Nanoliposome-mediated targeting of antibodies to tumors: IVIG antibodies as a model.

Monoclonal antibodies are routinely used as tools in immunotherapies against solid tumors. However, administration of monoclonal antibodies may cause undesired side effects due to their accumulation in non-targeted organs. Nanoliposomes of less than 200 nm can target antibodies to tumors by enhanced permeation and retention (EPR) mechanisms. To direct monoclonal antibodies to tumors, nanoliposomes encapsulating intravenous immunoglobulin (IVIG) as a model antibody were prepared. The liposomes had average diameters of 100 nm and encapsulation efficiencies of 31 to 46%. They showed less than 10% release in plasma at 37°C up to seven days. The secondary and tertiary structures of liposome-encapsulated antibodies were analyzed by circular dichroism (CD) spectroscopy. The near and far-UV spectra analyses revealed no obvious conformational changes in the structures of the encapsulated antibodies. The biodistribution of free and liposome-encapsulated iodinated antibodies was investigated in mice bearing C-26 colon carcinoma tumors. The accumulation of liposome-encapsulated antibodies in tumors was significantly greater than that of free antibodies due to the EPR effect. The PEGylated liposomes were more efficient in the delivery of antibodies to the tumor site than non-PEGylated liposomes. We conclude that administration of monoclonal antibodies in PEGylated liposomes is more efficient than administration of non-encapsulated monoclonal antibodies for solid tumor immunotherapy.

Preparation and biological evaluation of (177)Lu conjugated PR81 for radioimmunotherapy of breast cancer.

AIM: PR81 is a monoclonal antibody that binds with high affinity to MUC1 antigen that is over expressed in 80% of breast cancers. In this study, we developed a method for indirect labeling of PR81 with lutetium-177 and performed all preclinical qualifications in production of a biologic agent for radioimmunotherapy of breast cancer. MATERIALS AND METHODS: The radiochemical purity and in vitro stability of (177)Lu labeled PR81 was determined by instant thin layer chromatography. The immunoreactivity and cell toxicity of the complex were tested on MCF7 cell line. The biodistribution and scintigraphy studies were performed in BALB/c mice with breast tumor. RESULTS: The radiochemical purity was 91.2±3.8% after 2 h. The in vitro stabilities in phosphate buffer and human blood serum were 83.1±3.4% and 76.2±3.6% at 96 h, respectively. The immunoreactivity of the complex was 83.4±2.4%. The cell toxicity study showed that the complex inhibited 85.2±3.4% growth of MCF7 cells at a concentration of 2500 ng/ml after 96 h. The biodistribution and scintigraphy studies showed the accumulation of the complex at the site of tumors with high sensitivity and specificity. CONCLUSION: The results showed that one may consider (177)Lu-DOTA-PR81 as a potential radiopharmaceutical for therapy of human breast cancer, which needs further investigations.

Comparison of (99m)Tc-labeled PR81 and its F(ab')2 fragments as radioimmunoscintigraphy agents for breast cancer imaging.

OBJECTIVE: We digested anti-MUC1 monoclonal antibody PR81 to produce F(ab')2 fragments. A comparison was performed between the two radiolabeled PR81 and F(ab')2 fragments for breast tumor imaging in a mouse model. METHODS: The optimum conditions for pepsin digestion of PR81 were investigated in terms of enzymes: antibody ratio, digestion time duration and preserved immunoreactivity of the produced fragments. The F(ab')2 fragments were labeled with Technetium-99m using HYNIC as a chelator and tricine as a co-ligand. The immunoreactivity of the complexes was assessed by radioimmunoassay using MCF7 cells. Biodistribution and imaging studies were performed in female BALB/c mice with breast tumor xenograft at 4, 8 and 24 h post-administration. The PR81 was labeled with technetium-99m in the same way for comparison. RESULTS: The optimum time duration for PR81 digestion was found to be 28 h at an enzyme:antibody weight ratio of 1:20 that resulted in 95.2 ± 4.7% purity. The labeling of intact PR81 and its F(ab')2 fragments were 87.6 ± 4.2 and 76.1 ± 3.3% after 1 h, respectively (p value <0.05). The percentage of immunoreactivity of F(ab')2 fragments and intact PR81 were 75.4 ± 2.1% and 85.7 ± 2.9%, respectively (p value <0.05). The biodistribution and imaging studies demonstrated localization of the fragments at 4 h post-administration with high sensitivity and specificity. CONCLUSION: The results showed that F(ab')2 fragment of PR81 is more suitable than intact PR81 for safer and more rapid detection of human breast cancer.

177Lu labeling of Herceptin and preclinical validation as a new radiopharmaceutical for radioimmunotherapy of breast cancer.

INTRODUCTION: In the present study, Herceptin was labeled with lutetium-177 via DOTA, and the necessary preclinical quality control tests (in vitro and in vivo) were performed to evaluate its use as a radioimmunotherapy agent. MATERIAL AND METHODS: Herceptin was conjugated to DOTA as a chelator in three different conjugation buffers (ammonium acetate, carbonate and HEPES buffer); each of the resulting conjugates was compared with respect to in vitro characteristics such as number of chelates per antibody, incorporated activity, immunoreactivity and in vitro stability in PBS buffer and blood serum. The biodistribution study and gamma camera imaging were performed in mice bearing breast tumors. To assess the therapeutic effects of (177)Lu-Herceptin, cytotoxicity was investigated for 7 days in a SKBr3 breast cancer cell line. RESULTS: Carbonate buffer was the best conjugation buffer (number of chelates per antibody: 6; incorporated activity: 81%; immunoreactivity: 87%; buffer stability: 86%; serum stability: 81%, after 4 days). The efficient tumor uptake observed in the biodistribution studies was consistent with the gamma camera image results. At a concentration of 4 µg ml(-1), (177)Lu-Herceptin (surviving cells: 5 ± 0.6% of the total cells) of the total cells corresponded to an approximately eightfold increase in cytotoxicity in comparison to unmodified Herceptin (surviving cells: 43 ± 3.9%). CONCLUSION: The new complex described herein could be considered for further evaluation in animals and potentially in humans as a radiopharmaceutical for use in the radioimmunotherapy of breast cancer. These results may be important for patients who cannot tolerate the therapeutic dosage of Herceptin currently used because of heart problems.

Downregulation of IGF-IR expression by RNAi inhibits proliferation and enhances chemosensitization of human colon cancer cells.

PURPOSE: Colon cancer is the second leading cause of cancer death worldwide. Elevated expression of insulin-like growth factor-I receptor (IGF-IR) is a frequent genetic abnormality seen in this malignancy. For a better understanding of its role in maintaining the malignant phenotype, we used RNA interference (RNAi) directed against IGF-IR in our study. The aim of this study was to examine the anti-proliferation and chemosensitization effects elicited by a decrease in the transcription and protein levels of IGF-IR by RNAi in SW480 colon cancer cells. METHODS: A plasmid-based polymerase III promoter system was used to deliver and express short interfering RNA (siRNA) targeting IGF-IR to reduce its expression in SW480 cells. Western blot analysis was used to measure the protein level of IGF-IR. We assessed the effects of IGF-IR silencing on cancer cell growth by a cell growth curve. The effect of the 5-fluorouracil (5-FU)-induced cell death by knockdown of IGF-IR was also investigated by methyl thiazolyl tetrazolium assay. RESULTS: Transfection of siRNA targeting IGF-IR was shown to reduce IGF-IR messenger RNA levels by 95%. Western blotting detected a similar inhibition of IGF-IR protein levels in those cells. The cells transfected with PKD-short hairpin RNA-IGF-IR-V2 significantly decreased cell growth and rendered cells more sensitive to chemotherapy. The highest proliferation inhibitory and chemosensitization ratios were 53 +/- 2% and 1.78, respectively. CONCLUSION: This study indicates that downregulation of IGF-IR results in significant inhibition of tumor growth in vitro. It also provides a promising strategy to chemotherapy efficacy in human tumors and forming a basis for future in vivo trials.

Radiolabeling of trastuzumab with 177Lu via DOTA, a new radiopharmaceutical for radioimmunotherapy of breast cancer.

AIM: Trastuzumab is a monoclonal antibody that is used in treating breast cancer. We labeled this monoclonal antibody with lutetium-177 and performed in vitro quality control tests as a first step in the production of a new radiopharmaceutical. MATERIAL AND METHODS: Trastuzumab was labeled with lutetium-177 using DOTA as chelator. Radiochemical purity and stability in buffer and human blood serum were determined using thin layer chromatography. Immunoreactivity and toxicity of the complex were tested on MCF7 breast cancer cell line. RESULTS: The radiochemical purity of the complex was 96+/-0.9%. The stabilities in phosphate buffer and in human blood serum at 96 h postpreparation were 93+/-1.2% and 85+/-3.5%, respectively. The immunoreactivity of the complex was 89+/-1.4%. At a concentration of 1 nM, the complex killed 70+/-3% of MCF7 cells. At 1.9 nM, 90+/-5% of the cells were killed. CONCLUSIONS: The results showed that the new complex could be considered for further evaluation in animals and possibly in humans as a new radiopharmaceutical for use in radioimmunotherapy against breast cancer.

Breast tumor targeting with (99m)Tc-HYNIC-PR81 complex as a new biologic radiopharmaceutical.

Human epithelial mucin, MUC1, is commonly overexpressed in adenocarcinoma that includes more than 80% of breast cancers. The PR81 is a murine anti-MUC1 monoclonal antibody (MAb) that was prepared against the human breast cancer. We developed an indirect method for labeling of this antibody with (99m)Tc in order to use the new preparation in immunoscintigraphy studies of BALB/c mice bearing breast tumors. The (99m)Tc-PR81 complex was prepared using the HYNIC as a chelator and tricine as a coligand. The labeling efficiency determined by instant thin-layer chromatography (ITLC) was 89.2%+/-4.7%, and radiocolloides measured by cellulose nitrate electrophoresis were 3.4%+/-0.9%. The in vitro stability of labeled product was determined at room temperature by ITLC and in human serum by gel filtration chromatography - 88.3%+/-4.6% and 79.8%+/-5.7% over 24 h, respectively. The integrity of labeled MAb was checked by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis, and no significant fragmentation was seen. The results of cell binding studies showed that both labeled and unlabeled PR81 were able to compete for binding to MCF 7 cells. Biodistribution studies performed in female BALB/c mice with breast tumor xenografts at 4, 16 and 24 h after the (99m)Tc-HYNIC-PR81 injection demonstrated a specific localization of the compound at the site of tumors and minimum accumulation in non target organs. The tumor imaging was performed in BALB/c mice with breast xenograft tumors at 4, 8, 12, 16, 20, 24, 28, 32 and 36 h after the complex injection. The tumors were visualized with high sensitivity after 8 h. The findings showed that the new radiopharmaceutical is a promising candidate for radioimmunoscintigraphy of the human breast cancer.

[99mTc] HYNIC-hEGF, a potential agent for imaging of EGF receptors in vivo: preparation and pre-clinical evaluation.

Expression of epidermal growth factor receptors (EGFR) has prognostic and predictive value in many kinds of tumors. Imaging of expression of EGFR in vivo may give valuable diagnostic information. The epidermal growth factor (EGF), a natural ligand, is a possible candidate for the targeting of EGFR. The present study describes a method for preparation of (99m)Tc-EGF via the hydrazinopyridine-3-carboxylic acid (HYNIC) conjugation using tricine and ethylenediamine-N,N'-diacetic acid (EDDA) as co-ligands. Both conjugates bound EGFR expressing cells with nanomolar affinity, and demonstrated good intracellular retention. The complex with EDDA demonstrated much higher stability in blood serum and during cysteine challenge. Biodistribution of (99m)Tc-EDDA-HYNIC-EGF in normal mice demonstrated fast blood clearance of conjugate, and its ability to bind EGFR in vivo. (99m)Tc-EDDA-HYNIC-EGF is a promising candidate for visualization of EGFR expression in vivo.