No significant changes in circulating T- or B-cell populations were seen after treatment with NHS-IL12.

<p>Relative numbers of T- and B-cell populations were quantified in PBMCs by flow cytometric analysis on days 1, 2, 3, and 8 after treatment with NHS-IL12. Immune cell determination was based on labeling: B cells, CD21⁺MHCII⁺ (A),T cells, CD4⁺ (B) or CD8⁺ (C); regulatory T cells, CD4⁺Foxp3⁺ (D);. CD4⁺ T cells trended downward at day 8 compared to their baseline measurements (B). Other differences were not evident over the time points evaluated.</p

Serial serum pharmacokinetics and IFN-γ/cytokine panel collections.

<p>IFN: interferon.</p><p>Serial serum pharmacokinetics and IFN-γ/cytokine panel collections.</p

Serum IL-10 levels increased following treatment with NHS-IL12.

<p>Each line represents a different dog, with individual colors representing different treatment groups. Serum IL-10 levels at 48 and 192 hours were significantly different from time points 0–8 hours (Kruskal-Wallis test followed by Dunn’s multiple comparison test) when data from all dogs was pooled. There was no difference in IL-10 levels at 48 hours between any of the treatment groups (Kruskal-Wallis test, p = .06, the 0.4 mg/m2 group was not included because it was a single dog).</p

RGD-A-<i>TNF</i> trafficking resulted in selective tumor endothelial cell localization and TNF <i>α</i> expression.

<p>(A, B) RGD-A-<i>TNF</i> selectively targeted tumor-associated vasculature (arrows) and was absent from normal tissues at 4–6 hours (A) and at 4 days (B). Magnification, 400-fold; scale bar, 100 µM. Pre-treatment tumor biopsies, post-treatment tumor biopsies, and post-treatment normal tissues in dogs that received a single-dose of RGD-A-<i>TNF</i> double-stained with an anti-CD31 specific antibody plus an anti-bacteriophage specific antibody. Detection was performed with Alexa Fluor 488 (green, blood vessels), Alexa Fluor 594 (red, AAVP), and DAPI (blue, cell nuclei). (C) Pre-treatment tumor biopsies (day 0), post-treatment normal biopsies (day 4) and post-treatment tumor biopsies (day 4) were used for extraction of total RNA. RT-PCR was performed to measure transcript levels of human TNFα in quadruplicate. The Y-axis represents the relative TNFα expression levels in post-treatment normal biopsies and post-treatment tumor biopsies compared to pre-treatment tumor biopsies after normalization to GAPDH expression (Kruskal-Wallis Test, p = 0.0107). All data are presented as means±standard deviations. (D–F) Presence of RGD-A-<i>TNF</i> was evaluated in post-treatment (day 28) necropsy samples of tumor (D) and normal tissues (E, F). Tissues were stained for RGD-A-<i>TNF</i> as described earlier. RGD-A-<i>TNF</i> selectively targeted tumor-associated vasculature in post-treatment tumor samples (arrows). In contrast, the vector was not apparent in pre-treatment tumor samples or in post-treatment normal control necropsy samples (such as lung, liver, spleen or intestine) after serial administrations of RGD-A-<i>TNF</i>.</p

RGD-A-<i>TNF</i> Dose Escalation Study: Patient Population and RGD-A-<i>TNF</i> Trafficking Data

<p>TU: transducing units; IF: immunoflourescent staining for presence of RGD-A-<i>TNF</i> in tumors; OCS: osteochondrosarcoma; PCT: plasma cell tumor; BCT: basal cell tumor; NST: nerve sheath tumor; STS: soft tissue sarcoma; OS: osteosarcoma; FS: fibrosarcoma; N.A.: not analyzed due to improper shipment.</p>*<p>Patient I.D.: dog is identified as participating in study 1 (dose escalation study), followed by patient number (i.e. patient 1.2 is dog 2 in study 1 and so on);</p>**<p>These two tumors originally diagnosed as soft tissue sarcomas were reclassified upon histopathologic review by a single pathologist (SN).</p

Clinical activity of NHS-IL12 evident in dogs with malignant melanoma.

<p>Patient 0201 had a large malignant melanoma of the right maxilla and buccal surface. Prior to treatment on day 1, the tumor measured 4.4 cm in longest diameter (A). The dog received 0.8 mg/m² (1.04 mg) of NHS-IL12 administered subcutaneously. On day 29 RECIST response was assessed and a PR was noted. The tumor measured 2.4 cm, representing a 45% decrease (B). The dog continued monthly dosing of NHS-IL12 and had no evidence of disease after three cycles of therapy (C). On day 85, despite complete regression of the lesion, a formal CR could not be assigned to the observed clinical activity in this dog since canine malignant melanoma invades the underlying bone and a CT scan of the oral cavity was not performed at baseline. No gross disease was noted through nine cycles of therapy. PD eventually recurred in the neighboring nasal cavity.</p

Pharmacokinetics of NHS-IL12 in tumor-bearing dogs after subcutaneous administration.

<p>AUC: area under the time-concentration curve; Cl: clearance; C_max: maximum concentration; MRT: mean residence time; NA: not available, calculation not possible due to a small number of data points; ND, not detectable; T_1/2: half-life.</p><p>Pharmacokinetics of NHS-IL12 in tumor-bearing dogs after subcutaneous administration.</p

Serum IFN-γ induction was associated with NHS-IL12 dose and the observation of adverse events.

<p>IFN-γ levels were measured using ELISA techniques. Induction of IFN-γ was detectable in dogs treated with NHS-IL12 at a dose of 1.6 mg/m² or higher. IFN-γ levels spiked sharply at 24 hours post-treatment and returned to undetectable levels by 48 hours. Elevated serum IFN-γ (> 100 pg/ml) was associated with increased risk for toxicity. However the highest level of IFN-γ induction was not directly linked to the most severe (Grade 4 or 5) adverse events.</p

RGD-A-<i>TNF</i> administration resulted in objective tumor responses in dogs with spontaneous cancers.

<p>A large primary soft tissue sarcoma on the flank of Dog #2.11 is shown to feature the potential magnitude of the tumor response. Prior to therapy (day 0), the tumor measured 12.3 cm in longest diameter. At day 28, after 4 weekly infusions of RGD-A-<i>TNF</i>, the tumor measured 8.2 cm in longest diameter (a 33% regression) and a RECIST-based partial response (PR). At day 56, after a total of 8 weekly systemic infusions of RGD-A-<i>TNF</i>, the tumor measured 1.85 cm in longest diameter prior to resection. Therefore, this response equated to an 85% regression from baseline and a continued clinical PR. Upon surgical resection of the residual lesion, no viable tumor was found and a pathological complete response was determined.</p

Subcutaneous administration of NHS-IL12 resulted in measurable serum drug levels.

<p>Serum samples were collected from dogs to define systemic exposures of NHS-IL12 after subcutaneous administration. NHS-IL12 levels were measured pre-treatment and at 1, 2, 4, 8, 24, 36, and 48 hours following administration (8-point collection) and on days 8, 15, and 29. NHS-IL12 C_max was dose-dependent: 0.4 mg/m² and 0.8 mg/m² (A) and 1.6 mg/m² and 2.4 mg/m² (B). Clearance was prolonged in some dogs as NHS-IL12 was still measurable in five animals 14 days following treatment.</p