Locoregional Injection Research Articles

Heavy-atom enhanced synchrotron stereotactic radiotherapy of brain tumors: from DNA to preclinical studies

Gliomas are the most frequent primary brain tumors in adults. Despite multimodality treatment strategies that combine surgery, chemotherapy and radiotherapy, high grade gliomas are almost uniformly fatal. Heavy-atom-enhanced synchrotron stereotactic radiotherapy (SSR) is a novel therapeutic modality proposed to increase the toxicity to the tumor while protecting the surrounding healthy tissue. It consists in selective accumulation of high-Z elements in tumor followed by stereotactic irradiation, in tomography mode, with monochromatic X-rays from a synchrotron source, tuned at an optimal energy. Two complimentary SSR approaches have been successfully developed in the past 5 years, and may be promising in high-grade glioma management: iodine-enhanced SSR , with an iodinated contrast agent; and Pt-enhanced SSR , , a concomitant radio-chemotherapy treatment with locoregional injection of platinated chemotherapy drugs. Several in vitro and in vivo experiments have been carried out at the European Synchrotron Radiation Facility, either with iodine or platinum. In vivo experiments are performed on F98 glioma-bearing rats. The F98 glioma model simulates several characteristics of human glioblastoma including lethality following treatment with a variety of therapeutic modalities. Recently, we have shown that a significant increase in survival time is obtained when the platinated drug (cisplatin or carboplatin) is infused intratumoraly followed by X-rays irradiation. Furthermore, a significant percent of animals are cured. To optimise the treatment, work is in progress in order to determine and increase the incorporation of heavy-atoms in tumoral cells and also to improve the intracerebral drug distribution by developing new injection methods and using different chemotherapeutic drugs including oxaliplatin, chlorotertpyridineplatinum and 5-iodo-2’-deoxyuridine. In addition, efforts will be made to have a better understanding of the biological consequences at the molecular and cellular level of the SSR treatment. 1 - Adam JF et al, International Journal of Radiation Oncology, Biology, Physics 2006; 64:603-611. 2 Biston MC et al, Cancer Research 2004; 64:2317-2323. 3 Rousseau J et al, Clinical Cancer Research 2007;13:5195-5201.

(111)In]Bz-DTPA-hEGF: Preparation and in vitro characterization of a potential anti-glioblastoma targeting agent.

The overexpression of epidermal growth factor receptors, EGFR, in glioblastomas is well documented. Hence, the EGFR can be used as target structure for a specific targeting of glioblastomas. Both radiolabeled anti-EGFR antibodies and the natural ligand EGF are candidate agents for targeting. However, EGF, which has a rather low molecular weight (6 kDa), might have better tissue penetration properties through both normal tissue and tumors in comparison with anti-EGF antibodies and their fragments. The aim of this study was to prepare and evaluate in vitro an EGF-based antiglioma conjugate with residualizing label. Human recombinant EGF (hEGF) was coupled to isothiocyanate-benzyl-DTPA. The conjugate was purified from unreacted chelator using solid-phase extraction and labeled with (111)In. The labeling yield was 87% +/- 7%. The label was reasonably stable; the transchelation of (111)In to serum proteins was about 5% after incubation at 37 degrees C during 24 hours. The obtained [(111)In]benzyl-DTPA-hEGF conjugate was characterized in vitro using the EGFR expressing glioma cell line U343MGaCl2:6. The binding affinity, internalization, and retention of the conjugate were studied. The conjugate had receptor specific binding and the radioactivity was quickly internalized. The intracellular retention of radioactivity after interrupted incubation with conjugate was 71% +/- 1% and 59% +/- 1.5% at 24 and 45 hours, respectively. The dissociation constant was estimated to 2.0 nM. The results indicate that [(111)In]benzyl-DTPA-hEGF is a potential candidate for targeting glioblastoma cells, possibly using locoregional injection.

Targeted adenoviral vectors

Subgroup C human adenoviruses (Ad2 or Ad5) can transduce a wide array of dividing and quiescent adherent cell types in vitro and in vivo, essentially because their primary receptor (CAR) is widely expressed at the cellular surface. On the other hand, specific target cell types (eg some tumor cells, smooth muscle cells, fibroblasts) display limiting amounts of the CAR receptor so that large viral doses are needed for efficient infection, a constraint which favors vector dissemination in vivo, even after loco-regional injection. Abrogating the native virus-CAR interaction while allowing the virus to use a CAR-independent pathway for entry would thus represent a major milestone in order to better control the vector tropism in vivo. Towards this goal, we first engineered a series of capsid-modified adenoviruses by genetically inserting targeting peptides within protruding loops of the fiber and hexon capsid monomers (Vigne E, et al: J Virol 1999, 73:5156). The most interesting virus – AE43 – displays within the fiber HI loop a vitronectin-derived high affinity peptide for the urokinase-type plasminogen activator (uPAR or CD87), a cell surface receptor upregulated during cellular activation, and which controls cellular migration and invasion. Most interestingly, AE43 could increase transduction of cells normally refractory to adenovirus infection more than 100-fold in vitro. We could also demonstrate that AE43 retained its ability to enter the cell via a CAR-dependent pathway in vitro, and displayed a normal tropism following systemic injection in mice. To cripple the virus-CAR interaction, we then evaluated various strategies, including the introduction of CAR-ablating mutations within the fiber, and the shortening of the fiber shaft. Interestingly, some of these constructs could decrease transduction of CAR-positive cells at least 10-fold in vitro, with a similar reduction in liver transduction following systemic injection in naive mice. We are currently assessing the effect of combining within a single vector the uPAR-binding peptide of AE43 with our best CAR-ablating mutations.

Biodistribution and tumor localization of lymphokine-activated killer T cells following different routes of administration into tumor-bearing animals.

The efficiency of adoptive cellular immunotherapy of cancer might depend on the number of effector cells that reach the malignant tissues. In the present study, the biodistribution and tumor localization of ex vivo lymphokine-activated T killer (T-LAK) cells was investigated. T-LAK cells were labeled with 125I-dU or the fluorescent dye tetramethylrhodamine isothiocyanate (TRITC) and transferred by intravenous, -cardiac, -portal or -peritoneal injection into normal (C57BL/6) mice or mice with syngeneic day-7 to day-12 B16 melanoma metastases established in various organs. The overall biodistribution of the T-LAK cells was measured by gamma counting and their tumor localization by fluorescence microscopy. At 16 h after intravenous injection, the organ distribution of 125I-dU-labeled T-LAK cells was identical in normal and tumor-bearing animals. Fluorescence microscopy of lung tissue from animals receiving TRITC-labeled T-LAK cells revealed, however, a fivefold higher accumulation of T-LAK cells in lung metastases than in the surrounding normal lung tissue (1174 and 226 cells/mm2 respectively). Some pulmonary metastases were, however, resistant to infiltration. Very few intravenously injected cells redistributed to other organs or to tumors in these, since only 60 and 30 T-LAK cells/mm2 were found within metastases of the adrenal glands and the liver respectively. However, following injection of T-LAK cells via the left ventricle of the heart, a threefold increase (from 60 to 169 cells/mm2) in the number of transferred cells in metastases of the adrenal glands was observed. Moreover, following locoregional administration of T-LAK cells into the portal vein, tenfold higher numbers (from 30 to 400 cells/mm2) were found in hepatic metastases than were observed following intravenous or intracardiac injection. In the liver, a surprisingly large number of intraportally injected T-LAK cells (approx. 1300/mm2) were observed to accumulate in the perivascular spaces of the portal, but not the central veins. Even though some superficial ovarian and liver metastases were separated from the peritoneal cavity by only the peritoneal lining, no localization into these metastases was seen following intraperitoneal injection of the T-LAK cells. While treatment of tumor-bearing animals with T-LAK cells plus IL-2 reduced lung metastases by 76% as compared to treatment with IL-2 alone (P<0.03), no significant reduction of liver metastases was seen. T-LAK cells are able to localize substantially into tumor metastases in various anatomical locations, but mainly following locoregional injection. This finding might have important implications for the design of future clinical protocols of adoptive immunotherapy based on T cells.

Biodistribution of 111In-labelled SCN-bz-DTPA-BC-2 MAb following loco-regional injection into glioblastomas.

We analyzed the biodistribution of the 111In-labelled murine anti-tenascin-C MAb BC-2 after intralesional injection in 15 glioblastoma patients. The activated ligand DTPA was conjugated via the isothiocyanato-benzyl group onto BC-2. Conjugates were labelled with 111In, displaying immunoreactivity greater than 90% and labelling efficiency of 99 +/- 1%. In contrast to i.v. injections, excellent tumor uptake was obtained by direct intralesional injection of conjugates that showed only slow systemic release. In serum, conjugates were found to be intact; in urine, only low-molecular-weight decay products were detected. In 8 patients, outflow from the site of injection into systemic circulation was low; daily activity in the serum and urine was found to be below 2% of the total injected radioactivity; most of the injected activity was retained within the tumor, resulting in effective half-lives of 58 +/- 5 hr. In contrast, higher outflow up to 10% of regionally injected 111In-DTPA-BC-2 MAb into systemic circulation resulted in considerable shortening of the effective half-lives to 20 to 40 hr in 7 patients. This outflow was found to correlate with tumor size and blood/brain barrier disruption. In one patient, HPLC analysis of tumor cyst fluid 3 and 6 days after intralesional injection revealed conjugates to be intact and allowed the estimate of about 70% of the total injected 111In-DTPA-BC-2 to be confined to tumor tissue. We conclude that different outflow patterns can be observed following locoregional injection of 111In-DTPA-BC-2, leading to considerable variations in the effective half-lives of isotopes within the tumor, requiring adjustment of the radiation dose in therapeutic trials.