A synDNA vaccine delivering neoAg collections controls heterogenous, multifocal murine lung and ovarian tumors via robust T cell generation

Neoantigens are tumor-specific antigens that arise due to somatic mutations in the DNA of tumor cells. They represent ideal targets for cancer immunotherapy since there is minimal risk for on-target, off-tumor toxicities. Additionally, these are foreign antigens that should be immunogenic due to lack of central immune tolerance. Tumor neoantigens are predominantly passenger mutations, which do not contribute to tumorigenesis. In cases of multi-focal or metastatic tumors, different foci can have significantly different mutation profiles. This suggests that it is important to target as many neoantigens as possible to better control tumors and target multi-focal tumors within the same patient. Herein, we report a study targeting up to 40 neoantigens using a single DNA plasmid. We observed significant plasticity in the epitope strings arranged in the vaccine with regard to immune induction and tumor control. Different vaccines elicited T cell responses against multiple epitopes on the vaccine string and controlled growth of multi-focal, heterogeneous tumors in a therapeutic tumor challenge. Additionally, the multi-epitope antigens induced long-term immunity and rejected a tumor re-challenge several weeks after the final vaccination. These data provide evidence that DNA-encoded long antigen strings can be an important tool for immunotherapeutic vaccination against neoantigens with implications for other in vivo-delivered antigen strings

<i>p53</i> Mutagenesis by Benzo[<i>a</i>]pyrene Derived Radical Cations

Benzo­[<i>a</i>]­pyrene (B­[<i>a</i>]­P), a major human carcinogen in combustion products such as cigarette smoke and diesel exhaust, is metabolically activated into DNA-reactive metabolites via three different enzymatic pathways. The pathways are the <i>anti</i>-(+)-benzo­[<i>a</i>]­pyrene 7,8-diol 9,10-epoxide pathway (P450/epoxide hydrolase catalyzed) (B­[<i>a</i>]­PDE), the benzo­[<i>a</i>]­pyrene <i>o</i>-quinone pathway (aldo ketose reductase (AKR) catalyzed) and the B­[<i>a</i>]P radical cation pathway (P450 peroxidase catalyzed). We used a yeast <i>p53</i> mutagenesis system to assess mutagenesis by B­[<i>a</i>]P radical cations. Because radical cations are short-lived, they were generated <i>in situ</i> by reacting B­[<i>a</i>]P with cumene hydroperoxide (CuOOH) and horse radish peroxidase (HRP) and then monitoring the generation of the more stable downstream products, B­[<i>a</i>]­P-1,6-dione and B­[<i>a</i>]­P-3,6-dione. On the basis of B­[<i>a</i>]­P-1,6 and 3,6-dione formation, approximately 4 μM of radical cation was generated. In the mutagenesis assays, the radical cations produced <i>in situ</i> showed a dose-dependent increase in mutagenicity from 0.25 μM to 10 μM B­[<i>a</i>]P with no significant increase seen with further escalation to 50 μM B­[<i>a</i>]­P. However, mutagenesis was 200-fold less than with the AKR pathway derived B­[<i>a</i>]­P, 7-8-dione. Mutant <i>p53</i> plasmids, which yield red colonies, were recovered from the yeast to study the pattern and spectrum of mutations. The mutation pattern observed was G to T (31%) > G to C (29%) > G to A (14%). The frequency of codons mutated by the B­[<i>a</i>]P radical cations was essentially random and not enriched at known cancer hotspots. The quinone products of radical cations, B­[<i>a</i>]­P-1,6-dione and B­[<i>a</i>]­P-3,6-dione were more mutagenic than the radical cation reactions, but still less mutagenic than AKR derived B­[<i>a</i>]­P-7,8-dione. We conclude that B­[<i>a</i>]P radical cations and their quinone products are weakly mutagenic in this yeast-based system compared to redox cycling PAH <i>o</i>-quinones

Intraoperative near-infrared imaging can distinguish cancer from normal tissue but not inflammation.

Defining tumor from non-tumor tissue is one of the major challenges of cancer surgery. Surgeons depend on visual and tactile clues to select which tissues should be removed from a patient. Recently, we and others have hypothesized near-infrared (NIR) imaging can be used during surgery to differentiate tumors from normal tissue.We enrolled 8 canines and 5 humans undergoing cancer surgery for NIR imaging. The patients were injected with indocyanine green (ICG), an FDA approved non-receptor specific NIR dye that accumulates in hyperpermeable tissues, 16-24 hours prior to surgery. During surgery, NIR imaging was used to discriminate the tumor from non-tumor tissue.NIR imaging identified all tumors with a mean signal-to-background ratio of 6.7. Optical images were useful during surgery in discriminating normal tissue from cancer. In 3 canine cases and 1 human case, the tissue surrounding the tumor was inflamed due to obstruction of the vascular supply due to mass effect. In these instances, NIR imaging could not distinguish tumor tissue from tissue that was congested, edematous and did not contain cancer.This study shows that NIR imaging can identify tumors from normal tissues, provides excellent tissue contrast, and it facilitates the resection of tumors. However, in situations where there is significant peritumoral inflammation, NIR imaging with ICG is not helpful. This suggests that non-targeted NIR dyes that accumulate in hyperpermeable tissues will have significant limitations in the future, and receptor-specific NIR dyes may be necessary to overcome this problem

Preclinical evidence for NIR tumor labeling to detect primary and metastatic tumor deposits.

<p>(A) Five cancer cell types were injected into the flank of syngeneic mice. Once established (200 mm3), animals were dosed with 7.5 mg/kg of ICG and imaged. Tumors were harvested, imaged and stained for CD31 (Marked with black arrows). Histology images taken at 200x magnification. (B) C57bl/6 mice (n = 21) were injected with LLC cells in their flanks on Day 0. Starting on Day 12, the animals were euthanized, dosed with 7.5 mg/kg ICG 24 hours earlier and their thoracic cavities opened. Observers determined if the metastatic tumor nodules were visible in the lung. NIR imaging was then used to detect disease that was not visible to the un-assisted human eye. Histology images taken at 100x.</p

Intraoperative imaging of surgical margins during pulmonary resection.

<p>(A) Intraoperatively, the animal underwent right thoracotomy and palpation of the primary tumor. Intraoperative image of the pulmonary lobe as it is retracted from the canine chest revealed the dorsal portion of the lobe has significant compression (ie. atelectasis) from tumor obstruction. The ventral portion of the lung retained its normal appearance. Spectroscopic analysis of the tumor was performed <i>in situ</i>. All sites were recorded in triplicate and averaged. The graph shows the inflammatory tissue was highly fluorescent and could not be distinguished from tumor. (B) H&E demonstrated normal alveolar parenchyma (left panel), congestion lung with neutrophils and fibrotic plugs (middle panel) and tumor (right panel) from representative biopsies of the pulmonary lobe.</p

Representative intraoperative image of a canine lung cancer.

<p>(A) Signal-to-background ratio of tumor to surrounding normal lung tissue <i>in situ</i> and <i>ex vivo</i> in 8 canines. All values are reported in arbitrary units (a.u.). ^†Due to the large size of this tumor, no measurements of normal lung fluorescence could be obtained ex vivo. (B) After opening the chest, the tumor was visualized in the chest. The tumor was well-circumscribed and was highly fluorescent (signal-to-background ratio 11.3). The tumor lies in the caudad position and the hilum of the lung is cranial. (C) <i>Ex vivo</i>, the tumor was fluorescent (SBR 12.7) and the margins of the tumor were well-defined. (D) H&E confirmed a lung adenocarcinoma with 2+ CD31 staining. The signal-to-background ratio (SBR) was higher <i>ex vivo</i> than <i>in situ</i> because of superior control of the surrounding environment such as lighting conditions, exposure and lack of motion. Although the fluorescence from the tumors did not significantly change, the background fluorescence from the normal lung was reduced when the environment could be better controlled. Microscopically, the tumor microvascular density (MVD) did not seem to impact the degree of fluorescence.</p

Two representative human tumors.

<p>Computed tomography (CT) scan and positron emission tomography (PET) scan demonstrated an anterior mediastinal mass and a lung nodule in two patients. Patients were injected with ICG, and then underwent resection of their tumors. <i>Ex</i> vivo, NIR imaging demonstrated the tumors were highly fluorescent and the surrounding organ had minimal background noise. The optical images were easy to interpret by the surgeon and facilitated the identification of the tumor. Spectroscopy demonstrated a SBR of 8.1 for the thymoma and 7.9 for the carcinoid. The tumors were discrete and well-circumscribed and had minimal peritumoral inflammation.</p