Presentation_1_CD5 blockade, a novel immune checkpoint inhibitor, enhances T cell anti-tumour immunity and delays tumour growth in mice harbouring poorly immunogenic 4T1 breast tumour homografts.pptx

CD5 is a member of the scavenger receptor cysteine-rich superfamily that is expressed on T cells and a subset of B cells (B1a) cell and can regulate the T cell receptor signaling pathway. Blocking CD5 function may have therapeutic potential in treatment of cancer by enhancing cytotoxic T lymphocyte recognition and ablation of tumour cells. The effect of administering an anti-CD5 antibody to block or reduce CD5 function as an immune checkpoint blockade to enhance T cell anti-tumour activation and function in vivo has not been explored. Here we challenged mice with poorly immunogenic 4T1 breast tumour cells and tested whether treatment with anti-CD5 monoclonal antibodies (MAb) in vivo could enhance non-malignant T cell anti-tumour immunity and reduce tumour growth. Treatment with anti-CD5 MAb resulted in an increased fraction of CD8+ T cells compared to CD4+ T cell in draining lymph nodes and the tumour microenvironment. In addition, it increased activation and effector function of T cells isolated from spleens, draining lymph nodes, and 4T1 tumours. Furthermore, tumour growth was delayed in mice treated with anti-CD5 MAb. These data suggest that use of anti-CD5 MAb as an immune checkpoint blockade can both enhance activation of T cells in response to poorly immunogenic antigens and reduce tumour growth in vivo. Exploration of anti-CD5 therapies in treatment of cancer, alone and in combination with other immune therapeutic drugs, is warranted.</p

Combining COTI-2 with cetuximab and erlotinib synergistically enhances the efficacy of these drugs against human colorectal cancer cells.

<p>Human colorectal cancer cell lines HCT-15 (A), SW-620 (B), and COLO-205 (C) were treated with varying concentrations of COTI-2, cetuximab, erlotinib, or a combination of COTI-2 and either EGFR inhibitor. Tumor cells were allowed to proliferate for 4 days in the presence of drug(s) before cell viability was determined. All data points indicate the mean of 5 independent measures of viability ± SEM. *Significant difference from cells treated with COTI-2 alone using a Student’s <i>t</i>-test (<i>p</i><0.05).</p

The effect of COTI-2 treatment on U87-MG cells in combination with temsirolimus and rapamycin.

<p>U87-MG human glioma cells were cultured in the presence of various concentrations of temsirolimus plus COTI-2 (A) or rapamycin plus COTI-2 (B) for 4 days before cell viability was determined. Black circles indicate the combination of COTI-2 and temsirolimus (A) or rapamycin (B) and the white circles indicate treatment with COTI-2 alone. Data are the average mean of 6 independent experiments ± SEM.*Significant difference, Student’s <i>t</i>-test, <i>p</i><0.05.</p

COTI-2 enhances the cytotoxic activity of paclitaxel and cisplatin.

<p>DMS-114 (A and C) and SHP-77 cells (B and D) were cultured overnight then exposed to the indicated doses of paclitaxel and cisplatin plus or minus a pre-determined dose of COTI-2 (IC₂₅) for 4 days before cell viability was determined. The asterix (*) indicates a significant greater-than-additive effect in the combination therapy compared to single agent alone, <i>p</i><0.05, Student’s <i>t</i>-test. Data are the average mean of 3 independent experiments ± SEM. (E) AN3-CA human endometrial cells (1 x 10⁷) were injected into the right flanks of athymic nude mice (n = 10 mice per group). Xenografts were grown to an average volume of 170 mm³ before animals received treatment i.v. Vehicle control and COTI-2 (25 mg/kg) were administered 3 times a week on alternate days until study end. The schedule for paclitaxel was daily for 5 days (5 mg/kg). In the combination arm, animals received COTI-2 (25 mg/kg) 3 times a week for the entire study and 5 injections of paclitaxel (5 mg/kg). *Significantly different from the paclitaxel alone treatment group, Student’s <i>t</i>-test, <i>p</i><0.05. Error bars represent SEM.</p

Novel anti-cancer drug COTI-2 synergizes with therapeutic agents and does not induce resistance or exhibit cross-resistance in human cancer cell lines

<div><p>Emerging drug-resistance and drug-associated toxicities are two major factors limiting successful cancer therapy. Combinations of chemotherapeutic drugs have been used in the clinic to improve patient outcome. However, cancer cells can acquire resistance to drugs, alone or in combination. Resistant tumors can also exhibit cross-resistance to other chemotherapeutic agents, resulting in sub-optimal treatment and/or treatment failure. Therefore, developing novel oncology drugs that induce no or little acquired resistance and with a favorable safety profile is essential. We show here that combining COTI-2, a novel clinical stage agent, with multiple chemotherapeutic and targeted agents enhances the activity of these drugs <i>in vitro</i> and <i>in vivo</i>. Importantly, no overt toxicity was observed in the combination treatment groups <i>in vivo</i>. Furthermore, unlike the tested chemotherapeutic drugs, cancer cells did not develop resistance to COTI-2. Finally, some chemo-resistant tumor cell lines only showed mild cross-resistance to COTI-2 while most remained sensitive to it.</p></div

Chemo-resistant cancer cell lines often do not show cross-resistance to COTI-2.

<p>Paclitaxel-resistant (A) and cisplatin-resistant (B) A549, DMS-153, and SHP-77 cells were exposed to IC₅₀ concentrations of COTI-2 and tumor cell proliferation was measured after approximately 4 doublings of control cells. 5FUdR-resistant HeLa cells (C) and vincristine-resistant HN-5a cells (D) were exposed to IC₅₀ concentrations of COTI-2 as described in (A) and (B). Significant differences were assessed by Student’s <i>t</i>-test (<i>p</i><0.05). Data indicates mean values derived from 3 independent experiments ± SEM.</p

Only some chemotherapeutic drugs with similar molecular targets show enhanced activity when combined with COTI-2.

<p>DMS-114 (A and C) and SHP-77 (B and D) SCLC cells were treated with various concentrations of carboplatin (A and B) or vincristine (C and D) in combination with or without an IC₂₅ concentration of COTI-2 for 4 days before cell viability was determined. The asterix (*) indicates a significant greater-than-additive effect in the combination therapy compared to single agent alone, <i>p</i><0.05, Student’s <i>t</i>-test. Data are the average mean of 3 independent experiments ± SEM. (E and F) PANC-1 human pancreatic carcinoma cells (2 x 10⁶) were injected into each flank of NCr-<i>nu</i> mice (n = 12 mice per group). Xenografts were grown to ~100 mm³ before animals received treatment, which consisted of the vehicle control, COTI-2 (125 mg/kg), gemcitabine (100 mg/kg), or the combination (COTI-2 at 125 mg/kg and gemcitabine at 100 mg/kg) (E) or the vehicle control, COTI-2 (125 mg/kg), abraxane (15 mg/kg), or the combination (COTI-2 at 125 mg/kg and abraxane at 15 mg/kg) (F). COTI-2 was delivered <i>p</i>.<i>o</i>. with a schedule of 5 days on treatment and 2 days off weekly. Gemcitabine (100 mg/kg) was administered i.p., every second day, for a total of 6 injections. Abraxane (15 mg/kg) was administered i.v., once per day for 5 consecutive days. The dosing schedule for the combination treatments was identical to that of the single agent treatments for each drug. COTI-2 administration was initiated 1 day after treatment with either gemcitabine or abraxane. *Significantly different from single agent gemcitabine or abraxane treatment groups, Student’s <i>t</i>-test, <i>p</i><0.05.</p

Cancer cells do not develop acquired resistance to COTI-2 unlike treatment with paclitaxel and cisplatin.

<p>A549 NSCLC (A), DMS-153 SCLC (B) and SHP-77 SCLC (C) cells were cultured in IC₅₀ concentrations of COTI-2, paclitaxel, or cisplatin for 4 rounds of treatment (5 generations of cells including the parental cells). The surviving 50% of cells from the initial IC₅₀ tested were harvested and cultured for 5 days, after which time this new generation of cells was re-treated with the same agent and a new IC₅₀ value was established. Emerging resistance was identified by increasing IC₅₀ values in successive generations. Significant differences were assessed by Student’s <i>t</i>-test (<i>p</i><0.05). *Significantly different from parental cells treated with the mentioned drug. Data points indicate the mean from 3 independent experiments ± SEM.</p

Single-Walled Carbon Nanotubes Noncovalently Functionalized with Lipid Modified Polyethylenimine for siRNA Delivery <i>in Vitro</i> and <i>in Vivo</i>

siRNA can downregulate the expression of specific genes. However, delivery to specific cells and tissues <i>in vivo</i> presents significant challenges. Modified carbon nanotubes (CNTs) have been shown to protect siRNA and facilitate its entry into cells. However, simple and efficient methods to functionalize CNTs are needed. Here, noncovalent functionalization of CNTs is performed and shown to effectively deliver siRNA to target cells. Specifically, single-walled CNTs were functionalized by noncovalent association with a lipopolymer. The lipopolymer (DSPE-PEG) was composed of a phospholipid 1,2-distearoyl-<i>sn</i>-glycero-3-phosphoethanolamine (DSPE) and poly­(ethylene glycol) (PEG). Three different ratios of polyethylenimine (PEI) to DSPE-PEG were synthesized and characterized and the products were used to disperse CNTs. The resulting materials were used for siRNA delivery <i>in vitro</i> and <i>in vivo</i>. The structural, biophysical, and biological properties of DGI/C and their complexes formed with siRNA were investigated. Cytotoxicity of the materials was low, and effective gene silencing in B16–F10 cells was demonstrated <i>in vitro</i>. In addition, significant uptake of siRNA as well as gene silencing in the liver was found following intravenous injection. This approach provides a new strategy for siRNA delivery and could provide insight for the development of noncovalently functionalized CNTs for siRNA therapy

A549 clone sensitivity to methoxyamine (3 mM) before and after IDO induction.

<p>Proliferation of each of 5 individual A549 cell clonal populations before (<b>Panel A</b>) and after (<b>Panel B</b>) IDO induction with IFNγ. A549 clonal populations were cultured with or without IFNγ (25 ng/ml) for 48 h. Cultured medium was then replaced with fresh growth medium containing Methoxyamine (MX) (3 mM) and cells were allowed to proliferate for 72 h. Cells were then trypsinized and live cells were enumerated. <b>White bars</b>: A549 clones transfected with scrambled, non-targeting control shRNA. <b>Gray bars</b>: A549 cells transfected with anti-IDO shRNA. Each bar represents the mean of 3 values (<i>n</i> = 3 for determination of each value) ± SD. Results are normalized to control cells not treated with methoxyamine, without (panel A) or with (panel B) IFNγ treatment. <b>Panel C</b>: Induction of IDO in A549 clonal cell populations induces resistance to MX (3 mM). Results were obtained from 3 or 2 independent clonal cell populations with scrambled, non-targeting control shRNA or anti-IDO shRNA, respectively. Each bar represents a mean of 9 (white bars) or 6 (black bars) values ± SEM, *Significant difference, Student's <i>t</i>-test, <i>p</i><0.05. <b>Panel D</b>: Relationship between IDO protein level (relative to actin) and resistance to methoxyamine (MX)(proliferation relative to untreated control cells). The R² value of 0.83 represents a moderate positive relationship.</p