Understanding the Pathway article will provide an overview of the MET signaling pathway, discuss the role of MET in malignancy, and consider the potential for targeting MET in cancer therapy. MET is an integral plasma membrane protein that relays signals from the extracellular environment into the cytoplasm (Fig 1). MET is activated when its extracellular domain binds to hepatocyte growth factor (HGF), also known as scatter factor. This protein is primarily expressed on epithelial cells, whereas HGF is secreted by mesenchymal cells and bound in an inactive form to heparin proteoglycans within the extracellular matrix. The HGF polypeptide is inactive in its initial form and must be cleaved into a disulfide-linked heterodimer by an extracellular protease—HGF activator—to acquire MET-binding activity. HGF activator is a serine protease that is produced in the liver but localized and activated selectively at sites of tissue injury and in cancer. The mesenchymal-epithelial communication mediated by HGF-MET signaling is critical to the formation of threedimensional body structures (morphognesis) through cellular behaviors collectively described as invasive growth: proliferation and survival (resistance to apoptotic signals), increased cell motility, cell dissociation (scattering), epithelial tubulogenesis, infiltration of tissues, and stimulation of angiogenesis. These MET-dependent behaviors are essential for normal processes such as embryogenesis, organ regeneration, and wound healing. Cancers with increased MET activity commandeer this machinery to facilitate tumor growth and invasion/metastasis. The cascade of molecular events triggered by HGF begins with MET kinase activation within the receptor’s cytoplasmic tail. Two tyrosine residues within the kinase domain undergo transautophosphorylation to create a fully activated enzyme. Two additional tyrosine residues are subsequently phosphorylated, resulting in a high-affinity binding motif that recruits a range of Src homology 2 domain– containing proteins, which include nonenzymatic adaptor proteins such as growth factor receptor– bound protein 2 (GRB2) and GRB2-associated binding protein 1. GRB2-associated binding protein 1 is phosphorylated by MET and cooperates with GRB2 and other adaptors to serve as a scaffold for additional signaling proteins, including phosphoinositide 3-kinase, phospholipaseC1, STAT3, focal adhesion kinase (involved in motility), and the tyrosine MET

Immunomodulatory Activity of Nivolumab in Metastatic Renal Cell Carcinoma

Purpose: Nivolumab, an anti-PD-1 immune checkpoint inhibitor, improved overall survival versus everolimus in a phase 3 trial of previously treated patients with metastatic renal cell carcinoma (mRCC). We investigated immunomodulatory activity of nivolumab in a hypothesis-generating prospective mRCC trial. Experimental Design: Nivolumab was administered intravenously every 3 weeks at 0.3, 2, or 10 mg/kg to previously treated patients and 10 mg/kg to treatment-naïve patients with mRCC. Baseline and on-treatment biopsies and blood were obtained. Clinical activity, tumor-associated lymphocytes, PD-L1 expression (Dako immunohistochemistry; ≥5% vs. <5% tumor membrane staining), tumor gene expression (Affymetrix U219), serum chemokines, and safety were assessed. Results: In 91 treated patients, median overall survival [95% confidence interval (CI)] was 16.4 months [10.1 to not reached (NR)] for nivolumab 0.3 mg/kg, NR for 2 mg/kg, 25.2 months (12.0 to NR) for 10 mg/kg, and NR for treatment-naïve patients. Median percent change from baseline in tumor-associated lymphocytes was 69% (CD3+), 180% (CD4+), and 117% (CD8+). Of 56 baseline biopsies, 32% had ≥5% PD-L1 expression, and there was no consistent change from baseline to on-treatment biopsies. Transcriptional changes in tumors on treatment included upregulation of IFNγ-stimulated genes (e.g., CXCL9). Median increases in chemokine levels from baseline to C2D8 were 101% (CXCL9) and 37% (CXCL10) in peripheral blood. No new safety signals were identified. Conclusions: Immunomodulatory effects of PD-1 inhibition were demonstrated through multiple lines of evidence across nivolumab doses. Biomarker changes from baseline reflect nivolumab pharmacodynamics in the tumor microenvironment. These data may inform potential combinations. Clin Cancer Res; 22(22); 5461–71. ©2016 AACR.

First-in-human phase I study of copanlisib (BAY 80-6946), an intravenous pan-class I phosphatidylinositol 3-kinase inhibitor, in patients with advanced solid tumors and non-Hodgkin's lymphomas

This phase I study evaluated the safety, tolerability, and pharmacokinetics of copanlisib, an intravenously administered pan-phosphatidylinositol 3-kinase inhibitor in patients with advanced solid tumors or non-Hodgkins lymphoma. Copanlisib was well tolerated with a manageable safety profile, with anti-tumor activity in both advanced solid tumors and hematological malignancies.

Phase 2 trial of linifanib (ABT-869) in patients with advanced renal cell cancer after sunitinib failure.

PURPOSE This study assessed the efficacy and safety of linifanib in patients with advanced renal cell carcinoma (RCC) who were previously treated with sunitinib. MATERIALS AND METHODS This open-label, multicentre, phase 2 trial of oral linifanib 0.25 mg/kg/day enrolled patients who had prior nephrectomy and adequate organ function. The primary end-point was objective response rate (ORR) per response evaluation criteria in solid tumors (RECIST) by central imaging. Secondary end-points were progression-free survival (PFS), overall survival (OS) and time to progression (TTP). Safety was also assessed. RESULTS Fifty-three patients, median age 61 years (range 40-80) were enrolled (August 2007 to October 2008) across 12 North-American centres. Median number of prior therapies was 2 (range 1-4); 43 patients (81%) had clear-cell histology. ORR was 13.2%, median PFS was 5.4 months (95% Confidence Interval (CI): 3.6, 6.0) and TTP was the same; median OS was 14.5 months (95% CI: 10.8, 24.1). The most common treatment-related adverse events (AEs) were diarrhoea (74%), fatigue (74%) and hypertension (66%), and the most common treatment-related Grade 3/4 AE was hypertension (40%). CONCLUSIONS Linifanib demonstrated clinically meaningful activity in patients with advanced RCC after sunitinib failure. At 0.25 mg/kg/day, significant dose modifications were required. An alternative, fixed-dosing strategy is being evaluated in other trials.