Sensitive methods for evaluation of antibodies for host cell protein analysis and screening of impurities in a vaccine process.

BACKGROUND: Host cell proteins (HCP) should be carefully monitored in vaccine production. To achieve a reliable HCP estimation, a mixture of polyclonal antibodies (pAbs) with broad affinity would be of preference. Sensitive evaluations of the pAbs are therefore of value. METHODS: Column purification of specific HCPs with affinity to the anti-HCP pAbs was compared with Western blotting of the anti-HCP pAbs binding to filter bound total lysate. The anti-HCP pAbs were used in an HCP quantification analysis using surface plasmon resonance (SPR). Host cell derived impurities from an influenza vaccine process were analyzed using 2-D DIGE analysis. RESULTS: The Western blotting showed a similar HCP binding pattern of anti-HCP pAbs from immunizations using two adjuvants: CFA/IFA and AbISCO(®). From the column purification of HCPs, total proteins detectable were similar for all anti-HCP pAbs; however the immune response pattern differed significantly for the anti-HCP pAbs from the AbISCO(®) immunization. In the SPR HCP quantification assay the standard curve ranged from 0.3 to 40 µg/ml. The advantage of SPR compared with ELISA was the decreased hands on time and that the sample number was not limiting. The 2-D DIGE showed that most of the HCPs were removed at the clarification and virus capture step. DISCUSSION: Column purification of HCPs with affinity to the anti-HCP pAbs increased the sensitivity of affinity analysis compared with Western blotting and opened the possibility of further analysis. The anti-HCP pAbs did not interact with proteins in the virus; simplifying analysis of process samples using SPR. 2-D DIGE analysis gave a direct study of the impurity profile with the advantage of independence from antibody performance.

Enhancing the effect of radionuclide tumor targeting, using lysosomotropic weak bases.

PURPOSE: The aim of the present study was to investigate if treatment with lysosomotropic weak bases could increase the intracellular retention of radiohalogens and thereby increase the therapeutic effect of radionuclide tumor targeting. METHODS AND MATERIALS: Four different lysosomotropic bases, chloroquine, ammonium chloride, amantadine, and thioridazine, were investigated for their ability to increase radiohalogen retention in vitro. The two most promising substances, chloroquine and ammonium chloride, were studied in several cell lines (A431, U343MGaCl2:6, SKOV-3, and SKBR-3) in combination with radiolabeled epidermal growth factor (EGF) or the HER2 binding affibody (Z(HER2:4))(2). RESULTS: The uptake and retention of radionuclides was found to be substantially increased by simultaneous treatment with the lysosomotropic bases. The effect was, however, more pronounced in the epidermal growth factor:epidermal growth factor receptor (EGF:EGFR) system than in the (Z(HER2:4))(2):HER2 system. The therapeutic effect of ammonium chloride treatment combined with (211)At-EGF was also studied. The effect obtained after combined treatment was found to be much better than after (211)At-EGF treatment alone. CONCLUSIONS: The encouraging results from the present study indicate that the use of lysosomotropic weak bases is a promising approach for increasing the therapeutic effect of radionuclide targeting with radiohalogens.

Preparation and evaluation of (68)Ga-DOTA-hEGF for visualization of EGFR expression in malignant tumors.

UNLABELLED: Detection of epidermal growth factor receptor (EGFR) overexpression in many carcinomas provides important diagnostic information, which can influence patient management. The use of PET may enable such detection in vivo by a noninvasive procedure with high sensitivity. The aim of this study was to develop a method for preparation of a positron-emitting tracer based on a natural ligand to EGFR, the recombinant human epidermal growth factor (hEGF), and to perform a preclinical evaluation of the tracer. METHODS: DOTA-hEGF (DOTA is 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid) was prepared by coupling of a N-sulfosuccinimide ester of DOTA to hEGF. The conjugate was labeled with a generator-produced positron-emitting nuclide, (68)Ga (half-life = 68 min), using microwave heating. Binding specificity, affinity, internalization, and retention of (68)Ga-DOTA-hEGF was studied in 2 EGFR-expressing cell lines, U343 glioma cells and A431 cervical carcinoma cells. Biodistribution and microPET visualization studies were performed in BALB/c nu/nu mice bearing A431 carcinoma xenografts. RESULTS: A 1-min-long microwave-assisted labeling provided radioactivity incorporation of 77% +/- 4%. Both cell lines demonstrated receptor-specific uptake of the conjugate, rapid internalization of the tracer, and good retention of radioactivity. Binding to both cell lines occurred with high affinity, approximately 2 nmol/L. The biodistribution study demonstrated accumulation of radioactivity in xenografts and in EGFR-expressing organs. The microPET imaging study enabled visualization of tumors and demonstrated quick--within 5 min--localization of radioactivity in tumors. CONCLUSION: (68)Ga-DOTA-hEGF has potential for imaging EGFR overexpression in tumors.

Cellular uptake of radioiodine delivered by trastuzumab can be modified by the addition of epidermal growth factor.

PURPOSE: The purpose of this study was to analyse whether non-radiolabelled epidermal growth factor (EGF) can modify the cellular uptake of 125I when delivered as [125I]trastuzumab. 125I was used as a marker for the diagnostically and therapeutically more interesting isotopes 123I (SPECT), 124I (PET) and 131I (therapy). METHODS: The cell-associated radioactivity was measured in squamous carcinoma A431 cells following addition of [125I]trastuzumab. Different concentrations of [125I]trastuzumab and unlabelled EGF were used, and the total, membrane-bound and internalised radioactivity was measured. We also analysed how EGF and trastuzumab affected the cell growth. RESULTS: It was generally found that the cellular 125I uptake was decreased by the addition of EGF when [125I]trastuzumab was added for short incubation times. However, if the incubation times were longer, EGF increased the 125I uptake. This shift came earlier when higher [125I]trastuzumab concentrations were applied. The addition of EGF also influenced cell proliferation, and concentrations above 10 ng/ml reduced cell growth by approximately 20% after 24 h of incubation. CONCLUSION: By adding unlabelled EGF, it was possible to modify the cellular uptake of [125I]trastuzumab. This points towards new approaches for the modification of radionuclide uptake in EGFR- and HER2-positive tumours.

[177Lu]Bz-DTPA-EGF: Preclinical characterization of a potential radionuclide targeting agent against glioma.

Patients with glioblastoma multiforme have a poor prognosis due to recurrences originating from spread cells. The use of radionuclide targeting might increase the chance of inactivating single tumor cells with minimal damage to surrounding healthy tissue. As a target, overexpressed epidermal growth factor receptors (EGFR) may be used. A natural ligand to EGFR, the epidermal growth factor (EGF) is an attractive targeting agent due to its low molecular weight (6 kDa) and high affinity for EGFR. 177Lu (T(1/2) = 6.7 days) is a radionuclide well suited for treatment of small tumor cell clusters, since it emits relatively low-energy beta particles. The goal of this study was to prepare and preclinically evaluate both in vitro and in vivo the [177Lu]Bz-DTPA-EGF conjugate. The conjugate was characterized in vitro for its cell-binding properties, and in vivo for its pharmacokinetics and ability to target EGFR. [177Lu]Bz-DTPA-EGF bound to cultured U343 glioblastoma cells with an affinity of 1.9 nM. Interaction with EGFR led to rapid internalization, and more than 70% of the cell-associated radioactivity was internalized after 30 minutes of incubation. The retention of radioactivity was good, with more than 65% of the 177Lu still cell-associated after 2 days. Biodistribution studies of i.v. injected [177Lu]Bz-DTPA-EGF in NMRI mice demonstrated a rapid blood clearance. Most of the radioactivity was found in the liver and kidneys. The liver uptake was receptor-mediated, since it could be significantly reduced by preinjection of unlabeled EGF. In conclusion, [177Lu]Bz-DTPA-EGF seems to be a promising candidate for locoregional treatment of glioblastoma due to its high binding affinity, low molecular weight, and ability to target EGFR in vivo.

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

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

Combined effect of gefitinib ('Iressa', ZD1839) and targeted radiotherapy with 211At-EGF.

The EGFR-TKI (epidermal growth factor receptor tyrosine kinase inhibitor) gefitinib ['Iressa' (trademark of the AstraZeneca group of companies), ZD1839] increases the cellular uptake of radiolabelled epidermal growth factor (EGF). We investigated gefitinib treatment combined with astatine-211 EGF targeting in vitro using two cell lines expressing high levels of EGFR: A431 (sensitive to gefitinib) and U343MGaCl2:1 (resistant to gefitinib). In both cell lines, the uptake of 211At-EGF was markedly increased by concomitant treatment with gefitinib. Survival was investigated using both a clonogenic survival assay and a cell growth assay. Combined gefitinib and 211At-EGF treatment reduced the survival of U343 cells 3.5-fold compared with 211At-EGF alone. In A431 cells, 211At-EGF treatment resulted in very low survival, but combined treatment with gefitinib increased the survival by about 20-fold. These results indicate that combined treatment with gefitinib might increase the effect of ligand-mediated radionuclide therapy in gefitinib-resistant tumours and decrease the effect of such therapy in gefitinib-sensitive tumours.

Cellular retention of radioactivity and increased radiation dose. Model experiments with EGF-dextran.

Targeting of tumor cells with radiolabeled biomolecules is a possible approach to inactivate disseminated tumor cells. However, rapid degradation of the biomolecules after cellular internalization and subsequent excretion of the radioactivity is a problem. We studied the possibility of using dextran as a carrier of radionuclides to improve the intracellular retention. An EGF-dextran conjugate, aimed for targeting of tumor cells overexpressing the EGF-receptor, was used as model. Retention tests were performed with (125)I on different parts: [(125)I]-EGF-dextran-[(125)I], [(125)I]-EGF-dextran and EGF-dextran-[(125)I]. Comparisons were made with [(125)I]-EGF. The radiolabeled compounds were incubated with cultured glioma cells for different times. The cellular retention of radioactivity was then measured for up to 24 h. Expected radiation doses at the cellular level were calculated assuming that (131)I, instead of (125)I, was coupled to EGF and EGF-dextran. The results indicated that the EGF-part of the conjugate was degraded and the EGF-attached radioactivity was rapidly excreted, whereas radioactivity on dextran was retained intracellularly to a high degree, i.e. 70-80% of the radioactivity bound to dextran was still cell-associated after 24 h. The retention after 24 h was significantly higher (p < 0.001) when the radioactivity was on the dextran instead of the EGF-part. The radiolabeled EGF-dextran had a notably high specific radioactivity; up to 11 MBq/microg. There was potential for at least hundred times increased radiation dose per receptor interaction when the radioactivity was on the dextran part. The advantage with radioactivity on the dextran part was the high cellular retention and the high specific radioactivity (higher than previously reported for other residualizing labels) without severe loss of receptor specific binding. Thus, dextran seems suitable as a carrier of radionuclides aimed for therapy and gives potential for a highly increased radiation dose.

Treatment of cultured glioma cells with the EGFR-TKI gefitinib ("Iressa", ZD1839) increases the uptake of astatinated EGF despite the absence of gefitinib-mediated growth inhibition.

The EGFR-TKI (epidermal growth factor receptor tyrosine kinase inhibitor) gefitinib ("Iressa", ZD1839), a reversible growth inhibitor of EGFR-expressing tumour cells, has been shown to enhance the antitumour effect of ionising radiation, and also to increase the uptake of radioiodinated EGF. Thus, combination of gefitinib treatment and radionuclide targeting is an interesting option for therapy of brain tumours that are difficult to treat with conventional methods. The aim of this study was to evaluate how pre-treatment with gefitinib affects binding of astatinated EGF ((211)At-EGF) to cultured glioma U343 cells, which express high levels of EGFR. The growth of U343 cells in the presence of gefitinib was investigated, and it was found that gefitinib does not significantly inhibit the growth of these cells. Nevertheless, the uptake of (211)At-EGF in U343 cells was markedly increased (up to 3.5 times) in cells pre-treated with gefitinib (1 microM). This indicates that a combination of gefitinib treatment and radionuclide targeting to EGFR might be a useful therapeutic modality, even for patients who do not respond to treatment with gefitinib alone.