[en] The 4 specific aims of this project are: (1) Optimization of MST to increase tumor uptake; (2) Antigen heterogeneity; (3) Characterization and reduction of renal uptake; and (4) Validation in vivo of optimized MST targeted therapy. This proposal focussed upon optimizing multistep immune targeting strategies for the treatment of cancer. Two multi-step targeting constructs were explored during this funding period: (1) anti-Tag-72 and (2) anti-GD2.

[en] In the last grant period we have focused on multi-step targeting methodologies (MST), as a method for delivery of high dose to the tumor, with low dose to the bone marrow. We have explored uptake in colorectal, pancreatic and prostate cancer, using an special preparation, developed in collaboration with NeoRex A high tumor/bone marrow ratio is clearly achieved with MST, but with a cost, namely the higher dose to normal kidney. For this reason, we have in particular, (a) looked dosimetry for both tumor and normal organ, and especially renal dosimetry, which appears to be the target organ, for Y-90. (b) In parallel with this we have explored the dosimetry of very high dose rate radionuclides, including Holmium-166. (c) In addition, with NaiKong Cheung, we have developed a new MST construct based on the anti-GD2 targeting 5F11; (d) we have successfully completed development of s-factor tables for mice. In summary, renal dosimetry is dominated by about 4-5% of the injected dose being held long-term in the renal cortex, probably in the proximal tubule, due to the universal uptake of small proteins. This appears to be a function of a biotynlated protein binding of the strept-avidin construct, to HSP70. This cortical uptake has caused us to reconsider renal dosimetry as a whole, with the smaller mass of the cortex, rather than the whole kidney, as the target organ. These insights into dosimetry will be of great importance as MST, becomes more common in clinical practice

[en] P-glycoprotein (P-gp) has a role in multidrug resistance (MDR) encountered in human cancers. In this study, we used the colchicine-resistant cell line BE(2)-C/CHCb(0.2), a strain of neuroblastoma cell line BE(2)-C, as a model to measure variations of P-gp expression in cells grown in vitro and in vivo. Cells were cultured in the medium supplemented with colchicine. At the beginning of the study the drug was withdrawn and, after 22 days, added back to the culture medium. Cells were harvested at various time points and xenografted in nude mice. P-gp content in cells was measured by self-competitive binding assay and in tumors, by quantitative autoradiography (QAR). Both assays were carried out using ¹²⁵I-labeled monoclonal antibody MRK16, reactive with P-gp. Concentration of P-gp in cells varied from a maximum of 1,361 pmol/g in the presence of colchicine to a minimum of 374 pmol/g in the absence of colchicine in the culture medium. P-gp concentration in the tumors ranged from 929 to 188 pmol/g, which correlated with P-gp content in the cells at the time of their injection in the mice. QAR is an accurate and reliable method to quantify P-gp expression in tumors. Changes in colchicine concentration in the ambient medium of BE(2)-C/CHCb(0.2) cells growing in vitro resulted in a change in phenotype of P-gp expression, which was stable under conditions of in vivo growth over approximately 9 cell divisions in nude mice xenografts. Therefore, P-gp content in xenografts depends only on the level of resistance of the cells at the time of their injection in the mice

[en] Purpose: Due to the cytotoxicity of DNA-bound iodine-125, 5-[¹²⁵I]Iodo-2'-deoxyuridine ([¹²⁵I]IUdR), an analog of thymidine, has long been recognized as possessing therapeutic potential. In this work, the feasibility and potential effectiveness of hepatic artery infusion of [¹²⁵I]IUdR is examined. Methods: A mathematical model has been developed that simulates tumor growth and response to [¹²⁵I]IUdR treatment. The model is used to examine the efficacy and potential toxicity of prolonged infusion therapy. Treatment of kinetically homogeneous tumors with potential doubling times of either 4, 5, or 6 days is simulated. Assuming uniformly distributed activity, absorbed dose estimates to the red marrow, liver and whole-body are calculated to assess the potential toxicity of treatment. Results: Nine to 10 logs of tumor-cell kill over a 7- to 20-day period are predicted by the various simulations examined. The most slowly proliferating tumor was also the most difficult to eradicate. During the infusion time, tumor-cell loss consisted of two components: A plateau phase, beginning at the start of infusion and ending once the infusion time exceeded the potential doubling time of the tumor; and a rapid cell-reduction phase that was close to log-linear. Beyond the plateau phase, treatment efficacy was highly sensitive to tumor activity concentration. Conclusions: Model predictions suggest that [¹²⁵I]IUdR will be highly dependent upon the potential doubling time of the tumor. Significant tumor cell kill will require infusion durations that exceed the longest potential doubling time in the tumor-cell population

[en] 3F8 is a murine IgG₃ monoclonal antibody (MAb) selective for the ganglioside G_D2. Previous studies using ¹³¹I-3F8 have shown great potential in the imaging of neuroectodermal tumors and the therapy of human neuroblastoma. ¹³¹I is commonly used in radioimmunodiagnosis, but its relatively long half-life (8 days) and its high energy γ-emission (364 KeV) are suboptimal for imaging purposes when compared with ^99mTc (6 h and 140 KeV, respectively). To label 3F8 with ^99mTc, the antibody was first coupled with a heterobifunctional linker, succinimidyl-6-hydrazinonicotinate hydrochloride (SHNH), obtaining a hydrazinonicotinamide-antibody conjugate. Using ^99mTc-Tricine as the precursor complex, 3F8-SHNH was coupled efficiently to ^99mTc, resulting in >90% radiometal incorporation, with a specific activity >10 mCi/mg and retaining full immunoreactivity. Immunoscintigraphy at 6, 22, and 46 h after intravenous injection of 1 mCi of ^99mTc-3F8 showed selective neuroblastoma localization in xenografted nude mice, comparable to that obtained with the injection of 100 μCi of ¹³¹I-3F8. Biodistribution studies of ¹³¹I-3F8 and ^99mTc-3F8 in mice demonstrated comparable %ID/g uptake in tumor (with a T/B ratio: ∼2.5 at 24 h and ∼3.5 at 48 h) and normal organs, including blood, except for spleen and liver which had about a three times higher uptake of the ^99mTc conjugate. In conclusion, ^99mTc can be coupled conveniently at high specific activity to 3F8 without compromising immunoreactivity. SHNH appears to be a useful linker for ^99mTc in tumor diagnostic imaging and may have potential utility in coupling other radioisotopes (e.g., ^94mTc) for positron imaging and therapy

[en] To optimize the efficacy of radioimmunotherapy (RIT), the ideal antibody-radioisotope combinations should be used to deliver the highest tumor and the lowest normal tissue doses. In a mouse model, tumor and critical organ-absorbed doses delivered by different radioimmunoconjugates were calculated and compared. We used a Medical Internal Radiation Dosimetry (MIRD)-style mouse dosimetry model that incorporates cross-organ beta doses to make refined estimates of the radiation absorbed dose to tissues. Biodistribution data from neuroblastoma xenografted nude mice were used to estimate tumor, organ and bone marrow absorbed dose values for ⁹⁰Y-3F8, ¹³¹I-3F8 and ¹³¹I-F(ab')₂ fragments. Immunoreactive fractions of the radiolabeled antibodies were comparable. Although tumor uptake of the radioiodinated and radiometal labeled 3F8 was much higher than that of the radioiodinated F(ab')₂ fragments (maximum percent injected dose per gram values were 39.4, 33.2 and 20.1 for ¹³¹I-3F8, ⁹⁰Y-3F8 and ¹³¹I-F(ab')₂, respectively), tumor to nontumor ratios were higher for radioiodinated fragments (with the exception of tumor to kidney ratio). For the minimum tumor dose necessary for complete ablation, the bone marrow received 195, 278 and 401 cGy for ¹³¹I-F(ab')₂, ¹³¹I-3F8 and ⁹⁰Y-3F8, respectively. Tumor doses were 50.1, 232 and 992 cGy/MBq for ¹³¹I-F(ab')₂, ¹³¹I-3F8 and ⁹⁰Y-3F8, respectively. Tumor to bone marrow dose, which is defined as the therapeutic index, was 21.5, 14.7 and 10.4 for ¹³¹I-F(ab')₂, ¹³¹I-3F8 and ⁹⁰Y-3F8. ¹³¹I-F(ab')₂ fragments produced the highest therapeutic index but also the lowest tumor dose for radioimmunotherapy. Radiometal conjugated IgG produced the highest tumor dose but also the lowest therapeutic index

[en] Multidrug resistance (MDR) in tumors is associated with P-glycoprotein (Pgp) expression. In vivo quantitation of Pgp may allow MDR to be evaluated noninvasively prior to treatment planning. The purpose of this study was to radiolabel MRK-16, a monoclonal antibody that targets an external epitope of P-glycoprotein, and perform in vivo quantitation of P-glycoprotein in a MDR xenograft nude mouse model. MRK-16 was labeled with ¹²⁵I by the iodogen method, with subsequent purification by size exclusion chromatography. Groups of 10 Balb/c mice were each xenografted with colchicine-resistant or -sensitive neuroblastoma cell lines, respectively. Whole body clearance and tumor uptake over time was quantitated by gamma camera imaging, and biodistribution studies were performed with [¹²⁵]MRK-16 and an isotype matched control antibody, A33. Quantitative autoradiography and immunohistochemistry analysis of tumors was also evaluated to confirm specific targeting of [¹²⁵I]MRK-16. Peak tumor uptake was at 2-3 days post-injection, and was significantly greater in resistance compared to sensitive tumors (mean % injected dose/g ± SD) (18.76 ± 2.94 vs 10.93 ± 0.96; p < 0.05). Quantitative autoradiography verified these findings (19.13 ± 0.622 vs 12.08 ± 0.38, p < 0.05). Specific binding of [¹²⁵I]MRK-16 was confirmed by comparison to [¹³¹I]A33 in biodistribution studies, and localized to cellular components of tissue stroma by comparison of histologic and autoradiographic sections of sensitive and resistant tumors. Immunoblot analysis demonstrated a 4.5-fold difference in P-glycoprotein expression between sensitive and resistant cell lines without colchicine selective pressure. We conclude that in vivo quantitation of P-glycoprotein in MDR tumors can be performed with [¹²⁵I]MRK-16. These findings suggest a potential clinical application for radiolabeled MRK-16 in the in vivo evaluation of multidrug resistance in tumors

[en] Radionuclide labeled somatostatin analogues selectively target somatostatin receptor (SSTR)-expressing tumors as a basis for diagnosis and treatment of these tumors. Recently, a DOTA-functionalized somatostatin analogue, DOTATOC (DOTA-DPhe¹-Tyr³-octreotide) has been developed. This compound has been shown to be superior to the other somatostatin analogues as indicated by its uniquely high tumor-to-non-target tissue ratio. DOTATOC can be labeled with a variety of radiometals including gallium radioisotopes. Gallium-66 is a positron emitting radionuclide (T_1/2 =9.5 hr; β⁺=56%), that can be produced in carrier free form by a low-beam energy cyclotron. In this study we investigated SSTR targeting characteristics of ⁶⁶Ga-DOTATOC in AR42J rat pancreas tumor implanted nude mice as a potential agent for diagnosis and receptor-mediated internal radiotherapy of SSTR-expressing tumors. We compared our results with ⁶⁷Ga- and ⁶⁸Ga- labeled DOTATOC. The radiolabeling procedure gave labeling yield ranged from 85-95% and radiochemical and chemical purity was >95%. In-vitro competitive binding curves and in-vivo competitive displacement studies with an excess of unlabeled peptide indicates that there is specific binding of the radioligand to SSTR. Animal biodistribution data and serial microPET^TM images demonstrated rapid tumor uptake and rapid clearance from the blood and all tissues except kidney. Maximum % ID/g values for tumor were 10.0±0.7, 13.2±2.1 and 9.8±1.5 for ⁶⁶Ga-, ⁶⁷Ga-, and ⁶⁸Ga-DOTATOC, respectively. Calculated tumor, kidney and bone marrow doses for ⁶⁶Ga-DOTATOC based on biodistribution data were 178, 109 and 1.2 cGy/MBq, respectively. We conclude that ⁶⁶Ga labeled DOTATOC can be used for PET diagnosis and quantitative imaging-based dosimetry of SSTR positive tumors. ⁶⁶Ga-DOTATOC may also be used in higher doses for ablation of these tumors. However, kidney is the critical organ for toxicity (tumor/kidney ratio 1.64), and high kidney uptake must be eliminated before devising a therapy protocol

[en] Fourteen F-18 fluorodeoxyglucose (FDG) studies were carried out in 13 patients known to have bony metastases from carcinoma of the prostate. One patient was newly diagnosed. The remaining patients had various types of therapy and were considered hormonally resistant. The average age was 67. All patients had extensive bony metastases shown on the conventional Tc99m-MDP bone scans. Only about 18% of bony lesions apparent on the conventional bone scans showed corresponding increase of FDG uptake. Anatomical correlation was performed by using co-registered images of SPECT and PET in the same area. The positive FDG uptake was not related to the duration of illness, level of PSA, previous therapy, and magnitude of disease involvement. It appears that only a small percentage of bony metastases is associated with increased glycolysis. It is possible that other metabolic processes are more important than glycolysis for providing prostate cancer with a source of energy and nutrients

[en] Positron emission tomography (PET) utilizing fluorine-18 fluorodeoxyglucose (FDG) has been used in the evaluation of non-small cell lung cancer (NSCLC). Recently its use in the staging of small cell lung cancer (SCLC) has been reported. However, the prognostic value of FDG-PET imaging in SCLC has not been studied. We performed a retrospective analysis to assess this, with the following hypotheses: (1) PET-positive patients would have a less favorable prognosis than PET-negative patients and (2) a high standardized uptake value (SUV) would be associated with a poor prognosis. Retrospective review of a mixed population of treated and untreated patients imaged between 1995 and 2000 was performed. Results of 62 scans in 46 patients were analyzed. There were 8 untreated and 38 treated patients. Findings were correlated with pathology, computed tomography/magnetic resonance imaging and clinical data. The sensitivity of PET scanning was 100% with pathological correlation. The prognostic value of a positive PET study was determined. Overall survival in PET-positive cases was significantly worse than that in PET-negative cases (P=0.0108). Correlation of SUV_max with survival showed a significant negative correlation (P=0.0021). In the eight untreated patients, scans were strongly positive and in all cases the scan results concurred with the final clinical stage assigned on the basis of conventional methods. We conclude that FDG-PET imaging provides prognostic information in treated patients. A positive study and a high SUV_max are significantly associated with poor survival. Additionally, FDG-PET may be helpful in staging and follow-up. (orig.)