125I-labeled Monoclonal Antibody Research Articles

124I-Labeled Monoclonal Antibody and Fragment for the Noninvasive Evaluation of Tumor PD-L1 Expression In Vivo

Lung cancer is a highly heterogeneous cancer and is divided broadly into small and nonsmall cell lung cancer (SCLC or NSCLC). In all NSCLC patients, it is estimated that 50%-60% are programmed cell death ligand 1 (PD-L1) positive, and anti-PD-1/PD-L1 therapies have shown their clinical application prospects in advanced NSCLC. To avoid unnecessary adverse effects and provide anti-PD-1/PD-L1 therapy to the most appropriate patient population, the PD-L1 expression in patients preparing for treatment must be evaluated accurately and in real time. In this study, we noninvasively evaluate the PD-L1 expression in an NSCLC xenograft using 124I-labeled F(ab')2 fragments of durvalumab (Durva) and compared it with the 124I-labeled intact antibody in terms of the biodistribution and dosimetry. The aim is to develop a nuclide labeled molecular probe with better performance for PD-L1 immunoPET imaging. After cleaving using IdeS protease, the F(ab')2 fragments of Durva were labeled with 124I. The radioligand showed a high radiochemical purity (>96%) and outstanding stability. Western blot, quantitative real-time polymerase chain reaction, and flow cytometry were performed on the two selected NSCLC cell lines to measure the in vitro PD-L1 expression. The H460 cells showed a much higher PD-L1 expression than the A549 cells, both at the protein level and the mRNA level. In the following cell binding experiment and binding specificity assay, the labeled radioligand showed good affinity to high PD-L1 expression cells and could be blocked with excess unlabeled intact Durva. The results of the biodistribution and the positron emission tomography (PET) image showed that the peak tumor uptake of 124I-Durva-F(ab')2 was close to 124I-Durva, but much earlier (5.29 ± 0.42% ID/g for 124I-Durva-F(ab')2 at 12 h vs 5.18 ± 0.73% ID/g for 124I-Durva at 48 h). Compared with 124I-Durva, an accelerated blood clearance was observed for 124I-Durva-F(ab')2. The faster blood clearance allowed for a higher tumor-to-background ratio, which was reflected on the image in contrast. The H460 tumors showed excellent contrast as early as 4 h after injection with 124I-Durva-F(ab')2, and for 124I-Durva, the xenograft could not be distinguished clearly until 24 h after injection. Interestingly, 124I-Durva-F(ab')2 showed lower accumulations compared to other metal isotopes labeled PD-L1 antibodies in bone, liver, spleen etc., which will be beneficial for metastasis detection. Another benefit of accelerated blood clearance was a reduction in the radiation dose. According to the results of the OLINDA/EXM, the effective dose for the total body of 124I-Durva was 4.25-times greater than that of 124I-Durva-F(ab')2 (186 μSv/MBq vs 43.8 μSv/MBq). All of these data indicated that 124I-Durva-F(ab')2 is a promising immunoPET tracer for evaluating the in vivo PD-L1 levels in an NSCLC model and is expected to be successful in future clinical application.

Survival Advantage Following TAG-72 Antigen-Directed Cancer Surgery in Patients With Colorectal Carcinoma: Proposed Mechanisms of Action.

Patients with colorectal carcinoma (CRC) continue to have variable clinical outcomes despite undergoing the same surgical procedure with curative intent and having the same pathologic and clinical stage. This problem suggests the need for better techniques to assess the extent of disease during surgery. We began to address this problem 35 years ago by injecting patients with either primary or recurrent CRC with 125I-labeled murine monoclonal antibodies against the tumor-associated glycoprotein-72 (TAG-72) and using a handheld gamma-detecting probe (HGDP) for intraoperative detection and removal of radioactive, i.e., TAG-72-positive, tissue. Data from these studies demonstrated a significant difference in overall survival data (p < 0.005 or better) when no TAG-72-positive tissue remained compared to when TAG-72-positive tissue remained at the completion of surgery. Recent publications indicate that aberrant glycosylation of mucins and their critical role in suppressing tumor-associated immune response help to explain the cellular mechanisms underlying our results. We propose that monoclonal antibodies to TAG-72 recognize and bind to antigenic epitopes on mucins that suppress the tumor-associated immune response in both the tumor and tumor-draining lymph nodes. Complete surgical removal of all TAG-72-positive tissue serves to reverse the escape phase of immunoediting, allowing a resetting of this response that leads to improved overall survival of the patients with either primary or recurrent CRC. Thus, the status of TAG-72 positivity after resection has a significant impact on patient survival.

Clinical Experience in the Randomized Phase 3 Sierra Trial: Anti-CD45 Iodine (131I) Apamistamab [Iomab-B] Conditioning Enables Hematopoietic Cell Transplantation with Successful Engraftment and Acceptable Safety in Patients with Active, Relapsed/Refractory AML Not Responding to Targeted Therapies

Clinical Experience in the Randomized Phase 3 Sierra Trial: Anti-CD45 Iodine (131I) Apamistamab [Iomab-B] Conditioning Enables Hematopoietic Cell Transplantation with Successful Engraftment and Acceptable Safety in Patients with Active, Relapsed/Refractory AML Not Responding to Targeted Therapies

Radioimmunoimaging of 125I-labeled anti-CD93 monoclonal antibodies in a xenograft model of non-small cell lung cancer.

Lung cancer, especially non-small cell lung cancer (NSCLC), is the most common malignant tumor associated with poor prognosis. Angiogenesis plays a vital role in NSCLC, and could be used in tumor staging and therapy evaluation. CD93 (C1q receptor) is reportedly a key regulator of tumor angiogenesis. In the present study, the efficacy and specificity of a 125I-labeled CD93-specific monoclonal antibody (125I-anti-CD93 mAb) in detecting NSCLC xenografts were analyzed, and the association between CD93 expression and 125I-anti-CD93 mAb uptake by tumors was evaluated. The targeting ability of 125I-anti-CD93 mAb enabled its rapid, continuous and highly specific accumulation in CD93-expressing tumors in vivo. These results revealed the potential applicability of 125I-anti-CD93 mAb for non-invasive imaging diagnosis of CD93-positive NSCLC.

Intraventricular radioimmunotherapy targeting B7H3 for CNS malignancies.

e13592 Background: Tumors metastasizing to the central nervous system (CNS) are associated with significant mortality. We tested the toxicity and dosimetry of intraventricular 131I-labeled monoclonal antibody 8H9 targeting surface glycoprotein B7-H3 in patients with primary or metastatic CNS tumors. Methods: Tumor B7-H3 expression was assessed by immunohistochemistry. CSF flow was determined by 111Indium-DTPA cisternography. 131 patients received 2 mCi tracer of intra-Ommaya 124I- or 131I-8H9 for nuclear imaging followed by a therapeutic injection (10-80 mCi, dose levels 1-8 in 10 mCi increments for phase I patients; expanded cohort 50 mCi/injection) 131I-8H9. Pharmacokinetics were studied by serial CSF and blood samplings over 48 hours. Dosimetry was based on pharmacokinetics and region of interest analyses on serial PET. Toxicity was defined by the CTCAE v.3.0. 8H9 dosimetry and therapy injections were repeated after 1 month if no serious adverse events or progressive disease ensued. Tumor response was determined by clinical, radiographic, cytologic criteria; overall survival was noted. Results: 57 patients (ages 2 – 54 years, median age 11.7 years) received 158 injections Primary CNS diagnoses included medulloblastoma (n = 23), ependymoma (N = 8), chordoma (n = 1), rhabdoid tumor (n = 1), choroid plexus carcinoma (n = 3), ETMR (n = 3), glioblastoma multiforme (n = 1) , PXA (n = 1); metastatic tumors included sarcoma (n = 9), melanoma (n = 4), retinoblastoma (n = 2), and ovarian carcinoma (n = 1). Injections were well tolerated and routinely administered in the outpatient setting. Rare self-limited adverse events included grade 1 or 2 fever, headache, vomiting; 3 injections were associated with grade 3 toxicities requiring discontinuation of therapy including chemical meningitis (n = 2),and increasing communicating hydrocephalus (n = 1), Although not a dose limiting toxicity, myelosuppression occurred in patients who had received craniospinal radiation and at dose levels 6 and higher (≥60 mCi). 16 patients remain alive including patients with high-risk malignancies including choroid plexus carcinoma, ETMR, recurrent ependymoma and recurrent medulloblastoma. Conclusions: We conclude that intraventricular 131I-8H9 is safe, has favorable dosimetry to CSF, and may have clinical utility in the treatment of primary and metastatic CNS tumors. Clinical trial information: NCT00089245.

PDCT-04. SAFETY AND EFFICACY OF INTRAVENTRICULAR 131I-LABELED MONOCLONAL ANTIBODY 8H9 TARGETING THE SURFACE GLYCOPROTEIN B7-H3 IN PATIENTS WITH CNS/LM DISEASE

Tumors metastasizing to the central nervous system (CNS) are associated with significant mortality. Intraventricular 131I-labeled monoclonal antibody targeting tumor associated antigens may eradicate micrometastases. We tested the toxicity and dosimetry of intraventricular 131I-labeled monoclonal antibody 8H9 targeting surface glycoprotein B7-H3 in patients with CNS disease on a phase I/II trial. Adequate CSF flow was determined by 111Indium-DTPA cisternography through intraventricular devices. 131 patients [median age 5.4 years (1.2- 53.6 years)] with primary CNS malignancies or malignancies metastatic to the CNS received 383 injections involving 2 mCi 124I- or 131I-8H9 as a tracer for nuclear imaging and a therapeutic injection (10-80 mCi, dose levels 1-8 in 10 mCi increments for phase I patients; expanded cohort 50 mCi/injection) 131I-8H9. Pharmacokinetics were studied by serial CSF and blood samplings over 48 hours. Dosimetry was based on pharmacokinetics and ROI analyses on serial scans. Toxicity was defined by CTCAE v3.0. Injections were repeated after 1 month in the absence of serious adverse event or progressive disease. Tumor response was determined by clinical, radiographic, cytologic criteria; overall survival was noted. Injections were well tolerated and routinely administered in the outpatient setting. Rare self-limited adverse events included grade 1 or 2 fever, headache, vomiting, biochemical elevations in AST or ALT [1 injection with grade 3 ALT elevation (dose level 3)]. Although not a dose limiting toxicity, myelosuppression was observed in patients who had received craniospinal radiation. Mean absorbed CSF dose was 104.9 cGy/mCi and 2.6 cGy/mCi to the blood. Of 93 patients treated for metastatic CNS neuroblastoma, an improved overall survival was noted compared to median survival reported with conventional therapies. Intraventricular 131I-8H9 is safe, has favorable CSF and blood dosimetry, and has clinical utility in the treatment of B7-H3 -positive CNS cancers.

SCDT-38. SAFETY AND EFFICACY OF INTRAVENTRICULAR 131I-LABELED MONOCLONAL ANTIBODY 8H9 TARGETING THE SURFACE GLYCOPROTEIN B7-H3 IN PATIENTS WITH CNS/LM DISEASE

AbstractTumors metastasizing to the central nervous system (CNS) are associated with significant mortality. We tested the toxicity and dosimetry of intraventricular I-labeled monoclonal antibody 8H9 targeting surface glycoprotein B7-H3 in patients with CNS. Tumor B7-H3 expression was assessed by immunohistochemistry. CSF flow was determined by Indium-DTPA cisternography. 131 patients received 2 mCi tracer of intra-Ommaya I- or I-8H9 for nuclear imaging followed by a therapeutic injection (10-80 mCi, dose levels 1-8 in 10 mCi increments for phase I patients; expanded cohort 50 mCi/injection) I-8H9. Pharmacokinetics were studied by serial CSF and blood samplings over 48 hours. Dosimetry was based on pharmacokinetics and region of interest analyses on serial PET. Toxicity was defined by the CTCAE v.3.0. 8H9 dosimetry and therapy injections were repeated after 1 month if no serious adverse events or progressive disease ensued. Tumor response was determined by clinical, radiographic, cytologic criteria; overall survival was noted. 129 patients with primary or metastatic CNS tumors received 383 injections [median age 5.4 years (1.2- 53.6 years)] Injections were well tolerated and routinely administered in the outpatient setting. Rare self-limited adverse events included grade 1 or 2 fever, headache, vomiting, biochemical elevations in AST or ALT and 1 injection with grade 3 ALT elevation (dose level 3). Although not a dose limiting toxicity, myelosuppression occurred in patients who had received craniospinal radiation and at dose levels 6 and higher (>60 mCi). Of 93 patients treated for metastatic CNS neuroblastoma, an improved overall survival was noted compared to survival reported with conventional therapies. Interpatient variability for total absorbed dose to the CSF and blood was observed; mean absorbed CSF dose was 104.9 cGy/mCi by CSF sampling, and 2.6 cGy/mCi to the blood. We conclude that intraventricular I-8H9 is safe, has favorable dosimetry to CSF, and has clinical utility.

SPECT imaging of neuropilin receptor type-1 expression with 131I-labeled monoclonal antibody.

As a novel co-receptor for vascular endothelial growth factor (VEGF), neuropilin receptor type-1 (NRP-1) is overexpressed in several cancers and metastases, and serves as an attractive target for cancer molecular imaging and therapy. Previous single photon emission computerized tomography (SPECT) studies demonstrated that the small NRP-1-targeting peptides 99mTc-MA-ATWLPPR and 99mTc-CK3 showed poor tumor imaging quality, because of their rapid blood clearance and very low tumor uptake. Compared with small peptides, monoclonal antibodies (mAbs) can improve imaging of NRP-1-expression, due to their high affinity, specificity and slow extraction. A6-11-26 is a novel monoclonal antibody against NRP-1 b1b2 domain that exhibits inhibition of tumor growth in NPR-1-expressing preclinical models. The aim of the present study was to develop the 131I-labeled anti-NRP-1 monoclonal antibody A6-11-26 as a SPECT probe for imaging of NRP-1-positive tumor. An anti-NRP-1 monoclonal antibody (A6-11-26) was produced by hybridomas and was labeled with iodine-131 by the iodogen method. Invitro, the radiolabeling efficiency, radiochemical purity, immunoreactive fraction and stability were assessed. Binding affinity and specificity of 131I‑A6-11-26 to NRP-1 were evaluated using human glioblastoma U87MG cells. Invivo, biodistribution and SPECT/CT studies were conducted on mice bearing U87MG xenografts after the injection of 131I-A6-11-26 with or without co-injection of unlabeled A6-11-26 antibody. A6-11-26 was generated successfully by hybridoma with high purity (>95%) and was labeled with iodine-131 within 60min with high labelling efficiency (95.46±3.34%), radiochemical purity (98.23±1.41%). 131I-A6-11-26 retained its immunoreactivity and also displayed excellent stability in mouse serum and PBS solution during 1 to 96h. Cell uptake assays showed high NRP-1-specific uptake (15.80±1.30% applied activity at 6h) in U87MG cells. 131I-A6-11-26 bound to NRP-1 with low nanomolar affinity (KD=1.67±0.14nM) in U87MG cells. Invivo, biodistribution study demonstrated targeting of U87MG glioma xenografts was NRP-1 specific. The tumor uptake was 6.00±1.24%ID/g at 24h, and the tumor to muscle ratio was 3.20±0.30 at 24h, and reached the highest level of 6.13±0.24 at 120h after injection. SPECT imaging studies revealed that 131I-A6-11-26 could clearly identify U87MG tumors with good contrast, especially at 72-120h after injection. The present study demonstrates that 131I-A6-11-26 is capable of detecting lesions in an NRP-1-expressing tumor with high target selectivity, and may offer a promising SPECT agent for NRP-1 expression positive tumor and encourage further investigation.

Therapy to target renal cell carcinoma using 131I-labeled B7-H3 monoclonal antibody.

B7-H3 is a tumor-associated antigen that plays a critical role in potential tumor-targeted therapy. In this study, we aimed to assess the radiobiological effect of 131I-labeled B7-H3 monoclonal antibody (131I-4H7) in nude mice with human renal cell carcinoma (RCC) and evaluate the effect of 131I-4H7 on RCC treatment. The radiobiological activity and tumor uptake of 131I-4H7, and its effect on tumor growth were measured. 131I-4H7 was absorbed by the tumor and reached its maximal uptake rate (3.32% injected dose [ID]/g) at 24 h, at which point the drug concentration in the tumor was 7.36-, 2.06-, 1.80-, and 2.78-fold higher than that in muscle, kidneys, liver, and heart, respectively. Measurements and positron emission tomography–computed tomography imaging showed that tumor development was significantly inhibited by 131I-4H7. HE staining revealed that 131I-4H7 significantly injures tumor cells. Our results suggest that 131I-4H7 is markedly absorbed by the tumor and did suppress the development of RCC xenografted tumors in nude mice, which might provide a new candidate for antibody-mediated targeted radiotherapy in human RCC.

Characterization of a double‐sided silicon strip detector autoradiography system

The most commonly used technology currently used for autoradiography is storage phosphor screens, which has many benefits such as a large field of view but lacks particle-counting detection of the time and energy of each detected radionuclide decay. A number of alternative designs, using either solid state or scintillator detectors, have been developed to address these issues. The aim of this study is to characterize the imaging performance of one such instrument, a double-sided silicon strip detector (DSSD) system for digital autoradiography. A novel aspect of this work is that the instrument, in contrast to previous prototype systems using the same detector type, provides the ability for user accessible imaging with higher throughput. Studies were performed to compare its spatial resolution to that of storage phosphor screens and test the implementation of multiradionuclide ex vivo imaging in a mouse preclinical animal study. Detector background counts were determined by measuring a nonradioactive sample slide for 52 h. Energy spectra and detection efficiency were measured for seven commonly used radionuclides under representative conditions for tissue imaging. System dead time was measured by imaging (18)F samples of at least 5 kBq and studying the changes in count rate over time. A line source of (58)Co was manufactured by irradiating a 10 μm nickel wire with fast neutrons in a research reactor. Samples of this wire were imaged in both the DSSD and storage phosphor screen systems and the full width at half maximum (FWHM) measured for the line profiles. Multiradionuclide imaging was employed in a two animal study to examine the intratumoral distribution of a (125)I-labeled monoclonal antibody and a (131)I-labeled engineered fragment (diabody) injected in the same mouse, both targeting carcinoembryonic antigen. Detector background was 1.81 × 10(-6) counts per second per 50 × 50 μm pixel. Energy spectra and detection efficiency were successfully measured for seven radionuclides. The system dead time was measured to be 59 μs, and FWHM for a (58)Co line source was 154 ± 14 μm for the DSSD system and 343 ± 15 μm for the storage phosphor system. Separation of the contributions from (125)I and (131)I was performed on autoradiography images of tumor sections. This study has shown that a DSSD system can be beneficially applied for digital autoradiography with simultaneous multiradionuclide imaging capability. The system has a low background signal, ability to image both low and high activity samples, and a good energy resolution.

Targeting cancer stem cells with an 131I-labeled anti-AC133 monoclonal antibody in human colorectal cancer xenografts

IntroductionCancer stem cells (CSCs) are a subpopulation within a tumor, which possesses the characteristics of self-renewal, differentiation, tumorigenicity, and drug resistance. The aim of this study was to target the colorectal CSC marker CD133 with an131I-labeled specific monoclonal antibody (AC133 mAb) in a nude mouse xenograft model. MethodsColorectal adenocarcinoma cells (LoVo cell line) were separated into CD133(+) and CD133(−) cells by magnetic activated cell sorting. CD133(+), CD133(−), and unsorted LoVo cells were cultured and then implanted subcutaneously into the lower limbs of nude mice (n=5). AC133 mAb was labeled with 131I by the iodogen method. ResultsThe radiolabeled compound, 131I-AC133 mAb, showed high stability, specificity, and immunoactivity in vitro. Obvious accumulation of 131I-AC133 mAb was seen in nude mice bearing xenografts of CD133(+) and unsorted LoVo cells, but no uptake was found in mice bearing CD133(−) xenografts or specifically blocked xenografts. Biodistribution analysis showed that the tumor uptake of 131I-AC133 mAb was 6.97±1.40, 1.35±0.48, 6.12±1.91, and 1.61±0.44% ID/g (n=4) at day 7 after injection of 131I-AC133 mAb in CD133(+), CD133(−), unsorted LoVo cell and specifically blocked xenografts, respectively. The results of immunofluorescence, autoradiography, and western blotting further verified the specific binding of 131I-AC133 mAb to CD133(+) tumors. ConclusionsThis study demonstrates the possibility of targeting CSCs with a radiolabeled AC133 mAb in colorectal cancer xenografts based on in vitro, ex vivo, and in vivo experiments. Our findings suggest a new method for imaging CSCs non-invasively.

A phase II study of surgical excision, temozolomide, radiotherapy, and anti-EGFR radioimmunotherapy (EXTRA) as adjuvant therapy in high-grade gliomas

Despite evolution in surgical technique, radiotherapy technology, and targeted systemic therapies, prognosis for patients with high-grade gliomas remains poor. Standard treatment consisting of surgery, adjuvant chemoradiation, and chemotherapy has marginally improved survival in these patients. A recent novel approach has been the addition of adjuvant radioimmunotherapy with 125I-labeled anti-epidermal growth factor receptor monoclonal antibody 425 (125I-epidermal growth factor receptor (EGFR) monoclonal antibody (MAb) 425), which demonstrated safety and efficacy in patients with glioblastoma. Many institutions choose to treat grade 3 gliomas in an identical fashion to grade 4 tumors. In this phase II clinical trial, we tested the efficacy of adjuvant radioimmunotherapy in patients with pathologic diagnosis of high-grade glioma comprising either glioblastoma (GBM) or astrocytoma with anaplastic foci (AAF). Patients with newly diagnosed high-grade gliomas were eligible. Adjuvant radiotherapy was delivered with a median dose of 60 Gy after surgery. 125I-EGFR MAb 425 was given by three weekly intravenous injections of 1.85 GBq of radiolabeled 125I-EGFR MAb 425 following surgery and adjuvant radiation. When administered, temozolomide was given concominantly (75 mg/m2/day, 35–42 days) with radiotherapy followed by 6 cycles of adjuvant temozolomide (TMZ) (150–200 mg/m2/day × 5 days, every 28 days q28d). Median survival was the primary endpoint. We compared survival in three treatment groups: patients who underwent surgery followed by radiation (CTL), patients who underwent surgery and radiation followed by radioimmunotherapy (RIT), and patients who underwent RIT with the additional of temozolomide (RIT + TMZ). Between 1988 and 2008, a total of 390 patients with high-grade glioma underwent treatment at Hahnemann University Hospital with a median age of 48 years. Median survival in months was 7.3, 39.7, and 79.4 in the CTL, RIT, and RIT + TMZ arms, respectively. Multivariate analysis, which controlled for age and extent of surgery, demonstrated a persistent survival benefit of RIT. Combined results are reported as well as subgroup analysis of GBM and AAF patients. Adjuvant 125I-EGFR MAb 425 radioimmunotherapy is a safe and effective treatment for patients with high-grade glioma and warrants further study with a randomized trial.

New imaging agents targeting chemokine receptor CXCR4 for PET/SPECT and MRI

CXCR4 is a chemokine receptor overexpressed in numerous cancer types that plays a key role in invasion and metastases. For this reason, CXCR4 appears as an interesting biomarker for the field of diagnostic oncology and in the past few years it has gained interest for application in molecular imaging. Both peptide-based and small molecules have been developed as CXCR4 inhibitors. Among all existing organic molecules, cyclam derivatives AMD3100 and AMD3465 were described as strong CXCR4 inhibitors. To image CXCR4 expression in tumor models, macromolecular agents such as 111In- and 18F-labeled peptides and 125 I-labeled monoclonal antibodies have been investigated using either SPECT or PET. Recently, AMD3100 and AMD3465 were directly radiolabeled with 99mTc and 64Cu, but these complexes have a low in vivo stability leading to the decomplexation of the metal. In this context, we decided to use the high affinity of these molecules for CXCR4 and to attach to these systems an optimized chelating agent in order to image CXCR4 by SPECT/PET imaging or MRI using different metals such as 111In, 64Cu, 68Ga or Gd. AMD3100 and AMD3465 were modified in order to introduce an adapted chelator. Our new analogues were then metalated with Gd, cold Ga, In, and Cu, but also 68Ga and 64Cu. Biological experiments on Jurkat and MCF-7 cell lines were performed to determine the affinity of our new imaging agents toward the CXCR4 receptor. We will present the synthetic pathway toward our new AMD analogues together with flow cytometry and radiolabelling experiments. Two compounds show promising results and will be used for in-vivo experiments. We report here new imaging agents targeting the chemokine receptor CXCR4 that could be use either for PET/SPECT and MRI. Our concept also offers the possibility of attaching a fluorescent tag for optical imaging or bimodal systems.

DNA damage-centered signaling pathways are effectively activated during low dose-rate Auger radioimmunotherapy

IntroductionLow dose-rate radioimmunotherapy (RIT) using 125I-labelled monoclonal antibodies (125I-mAbs) is associated with unexpected high cytotoxicity per Gy. MethodsWe investigated whether this hypersensitivity was due to lack of detection of DNA damage by the targeted cells. DNA damage was measured with the alkaline comet assay, gamma-H2AX foci and the micronucleus test in p53−/− and p53+/+ HCT116 cells exposed to increasing activities of internalizing anti-HER1 125I-mAbs or non-internalizing anti-CEA 125I-mAbs. The expression of proteins involved in radiation response and progression of cells through the cycle were determined. ResultsCell hypersensitivity to low absorbed doses of anti-CEA 125I-mAbs was not due to defect in DNA damage detection, since ATM (ataxia telangiectasia mutated gene), gamma-H2AX, p53 and p21 were activated in RIT-treated HCT116 cells and G2/M cell cycle arrest was observed. Moreover, the alkaline comet assay showed that DNA breaks accumulated when cells were placed at 4°C during exposure but were repaired under standard RIT conditions (37°C), suggesting that lesions detected under alkaline conditions (mostly DNA single strand breaks and alkali-labile sites) are efficiently repaired in treated cells. The level of gamma-H2AX protein corroborated by the level of foci measured in nuclei of treated cells was shown to accumulate with time thereby suggesting the continuous presence of DNA double strand breaks. This was accompanied by the formation of micronuclei. ConclusionHypersensitivity to non-internalizing 125I-mAbs is not due to lack of detection of DNA damage after low absorbed dose-rates. However, DNA double strand breaks accumulate in cells exposed both to internalizing and non-internalizing 125I-mAbs and lead to micronuclei formation. These results suggest impairment in DNA double strand breaks repair after low absorbed doses of 125I-mAbs.

Mean platelet volume: interactions with platelet aggregation activity and glycoprotein IIb-IIIa and Ib expression levels

Increased mean platelet volume (MPV) is an independent risk factor of thrombotic events in patients with cardiovascular diseases. Interactions of MPV with platelet aggregation activity and contents of glycoprotein (GP) IIb-IIIa (alphaIIb/beta3 integrin, fibrinogen receptor) and GP Ib (von Willebrand factor receptor) were investigated in this study. Investigation was performed in a group of healthy volunteers (n = 38) and in a group of patients with acute coronary syndrome (ACS). In patients blood was collected at days 1, 3-5 and 8-12 after ACS development. As an antiaggregant therapy all patients received acetylsalicylic acid (ASA, inhibitor of thromboxane A2 synthesis) and most of them--clopidogrel (ADP receptor antagonist) with the exception of part of the patients (n = 44) at day 1 who had not taken clopidogrel before first blood collection. In volunteers platelet aggregation was stimulated by 1.25, 2.5, 5 and 20 M ADP, and in patients--by 5 and 20 M ADP. GP IIb-IIIa and GP Ib content on platelet surface was measured using 125I-labelled monoclonal antibodies. GP IIb-IIIa and GP Ib genetic polymorphisms were determined in ACS patients. In healthy donors significant correlations between MPV and aggregation levels were revealed at 1.25 and 2.5 M ADP (coefficients of correlation (r)--0.396 and 0.373, p < 0.05) and at 5 and 20 those interactions did not reach significant level (r--0.279 and 0.205, p > 0.05). Correlations between MPV and aggregation levels were observed at day 1 of ACS in a subgroup of patients who received ASA but had not started clopidogrel treatment (r--0.526, p < 0.01 and 0.368, p < 0.05 for 5 and 20 M ADP respectively). Interactions between these parameters were not registered upon combined treatment with ASA and clopidogrel. Strong direct correlations between MPV and GP IIb-IIIa and GP Ib contents were detected in healthy donors and ACS patients (at all time points) -r from 0.439 to 0.647 (p < or = 0.001 for all correlations). Genetic polymorphisms of GP IIb-IIIa (GP IIIa Leu33Pro) and GP Ib ((-5)T/C (Kozak) and Thr145Met) identified in ACS patients did not affect expression levels of corresponding glycoproteins. The data obtained indicated that increased MPV values correlate with increased platelet aggregation activity and enhanced GP IIb-IIIa and GP Ib expression.

Design and preliminary assessment of 99mTc-labeled ultrasmall superparamagnetic iron oxide-conjugated bevacizumab for single photon emission computed tomography/magnetic resonance imaging of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) has a very high incidence and mortality. Early diagnosis and timely treatments are therefore required to improve the quality of life and survival rate of HCC patients. Here, we developed a vascular endothelial growth factor (VEGF)-based multimodality imaging agent for single photon emission computed tomography (SPECT), computed tomography (CT) and magnetic resonance imaging (MRI) and used it to assess HCC mice and explore the combinative value of CT/MRI-based morphological imaging and SPECT functional imaging. HCC targeting with 125I-labeled bevacizumab monoclonal antibody (mAb) was examined using SPECT/CT in HepG2 tumor-bearing mice after intravenous mAb injection. Based on this, an integrated, bimodal, VEGF-targeted, ultrasmall superparamagnetic iron oxide (USPIO)-conjugated 99mTc-labeled bevacizumab mAb was synthesized to increase tumor penetration and accumulations. The in vivo pharmacokinetics and HepG2 tumor targeting were explored through in vivo planar imaging and SPECT/CT using a mouse model of HepG2 liver cancer. The specificity of the radiolabeled nanoparticles for HepG2 HCC was verified using in vitro immunohistochemistry and Prussian blue staining. With diethylenetriamine pentaacetic acid as a bifunctional chelating agent, USPIO-bevacizumab achieved a 99mTc labeling efficiency of >90 %. The in vivo imaging results also exhibited the targeting of USPIO on HepG2 HCC. The specificity of these results was confirmed using in vitro immunohistochemistry and Prussian blue staining. Our preliminary findings showed the potential of USPIO as an imaging agent for the SPECT/MRI of HepG2 HCC.

Treatment of 131I-labeled anti-CD147 monoclonal antibody in VX2 carcinoma-induced liver tumors

Hepatocellular carcinoma (HCC) is a major health problem worldwide. CD147 has been reported to be overexpressed in HCC and blocking CD147 expression can decrease tumor growth. (131)I is often used in combination with other drugs to treat HCC and yields positive results. In this study, we combined the (131)I and CD147 monoclonal antibody to treat HCC in a rabbit VX2 animal model. In the (131)I-labeled CD147 antibody ((131)I-CD147-Ab) treatment group, the animals lived considerably longer than the animals in the other treatment groups. Metastasis and tumor growth in the (131)I-CD147-Ab treatment group were also inhibited. MMP2 and CD31 expression were significantly lower in the treatment group, whereas Tunel staining was overexpressed. These findings suggest that (131)I-CD147-Ab is a promising drug in the treatment of HCC, by inhibiting metastasis and growth and by decreasing the expression of MMP2 and CD31 or by inducing tumor necrosis. After testing the biochemical parameters, (131)I-CD147-Ab caused fewer side-effects in the animals.

Apoptosis and p53 are not involved in the anti-tumor efficacy of 125I-labeled monoclonal antibodies targeting the cell membrane

(125)I-labeled monoclonal antibodies ((125)I-mAbs) can efficiently treat small solid tumors. Here, we investigated the role of apoptosis, autophagy and mitotic catastrophe in (125)I-mAb toxicity in p53(-/-) and p53(+/+) cancer cells. We exposed p53(-/-) and p53(+/+) HCT116 cells to increasing activities of internalizing (cytoplasmic location) anti-HER1 (125)I-mAbs, or non-internalizing (cell surface location) anti-CEA (125)I-mAbs. For each targeting model we established the relationship between survival and mean nucleus absorbed dose using the MIRD formalism. In both p53(-/-) and p53(+/+) HCT116 cells, anti-CEA (125)I-mAbs were more cytotoxic per Gy than anti-HER1 (125)I-mAbs. Sensitivity to anti-CEA (125)I-mAbs was p53-independent, while sensitivity to anti-HER1 (125)I-mAbs was higher in p53(-/-) HCT 116 cells, suggesting that they act through different signaling pathways. Apoptosis was only induced in p53(+/+) HCT116 cells and could not explain cell membrane radiation sensitivity. Inhibition of autophagy did not modify the cell response to (125)I-mAbs. By contrast, mitotic death was similarly induced in both p53(-/-) and p53(+/+) HCT116 cells by the two types of (125)I-mAbs. We also showed using medium transfer experiments that γ-H2AX foci were produced in bystander cells. Cell membrane sensitivity to (125)I-mAbs is not mediated by apoptosis and is p53-independent. Bystander effects-mediated mitotic death could be involved in the efficacy of (125)I-mAbs binding cell surface receptors.

Targeting Free Prostate-Specific Antigen forIn VivoImaging of Prostate Cancer Using a Monoclonal Antibody Specific for Unique Epitopes Accessible on Free Prostate-Specific Antigen Alone

This study investigated the feasibility of targeting the free, unbound forms of prostate-specific antigen (fPSA) for in vivo imaging of prostate adenocarcinomas (PCa), as PSA is produced and secreted at abundance during every clinical stage and grade of PCa, including castration-resistant disease. We injected (125)I-labeled monoclonal antibody PSA30 (specific for an epitope uniquely accessible on fPSA alone) intravenously in male nude mice carrying subcutaneous xenografts of LNCaP tumors (n=36). Mice were sacrificed over a time course from 4 hours to 13 days after injecting (125)I-labeled PSA30. Tissue uptake of (125)I-PSA30 at 48 and 168 hours after intravenous injection was compared with two clinically used positron emission tomography radiopharmaceuticals, (18)F-fluoro-deoxy-glucose ((18)F-FDG) or (18)F-choline, in cryosections using Digital AutoRadiography (DAR) and also compared with immunohistochemical staining of PSA and histopathology. On DAR, the areas with high (125)I-PSA30 uptake corresponded mainly to morphologically intact and PSA-producing LNCaP cells, but did not associate with the areas of high uptake of either (18)F-FDG or (18)F-choline. Biodistribution of (125)I-PSA30 measured in dissected organs ex vivo during 4 to 312 hours after intravenous injection demonstrated maximum selective tumor uptake 24-48 hours after antibody injection. Our data showed selective uptake in vivo of a monoclonal antibody highly specific for fPSA in LNCaP cells. Hence, in vivo imaging of fPSA may be feasible with putative usefulness in disseminated PCa.

Bone Marrow Dosimetry Using 124I-PET

Bone marrow is usually dose-limiting for radioimmunotherapy. In this study, we directly estimated red marrow activity concentration and the self-dose component of absorbed radiation dose to red marrow based on PET/CT of 2 different (124)I-labeled antibodies (cG250 and huA33) and compared the results with plasma activity concentration and plasma-based dose estimates. Two groups of patients injected with (124)I-labeled monoclonal antibodies (11 patients with renal cancer receiving (124)I-cG250 and 5 patients with colorectal cancer receiving (124)I- huA33) were imaged by PET or PET/CT on 2 or 3 occasions after infusion. Regions of interest were drawn over several lumbar vertebrae, and red marrow activity concentration was quantified. Plasma activity concentration was also quantified using multiple patient blood samples. The red marrow-to-plasma activity concentration ratio (RMPR) was calculated at the times of imaging. The self-dose component of the absorbed radiation dose to the red marrow was estimated from the images, from the plasma measurements, and using a combination of both sets of measurements. RMPR was observed to increase with time for both groups of patients. Mean (±SD) time-dependent RMPR (RMPR(t)) for the cG250 group increased from 0.13 ± 0.06 immediately after infusion to 0.23 ± 0.09 at approximately 6 d after infusion. For the huA33 group, mean RMPR(t) was 0.10 ± 0.04 immediately after infusion, 0.13 ± 0.05 approximately 2 d after infusion, and 0.20 ± 0.09 approximately 7 d after infusion. Plasma-based estimates of red marrow self-dose tended to be greater than image-based values by, on average, 11% and 47% for cG250 and huA33, respectively, but by as much as -73% to 62% for individual patients. The hybrid method combining RMPR(t) and plasma activity concentration provided a closer match to the image-based dose estimates (average discrepancies, -2% and 18% for cG250 and huA33, respectively). These results suggest that the assumption of time-independent proportionality between red marrow and plasma activity concentration may be too simplistic. Individualized imaged-based dosimetry is probably required for the optimal therapeutic delivery of radiolabeled antibodies, which does not compromise red marrow and may allow, for some patients, a substantial increase in administered activity and thus tumor dose.