Unlabeled Antibody Research Articles

Development of a quantum dot-based fluorescent immunoassay for progesterone determination in bovine milk

The use of semiconductor quantum dots (QDs) as fluorescent labels to develop a competitive immunoassay for sensitive detection and quantification of progesterone in cow's milk is described. Colloidal water-soluble CdSe/ZnS QDs are conjugated to an antigen derivative (progesterone-BSA conjugate) and a simple methodology is optimised to determine the antigen concentration in the final bioconjugate. The obtained QD-linked antigens were then employed together with unlabelled anti-progesterone monoclonal antibodies, as the biological recognition elements, in the development of the quantitative QDs-based fluorescent immunoassay for progesterone in bovine milk. After optimization, the developed immunoassay proved to cover a progesterone concentration range from 0.3 to 14.5 ng/mL in cow milk. Milk samples were just diluted 10-fold with deionised water and directly analysed with the proposed immunoassay, without additional sample pre-treatment or analyte extraction. The minimum detectable level (IC 10) of the developed immunoassay turned out to be 0.1 ng/mL of progesterone in bovine milk. The sensitivity (IC 50) achieved was 2.2 ng/mL with a reproducibility of 3.5% RSD as obtained from the results of the analysis of the triplicate of same samples but in three different days. Applicability of the proposed methodology was evaluated by analyzing cow's milk samples enriched with known concentrations of progesterone and recoveries better than 90% were achieved.

Reflecting on Stem Cell Differentiation

Reflecting on Stem Cell Differentiation

Optimal preloading in radioimmunotherapy with anti-CD45 antibody

Anti-CD45 antibody is predominantly used in the treatment of acute leukemia. CD45 is stably expressed on all leukocytes and their precursors, and therefore the liver and spleen constitute major antigen sinks. Thus, as the red marrow is the target organ, in radioimmunotherapy with anti-CD45 antibody, preloading with unlabeled antibody is a method to increase the absorbed dose to the target cells. In a previous study, a method to individually determine the optimal preload for five patients with acute leukemia was developed. Here, this method is examined and improved using two pretherapeutic measurement series and a refined pharmacokinetic model. To obtain the biodistribution of 111In-labeled anti-CD45 antibody under different saturation conditions, two measurement series one with and one without preloading were conducted in five patients. For each patient, two physiologically based pharmacokinetic models were fitted to the data and the corrected Akaike information criterion was used to identify the model, which was empirically most supported. The resultant parameter values were compared to values reported in the literature. To individually determine the optimal amount of unlabeled antibody for therapy, computer simulations for preloads ranging from 0 to 60 mg were performed based on the estimated parameters of each patient. The prediction power of the model was assessed by comparing the simulated therapeutic serum curves to the actual 90Y measurements. Visual inspection showed good fits and the adjusted R2 was >0.90 for all patients. All parameters were in a physiologically reasonable range. The relative deviation of the predicted area under the therapeutic serum curve and the measured curve was 15%-33%. The optimal preloading increased the marrow-over-liver selectivity up to 3.9 fold compared to the simulated biodistribution using a standard dose (0.5 mg/kg). The presented method can be used to individually determine the optimal preload and the corresponding residence times in radioimmunotherapy with anti-CD45 antibody.

Abstract 5301: Development of CAIX and CAXII targeting imaging probes for non-invasive detection of metastatic breast cancer in axillary lymph nodes

Abstract Axillary lymph node (ALN) status is the most important prognostic factor for staging breast cancer. ALNs are routinely excised for evaluation which is an invasive procedure associated with serious morbidities. The current study aims to develop a targeting platform for the non-invasive detection of metastatic breast cancer in lymph nodes. If successful, such an approach would decrease unnecessary surgery and significantly reduce associated morbidity. Two cell surface carbonic anhydrase isozymes (CAIX and/or CAXII) were identified by comparison of the gene expression profiles of lesions from breast cancer patients and benign breast tissue and confirmed with public databases and literature. Protein expression was validated by immunohistochemistry of patient tissue samples in tissue microarray. Together, these two markers detect 100% of the metastatic breast cancer to lymph nodes surveyed (n = 37). Targeted probes were developed by conjugation of CAIX and CAXII specific monoclonal antibodies (mAbs) to a near-infrared fluorescent dye (VivoTag 680, VisEn). The selectivity of the probes were determined by comparing their binding to expressing and non-expressing breast cancer cells in vitro and by intravenous injection of the probes into nude mice bearing mammary fat pad (MFP) xenograft tumors of marker expressing cells, and non-expressing cells or by pre-injection of unlabeled antibody. To determine whether the probes can be delivered through the lymphatics and detect ALN metastases, luciferase expressing MDA-mb-231 cells that endogenously express CAIX, and luciferase expressing MDA-mb-231 engineered cells to express CAXII were used to form spontaneous ALN metastases from primary mammary fat pad tumors. Once ALN metastasis was observed by bioluminescence imaging, the probes were injected pri-tumorally into the MFP and observed to traverse through the MFP into the lymph node. A specific and durable fluorescence signal was observed in target positive metastases out to at least 48 h post-injection, long after completely clearing from the MFP. Sensitivity was determined by implanting varying numbers of CAIX and CAXII expressing cells into the ALN using ultrasound guidance. As few as 1000 cells were successfully detected indicating the high sensitivity of this method. Hence, these imaging probes have potential use for clinical staging of breast cancer. In the future, this approach may be used to deliver nanoparticles for combined imaging and therapeutic applications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5301. doi:10.1158/1538-7445.AM2011-5301

Comparative analysis of fluorescence resonance energy transfer (FRET) and proximity ligation assay (PLA)

Both fluorescence resonance energy transfer (FRET) and proximity ligation assay (PLA) are techniques used in the investigation of protein interactions but the latter has not been evaluated in a systematic way, prompting us to compare their performance quantitatively. Proteins were labeled with oligonucleotide- or fluorophore-conjugated antibodies and their proximity was analyzed by flow cytometry in order to obtain statistically robust data. Both intermolecular and intramolecular PLA signals reached saturation at high expression levels. At the same time, the FRET efficiency was independent of, while the FRET signal exhibited a strict linear correlation with the expression levels of proteins. When the density of oligonucleotide- and fluorophore-conjugated antibodies was systematically changed by competition with unlabeled antibodies the FRET signal was linearly proportional to the amount of bound fluorophore-tagged antibodies, whereas the PLA signal was again saturated. The saturation phenomenon in PLA could not be eliminated by decreasing the duration of the rolling circle amplification reaction. Our data imply that PLA is a semiquantitative measure of protein colocalizations due to non-linear effects in the reaction and that caution should be exercised when interpreting PLA data in a quantitative way.

Fractal-like Silver Aggregates Enhance the Brightness and Stability of Single-Molecule Fluorescence

This paper describes a method to significantly enhance single-molecule fluorescence detection in confocal microscopy, demonstrating that fractal-like silver structures significantly improve dye stability and brightness. The experiments compared two immunoassay models based on the deposition of rabbit IgG on silver structures. The experiments were performed with the fluorophore-labeled protein at low picomolar concentrations. Well-separated bright spots were still easily distinguishable. Under standardized conditions we observed increased photostability and brightness enhancement for the dyes that were immobilized on the surface using a primary antibody. In contrast, when the unlabeled primary antibody was immobilized on the surface and the labeled secondary antibody was placed at a larger distance, we observed only a modest enhancement of fluorescence. Furthermore, based on backscattered reflected light images, it was proven that the observed fluorescence enhancements originate from the areas with deposited silver nanostructures. Fractal-like substrates are relatively easy to prepare. We believe that with their superior performance, they should find wide applications in single-molecule studies in which a longer observation time is required.

211At-AntiCD33 in NMRI nu/nu mice

SummaryThe aim of this study is to verify the in vivo stability, to determine the biodistribution and to estimate the unspecific radiotoxicity of an 211At-labelled CD33-antibody (211At-anti-CD33) in mice with a view to therapeutic application in treating leukaemia. Animals, methods: 211At was produced via the 209Bi(α,2n)211At reaction and was linked via 3-211At-succinimidyl-benzoate to the anti-CD33-antibody. The biodistribution and the in vivo stability in serum were determined after i.v.-injection in NMRI nu/nu-mice. For toxicity experiments, mice received either three times 315–650 kBq 211At-antiCD33 or unlabelled antibody and NaCl-solution respectively. Results: 211At-antiCD33 showed a characteristic biodistribution complying with the unspecific antibody retention in the reticular endothelial system. The largest proportion of radioactivity remained in blood and blood-rich tissues with a minor accumulation in the thyroid and stomach. After 21 h, > 85% of activity in serum still represented intact antibody. Mice showed no difference in unspecific toxicity of 211At-labelled antibodies over six months compared to those treated with unlabelled antibody and NaCl-solution respectively, with regard to histopathologic lesions, survival time, behaviour and haemograms. Conclusion: The radiolabelling method yielded adequate in vivo stability of 211At-antiCD33. Biodistribution with rapid elimination of free 211At via kidneys and urine complies with requirements for targeted therapy. Activity doses potentially required for treatment do not elicit radiotoxicity to normal organs in mice. Further development is required to enhance the apparent specific activity and to verify the efficacy in an adequate animal model before phase I clinical studies in leukaemia can be envisaged.

Radioimmunotherapy of solid tumors: searching for the right target.

Radioimmunotherapy of solid tumors remains a challenge despite the tremendous success of ⁹⁰Y ibritumomab tiuxetan (Zevalin) and ¹³¹I Tositumomab (Bexxar) in treating non-Hodgkin's lymphoma. For a variety of reasons, clinical trials of radiolabeled antibodies against solid tumors have not led to responses equivalent to those seen against lymphoma. In contrast, promising responses have been observed with unlabeled antibodies that target solid tumor receptors associated with cellular signaling pathways. These observations suggest that anti-tumor efficacy of the carrier antibody might be critical to achieving clinical responses. Here, we review and compare tumor antigens targeted by radiolabeled antibodies and unlabeled antibodies used in immunotherapy. The review shows that the trend for radiolabeled antibodies under pre-clinical development is to also target antigens associated with signaling pathways that are essential for the growth and survival of the tumor.

Immunohistochemical expression of EGP40, a tumor marker, in different grades of oral squamous cell carcinoma

Aim:The aim of this study was to see the distribution and pattern of staining of epithelial glycoprotein 40 EGP-40 (also known as GA733-2, ESA, KSA, 17-1A antigen) in the different grades of OSCC.Materials and Methods:30 biopsy reports retrieved from the files of the Department of Oral Pathology and Microbiology, College of Dental Surgery, Manipal were used. These comprised of 10 microslides each of 3 different histological grades of Oral Squamous Cell Carcinoma, namely Well Differentiated, Moderately Differentiated and Poorly Differentiated carcinomas. Immunoperoxidase staining for IgG, was performed by the unlabelled antibody peroxidase-antiperoxidase complex (PAP) method. The criteria used to define an antigen positive area were: Homogenous /Patchy staining of the section and Cytoplasmic/ Membranous staining of the tumor cells.Results:The expression of EGP 40 in different grades of OSCC showed an inverse relationship to differentiation and a direct relationship with the proliferation of the tumor cells and its expression became more pronounced as the grade worsened i.e. from well to poorly differentiated OSCC.Conclusion:In the present study, the surface antigen EGP40 (Ep-CAM) was detected in the different grades of OSCC with its expression becoming more pronounced as the grade worsened i.e. from well to poorly differentiated OSCC. However, further studies are required to understand the dualistic role of EGP40 (Ep-CAM) in mediating cell to cell adhesion preventing cell scattering and its heterogeneous expression in promoting tumor invasion and metastasis and also to determine its exact role and significance at a practically applicable level.

Development of 99mTcHYNIC-Tocilizumab as Imaging Agent In Multiple Myeloma

AbstractAbstract 4081 Introduction:Multiple myeloma (MM) accounts for 10% of hematological malignancies and 1% of cancer. It represents the second most common hematologic malignancy. The disease is characterised by plasma cell infiltration of the bone marrow, osteolytic bone lesions and the presence of a monoclonal paraprotein in the serum or urine. Interleukin-6 (IL-6) is a key molecule in the pathogenesis of MM. Its secretion occurs from the tumoral plasma cell as well as from the microenvironment. This results in myeloma cell proliferation. Tocilizumab, a humanized anti IL-6 receptor monoclonal antibody that blocks IL-6 cell-to-cell signaling, is currently being studied in MM. Most MM patients experience bone lesions. In spite of the new imaging techniques, the distinction between malignant and benign lesions remains sometimes difficult in clinical practice. The development of a more specific imaging agent may improve the accuracy of this diagnosis. Objective:Development of 99mTcHYNIC-Tocilizumab conjugate. Chemical and biological evaluation as a potential imaging agent in multiple myeloma. Methodology:10 mg Tocilizumab (Roche) were added to 20 mg HYNIC in NaHCO3 1M, incubated for 30 min at room temperature and purified by means of PD10 column. 1 ml fractions were measured at 280nm. Conjugation of HYNIC-Tocilizumab was evaluated by mass spectrometry MALDITOF and acrylamide gel electrophoresis. 20 uL Tricine/stannous chloride in a 1:1 mass relation were added to 1 mg HYNIC-Tocilizumab in 100 uL of 0.9% NaCl. 2 mL of a solution containing 370 MBq 99mTcO4- was added. Radiochemical purity was controlled by three chromatographic systems: ITLC/NaCl 0.9%, Whatman 1MM/MEK, ITLC in BSA/EtOH-NH3-H2O (2:1:5) as stationary and mobile phase, respectively. Binding to IL-6 receptor in U266 myeloma cells was evaluated in vitro at 15, 30, 60 and 120 min.Tocilizumab was derivatized with FTIC and purified by PD10 column. Laser scanning confocal microscopy was done with an excitation/emission wavelength of 488/530 nm. Fluorescent images were obtained. Biodistribution studies were performed at 24 h in CD1 normal mice (n=3). Each mice was injected with 37 MBq of 99mTcHYNIC-Tocilizumab and sacrificed 24 hs after injection. Organs of interest were collected. Results:Mass spectroscopy MALDITOF (MH+Tocilizumab 147626 Da, M+HYNIC-Tocilizumab 148375 Da) confirmed derivatization of the antibody. This represents 5 molecules of HYNIC per molecule of the antibody. Poliacrylamide gel electrophoresis confirmed that the antibody retained its integrity after derivatization. Radiochemical purity was 88±3 %. In vitro cell binding assays confirmed the binding ability of the radiolabeled antibody to IL-6 receptor. Specificity of binding was supported by competition experiments using unlabelled antibody. Confocal microscopy proved the ability of the fluorescent antibody to recognize the IL-6 receptor in U266 myeloma cells. Biodistribution at 24 h showed blood (8.0±2.0) %act/g, liver (11±1.5) %act/g, kidney (4.0±1.0)%act/g and spleen (6±1.4) %act/g uptake with hepatic and renal elimination. Animal models of MM are being evaluated using xenografted MM cells. Conclusion:99mTcHYNIC-Tocilizumab labeling was performed. The labeled antibody retained adequate biochemical and biological properties, which included maintenance of specific binding to IL-6 receptor. These findings indicate that this radiolabeled antibody could be used as an imaging agent in multiple myeloma. Acknowledgments:OIEA, Laboratorios Roche, Dr Gustavo Arroyo. Disclosures:No relevant conflicts of interest to declare.

Immunocapture enzyme-linked immunosorbent assay for assessment of in vitro potency of recombinant hepatitis B vaccines.

Quantification of hepatitis B surface antigen (HBsAg) or relative in vitro potency in the final vaccines is a prerequisite for hepatitis B vaccine batch release. The commercial kit for automated analysis (AxSYM) is expensive, and an alternative is required for the estimation of HBsAg in hepatitis B vaccines. Mouse monoclonal antibodies (MAbs) specific for HBsAg were developed and characterized. One of the monoclonal antibodies (HBs06) was used in development of an immunocapture ELISA (IC-ELISA) as an unlabeled capture antibody and biotin-labeled detection antibody. The IC-ELISA was standardized and validated using experimental hepatitis B vaccine batches with various HBsAg concentrations per dose and commercial vaccines. The vaccine was treated with an alkaline solubilizer to desorb the HBsAg from Algel-adjuvanted vaccines before testing, and the sensitivity of the test was 5 ng/ml. A good correlation could be observed between the HBsAg estimates derived by both formats, except for the higher HBsAg concentration range, where the IC-ELISA format could estimate closer to the actual values than AxSYM. There was a significant correlation between the estimated relative potencies of the two methods. There was lack of correlation between the in vivo potency and the relative in vitro potency. However, the estimates of IC-ELISA were comparable to the in vivo values when compared with the estimates of AxSYM. The IC-ELISA can therefore be considered to be a reliable test for deriving in vitro relative potency and antigen concentration in vaccine batches for batch control and release.

Potential of Optimal Preloading in Anti-CD20 Antibody Radioimmunotherapy: An Investigation Based on Pharmacokinetic Modeling

Recently, it has been suggested that the concept of preloading is limited by using a standard amount of unlabeled antibody. To identify the potential of optimal preloading, a pharmacokinetic model that describes the biodistribution of anti-CD20 antibody was developed. Simulations were conducted for different tumor burdens, spleen sizes, and tumor permeabilities. The optimal amount of unlabeled antibody was determined for each scenario. These simulations show that the currently administered standard amount is not optimal. A preload of 150 mg or lower would result in equal or higher tumor uptake in all cases. For tumors with high permeability, the uptake of labeled antibody could be increased by a factor of 8.5 using the considerably reduced optimal preload. The most sensitive parameter for the choice of the optimal amount of unlabeled antibody is the tumor uptake index. The results indicate that a personalized approach for radioimmunotherapy (RIT) with anti-CD20 antibody is required to account for the interpatient variability. The optimal amount of unlabeled antibody, which has to be determined by using a pharmacokinetic model, could substantially improve tumor uptake and thus RIT with anti-CD20 antibody.

Abstract 5344: The contribution of unlabeled antibodies to intracellular antigens to efficacy of radioimmunotherapy in experimental metastatic melanoma and cervical cancer

Abstract Introduction: Radioimmunotherapy (RIT) targeting intracellular antigens released in rapidly growing tumors as a result of a cellular turnover is an attractive modality for treatment of aggressive solid tumors. We are developing RIT for metastatic melanoma and HPV-related cervical cancer by targeting the intracellular antigens melanin and E6 oncoprotein, respectively, with radiolabeled specific antibodies (mAbs). In prior studies we observed that administration of unlabeled mAbs to melanin and E6 alone to tumor-bearing mice caused retardation of tumor growth. Objective: To investigate the immunological mechanisms by which unlabeled mAbs to intracellular antigens melanin and E6 oncoprotein contribute to the efficacy of RIT of melanoma and cervical cancer. Methods: The CasKi human cervical tumors and A2058 human metastatic melanoma tumors were induced in female nude mice and when the tumors diameter reached 0.5-0.7 cm, the mice with cervical tumors were treated with C1P5 mAb (IgG1 isotype) to E6 or isotype-matching control mAbs, or left untreated; and mice with melanoma tumors were given 6D2 mAb (IgM isotype) to melanin or isotype matching control mAbs; or left untreated. The tumor growth was monitored for 30 days. On day 5 and 10 post-treatment 4 mice out of each group were sacrificed and their tumors analyzed for C3 complement by Western blot and immunohistochemistry (IHC). On day 10 the tumors were analyzed by flow cytometry for the infiltration by inflammatory cells. Results: There was significant retardation of melanoma and cervical tumor growth during the observation period by 6D2 mAb to melanin and C1P5 mAb to E6 compared with untreated controls (P<0.05). For both types of tumors the isotype-matching control mAbs had the same in magnitude effect on the tumor growth as specific mAbs (P>0.05). On days 5 and 10 C3 complement deposition was observed by Western blot and IHC in specific and non-specific mAbs-treated tumors but not in the untreated controls. The expression of C3 was more prominent on day 10. On day 10 there was infiltration of mAb-treated melanoma and cervical tumors by CD45+, CD4+ and CD8+ inflammatory cells with their levels being 30-100% greater than in untreated tumors. Conclusions: Our results of the treatment of melanoma and cervical tumors with unlabeled mAbs to intracellular antigens suggest that these mAbs can initiate complement dependent toxicity (CDC) and antibody dependent cellular toxicity (ADCC). The effect of mAbs did not depend on a mAb specificity for a particular antigen. It appeared to be dependent on the affinity of the Fc portion of mAbs for C1q molecule (CDC case) or for the Fc receptors on inflammatory cells (ADCC case). Further investigation of the contribution of unlabeled mAbs to efficacy of RIT will help to design effective treatment regiments for patients with metastatic melanoma and cervical cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5344.

Syntaxin-4 Defines a Domain for Activity-Dependent Exocytosis in Dendritic Spines

Changes in postsynaptic membrane composition underlie many forms of learning-related synaptic plasticity in the brain. At excitatory glutamatergic synapses, fusion of intracellular vesicles at or near the postsynaptic plasma membrane is critical for dendritic spine morphology, retrograde synaptic signaling, and long-term synaptic plasticity. Whereas the molecular machinery for exocytosis in presynaptic terminals has been defined in detail, little is known about the location, kinetics, regulation, or molecules involved in postsynaptic exocytosis. Here, we show that an exocytic domain adjacent to the postsynaptic density (PSD) enables fusion of large, AMPA receptor-containing recycling compartments during elevated synaptic activity. Exocytosis occurs at microdomains enriched in the plasma membrane t-SNARE syntaxin 4 (Stx4), and disruption of Stx4 impairs both spine exocytosis and long-term potentiation (LTP) at hippocampal synapses. Thus, Stx4 defines an exocytic zone that directs membrane fusion for postsynaptic plasticity, revealing a novel specialization for local membrane traffic in dendritic spines.

Down-regulation of PROS1 Gene Expression by 17β-Estradiol via Estrogen Receptor α (ERα)-Sp1 Interaction Recruiting Receptor-interacting Protein 140 and the Corepressor-HDAC3 Complex

Pregnant women show a low level of protein S (PS) in plasma, which is known to be a risk for deep venous thrombosis. 17Beta-estradiol (E(2)), an estrogen that increases in concentration in the late stages of pregnancy, regulates the expression of various genes via the estrogen receptor (ER). Here, we investigated the molecular mechanisms behind the reduction in PS levels caused by E(2) in HepG2-ERalpha cells, which stably express ERalpha, and also the genomic ER signaling pathway, which modulates the ligand-dependent repression of the PSalpha gene (PROS1). We observed that E(2) repressed the production of mRNA and antigen of PS. A luciferase reporter assay revealed that E(2) down-regulated PROS1 promoter activity and that this E(2)-dependent repression disappeared upon the deletion or mutation of two adjacent GC-rich motifs in the promoter. An electrophoretic mobility shift assay and DNA pulldown assay revealed that the GC-rich motifs were associated with Sp1, Sp3, and ERalpha. In a chromatin immunoprecipitation assay, we found ERalpha-Sp protein-promoter interaction involved in the E(2)-dependent repression of PROS1 transcription. Furthermore, we demonstrated that E(2) treatment recruited RIP140 and the NCoR-SMRT-HDAC3 complex to the PROS1 promoter, which hypoacetylated chromatin. Taken together, this suggested that E(2) might repress PROS1 transcription depending upon ERalpha-Sp1 recruiting transcriptional repressors in HepG2-ERalpha cells and, consequently, that high levels of E(2) leading to reduced levels of plasma PS would be a risk for deep venous thrombosis in pregnant women.

Fluorescent-labeled antibodies: Balancing functionality and degree of labeling

A critical assumption in using labeled antibodies is that the conjugation reaction has no deleterious effects on antibody avidity. This study demonstrates that this assumption need not hold true and presents a methodology to quantitatively determine the degree of inactivation and/or changes in antibody–antigen binding that can occur with conjugation. Fluorescein isothiocyanate (FITC) was conjugated to a mouse monoclonal antibody, Fc125, against hemagglutinin (HA) using varying fluorophore/protein (F:P) labeling ratios. Antibody binding, as a function of the F:P labeling ratio, was evaluated using a kinetic enzyme-linked immunosorbent assay (ELISA) and analyzed using global fitting. A two-parameter adjustment of the antibody concentration and the maximum rate was sufficient to describe the rate changes. The concentration parameter dominated the rate changes, consistent with the hypothesis that the coupling reaction inactivated an increasing fraction of the antibody population with a smaller change (∼15% at the highest F:P ratio) in antibody–antigen binding. An optimal F:P ratio that minimized both inactivation and unlabeled antibody was calculated. This procedure can be used to prepare functional, labeled antibody reagents with defined activity and can aid in quantitative applications where the stoichiometry and functionality of the labeled antibody are critical.

Methodology to Incorporate Biologically Effective Dose and Equivalent Uniform Dose in Patient-Specific 3-Dimensional Dosimetry for Non-Hodgkin Lymphoma Patients Targeted with 131I-Tositumomab Therapy

A 3-dimensional (3D) imaging-based patient-specific dosimetry methodology incorporating antitumor biologic effects using biologically effective dose (BED) and equivalent uniform dose (EUD) was developed in this study. The methodology was applied to the dosimetry analysis of 6 non-Hodgkin lymphoma patients with a total of 10 tumors. Six registered SPECT/CT scans were obtained for each patient treated with (131)I-labeled antibody. Three scans were obtained after tracer administration and 3 after therapy administration. The SPECT/CT scans were used to generate 3D images of cumulated activity. The cumulated activity images and corresponding CT scans were used as input to Monte Carlo dose-rate calculations. The dose-rate distributions were integrated over time to obtain 3D absorbed dose distributions. The time-dependent 3D cumulative dose distributions were used to generate 3D BED distributions. Techniques to incorporate the effect of unlabeled antibody (cold protein) in the BED analysis were explored. Finally, BED distributions were used to estimate an EUD for each tumor volume. Model parameters were determined from optimal fits to tumor regression data. The efficiency of dose delivery to tumors--the ratio of EUD to cumulative dose--was extracted for each tumor and correlated with patient response parameters. The model developed in this study was validated for dosimetry of non-Hodgkin lymphoma patients treated with (131)I-labeled antibody. Correlations between therapy efficiency generated from the model and tumor response were observed using averaged model parameters. Model parameter determination favored a threshold for the cold effect and typical magnitude for tumor radiosensitivity parameters. The inclusion of radiobiologic effects in the dosimetry modeling of internal emitter therapy provides a powerful platform to investigate correlations of patient outcome with planned therapy.

The influence of proteasome inhibitor MG132, external radiation, and unlabeled antibody on the tumor uptake and biodistribution of (188)re-labeled anti-E6 C1P5 antibody in cervical cancer in mice.

Human papillomavirus (HPV) infection is considered a necessary step for the development of cervical cancer, and >95% of all cervical cancers have detectable HPV sequences. The authors of this report recently demonstrated the efficacy of radioimmunotherapy (RIT) targeting viral oncoprotein E6 in the treatment of experimental cervical cancer. They hypothesized that the pretreatment of tumor cells with various agents that cause cell death and/or elevation of E6 levels would increase the accumulation of radiolabeled antibodies to E6 in cervical tumors. HPV type 16 (HPV-16)-positive CasKi cells were treated in vitro with up to 6 grays of external radiation, or with the proteasome inhibitor MG-132, or with unlabeled anti-E6 antibody C1P5; and cell death was assessed. The biodistribution of (188)Re-labeled C1P5 antibody was determined in both control and radiation MG-132-treated CasKi tumor-bearing nude mice. (188)Re-C1P5 antibody demonstrated tumor specificity, very low uptake, and fast clearance from the major organs. The amount of tumor uptake was enhanced by MG-132 but was unaffected by pretreatment with radiation. In addition, in vitro studies demonstrated an unanticipated effect of unlabeled antibody on the amount of cell death, a finding that was suggested by the authors' previous in vivo studies in a CasKi tumor model. The current results indicated that pretreatment of cervical tumors with the proteasome inhibitor MG-132 and with unlabeled antibody to E6 can serve as a means to generate nonviable cancer cells and to elevate the levels of target oncoproteins in the cells for increasing the accumulation of targeted radiolabeled antibodies in tumors. These results favor the further development of RIT for cervical cancers targeting viral antigens.

Improving the efficacy of radioimmunotherapy for non‐Hodgkin lymphomas

Approximately 66,000 Americans develop non-Hodgkin lymphoma (NHL) each year. Although the use of unlabeled antibodies such as rituximab has significantly improved survival when combined with standard chemotherapy regimens, approximately two-thirds of lymphoma patients eventually develop disease recurrence and die of their disease. Novel treatments are urgently needed to cure these patients. One strategy involves the use of radiolabeled immunoconjugates that specifically localize radiation delivery to sites of lymphoma while minimizing toxicity to normal tissues. A growing number of studies support the contention that radiolabeled antibody therapy can improve overall survival of lymphoma patients and lead to durable remissions, with probable cures, in many patients. Various approaches for enhancing the effectiveness of radioimmunoconjugates have been studied, including: use in newly diagnosed lymphoma patients, combination with chemotherapy or other monoclonal antibodies, use with hematopoietic stem cell transplantation, multistep pretargeting strategies to further minimize toxicity, and simultaneous targeting of multiple B-cell antigens. This article summarizes the current knowledge supporting the use of radioimmunotherapy, an underused but effective treatment modality in NHL patients.

In vitro experimental 211At-anti-CD33 antibody therapy of leukaemia cells overcomes cellular resistance seen in vivo against gemtuzumab ozogamicin

Monoclonal anti-CD33 antibodies conjugated with toxic calicheamicin derivative (gemtuzumab ozogamicin, GO) are a novel therapy option for acute myeloid leukaemia (AML). Key prognostic factors for patients with AML are high CD33 expression on the leukaemic cells and the ability to overcome mechanisms of resistance to cytotoxic chemotherapies, including drug efflux or other mechanisms decreasing apoptosis. Alpha particle-emitting radionuclides overwhelm such anti-apoptotic mechanisms by producing numerous DNA double-stranded breaks (DSBs) accompanied by decreased DNA repair. We labelled anti-CD33 antibodies with the alpha-emitter (211)At and compared survival of leukaemic HL-60 and K-562 cells treated with the (211)At-labelled antibodies, GO or unlabelled antibodies as controls. We also measured caspase-3/7 activity, DNA fragmentation and necrosis in HL-60 cells after treatment with the different antibodies or with free (211)At. The mean labelling ratio of (211)At-labelled antibodies was 1:1,090 +/- 364 (range: 1:738-1:1,722) in comparison to 2-3:1 for GO. Tumour cell binding of (211)At-anti-CD33 was high in the presence of abundant CD33 expression and could be specifically blocked by unlabelled anti-CD33. (211)At-anti-CD33 decreased survival significantly more than did GO at comparable dilution (1:1,000). No significant differences in induction of apoptosis or necrosis or DNA DSB or in decreased survival were observed after (211)At-anti-CD33 (1:1,090) versus GO (1:1) treatment. Our results suggest that (211)At is a promising, highly cytotoxic radioimmunotherapy in CD33-positive leukaemia and kills tumour cells more efficiently than does calicheamicin-conjugated antibody. Labelling techniques leading to higher chemical yield and specific activities must be developed to increase (211)At-anti-CD33 therapeutic effects.