Translational imaging of the fibroblast activation protein (FAP) using the new ligand [68Ga]Ga-OncoFAP-DOTAGA.

PURPOSE: The fibroblast activation protein (FAP) is an emerging target for molecular imaging and therapy in cancer. OncoFAP is a novel small organic ligand for FAP with very high affinity. In this translational study, we establish [68Ga]Ga-OncoFAP-DOTAGA (68Ga-OncoFAP) radiolabeling, benchmark its properties in preclinical imaging, and evaluate its application in clinical PET scanning. METHODS: 68Ga-OncoFAP was synthesized in a cassette-based fully automated labeling module. Lipophilicity, affinity, and serum stability of 68Ga-OncoFAP were assessed by determining logD7.4, IC50 values, and radiochemical purity. 68Ga-OncoFAP tumor uptake and imaging properties were assessed in preclinical dynamic PET/MRI in murine subcutaneous tumor models. Finally, biodistribution and uptake in a variety of tumor types were analyzed in 12 patients based on individual clinical indications that received 163 ± 50 MBq 68Ga-OncoFAP combined with PET/CT and PET/MRI. RESULTS: 68Ga-OncoFAP radiosynthesis was accomplished with high radiochemical yields. Affinity for FAP, lipophilicity, and stability of 68Ga-OncoFAP measured are ideally suited for PET imaging. PET and gamma counting-based biodistribution demonstrated beneficial tracer kinetics and high uptake in murine FAP-expressing tumor models with high tumor-to-blood ratios of 8.6 ± 5.1 at 1 h and 38.1 ± 33.1 at 3 h p.i. Clinical 68Ga-OncoFAP-PET/CT and PET/MRI demonstrated favorable biodistribution and kinetics with high and reliable uptake in primary cancers (SUVmax 12.3 ± 2.3), lymph nodes (SUVmax 9.7 ± 8.3), and distant metastases (SUVmax up to 20.0). CONCLUSION: Favorable radiochemical properties, rapid clearance from organs and soft tissues, and intense tumor uptake validate 68Ga-OncoFAP as a powerful alternative to currently available FAP tracers.

F8-SIP mediated targeted photodynamic therapy leads to microvascular dysfunction and reduced glioma growth.

The extra domain A (ED A) of fibronectin has been identified as a tumor vessel specific neovascular marker in glioma. Antibody based vascular targeting against ED A of fibronectin allows precise accumulation of photosensitizer in glioma microvasculature and thereby promises to overcome drawbacks of current photodynamic therapy (PDT) for glioma treatment. Our aim was to characterize microcirculatory consequences of F8-small immunoprotein (SIP) mediated PDT by intravital microscopy (IVM) and to analyze the effects on glioma growth. For IVM SF126 glioma cells were implanted into dorsal skinfold-chamber of nude mice. PDT was performed after intravenous injection of photosensitizer (PS)-coupled F8-SIP or PBS (n = 4). IVM was performed before and after PDT for 4 days. Analysis included total and functional (TVD, FVD) vessel densities, perfusion index (PI), microvascular permeability and blood flow rate (Q). To assess tumor growth SF126 glioma cells were implanted subcutaneously. PDT was performed as a single and repetitive treatment after PS-F8-SIP injection (n = 5). Subcutaneous tumors were treated after uncoupled F8-SIP injection as control group (n = 5). PDT induced microvascular stasis and thrombosis with reduced FVD (24 h: 115.98 ± 0.7 vs. 200.8 ± 61.9 cm/cm(2)) and PI (39 ± 11 vs. 70 ± 10 %), whereas TVD was not altered (298 ± 39.2 vs. 278.2 ± 51 cm/cm(2)). Microvascular dysfunction recovered 4 days after treatment. Microvascular dysfunction led to a temporary reduction of glioma growth in the first 48 h after treatment with complete recovery 5 days after treatment. Repetitive PDT resulted in sustained reduction of tumor growth. F8-SIP mediated PDT leads to microvascular dysfunction and reduced glioma growth in a preclinical glioma model with recovery of microcirculation 4 days after treatment. Repetitive application of PDT overcomes microvascular recovery and leads to prolonged antiglioma effects.

Severe community-acquired pneumonia and positive urinary antigen test for S. pneumoniae: amoxicillin is associated with a favourable outcome.

Positive urinary antigen tests (UAT) for pneumococcal infection in community-acquired pneumonia (CAP) may lead to targeted antibiotic therapy. We report an audit aimed at defining the link between mortality and targeted therapy. We conducted a retrospective multicentre audit of patients with severe CAP for whom a UAT was positive for S. pneumoniae. Patients admitted from January 2010 to December 2013 to 8 medical centres (from A to H) were included. Co-morbidities were defined by the specific treatment administered before hospital care, or if the diagnosis was newly established during the hospital stay. We used the Pneumonia Severity Index (PSI) to assess disease severity. Only patients with PSI > 90 were included. Antibiotic treatments and the PSI were extracted from patients' charts. Amoxicillin had to be prescribed as a targeted antibiotic treatment or at the time of antibiotic reassessment. A total of 389 patients were included. The mean (±STD) PSI score was 128 ± 29; 38.9% of the patients had a class 5 PSI score. Intensive care was required for 36.6% of the patients. Amoxicillin was initially prescribed in 47 cases (12.1%) and in 34 cases after reassessment (8.7%). In logistic regression analysis, we found three parameters associated with mortality: being hospitalised in institution D, class 5 PSI score, and metastatic cancer. In contrast, three antibiotic regimens were protective factors, including targeted therapy: OR = 0.09, p < 0.001. In the context of severe CAP with positive UAT for S. pneumoniae, targeted therapy was associated with a reduction in mortality.

Trypan blue/giemsa staining to assess sperm membrane integrity in salernitano stallions and its relationship to pregnancy rates.

Aim of this study was to test the reliability of Trypan blue/Giemsa staining to evaluate sperm membrane integrity, acrosomal intactness and morphology in stallion to verify whether it could be applied in vitro as useful tool for sperm fertilizing ability. Fertility data on inseminated mares were collected to evaluate the relationship of sperm quality to pregnancy rates. Forty-one ejaculates were collected from 3 stallions of Salernitano Horse Breed and evaluated for gross appearance, volume, visual motility and membrane integrity with Trypan blue/Giemsa staining and thirty-five mares were inseminated during the breeding season from April to July. Differences among stallions were found in volume, sperm concentration (p < 0.05) and visual motility (p < 0.01). A decrease in sperm motility, concentration (p < 0.05) and total sperm number was found in June-July (p < 0.01). Live sperm with intact acrosome (LSIA) and proximal droplets (PD) were lower (p < 0.01) in June-July, while acrosome reacted sperm (ARS) percentage increased (p < 0.05). No fertility differences were found among stallions with an average fertility per cycle of 44.6% and a pregnancy rate of 68.6%. Higher percentages of LSIA were found in the ejaculates used to inseminate mares that became pregnant vs those used in mares not pregnant (p < 0.05). The significance of LSIA as test variable to verify the reliability of Trypan blue/Giemsa staining was confirmed by Receiver operating characteristic ROC analysis and the sensitivity of the test was 85% at a cut-off value of 48% LSIA. Trypan blue-Giemsa showed to be an accurate method that can be applied on field to evaluate sperm membrane integrity and to identify poor-quality ejaculates.

The antibody-based targeted delivery of TNF in combination with doxorubicin eradicates sarcomas in mice and confers protective immunity.

BACKGROUND: Soft-tissue sarcomas are a group of malignancies of mesenchymal origin, which typically have a dismal prognosis if they reach the metastatic stage. The observation of rare spontaneous remissions in patients suffering from concomitant bacterial infections had triggered the clinical investigation of the use of heat-killed bacteria as therapeutic agents (Coley's toxin), which induced complete responses in patients in the pre-chemotherapy era and is now known to mediate substantial elevations in serum TNF levels. METHODS: We designed and developed a novel immunocytokine based on murine TNF sequentially fused to the antibody fragment F8 (specific to extra-domain A of fibronectin). The antitumor activity was studied in two syngeneic murine sarcoma models. RESULTS: The L19 antibody (specific to extra-domain B of fibronectin) has shown by SPECT imaging procedures to selectively localise on sarcoma in a patient with a peripheral nerve sheath tumour, and immunohistochemical analysis of human soft-tissue sarcoma samples showed comparable antigen expression of EDA and EDB. The antibody-based pharmacodelivery of TNF by the fusion protein 'F8-TNF' to oncofetal fibronectin in sarcoma-bearing mice leads to complete and long-lasting tumour eradications when administered in combination with doxorubicin, the first-line drug for the treatment of sarcomas in humans. Doxorubicin alone did not display any therapeutic effect in both tested models of this study. The cured mice had acquired protective immunity against the tumour, as they rejected subsequent challenges with sarcoma cells. CONCLUSION: The findings of this study provide a rationale for the clinical study of the fully human immunocytokine L19-TNF in combination with doxorubicin in patients with soft-tissue sarcoma.

A chemically modified antibody mediates complete eradication of tumours by selective disruption of tumour blood vessels.

BACKGROUND: The possibility of eradicating cancer by selective destruction of tumour blood vessels may represent an attractive therapeutic avenue, but most pharmaceutical agents investigated so far did not achieve complete cures and are not completely specific. Antibody conjugates now allow us to evaluate the impact of selective vascular shutdown on tumour viability and to study mechanisms of action. METHODS: We synthesised a novel porphyrin-based photosensitiser suitable for conjugation to antibodies and assessed anticancer properties of its conjugate with L19, a clinical-stage human monoclonal antibody specific to the alternatively spliced EDB domain of fibronectin, a marker of tumour angiogenesis. RESULTS: Here we show in two mouse model of cancer (F9 and A431) that L19 is capable of highly selective in vivo localisation around tumour blood vessels and that its conjugate with a photosensitiser allows selective disruption of tumour vasculature upon irradiation, leading to complete and long-lasting cancer eradication. Furthermore, depletion experiments revealed that natural killer cells are essential for the induction of long-lasting complete responses. CONCLUSIONS: These results reinforce the concept that vascular shutdown can induce a curative avalanche of tumour cell death. Immuno-photodynamic therapy may be particularly indicated for squamous cell carcinoma of the skin, which we show to be strongly positive for markers of angiogenesis.

Combination of temozolomide with immunocytokine F16-IL2 for the treatment of glioblastoma.

BACKGROUND: Glioblastoma patients are still not cured by the treatments available at the moment. We investigated the therapeutic properties of temozolomide in combination with F16-IL2, a clinical-stage immunocytokine consisting of human interleukin (IL)-2 fused to the human antibody F16, specific to the A1 domain of tenascin-C. METHODS: We conducted three preclinical therapy studies, using subcutaneous and intracranial U87MG glioblastoma tumours xenografted in BALB/c nude mice. The same therapeutic schedule was used, consisting of five total administrations every third day, of 0.525 mg temozolomide, 20 microg F16-IL2, the combination, or the control solution. RESULTS: Immunohistochemical analysis of U87MG xenografts and of human glioblastoma specimens showed selective tumour staining of F16. A quantitative biodistribution confirmed the preferential tumour accumulation of radiolabelled F16-IL2. In the study with subcutaneous xenografts, the combination of F16-IL2 with temozolomide induced complete remission of the animals, which remained tumour free for over 160 days. The same treatment led to a consistent size reduction of intracranial xenografts and to a longer survival of animals. The immunocytokine promoted the recruitment of leukocytes into tumours of both models. CONCLUSION: The combined use of temozolomide with F16-IL2 deserves clinical investigations, which will be facilitated by the excellent safety profile in cynomolgus monkeys, and by the fact that F16-IL2 is in clinical trials in patients with cancer.

Extra cellular matrix remodelling after heterotopic rat heart transplantation: gene expression profiling and involvement of ED-A+ fibronectin, alpha-smooth muscle actin and B+ tenascin-C in chronic cardiac allograft rejection.

Chronic cardiac rejection is represented by cardiac allograft vasculopathy (CAV) and cardiac interstitial fibrosis (CIF) known to cause severe complications. These processes are accompanied by remarkable changes in the cardiac extra cellular matrix (cECM). The aim of our study was to analyse the cECM remodelling in chronic rejection and to elucidate a potential role of ED-A domain containing fibronectin (ED-A(+) Fn), alpha smooth muscle actin (ASMA) and B domain containing tenascin-C (B(+) Tn-C). A model of chronic rejection after heterotopic rat heart transplantation was used. Allografts, recipient and control hearts were subjected to histological assessment of rejection grade, to real-time PCR based analysis of 84 genes of ECM and adhesion molecules and to immunofluorescence labelling procedures, including ED-A(+) Fn, ASMA and B(+) Tn-C antibodies. Histological analysis revealed different grades of chronic rejection. By gene expression analysis, a relevant up-regulation of the majority of ECM genes in association with chronic rejection could be shown. For 8 genes, there was a relevant up-regulation in allografts as well as in the corresponding recipient hearts. Association of ASMA positive cells with the grade of chronic rejection could be proven. In CAV and also in CIF there were extensive co-depositions of ED-A(+) Fn, ASMA and B(+) Tn-C. In conclusion, chronic cardiac allograft rejection is associated with a cECM remodelling. ASMA protein deposition in CAV, and CIF is a valuable marker to detect chronic rejection. Interactions of VSMCs and Fibro-/Myofibroblasts with ED-A(+) Fn and B(+) Tn-C might functionally contribute to the development of chronic cardiac rejection.

Human monoclonal antibodies targeting carbonic anhydrase IX for the molecular imaging of hypoxic regions in solid tumours.

BACKGROUND: Hypoxia, which is commonly observed in areas of primary tumours and of metastases, influences response to treatment. However, its characterisation has so far mainly been restricted to the ex vivo analysis of tumour sections using monoclonal antibodies specific to carbonic anhydrase IX (CA IX) or by pimonidazole staining, after the intravenous administration of this 2-nitroimidazole compound in experimental animal models. METHODS: In this study, we describe the generation of high-affinity human monoclonal antibodies (A3 and CC7) specific to human CA IX, using phage technology. RESULTS: These antibodies were able to stain CA IX ex vivo and to target the cognate antigen in vivo. In one of the two animal models of colorectal cancer studied (LS174T), CA IX imaging closely matched pimonidazole staining, with a preferential staining of tumour areas characterised by little vascularity and low perfusion. In contrast, in a second animal model (SW1222), distinct staining patterns were observed for pimonidazole and CA IX targeting. We observed a complementary pattern of tumour regions targeted in vivo by the clinical-stage vascular-targeting antibody L19 and the anti-CA IX antibody A3, indicating that a homogenous pattern of in vivo tumour targeting could be achieved by a combination of the two antibodies. CONCLUSION: The new human anti-CA IX antibodies are expected to be non-immunogenic in patients with cancer and may serve as broadly applicable reagents for the non-invasive imaging of hypoxia and for pharmacodelivery applications.

The impact of hepatitis C and biliary complications on patient and graft survival following liver transplantation.

Recurrent hepatitis C (HCV) and biliary complications (BC) are major causes of post liver transplant morbidity and mortality. The impact of these complications may be additive or synergistic. We performed a retrospective cohort study to analyze the effects of HCV and BC on all patients transplanted at two institutions over 6 years. BC was defined by imaging findings in the setting of abnormal liver function tests that required intervention. The primary outcomes were graft and patient survival over a mean 3.4 years. 709 patients (619 deceased, 90 living donor) were included, 337 with HCV and 372 without. BC was diagnosed more frequently in patients with HCV, 26% versus 18% (p = 0.008). One-year and overall patient and graft survival were significantly lower in patients with HCV, but BC impacted only 1-year graft survival. The combination of BC and HCV had no additional impact on survival or fibrosis rates on 1-year protocol biopsies. Multivariate analysis revealed HCV (HR 2.1) and HCC (HR 1.9) to be independent predictors of mortality. Since BC are diagnosed more frequently in HCV patients and only affect early graft loss, it is likely that recurrent HCV rather than BC accounts for the majority of adverse graft outcomes.

Internalization via Antennapedia protein transduction domain of an scFv antibody toward c-Myc protein.

We constructed a single-chain variable fragment miniantibody (G11-scFv) directed toward the transactivation domain of c-Myc, which is fused with the internalization domain Int of Antennapedia at its carboxyl terminus (a cargo-carrier construct). In ELISA experiments, an EC(50) for binding saturation was achieved at concentrations of G11-scFv-Int(-) of approximately 10(-8) M. Internalization of a fluoresceinated Fl-G11-scFv-Int(+) construct was observed in intact human cultured cells with confocal microscopy. After 5 h of incubation in medium containing 1 microM Fl-G11-scFv-Int(+) or Fl-G11-scFv-Int(-), fluorescence intensity was determined in individual cells, both for cytoplasmic and nuclear compartments: concentration levels of Fl-G11-scFv-Int(+), relative to the extracellular culture medium concentration, were 4-5 times higher in the cytoplasm, 7-8 times higher in the nucleus, and 10 times higher in the nucleoli. In the same experimental conditions, the Fl-G11-scFv-Int(-) construct was 3-4 times more concentrated outside of the cells than inside. Cell membranes kept their integrity after 5 h of incubation. The antiproliferative activity of our miniantibody was studied on HCT116 cells. Incubation with 4 microM G11-scFv-Int(+) for 4 days induced very significant statistical and biological growth inhibition, whereas Int alone was completely inactive. Miniantibodies capable of penetrating cell membranes dramatically broaden the potential for innovative therapeutic agents and attack of new targets.

NMR determination of residual structure in a urea-denatured protein, the 434-repressor.

A nuclear magnetic resonance (NMR) structure determination is reported for the polypeptide chain of a globular protein in strongly denaturing solution. Nuclear Overhauser effect (NOE) measurements with a 7 molar urea solution of the amino-terminal 63-residue domain of the 434-repressor and distance geometry calculations showed that the polypeptide segment 54 to 59 forms a hydrophobic cluster containing the side chains of Val54, Val56, Trp58, and Leu59. This residual structure in the urea-unfolded protein is related to the corresponding region of the native, folded protein by simple rearrangements of the residues 58 to 60. Based on these observations a model for the early phase of refolding of the 434-repressor(1-63) is proposed.

HCV histological recurrence and survival following liver transplantation in patients with and without hepatocellular carcinoma.

BACKGROUND AND AIM: Hepatitis C virus (HCV)-related cirrhosis is one of the leading indication for liver transplantation (LT) and a major risk factor for the development of hepatocellular carcinoma (HCC). HCV recurrence after LT is universal. This study evaluated HCV recurrence and survival in patients transplanted for HCV and HCC. METHODS: We evaluated all adults transplanted for HCV cirrhosis between January 1999 and December 2006, HCC was diagnosed on the explant and HCV recurrence confirmed on protocol liver biopsies performed at 6 months and yearly after LT. The sustained viral response (SVR) was defined as HCV-RNA undetectable at 6 months after therapy discontinuation. The patient survival rates were assessed with Kaplan-Meier curves and the chi-square test was used when appropriate. RESULTS: Two hundred sixteen patients underwent LT for HCV including 153 men and 63 women of mean age 54 years with a mean follow-up of 35 months. There were 71 (33%) HCC(+) patients. At 1, 3, and 5 years from LT severe fibrosis (Scheuer 3-4) due to the HCV recurrence was reported in 18%, 14%, and 11% for HCC(+) and 14%, 16%, and 28% for HCC(-) patients respectively (P=NS). HCC recurred only in 3 (4%) patients at a mean follow-up of 3 years. Patients who received antiviral treatment after LT were 10% HCC(+) and 12% HCC(-) patients (P=NS). SVR was seen in 3/7 (43%) of HCC(+) and in 10/18 (55%) of HCC(-) patients (P=NS). At 1, 3, and 5 years the patient survivals was 91%, 86%, and 86% for HCC(+) and 94%, 86%, and 83% for HCC(-) patients, respectively (P=NS). CONCLUSIONS: Severe fibrosis due to HCV recurrence, which increases over time, involves one third of transplanted patients at 5 years after LT. The long-term survival was identical among HCC(+) compared to HCC(-) recipients. The recurrence of HCC was negligible and did not affect patient survival.

An atlas of bloodstream-accessible bone marrow proteins for site-directed therapy of acute myeloid leukemia.

The concept of arming antibodies with bioactive payloads for a site-specific therapy of cancer has gained considerable interest in recent years. However, a successful antibody-based targeting approach critically relies on the availability of a tumor-associated target that is not only preferentially expressed in the tumor tissue but is also easily accessible for antibody therapeutics coming from the bloodstream. Here, we perfused the vasculature of healthy and acute myeloid leukemia (AML)-bearing rats with a reactive ester derivative of biotin and subsequently quantified the biotinylated proteins to identify AML-associated bone marrow (BM) antigens accessible from the bloodstream. In total, >1400 proteins were identified. Overall, 181 proteins were >100-fold overexpressed in AML as compared with normal BM. Eleven of the most differentially expressed proteins were further validated by immunohistochemistry and confocal microscopic analyses, including novel antigens highly expressed in AML cells (for example, adaptor-related protein complex 3 ß2) and in the leukemia-modified extracellular matrix (ECM) (for example, collagen-VI-α-1). The presented atlas of targetable AML-associated BM proteins provides a valuable basis for the development of monoclonal antibodies that could be used as carriers for a site-specific pharmacodelivery of cytotoxic drugs, cytokines or radionuclides to the BM in AML.

A monoclonal antibody prevents aggregation of the NBD1 domain of the cystic fibrosis transmembrane conductance regulator.

The homozygous deletion of the phenylalanine at position 508 (DeltaPhe508) in the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the most common CF-causing genetic defect. It has been proposed that the propensity of NBD1 to aggregate may lead to a lower display of the CFTR chloride channel to the cell membrane and to the disease, thus opening an avenue for the pharmacological development of CFTR folding correctors. Here, we show that a human monoclonal antibody fragment specific to the folded conformation of NBD1 inhibits the aggregation of NBD1 in vitro. However, in contrast to the previously published observations, we proved experimentally that NBD1 of wild-type and DeltaPhe508 version of CFTR display comparable propensities to aggregate in vitro and that the corresponding full-length CFTR protein reaches the cell membrane with comparable efficiency in mammalian cell expression systems. On the basis of our results, the 'folding defect' hypothesis seems unlikely to represent the causal mechanism for the pathogenesis of CF. A solid understanding of how the DeltaPhe508 deletion leads to the disease represents an absolute requirement for the development of effective drugs against CF.

Nitric oxide generation is associated with an unbalance of protein tyrosine phosphatases during liver transplantation.

Organ dysfunction secondary to ischemia-reperfusion (I/R) injury still represents a major problem in liver transplantation. Apoptosis has been observed in hepatocytes and sinusoidal endothelial cell, following I/R injury and it has been postulated as a contributing factor in ischemia-reperfusion graft dysfunction, involving a complex series of events, as changes of protein tyrosine-kinase phosphorylation. We evaluated hepatic purine metabolites, protein tyrosine phosphatases (PTPs), nitrate plus nitrite levels (NOx), caspase-3 (C-3) activity and DNA fragmentation in the time course of twelve pig orthotopic liver transplantation. Biopsies were taken before explantation (t0), after cold ischemic storage (t1) and 30 min from reperfusion (t2). During the ischemic period we observed a reduction of high energy phosphates and an increase of purine bases; PTP activity was largely increased. At t2 high energy phosphates showed a tendency to increase with respect to t1, with a partial restoration of phosphorylation potential, measured as ATP/ADT ratio. PTP activity was significantly reduced, with a concomitant increase of NOx production and C-3 activity; in a considerable number of cases we observed a sustained DNA fragmentation. We speculate that NOx production could be related to nitrosative stress, which in turn leads to dynamic alteration in PTP balance and cell signalling, regulating the activity of a number of proteins implicated in apoptotic cell death. These findings could be of interest in new potential strategy to prevent and treat I/R injury.

Selective targeting and photocoagulation of ocular angiogenesis mediated by a phage-derived human antibody fragment.

Molecules that selectively target and occlude new blood vessels would be useful for diagnosis and treatment of pathologies associated with angiogenesis. We show that a phage-derived human antibody fragment (L19) with high affinity for the ED-B domain of fibronectin, a marker of angiogenesis, selectively localizes to newly formed blood vessels in a rabbit model of ocular angiogenesis. The L19 antibody, chemically coupled to a photosensitizer and irradiated with red light, mediates complete and selective occlusion of ocular neovasculature and promotes apoptosis of the corresponding endothelial cells. These results demonstrate that new ocular blood vessels can be distinguished immunochemically from preexisting ones and suggest that the targeted delivery of photosensitizers may be effective in treating angiogenesis-related pathologies.

Radioactive labeling of recombinant antibody fragments by phosphorylation using human casein kinase II and [gamma-32P]-ATP.

A wide range of antibody fragments can be expressed in bacteria and detected immunochemically via peptide tags. Using specially designed tags, we have developed a strategy for radiolabeling antibody fragments secreted from bacteria. Tagged antibody fragments were secreted either into the bacterial periplasm or the culture medium. The tag was not subject to proteolysis either in the broth or in human plasma. After affinity purification the antibody fragments were phosphorylated with [gamma-32P]ATP and casein kinase II. The labeled fragments were used in a gel band-shift assay to measure antigen binding affinities. In contrast to non site-specific methods such as radioiodination, antibodies labeled with casein kinase II retain full immunoreactivity. Radioactively phosphorylated antibody fragments may have many other applications, including radioimmunoassays and radioimmunotherapy.