Purpose: The ubiquitously expressed transmembrane glycoprotein CD47 delivers an anti-phagocytic (do not eat) signal by binding signal-regulatory protein α (SIRPα) on macrophages. CD47 is overexpressed in cancer cells and its expression is associated with poor clinical outcomes. TTI-621 (SIRPαFc) is a fully human recombinant fusion protein that blocks the CD47–SIRPα axis by binding to human CD47 and enhancing phagocytosis of malignant cells. Blockade of this inhibitory axis using TTI-621 has emerged as a promising therapeutic strategy to promote tumor cell eradication. Experimental Design: The ability of TTI-621 to promote macrophage-mediated phagocytosis of human tumor cells was assessed using both confocal microscopy and flow cytometry. In vivo antitumor efficacy was evaluated in xenograft and syngeneic models and the role of the Fc region in antitumor activity was evaluated using SIRPαFc constructs with different Fc tails. Results: TTI-621 enhanced macrophage-mediated phagocytosis of both hematologic and solid tumor cells, while sparing normal cells. In vivo, TTI-621 effectively controlled the growth of aggressive AML and B lymphoma xenografts and was efficacious in a syngeneic B lymphoma model. The IgG1 Fc tail of TTI-621 plays a critical role in its antitumor activity, presumably by engaging activating Fcγ receptors on macrophages. Finally, TTI-621 exhibits minimal binding to human erythrocytes, thereby differentiating it from CD47 blocking antibodies. Conclusions: These data indicate that TTI-621 is active across a broad range of human tumors. These results further establish CD47 as a critical regulator of innate immune surveillance and form the basis for clinical development of TTI-621 in multiple oncology indications. Clin Cancer Res; 23(4); 1068–79. ©2016 AACR.

Purpose: To evaluate safety and preliminary efficacy of the humanized anti-epidermal growth factor receptor monoclonal antibody h-R3 in combination with radiotherapy (RT) in unresectable head and neck cancer patients. Secondary end points were the measurement of h-R3 serum levels and the assessment of the potential mechanisms of antitumor effect on patient biopsies. Anti-idiotypic response to h-R3 was assessed. To predict pharmacologic effect, a mathematical model for antibodies recognizing antigens expressed in tumors and normal tissues was built. Patients and Methods: Twenty-four patients with advanced carcinomas of the head and neck received six once-weekly infusions of h-R3 at four dose levels in combination with RT. Pretreatment tumor biopsies were obtained to evaluate epidermal growth factor receptor expression as an enrollment criterion. Second biopsies were taken to evaluate the proliferative activity and angiogenesis in comparison with the pretreatment samples. Patient serum samples were collected to measure h-R3 levels and anti-idiotypic response. Results: The combination of h-R3 and RT was well tolerated. Antibody-related adverse events consisted in infusion reactions. No skin or allergic toxicity appeared. Overall survival significantly increased after the use of the higher antibody doses. Immunohistochemistry studies of tumor specimens before and after treatment revealed that antitumor response correlated with antiproliferative and antiangiogenic effect. One patient developed antibodies to h-R3. The mathematical model predicted that the maximum difference between the area under the curve in tumors and normal tissues is reached when the antibody has intermediate affinity. Conclusion: h-R3 is a well-tolerated drug that may enhance radiocurability of unresectable head and neck neoplasms.

CD44 is an integral hyaluronan receptor that can promote or inhibit motogenic signaling in tumor cells. Rhamm is a nonintegral cell surface hyaluronan receptor (CD168) and intracellular protein that promotes cell motility in culture. Here we describe an autocrine mechanism utilizing cell surface Rhamm-CD44 interactions to sustain rapid basal motility in invasive breast cancer cell lines that requires endogenous hyaluronan synthesis and the formation of Rhamm-CD44-ERK1,2 complexes. Motile/invasive MDA-MB-231 and Ras-MCF10A cells produce more endogenous hyaluronan, cell surface CD44 and Rhamm, an oncogenic Rhamm isoform, and exhibit more elevated basal activation of ERK1,2 than less invasive MCF7 and MCF10A breast cancer cells. Furthermore, CD44, Rhamm, and ERK1,2 uniquely co-immunoprecipitate and co-localize in MDA-MB-231 and Ras-MCF10A cells. Combinations of anti-CD44, anti-Rhamm antibodies, and a MEK1 inhibitor (PD098059) had less-than-additive blocking effects, suggesting the action of all three proteins on a common motogenic signaling pathway. Collectively, these results show that cell surface Rhamm and CD44 act together in a hyaluronan-dependent autocrine mechanism to coordinate sustained signaling through ERK1,2, leading to high basal motility of invasive breast cancer cells. Therefore, an effect of CD44 on tumor cell motility may depend in part on its ability to partner with additional proteins, such as cell surface Rhamm.

In light of the continuing need for effective anticancer agents, and the association of fruit and vegetable consumption with reduced cancer risk, edible plants are increasingly being considered as sources of anticancer drugs. Cranberry presscake (the material remaining after squeezing juice from the berries), when fed to mice bearing human breast tumor MDA-MB-435 cells, was shown previously to decrease the growth and metastasis of tumors. Therefore, further studies were undertaken to isolate the components of cranberry that contributed to this anticancer activity, and determine the mechanisms by which they inhibited proliferation. Using standard chromatographic techniques, a warm-water extract of cranberry presscake was fractionated, and an acidified methanol eluate (Fraction 6, or Fr6) containing flavonoids demonstrated antiproliferative activity. The extract inhibited proliferation of 8 human tumor cell lines of multiple origins. The androgen-dependent prostate cell line LNCaP was the most sensitive of those tested (10 mg/L Fr6 inhibited its growth by 50%), and the estrogen-independent breast line MDA-MB-435 and the androgen-independent prostate line DU145 were the least sensitive (250 mg/L Fr6 inhibited their growth by 50%). Other human tumor lines originating from breast (MCF-7), skin (SK-MEL-5), colon (HT-29), lung (DMS114), and brain (U87) had intermediate sensitivity to Fr6. Using flow cytometric analyses of DNA distribution (cell cycle) and annexin V-positivity (apoptosis), Fr6 was shown in MDA-MB-435 cells to block cell cycle progression (P < 0.05) and induce cells to undergo apoptosis (P < 0.05) in a dose-dependent manner. Fr6 is potentially a source of a novel anticancer agent.