Preclinical Evaluation and Dosimetry of [111In]CHX-DTPA-scFv78-Fc Targeting Endosialin/Tumor Endothelial Marker 1 (TEM1)

Purpose

Endosialin/tumor endothelial marker-1 (TEM1) is an attractive theranostic target expressed by the microenvironment of a wide range of tumors, as well as by sarcoma and neuroblastoma cells. We report on the radiolabeling and preclinical evaluation of the scFv78-Fc, a fully human TEM1-targeting antibody fragment cross-reactive with mouse TEM1.

Procedures

The scFv78-Fc was conjugated with the chelator p-SCN-Bn-CHX-A”-DTPA, followed by labeling with indium-111. The number of chelators per molecule was estimated by mass spectrometry. A conventional saturation assay, extrapolated to infinite antigen concentration, was used to determine the immunoreactive fraction of the radioimmunoconjugate. The radiopharmaceutical biodistribution was assessed in immunodeficient mice grafted with Ewing’s sarcoma RD-ES and neuroblastoma SK-N-AS human TEM1-positive tumors. The full biodistribution studies were preceded by a dose-escalation experiment based on the simultaneous administration of the radiopharmaceutical with increasing amounts of unlabeled scFv78-Fc. Radiation dosimetry extrapolations to human adults were obtained from mouse biodistribution data according to established methodologies and additional assumptions concerning the impact of the tumor antigenic sink in the cross-species translation.

Results

[¹¹¹In]CHX-DTPA-scFv78-Fc was obtained with a radiochemical purity >?98 % after 1 h incubation at 42 °C and ultrafiltration. It showed good stability in human serum and >?70 % immunoreactive fraction. Biodistribution data acquired in tumor-bearing mice confirmed fast blood clearance and specific tumor targeting in both xenograft models. The radiopharmaceutical off-target uptake was predominantly abdominal. After a theoretical injection of [¹¹¹In]CHX-DTPA-scFv78-Fc to the reference person, the organs receiving the highest absorbed dose would be the spleen (0.876 mGy/MBq), the liver (0.570 mGy/MBq) and the kidneys (0.298 mGy/MBq). The total body dose and the effective dose would be 0.058 mGy/MBq and 0.116 mSv/MBq, respectively.

Conclusions

[¹¹¹In]CHX-DTPA-scFv78-Fc binds specifically to endosialin/TEM1 in vitro and in vivo. Dosimetry estimates are in the range of other monoclonal antibodies radiolabeled with indium-111. [¹¹¹In]CHX-DTPA-scFv78-Fc could be potentially translated into clinic.

     

Doppler velocimetry in prolonged pregnancy

Eighty-two patients at 287 days' gestation or longer were tested by nonstress test (NST), amnioscopy, ultrasound assessment of amniotic fluid volume, and Doppler velocimetry. Several maternal and fetal arteries were analyzed: uterine, umbilical, descending thoracic aorta, renal, and middle cerebral. During the study, other maternal-fetal functional indices were recorded: hPL, estriol, hematocrit, platelets, mean platelet volume, and uric acid. No abnormalities were found in the uterine, umbilical, middle cerebral, thoracic descending aorta, and renal artery velocimetry in post-dates gestations. However, a significant reduction of the time-averaged mean velocity in the descending thoracic aorta was associated with an increased incidence of oligohydramnios, meconium-stained fluid, abnormal NST, and cesarean delivery for fetal distress. The present study suggests that serial Doppler flow measurements of mean velocity of the fetal descending thoracic aorta may be a simple and rapid technique for identifying prolonged pregnancies at increased risk for perinatal complications.     

Ovarian carcinoma expresses the NKG2D ligand Letal and promotes the survival and expansion of CD28- antitumor T cells

The role of the NKG2D immunoreceptor and its ligands in antitumor immune response is incompletely understood. Here, we report that effector immune cells infiltrating ovarian carcinoma are mostly CD8+ lymphocytes lacking CD28 but expressing the NKG2D costimulatory receptor. Human ovarian carcinoma expresses the novel NKG2D ligand lymphocyte effector cell toxicity-activating ligand (Letal). Letal was found to be an independent prognosticator of improved survival in advanced ovarian cancer. Higher levels of tumor-derived Letal were associated with stronger lymphocyte infiltration. Letal exerted marked costimulatory effects and induced type-1 polarization in CD8+CD28- tumor-infiltrating lymphocytes ex vivo. Letal engagement increased the expression of the glucose transporter Glut-1, enhanced glucose up-take, and protected CD8+ lymphocytes from cisplatin-induced killing. Letal also down-regulated the expression of Fas in CD8+ cells and rendered them resistant to Fas ligand-induced apoptosis. Our results indicate that Letal promotes tumor immune surveillance by promoting the survival and intratumoral expansion of antitumor cytotoxic lymphocytes. We propose that Letal could be used for the ex vivo expansion of apoptosis-resistant tumor-reactive cytotoxic lymphocytes for adoptive transfer.     

The oncogene phosphatidylinositol 3'-kinase catalytic subunit alpha promotes angiogenesis via vascular endothelial growth factor in ovarian carcinoma

The gene of phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) has been implicated as an oncogene in ovarian cancer [L. Shayesteh et al., Nat. Genet., 21: 99-102, 1999]. In this study, we examined the expression of PIK3CA mRNA and its p110alpha protein product in human ovarian carcinoma and investigated its role in regulating angiogenesis via vascular endothelial growth factor (VEGF). PIK3CA mRNA was detected in 66.6% of stage I and 93.9% of advanced stage ovarian cancer specimens and in all 17 ovarian cancer cell lines. PIK3CA mRNA levels were significantly higher in invasive carcinomas compared with benign and low malignant potential neoplasms (P = 0.007), but no significant difference was seen between early and advanced stage carcinomas (P = 0.812). Strong expression of immunoreactive p110alpha was detected in tumor cells and/or stroma endothelium. PIK3CA expression in vivo positively correlated, both at the mRNA and the protein level, with the expression of VEGF as well as with the extent of microvascular development. Furthermore, PIK3CA mRNA overexpression positively correlated with increased proliferation and decreased apoptosis of tumor cells in vivo. In vitro, PIK3CA expression positively correlated with the expression of VEGF in ovarian cancer cells, whereas the phosphatidylinositol 3'-kinase inhibitor Ly294002 reduced both the constitutive and inducible expression of hypoxia-inducible factor-1alpha at the mRNA and protein levels and abrogated VEGF up-regulation by glucose starvation. Furthermore, Ly294002 suppressed cell proliferation and, at higher doses, induced marked apoptosis in ovarian cancer cells. Collectively, these data strongly indicate that PIK3CA supports ovarian cancer growth through multiple and independent pathways affecting cell proliferation, apoptosis and angiogenesis, and plays an important role in ovarian cancer progression.     

Immunotherapy for gynaecological malignancies

Gynaecological malignancies, excluding breast cancer, cause approximately 25,000 deaths yearly among women in the US. Therefore, novel approaches for the prevention or treatment of these diseases are urgently required. In the case of cervical cancer, human papillomavirus (HPV) xenoantigens are readily recognised by the immune system, and their targeting has shown great promise in preclinical models of therapeutic vaccination and in clinical studies of preventative vaccination. A growing body of evidence indicates that ovarian cancer is also immunogenic and can thus be targeted through immunotherapy. This review outlines the principles and problems of immunotherapy for cervical and ovarian cancer, including the authors' personal assessment.     

Vaccinia virus preferentially infects and controls human and murine ovarian tumors in mice

Vaccinia virus has been shown to efficiently infect tumor cells. Therefore, vaccinia virus represents a potentially safe and effective antitumor agent against ovarian cancer. Here, we assessed the ability of vaccinia virus to preferentially infect and control both human and murine ovarian tumors in vivo. We used the non-invasive luminescence imaging system to monitor the infection and suppression of ovarian tumors by vaccinia in live mice. Our data indicated that vaccinia was able to effectively infect and kill both human and murine ovarian tumors. Vaccinia virus administered to mice intraperitoneally was specifically targeted to the murine or human ovarian tumors and led to antitumor responses. These findings suggest that vaccinia virus is capable of selectively targeting and controlling ovarian tumors. Thus, intraperitoneal injection with vaccinia virus may provide a potentially effective strategy for treating advanced-stage ovarian cancers.     

Rapid isolation of high-affinity human antibodies against the tumor vascular marker Endosialin/TEM1, using a paired yeast-display/secretory scFv library platform

Endosialin/TEM1 is predominantly expressed on neovasculature, thus ideally suited for diagnostic, targeted imaging and therapy of cancer. To isolate TEM1-specific affinity reagents, we thought to screen a recombinant antibody (scFv) library derived from the repertoire of a patient with thrombotic thrombocytopenic purpura (TTP), as autoimmune disorders may produce self-reactive specificities. The yeast-display scFv library was constructed by homologous recombination of the TTP patient repertoire originally expressed on M13 bacteriophage in the novel vector pAGA2 for yeast-display expression. The TTP yeast-display library (10⁹ members) was screened by magnetic and flow sorting with human TEM1 recombinant protein. A pool of yeast-display scFv able to detect 2 nM of TEM1 was obtained and transformed into yeast-secreted scFv by homologous recombination using the novel p416 BCCP vector for yeast secretion of biotinylated scFv. Anti-TEM1 yeast-secreted scFv were independently validated in vitro by flow cytometry analysis and ELISA assays, then in vivo biotinylated in N-termini to produce biobodies. Biobody-78 bound specifically to Endosialin/TEM1-expressing ovarian tumor in vivo, with functional stability over 48 h. Our results suggest that our novel paired display-secretory yeast libraries can serve as an ideal platform for the rapid isolation of high-affinity reagents, and that anti-TEM1 biobody-78 can be used for in vitro assays including flow cytometry analysis, as well as in vivo for targeted imaging and therapy of cancer.