TTI-621 (SIRPαFc): A CD47-Blocking Innate Immune Checkpoint Inhibitor with Broad Antitumor Activity and Minimal Erythrocyte Binding.

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.

A novel small molecule with potent anticancer activity inhibits cell growth by modulating intracellular labile zinc homeostasis.

ML-133 is a novel small molecule with potent antiproliferative activity, as shown in cancer cell lines and in a human colon tumor xenograft model. ML-133 reduces the concentration of intracellular labile zinc in HT-29 colon cancer cells, leading to induction of the Krüppel-like factor 4 transcription factor. Krüppel-like factor 4 displaces the positive regulator SP1 from the cyclin D1 promoter, thereby negatively regulating the expression of cyclin D1 and promoting the G(1)-S phase arrest of cell proliferation. The antiproliferative and antitumor activity of ML-133 described in the present study suggests modulation of intracellular zinc homeostasis as a potential strategy for the treatment of several cancer types, and ML-133 represents a promising new class of antitumor agents that deserves further development.

GTI-2040 displays cooperative anti-tumor activity when combined with interferon alpha against human renal carcinoma xenografts.

GTI-2040, an antisense oligonucleotide targeting the small subunit of ribonucleotide reductase, acts as an anti-tumor agent in animal models of human cancer. In the present study, the anti-tumor activity of GTI-2040, in combination with interferon alpha (IFNalpha) was investigated against human renal cell carcinoma tumors xenografted into mice. The human renal cell carcinoma cell lines, Caki-1 and A498 were sensitive to IFNalpha both in vitro and when implanted into mice. In combination with GTI-2040 there were cooperative effects at intermediate doses of the two agents and complete tumor regression at higher combination doses. A control oligonucleotide was not effective as a monotherapy and did not improve the efficacy of IFNalpha. The effect of combination treatment on apoptosis and proliferation of tumor cells, isolated from xenografted tumors, was examined by histochemistry. GTI-2040 increased the percentage of cells undergoing apoptosis with a concomitant decrease in proliferation. IFNalpha alone had no effect but in combination with GTI-2040 resulted in increased apoptosis and decreased proliferation compared to GTI-2040 alone. Taken together these results expand the potential clinical applications of GTI-2040 to include combination therapy with IFNalpha.

11-Phenyl-[b,e]-dibenzazepine compounds: novel antitumor agents.

A series of 11-phenyl-[b,e]-dibenzazepine compounds were synthesized and shown to be inhibitors of tumor cell proliferation with IC(50) values ranging from submicromolar to micromolar concentrations. Flow cytometric analyses of several active compounds demonstrated inhibition of cell cycle progression at the G(0)-G(1) phase transition resulting in G(0)-G(1) arrest.

Anti-proliferative and anti-tumor effects of antisense oligonucleotide GTI-2601 targeted against human thioredoxin.

Human thioredoxin has been implicated in cancer as a growth stimulator through regulation of DNA replication and growth factor activity, as a modulator of transcription factor activity, and as an inhibitor of apoptosis. In the present study, the steady-state level of thioredoxin protein was examined in a number of cancer cell lines. Interestingly, thioredoxin expression is elevated in a variety of human tumor cell lines compared with normal cell lines. The altered expression of thioredoxin in tumor cells suggests it may be a target in the development of novel therapeutic agents for the treatment and prevention of cancer. Further to this possibility, 26 phosphorothioate antisense oligodeoxynucleotides (PS-AS-ODNs) were evaluated for the ability to inhibit thioredoxin expression in cell culture. One PS-AS-ODN, GTI-2601, specifically reduced the levels of thioredoxin mRNA and protein, exhibited potent anti-proliferative effects on colony formation in vitro, and had anti-tumor effects in human tumor xenograft mouse models in vivo. Sequence-specific decreases in thioredoxin expression levels were accompanied by significant suppression of tumor growth in mice. Taken together, these data suggest that thioredoxin may be a useful target for developing PS-AS-ODNs as drug candidates against human cancer.

NC381, a novel anticancer agent, arrests the cell cycle in G0-G1 and inhibits lung tumor cell growth in vitro and in vivo.

Although clotrimazole (CLT), an antifungal drug, inhibits tumor cell proliferation and angiogenesis, its clinical application is hampered by significant hepatotoxicity due to the presence of an imidazole moiety. In our attempts to develop CLT analogs that are devoid of imidazole and are as efficacious as CLT, one pharmacophore designated NC381 was generated and shown to inhibit tumor cell growth via a mechanism similar to that of CLT. In vitro, treatment of NCI-H460 nonsmall cell lung cancer (NSCLC) cells with NC381 inhibited growth in a time-dependent manner. Flow cytometric analysis demonstrated that the decrease in cell growth was associated with inhibition of cell cycle progression at the G(1)-S phase transition, resulting in G(0)-G(1) arrest. There was a concomitant inhibition of cyclin D1 expression and subsequent reduction in the formation of the cyclin D1-CDK4 complex. Consistent with a decrease in the cyclin D1-CDK4 complex, NC381 treatment resulted in significant inhibition of pRb phosphorylation. There also were changes in the activity of cell cycle-related proteins, including p16(Ink4) and p27(Kip1). Together, these results are consistent with a model in which NC381 arrests cell cycle progression via inhibition of the pathway that promotes exit from the G(1) phase of the cell cycle. Furthermore, the clinical applicability of NC381 was evaluated in an in vivo murine xenograft model of human NSCLC (NCI-H460). NC381 treatment resulted in significant inhibition of tumor growth. Given the poor prognosis and the limited treatment options available, the present results underscore the potential of NC381 in the treatment of human NSCLC.

Adenovirus-mediated ribonucleotide reductase R1 gene therapy of human colon adenocarcinoma.

Ribonucleotide reductase is the enzyme responsible for the reduction of ribonucleotides to their corresponding deoxyribonucleotides for DNA synthesis. Ribonucleotide reductase is a multisubunit complex containing two polypeptides, R1 and R2. In addition to catalytic and allosteric regulatory functions, the R1 subunit appears to act as a novel tumor suppressor. Previous studies demonstrated that overexpression of mouse R1 resulted in suppression of tumorigenicity and metastatic potential, whereas expression of antisense RNA, complementary to R1 mRNA, increased anchorage-independent growth of ras-transformed NIH 3T3 cells. The current study investigated the potential of R1 gene therapy for human cancer using a recombinant adenovirus encoding the human R1 gene (rAd5-R1). Recombinant viruses were constructed by FLP-mediated site-specific recombination and demonstrated high infectivity of a human colon carcinoma cell line (Colo320 HRS), as assessed by expression of a viral encoded beta-Gal gene (rAd5-LacZ). R1mRNA and protein were overexpressed in Colo320 HRS cells infected with rAd5-R1 compared with untreated or rAd5-LacZ-infected cells. Infection with rAd5-R1 inhibited Colo320 HRS cell proliferation, in vitro, in a time- and dose-dependent manner. When Colo320 HRS cells were treated with rAd5-R1, before injection into CD-1 mice, there was complete inhibition of tumor growth compared with treatment with rAd5-LacZ. Furthermore, intratumoral injection of rAd5-R1 into Colo320 HRS tumor xenografts inhibited tumor growth in CD-1 mice compared with rAd5-LacZ treated mice (P = 0.0001). These results demonstrate gene-specific antitumor effects of R1 and suggest that rAd5-R1 gene therapy has the potential to improve currently available treatments for colon cancer.