An enzyme-linked immunosorbent assay (ELISA) was developed to detect different hantavirus antigens in cell culture; i.e. Puumala (PUU), Hantaan (HTN), and Dobrava (DOB) viruses. The assay was based on binding human serum immunoglobulin M (IgM) antibodies to the solid phase by use of goat anti-IgM antibodies. The captured IgM antibodies were present in the acute phase serum from two patients: one infected in Sweden and the other in Bosnia. Antigens being bound to the solid phase by the human anti-PUU and anti-DOB/HTN IgM antibodies were detected by a broadly reacting polyclonal rabbit anti PUU-recombinant nucleocapsid protein antiserum. The IgM isotype was proven to be at least five times more efficient than IgG when used as the capturing antibody. The sensitivity of the PUU antigen ELISA was approximately 0.5 ng/ml, as measured by titration with a PUU recombinant nucleoprotein antigen. Cell-associated PUU antigen in tissue culture was seen after 48 hr by the PUU-ELISA and after 96 hr by immunofluorescent assay. When tested for capacity to discriminate between PUU, DOB, and HTN viruses, significant differences were found: the Swedish serum detected PUU antigen at high titers, whereas no reactivity was found against DOB and HTN; the Bosnian serum detected both DOB and HTN at high titers but had a low reactivity to PUU. The method was also tested for its usefulness in detecting PUU antigen in bank vole (clethrionomys glareolus) lungs. Of 59 animals captured from the surroundings of patients with nephropathia epidemica, three became positive with a high activity in the PUU-ELISA, but with low reactivity in the DOB/HTN-ELISA. It is concluded that a sensitive ELISA has been developed to detect different hantaviruses in cell culture and lungs of bank voles.
Iodine-131 (131I) has been used both in unconjugated form and conjugated to antibody derivates (i.e., radioimmunotherapy; RIT) to treat malignant diseases. The mechanisms by which 131I-irradiation causes growth retardation are, however, inadequately understood. The aim of this study was to elucidate the sequential molecular and cellular events that initiate cell death in HeLa Hep2 cells exposed to 131I. In this paper, HeLa Hep2 cells were found to display a transient G2-M arrest following irradiation, but then reentered the cell cycle still containing unrepaired cellular damage. An increase of multipolar mitotic spindles, as well as a significant increase in centrosome numbers from 8.8% +/- 1.9% in controls to 54.7% +/- 2.2% in irradiated cells, was observed (p < 0.0001). A subsequent failure of cytokinesis caused the cells to progress into mitotic catastrophe. This was accompanied by the formation of giant cells with multiple nuclei, multilobulated nuclei, and an increased frequency of polyploidy cells. A fraction of the cells also displayed apoptotic features, including the activation of initiator caspases-2, -8, -9, and effector caspase-3, as well as cleavage of poly(ADP-ribose) polymerase, a cell-death substrate for active caspase-3. These findings demonstrate that mitotic catastrophes and the activation of a delayed type of apoptosis might be important mechanisms involved in cell death following the RIT of solid tumors with -emitting radionuclides, such as 131I.
BACKGROUND: The apoptotic signalling pathways involved in the delayed type of apoptosis occurring in HeLa Hep2 cells following radiation were investigated. MATERIALS AND METHODS: HeLa Hep2 cells were exposed to 5 Gy of cobalt-60 radiation. The activation of caspase-2, caspase-8, caspase-9 and effector caspase-3 was investigated by caspase assay plates and Western blots. Cleavage of poly (ADP-ribose) polymerase (PARP) was analysed on Western blots. HeLa Hep2 cells were irradiated with or without preincubation with inhibitors of protein synthesis (cycloheximide, CHX) and caspases, followed by TUNEL staining and caspase assay plate evaluation. RESULTS: Initiator caspases-2, -8, -9, and effector caspase-3, were found to be activated and PARP cleaved following irradiation. CHX completely inhibited the caspase activation and the associated apoptosis. Pretreatment with caspase-2 inhibitor indicated that caspase-2 was involved in the execution of the apoptosis. CONCLUSION: Activation of the apoptotic signalling pathways following irradiation of HeLa Hep2 cells includes components from the intrinsic as well as the extrinsic pathways and seems to require de novo protein synthesis.
PURPOSE: Experimental radioimmunotherapy delivering absorbed doses of 2.5 to 10 Gy has been shown to cause growth retardation of tumors. The purpose of this study was to elucidate the sequential molecular and cellular events occurring in HeLa Hep2 cells exposed to such doses. METHODS: Dose-response curves, activation of cell cycle checkpoints, and mitotic behavior were investigated in HeLa Hep2 cells following 2.5- to 10-Gy irradiation by carrying out 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, Western blots, fluorescence-activated cell sorting analysis, and immunofluorescence stainings. Terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling staining was used to detect apoptosis. RESULTS: A G2-M arrest was shown by fluorescence-activated cell sorting analysis. p53 and p21 were found to be up-regulated but were not immediately related to the arrest. The G2-M arrest was transient and the cells reentered the cell cycle still containing unrepaired cellular damage. This premature entry caused an increase of anaphase bridges, lagging chromosomal material, and multipolar mitotic spindles as visualized by propidium iodide staining and immunofluorescence staining with alpha-tubulin and gamma-tubulin antibodies. Furthermore, a dose-dependent significant increase in centrosome numbers from 12.6+/-6.6% to 67+/-5.3% was identified as well as a dose-dependent increase of polyploid cells from 2.8+/-1.3% to 17.6+/-2.1% with the highest absorbed dose of 10 Gy. These disturbances caused the cells to progress into mitotic catastrophe and a fraction of these dying cells showed apoptotic features as displayed by terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling staining 5 to 7 days after irradiation. CONCLUSION: An absorbed dose of 2.5 to 10 Gy was shown to force HeLa Hep2 cells into mitotic catastrophe and delayed apoptosis. These might be important cell death mechanisms involved in tumor growth retardation following radioimmunotherapy of solid tumors.
The main goal when treating malignancies with radiation therapy is to deprive tumor cells of their reproductive potential. One approach to achieve this is by inducing tumor cell apoptosis. Accumulating evidences suggest that induction of apoptosis alone is insufficient to account for the therapeutic effect of radiotherapy. It has become obvious in the last few years that inhibition of the proliferative capacity of malignant cells following irradiation, especially with solid tumors, can occur via alternative cell death modalities or permanent cell cycle arrests, i.e., senescence. In this review, apoptosis and mitotic catastrophe, the two major cell deaths induced by radiation, are described and dissected in terms of activating mechanisms. Furthermore, treatment-induced senescence and its relevance for the outcome of radiotherapy of cancer will be discussed. The importance of p53 for the induction and execution of these different types of cell deaths is highlighted. The efficiency of radiotherapy and radioimmunotherapy has much to gain by understanding the cell death mechanisms that are induced in tumor cells following irradiation. Strategies to use specific inhibitors that will manipulate key molecules in these pathways in combination with radiation might potentiate therapy and enhance tumor cell kill.
At immunolocalization of experimental tumors, idiotypic monoclonal antibodies, such as TS1 against cytokeratin 8, can be used to carry and deposit in vivo terapeutics in the tumor. These carriers also remain in the circulation and may cause negative side-effects in other tissues. In this report, several derivatives of the antiidiotypic antibody alphaTS1 were produced and tested for their clearing capacity of the idiotypic carrier antibody TS1. Intact monoclonal alphaTS1, scFv of a alphaTS1 and alphaTS1 Fab'2 and fragments were produced by recombinant technology or by cleavage with Ficin. The scFv was tailored by use of the variable domain genes of the light and heavy chain from the hybridoma clone in combination with a (Gly4Ser)3-linker, followed by expression in E. coli. When tested for clearing capacity, the intact divalent antiidiotypic IgG was found to be the most efficient. The divalent and the monovalent Fab fragment also demonstrated significant clearing, but lower than the intact antiidiotypic IgG. The alphaTS1 scFv antibody when injected separately was not found to clear the idiotype, but could do so when preincubated with the idiotype. Rapid excretion and in vivo instability of this low molecular weight antibody fragment may be the major reasons. Similar results were obtained when the system was reversed and the 131I-labeled antiidiotype IgG was cleared with the idiotype fragment. It is concluded that both intact antiidiotypic IgG, and Fab'2 fragments are able to clear the idiotypic antibodies. The experimental data support the conclusion that the Fc parts from both the idiotype and the antiidiotype may contribute to this elimination.
Recombinant antibodies may be engineered to obtain improved functional properties. Functional mapping of the residues in the binding surfaces is of importance for predicting alterations needed to yield the desired properties. In this investigation, 17 single mutation mutant single-chain variable fragments (scFvs) of the anti-idiotypic antibody anti-TS1 were generated in order to functionally map amino acid residues important for the interaction with its idiotype TS1. Residues in anti-TS1 determined to be very important for the interaction were identified, Y32L, K50L, K33H, and Y52H, and they were distributed adjacent to a centrally located hydrophobic area, and contributed extensively to the interaction energy (≥2.5 kcal/mol) in the interaction. Quantitative ELISA assays, BIAcore technologies and three-dimensional surface analysis by modeling were employed to visualize the consequences of the mutations. The expression levels varied between 2 - 1,800 nM as determined by ELISA. All the 17 scFvs displayed higher dissociation rates (60 - 1,300 times) and all but two of them also faster association rates (1.3 - 56 times). The decrease in affinity was determined to be 1.6 - 12,200 times. Two of the mutants displayed almost identical affinity with the wild type anti-TS1, but with a change in both association and dissociation rates. The present investigation demonstrates that it is possible to generate a large panorama of anti-idiotypic antibodies, and single out a few that might be of potential use for future clearing and pre-targeting purposes of idiotypic-anti-idiotypic interactions.
Recombinant single-chain fragment variable (scFv) antibodies with specificity to tumor antigens can be used to target tumors in vivo. The approach to use administration of complexes of idiotypic-anti-idiotypic scFvs when targeting tumors has not been tested earlier, and from a theoretical point it could contribute to longer in vivo circulation and improved targeting efficiency by dissociation, when in contact with the target antigen. In this study two models to evaluate the targeting efficiency of such complexes were used. HeLa HEp-2 tumor cells were grown as multicellular tumor spheroids (MCTS) and exposed to the antibody constructs in vitro. The behavior in vivo was tested in an in vivo tumor xenograft model. To increase the size of the anticytokeratin 8 scFv, TS1-218, complexes were formed between TS1-218 and its anti-idiotype, alphaTS1 scFv. The functionality of (125)I-labeled TS1-218 alone and in complex was studied in both models. The uptake patterns were similar in both models. The idiotypic TS1-218 was able to localize to the MCTS and xenografted tumors, both alone and in complex with alphaTS1 scFv. TS1-218 in complex, however, demonstrated a significantly higher uptake than the monomeric TS1-218 in both models (p < 0.0005 and p < 0.0089, respectively). When complexes were administered in vivo, a slower clearance and an increased tumor half-life could be observed. The present investigation indicates that administration of targeting antibodies, with initially blocked antigen-binding sites by complex formation with their anti-idiotypes, may improve targeting efficiency.
The immunoreactivity, stability and in vivo kinetics of an anticytokeratin 8 monoclonal antibody, TS1, were investigated following different degrees of labeling with 125I (0.2, 1 and 2-3 125I/TS1 MAb). By testing with ELISA, it was demonstrated that a high degree of iodination, i.e. > 2 125I/TS1, caused a rapid decrease in immunoreactivity to almost zero within 10 days. Furthermore, a complete degradation to low molecular weight fragments and free iodine was seen, as shown by SDS PAGE and autoradiography. The differently labeled radionuclide conjugates were injected into nude mice inoculated with HeLa Hep2 cells and tumor doses (estimated by MIRD formalism), tumor:non-tumor dose ratios, % I.D./gram tissue, Gy/MBq and in vivo kinetics of the differently labeled MAbs were determined. Despite the in vitro instability of the highest iodinated radionuclide conjugate, it was possible to deliver high doses to the tumors if the conjugate was injected into the animal immediately after completion of the iodination procedure. Increases from 1.4 Gy to 15.2 Gy delivered tumor dose were obtained with a tenfold increase in the specific activity, without alterations in the tumor:non-tumor tissue dose ratios. There is room for significant improvements in efficacy at radioimmunotherapy, which can be gained by optimizing the degree of iodination. For therapeutical applications a high degree of iodination may be an advantage.
Purpose: To explore kinetic changes in the gene expression profile during radiation induced mitotic catastrophes.
Methods and Materials: We measured temporal global gene expression in HeLa Hep2 tumor cells using bead chip arrays (Illumina) following exposure to 5 Gy of ionizing radiation (60Co). Genes with less than a 2-fold change in expression and a p-value > 0.05 were discarded. Signaling pathways significantly altered following irradiation were explored using Metacore. Furthermore, biological responses linked to mitotic catastrophe including cell cycle arrests, anaphase bridge formation and centrosome amplification were analyzed and correlation to gene expression changes evaluated.
Results: Following irradiation a G2-arrest was induced. This arrest was transient and cells entered mitosis before DNA damage was repaired causing anaphase bridge formation. Furthermore, radiation induced hyperamplification of centrosomes was observed. These phenotypical changes correlated well with the observed gene expression changes. At 6 h following irradiation the expression was changed only for a few genes including histone H2 and H4, essential for activation of a DNA-damage checkpoint. Striking changes appeared at later time-points. From 12-96 hours post irradiation a significant fraction of the genes with altered expression were found to be involved in cell cycle progression and its regulation. The significant changes were seen for genes important for several mitotic processes, and those involved in the G2- and spindle assembly checkpoints. Also centrosome associated genes displayed an increased expression. Furthermore, 96 hours after irradiation pathways involved in immune and inflammatory responses were significantly altered.
Conclusions: This study elucidates specific characteristics in the altered gene expression pattern induced by irradiation, which can be linked to the sequential steps observed in HeLa Hep2 cells during mitotic catastrophes. Therapeutic strategies employing these alterations might potentiate future therapy and enhance tumor cell killing.
Aim: To explore kinetic changes in the gene expression profile during radiation-induced mitotic catastrophes. Materials and Methods: Gene expression changes were measured in HPV-infected HeLa Hep2 tumor cells following exposure to 5 Gy of ionizing radiation (Co-60). Signaling pathways were explored and correlated to the biological responses linked to mitotic catastrophe. Results: Following irradiation a transient G(2)-arrest was induced. Anaphase bridge formation and centrosome hyperamplification was observed. These phenotypical changes correlated well with the observed gene expression changes. Genes with altered expression were found to be involved in mitotic processes as well as G(2)- and spindle assembly checkpoints. Also centrosome-associated genes displayed an increased expression. Conclusion: This study elucidates specific characteristics in the altered gene expression pattern induced by irradiation, which can be correlated to the events of mitotic catastrophe in HeLa Hep2 cells. Therapeutic strategies modulating these alterations might potentiate future therapy and enhance tumor cell killing.
This study aims to identify the temporal changes in gene expression in MOLT-4, a leukemia cell line, in response to radiation and to present a comprehensive description of the pathways and processes that most significantly relate to the cellular biological responses. A global gene expression profile of 24,500 genes was performed on MOLT-4 tumor cells following exposure to 5 Gy of ionizing radiation (Co-60) using a bead chip array (Illumina). Signaling pathways and processes significantly altered following irradiation were explored using MetaCore. Cellular viability [3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], activation of cell cycle checkpoints [fluorescence activated cell sorting (FACS)], and induction of apoptosis (FACS, caspase assays) were evaluated to correlate these biological responses to the gene expression changes. Totally, 698 different genes displayed a significantly altered expression following radiation, and out of these transcripts, all but one showed increased expression. One hour following irradiation, the expression was changed only for a few genes. Striking changes appeared at later time-points. From 3 to 24 h post-irradiation, a significant fraction of the genes with altered expression were found to be involved in cell cycle checkpoints and their regulation (CDKN1A), DNA repair (GADD45A, DDB2, XPC), apoptosis induction (DR5, FasR, Apo-2L, Bax), and T-cell activation/proliferation (CD70, OX40L). Irradiated MOLT-4 cells were arrested at the G2-checkpoint, followed by a decrease in cell viability, most pronounced 48 h after exposure. The cell death was executed by induced apoptosis and was visualized by an increase in subG1 cells and an increased activation of initiator (caspase-8 and caspase-9) and execution (caspase-3) caspases. Activation of cell cycle arrest and apoptosis correlated well in time with the changes in gene expression of those genes important for these biological processes. Activation of the apoptotic signaling pathways in MOLT-4 cells following irradiation includes components from the intrinsic as well as the extrinsic apoptotic pathways. This study indicates that the altered gene expression pattern induced by irradiation is important for the sequential steps observed in MOLT-4 cells during apoptosis induction.
Purpose : To study the kinetics of gene expression alterations following radiation exposure of isogenic HCT116 p53+/+ and HCT116 p53-/- cell lines. Materials and methods : Cells were exposed to 5 Gy of irradiation (Cs-137) and genome-wide temporal expression analysis using Illumina bead chip arrays was performed. Signalling pathways were explored using Metacore (Genego). Biological responses including cell cycle checkpoint activation, centrosome amplification and senescence induction were analyzed. Results : Significant differences in the radiation response were observed between the p53+/+ and the p53-/- cell lines. In p53+/+ cells concurrent G1- and G2-arrests were activated followed by senescence induction. Increased expression of genes associated with senescence, senescence associated secretory phenotype (SASP) and repression of genes essential for G2-M transition were detected. P53-/- cells arrested mainly in G2 followed by centrosome amplification, mitotic slippage and a subsequent increase of polyploid cells. Furthermore, changes in expression correlated well with these signs of mitotic catastrophe. Conclusions : The presence or absence of p53 triggers different signalling cascades with different endpoints. Elucidating these differences is important as it enables improvement of radiation treatment and could be used to develop new combination treatments with specific inhibitors of key regulators of these cell death modalities.
Neurofilaments are necessary for the maintenance of axonal caliber and structural organization of nerve cells. The low molecular weight form of neurofilament, the neurofilament light protein, which serves as the core of the filament, was used as immunogen for generation of hybridomas with selective reactivity with this form of the filament. Six hybridomas, out of approximately 100 tested clones, were highly discriminatory. All involved epitopes were localized to the rod region of the antigen, as determined by alpha-chymotrypsin digestion of the purified filament and enzymatic peptide mapping. Synthetic peptides (20 mers) covering the entire rod region did not react with the antibodies. A phage display peptide library was used to identify four consensus sequences for the antibodies. The results indicate that all epitopes were of conformational type. Pair-wise BIAcore data furthermore indicated that the epitopes were independent. The access to such specific reagents is a prerequisite for further elucidation of the biology of the low molecular weight form of neurofilaments proteins.
Neurofilaments, a major cytoskeletal constituent of neuronal cells, can be released into the cerebrospinal fluid during several neurodegenerative diseases. By means of a new sensitive ELISA capable of measuring 60 ng/l of neurofilament light, significant elevations were observed for different neurological disorders. Cerebral infarction presented levels of 19800+/-9100 ng/l, amyothropic lateral sclerosis 3600+/-1200 ng/l, 'relapsing-remitting' MS 2500+/-1500 ng/l, extrapyramidal symptoms 1100+/-300 ng/l, late onset AD 300+/-100 ng/l and vascular dementia 1400+/-800 ng/l. In patients with no signs of neurological diseases the upper normal level and cut-off values was determined to be below 100 ng/l. NF-L determinations will be a valuable complement in identifying neuronal degradation and can be used clinically for diagnostic and monitoring purposes.
Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) is a chronic relapsing-remitting animal model of multiple sclerosis (MS). Neurofilament light (NF-L), a structural protein expressed in neuronal cells can be used to quantify the amount of neuronal damage in MS patients. An immunoassay was used to measure levels of neurofilament light in cerebrospinal fluid (CSF) in rats with myelin oligodendrocyte glycoprotein-induced EAE. Significantly increased levels of neurofilament were found in the immunized animals compared to the controls, strengthening the similarities in the diseases and the progression pattern between the animal model and MS. The turnover of NF-L during this disease is increased since significantly elevated levels also were identified in the spinal cord of the diseased animals and immunohistochemistry gave support for this observation. Monitoring neurofilament levels in EAE can be used to follow disease progression and effects of therapy.
Objective: To evaluate levels of neurofilament light (NFL) and glial fibrillary acidic protein (GFAP) in CSF from patients with multiple sclerosis (MS) in relation to clinical progress of the disease.
Methods: CSF levels of NFL and GFAP were determined by sensitive ELISAs in 99 patients with different subtypes of MS, classified in terms of “ongoing relapse” or “clinically stable disease,” and 25 control subjects. Levels were compared with paraclinical data such as immunoglobulin G index and inflammatory cell count in the CSF, and the levels were related to Expanded Disability Status Scale score and progression index at clinical follow-up evaluations later in the disease course.
Results: NFL and GFAP levels were elevated in MS patients as compared with control subjects (p < 0.001). The NFL levels were higher at relapses, whereas GFAP levels were unaffected. High NFL levels correlated with progression in patients with an active relapse (r = 0.49; p < 0.01) and in clinically stable patients (r = 0.29; p < 0.05). GFAP correlated to progression in the total patient cohort (r = 0.24; p < 0.05). Moreover, a strong correlation between NFL levels and inflammatory cell counts was evident in the group of patients with an ongoing relapse (r = 0.52; p = 0.001).
Conclusions: CSF levels of neurofilament light and glial fibrillary acidic protein may have prognostic value in multiple sclerosis.
Fourteen monoclonal antibodies with specificity against native or recombinant antigens within the S100 family were investigated with regard to immunoreactivity. The specificities of the antibodies were studied using ELISA tests, Western blotting epitope mapping using competitive assays, and QCM technology. The mimotopes of antibodies against S100A4 were determined by random peptide phage display libraries. Antibody specificity was also tested by IHC and pair combinations evaluated for construction of immunoradiometric assays for S100B. Out of the 14 antibodies included in this report eight demonstrated specificity to S100B, namely MAbs 4E3, 4D2, S23, S53, 6G1, S21, S36, and 8B10. This reactivity could be classified into four different epitope groups using competing studies. Several of these MAbs did display minor reactivity to other S100 proteins when they were presented in denatured form. Only one of the antibodies, MAb 3B10, displayed preferential reactivity to S100A1; however, it also showed partial cross-reactivity with S100A10 and S100A13. Three antibodies, MAbs 20.1, 22.3, and S195, were specific for recombinant S100A4 in solution. Western blot revealed that MAb 20.1 and 22.3 recognized linear epitopes of S100A4, while MAb S195 reacted with a conformational dependent epitope. Surprisingly, MAb 14B3 did not demonstrate any reactivity to the panel of antigens used in this study.
This multi-center validation study identified the lack of preparation of accurate and consistent protein standards as the main reason for a poor inter-laboratory CV. This issue is also relevant to other protein biomarkers based on this type of assay and will need to be solved in order to achieve an acceptable level of analytical accuracy. The raw data of this study is available online.
Radioimmunoscintigraphy (RIS) was performed in 20 patients with gynecologic tumors, 14 ovarian, 5 cervical, and one endometrial carcinoma. One murine monoclonal antibody (mab) against placental alkaline phosphatase (H7) was used after radiolabeling with 131I. The labeling procedure yielded antibodies with specific activity varying between 60 and 73 MBq/mg mab. Each patient received 57 to 100 MBq of the preparation. RIS was performed 7 to 35 days later. Patients with ovarian adenocarcinoma had an accumulation of activity on RIS at tumor sites (79%, 11/14) verified by ultrasonography, CT, and clinical examination. A low or absent accumulation of activity was seen in patients with cervical tumors. The patient with an endometrial adenocarcinoma was seen to have an activity accumulation at RIS corresponding to tumor sites determined by ultrasound and/or CT. It is concluded that RIS using monoclonal antibodies against placental alkaline phosphatase can provide information which will supplement that gained from other investigations of patients with ovarian adenocarcinomas.
Radioimmunoscintigraphy (RIS) is a potentially valuable method for the detection of primary, secondary and recurrent malignant tumours. Antigens that have been used for monitoring as well as for RIS of ovarian carcinomas include CA 125, PLAP, HMFG, and CA 19-9. Between 70 and 100% of the tumours have been detected at RIS when these antigens have been used. Conventional methods, e.g., computerized tomography (CT) and ultrasonography (US), demonstrate similar or lower detection rate than RIS for tumour diagnosis. RIS gives additional information to conventional radiological methods (CT and US) for the detection of occult ovarian carcinomas. A review of earlier investigations is given and our own recent results using PLAP as a target antigen are presented. The future potential of the technology is discussed.
The radioimmunotherapeutic potential of 131I-labeled monoclonal antibodies was investigated in 36 nude mice (BALB/c nu/nu) inoculated s.c. with the HeLa Hep 2 human adenocarcinoma cell line. The membrane bound tumour associated antigen placental alkaline phosphatase and several intracellular cytokeratins served as targets for the antibodies. The specific radioactivity in each organ was determined after i.p. injection of the 131I-labeled antibodies (0.2-0.3 mg, approximately 15 MBq/animal), and high localization to the tumours was seen. Significant growth inhibition was observed after injection of the radiolabeled monoclonal antibody H7 against the placental alkaline phosphatase, which reduced the tumour growth to only 12% during a 3 week period compared to a growth of more than 100% for the controls. Animal weight losses were seen. Synthesis of endogenous antibodies to the target antigens was found to be significant. Morphometric evaluation of the relations between stroma, tumour cells and necrotic areas in the tumours after radioimmunotherapy demonstrated a significant increase of the mean relative connective tissue volume and a significant decreased mean of relative volume of tumour cells in the group treated with iodinated antiplacental alkaline phosphatase antibody. This therapeutic principle is encouraging and may offer new possibilities for future treatment of some malignant diseases.
Non-specific testicular accumulation of radiolabelled intact anti-CEA monoclonal antibody (MAb), (A431/26, Behringwerke AG) was observed in 11 out of 12 patients with the testes and prostate included in the examination field at radioimmunoscintigraphy (RIS). Previous studies have shown that placental alkaline phosphatase (PLAP) serves as an Fc-receptor, mediating IgG transport through the placenta. A closely related protein, the germ cell alkaline phosphatase (GCAP), is expressed in the testes. The testicular uptake of IgG is observed only when intact but not fragmented MAbs are used, indicating involvement of Fc-receptors. MDCK cells (dog kidney cell line) transfected with the plasmid pSVT7 containing the GCAP gene were shown to acquire the capacity to both express membrane bound GCAP and to bind IgG on the cell surface. This might indicate that GCAP is responsible for the non-specific accumulation of intact MAb in the testes and prostate often observed when intact murine MAbs are used for radioimmunolocalization (RIL).
The radiommunotherapeutic potential of 125I-labeled monoclonal antibodies was investigated in 48 nude mice (BALB/c, nu/nu) inoculated s.c. with the HeLa Hep 2 human adenocarcinoma cell line. This isotope, 125I, which is not commonly used for therapeutic purposes caused significant decrease in tumour growth from day 10 to day 42, when coupled to monoclonal antibodies directed against placental alkaline phosphatase (H7) or cytokeratins (TS1). The average growth rate was approximately 50-60% of that observed in the untreated control group after 42 days. The specific radioactivity in each organ 42 days after injection of radiolabeled monoclonal antibodies, indicated that these target antigens retain significant amounts of radiolabeled antibody in the tumours for at least 6 weeks after injection. No weight loss was seen in the animals during this experiment. By use of autoradiographic techniques, the labeled monoclonal antibodies were visualized deep in tumours in characteristic patterns representative of viable tumour cells (H7) and necrotic areas (TS1). The therapeutic approach using 125I labeled antibodies is encouraging and may offer new dimensions in radioimmunotherapy.
Interleukin-2 (IL-2) is an important cytokine in the autoimmune process proceeding Type 1 diabetes. Our aim was to investigate, in two previously used animal models, the NOD mouse and the BB/W rat, the in vivo tissue distribution of radio-labeled IL-2. If the radio-labeled IL-2 accumulated significantly in the pancreas compared to surrounding organs it could allow imaging of lymphocyte infiltration of the islets of Langerhans by scintigraphic methods. IL-2 was labeled enzymatically with(125)Iodine. Radio-labeled IL-2 was injected iv in prediabetic NOD mice, diabetic NOD mice and Balb/c mice in the first animal model and in BB rats in the second model. Animals were sacrificed at different time points and the activity in different organs was measured. It was found that the mean activity in the pancreas in both diabetic and prediabetic NOD mice was significantly higher compared to pancreas from Balb/c mice (P< 0.001 and P=0.005, respectively). However, the mean activity in the pancreas was at the lower range of the surrounding organs in both animal models, thereby excluding the possibility of imaging the autoimmune process by scintigraphic methods. It is concluded that radio-labeled IL-2 did accumulate significantly in the pancreas of NOD mice compared to control mice but there is a need to develop new techniques in order to visualize the localized activity.
The fractionated strategy can contribute to a significant accumulation of radiolabeled TS1 in the tumors. Furthermore, the use of alphaTS1 makes it possible to increase the tumor-to-nontumor dose ratio and maintain a prolonged high activity accumulation in the tumor.
This study confirms an extensive accumulation of TS1 in the tumor, with peak values as late as 30 days after injection of labeled TS1. Furthermore, both preinjection of nonlabeled TS1 and postinjection of alphaTS1 can improve radioimmunotargeting.
The anti-tumour effect of the 131I-labelled antiprostate monoclonal antibody (MAb) E4 was studied in an experimental model with 41 nude mice, subcutaneously xenografted with a human prostate cancer cell line (DU-145). The mice were divided into four study groups, i.e. one receiving single and another repeated injections of the radiolabelled MAb. A third group was injected with non-labelled MAb, and the fourth served as an untreated control group. The tumour volumes increased similarly in all groups during the 27-day observation period. The tumour tissue was morphologically disintegrated in the group that received repeated radioimmunotherapy (RIT). The tumours from this group contained large fluid-filled cystic parts and demonstrated pronounced cellular and subcellular polymorphism in the remaining viable tumour tissue. The untreated control tumours and single therapy tumours remained solid. The proportion of the total tumour volume that consisted of viable tumour cells, as determined by morphometric techniques, was significantly lower in the 131I-E4-treated groups. The use of 131I-labelled E4 MAb has thus demonstrated a promising therapeutic potential.
The almost overwhelming volume of information and new technological developments that has demanded so much of our scientific attention over the last decade will shortly revolutionize clinical diagnostics. Some of these developments are already affecting the working lives of scientists and clinicians alike, but will eventually require a greater understanding and acceptance from a much wider audience. Therefore it is important in our current scientific endeavor and commercial enthusiasm for molecular diagnostics that we maintain some awareness of the significant obstacles that must be overcome if we are to see an appropriate, timely and widespread adoption of molecular diagnostic testing in oncology. This article presents a brief commentary on the current state of the art in molecular diagnostics in oncology and how this relates to a more personalized approach to treatment.
RNA interference (RNAi) is a potent and ubiquitous gene-silencing mechanism that is generating considerable excitement in the fields of molecular biology and gene therapy. It is now in widespread use for loss-of-function analysis in many diseases including cancer. Nevertheless, RNAi is still in its infancy, with new discoveries appearing on a monthly basis. This article presents a brief outline of the history and recent advances in RNAi with a specific focus on its potential in oncology.
Idiotypic-anti-idiotypic antibody interactions can be used in vivo to regulate the serum levels of specific radiolabeled antibodies. Anti-idiotypic antibodies can also be used as clearing agents for radiolabeled antibodies in radioimmunolocalization and radioimmunotherapy. The present study describes the immunochemical interactions between the monoclonal idiotype (H7) and three generated monoclonal anti-idiotypic antibodies (alphaH7:1, alphaH7:35, and alphaH7:38). An unexpected variability in complex formation could be demonstrated in vitro, revealing three different stable complex patterns, i.e., low molecular weight 1:1 complexes, ladder formation with oligomeric, consecutively added constituents, and large linear polymeric complexes of high molecular weight. Within 24 h, the anti-idiotypes were able to cause a significant decrease in total body radioactivity, and the antibody generating a ladder formation (alphaH7:38) was found to be the most efficient at removing radiolabeled idiotypes from the circulation. It is concluded that monoclonal anti-idiotypic antibodies may be valuable tools in improving radioimmunolocalization and radioimmunotargeting.
We have shown previously that a bispecific antibody (BsAb) directed against both germ-cell alkaline phosphatase (GCAP) and the CD3 complex on mouse T cells could effectively eliminate GCAP-positive tumor cells in vivo using an immunocompetent mouse model. However, some GCAP-negative tumor cells were still able to grow, suggesting that BsAb therapy, when used in a clinical setting, could benefit from targeting several tumor markers to prevent outgrowth of tumor cells lacking a targeted marker. To test this hypothesis, we developed an in vitro model based on primary human ovarian carcinoma (OC) cultures and BsAbs directed against human T cells and several tumor markers [placental alkaline phosphatase (PLAP), GCAP, folate-binding protein (FBP) and CA19.9]. OC cells, isolated from primary tumors, were co-cultured with human peripheral blood mononuclear cells in the presence or absence of various concentrations of BsAbs against PLAP/GCAP, FBP and CA19.9 administered separately or in combination. Results derived from 18 primary OC samples showed that the combination treatment was better than or equally effective as the best single BsAB treatment in 60% of cases. Sometimes targeting FBP, PLAP/GCAP or CA19.9 alone was superior to targeting all simultaneously. Combining each BsAb with a low dose of IL-2 was always beneficial. These results indicate that before using a specific BsAb in the clinic, it is important to determine the optimal BsAb for each patient using this in vitro assay on cells from the removed tumor mass.
Three monoclonal antibodies and their Fab and Fab'2 fragments with specificity against human placental alkaline phosphatase (PLAP) were evaluated for tumour immunolocalization of human PLAP-producing Hela Hep 2 tumours in nude mice. The antibodies and their fragments were labelled with 125I and injected intraperitoneally in mice with developing Hep 2 tumours. The animals were followed individually for 14 days with repetitive computerized gamma-camera recordings, which enable quantitation of several crucial parameters, i.e. the time-dependent antibody uptake in the tumours, decrease in background activity and tumour/background ratio. Excellent radioimmunolocalization was obtained with both the intact PLAP-specific immunoglobulins and their fragments but not with the nonspecific antibodies. No background subtraction had to be used. As much as 15% of the initially injected dose could be visualized in the tumours and for the uncleaved mab up to 80% of the radioactivity in the animals was retained in the tumours after 14 days, a considerably longer observation time than usually reported in such tumour xenograft models. The Fab and Fab'2 fragments were found to be excreted fast with less than 5% of the injected dose remaining in the animals after 48 h, but still with positive specific localization to the tumours after an initial high uptake in the kidneys. The results are encouraging and indicate significant potentials of the PLAP-antiPLAP mab system for immunolocalization studies in patients.
Several new technologies to generate and modify established hybridomas that produce monoclonal antibodies have recently been presented and further development should make them more suitable for diagnostic and therapeutic techniques. Different proteolytic procedures have been used for the fragmentation of intact antibodies to Fab2' and Fab fragments and recombinant DNA techniques have made it possible to obtain chimeric, humanized, Fv fragments and single-chain Fvs. A review of the new approaches is presented and the future implications are discussed.
The significance of the PLAP (Placental alkaline phosphatase)/PLAP-like isozyme as tumour marker in relation to CA 125 and TPA for the monitoring of patients with malignant ovarian epithelial tumours was evaluated. Of all patients (n = 85), 40% had all three markers elevated. CA 125 being the most sensitive (60%), and the PLAP/PLAP-like isozyme and TPA both 40%. A tendency to certain tumour marker patterns of these three antigens in serum can be seen with regard to histopathology. Serous and anaplastic adenocarcinomas usually have all three markers moderately elevated, mucinous and mesonephric adenocarcinomas both have low incidences and low average levels of all three markers. Endometrioid and non-mucinous adenocarcinomas are often associated with high levels of the PLAP/PLAP-like isozyme and CA 125, while TPA shows moderate elevation. The PLAP/PLAP-like isozyme is positively correlated to tumour burden and the outcome of the disease. It may provide additional information on CA 125 in the monitoring of patients with ovarian cancer.
In this review, we have selected some parameters with the potential to improve the efficacy of RIL and RIT. Focus has partially been on the behaviour of radiolabelled antibodies in vivo in relation to properties and amounts of both target antigen and the antibodies used. If, out of the 28 factors listed in Table 1, some should be given preference in future work, it is our opinion that after the initial saturation of the tumour site a rapid decrease in redundant antibody is of significant importance. Furthermore, quantitative aspects of both antigens and antibodies should be more carefully evaluated when possible. By combining several of the listed approaches toward increasing efficiency, a more extensive use of RIL and RIT could be expected in the future.
Significant improvements in tumor/nontumor ratio can be achieved by injections of nonlabeled anti-idiotypic monoclonal antibodies (MAbs) during radioimmunolocalization and radioimmunotherapy using MAbs to target experimental tumors. The in vivo effects of an anti-idiotypic MAb (alpha H7) against a radioiodinated, high affinity, low dissociation rate, monoclonal antiplacental alkaline phosphatase antibody (H7) was investigated. Following in vivo injection of the anti-idiotypic MAb, the radioactivity in experimental tumors was found to decrease only 25% while the reduction of corresponding radioactivity in nontumor tissues amounted to 65-85%, compared to the group receiving no anti-idiotypic MAbs. These results indicate that it is possible to partially clear the circulation and nontumor tissues from excess of radiolabeled idiotypic antibody, without significant decrease in specific tumor localization, increasing the tumor/nontumor ratio three- to fourfold. Circulating nontumor targeting radiolabeled antibodies is one of the major limiting factors in radioimmunotherapy today. Injection of anti-idiotypic MAbs could selectively significantly reduce the radiation dose to radio-sensitive tissues, i.e., bone marrow and intestine, thus improving efficiency in radioimmunoscintigraphy and radioimmunotherapy.
A syngeneic, high-affinity, anti-idiotypic monoclonal antibody (MAb; alpha TS1) raised against an anticytokeratin monoclonal antibody (TS1) was evaluated as a second antibody to promote the rapid clearance of radiolabeled TS1 from the blood during experimental radioimmunolocalization. By using a novel biosensor technology (BIAcore), association rate dissociation rate, and affinity constants between the idiotype and the anti-idiotype could be determined. The in vivo results in nude mice carrying HeLa Hep 2 tumors demonstrate the possibility of selectively regulating the amount of the idiotypic 125I-labeled circulating MAb by in vivo injection of this high-affinity, anti-idiotypic antibody. Injection of the anti-idiotype in a molar ratio of 0.75 to the idiotype cleared the blood pool from circulating radiolabeled idiotype within 24 h, with a concomitant rapid excretion of 125I in urine. The total amount of remaining radioactivity in the animals decreased to 15-20% during these 24 h, with the tumors still retaining 60-65% of their initial radioactivity. This approach, using syngeneic primary and secondary MAbs with minimized immunogenicity, significantly improves the tumor:nontumor ratio, thus improving efficiency in experimental radioimmunolocalization and radioimmunotherapy, leaving the endogenous antibody repertoire of the host unaffected.
A monoclonal antiidiotypic antibody alpha H7, was generated against a monoclonal antibody H7 with specificity towards placental alkaline phosphatase. The in vitro and in vivo effects of alpha H7 were investigated. The antiidiotypic antibody was found to generate stable complexes with the radiolabeled idiotypic antibody, visualized both in vivo and in vitro, as revealed by PAGE and autoradiography. Using biosensor technology (BIAcore, Pharmacia) the interactions were followed in real time and the association rate, dissociation rate, and affinity constants between the reactants were determined (KA H7/PLAP 6.7 x 10(9) M-1, KA H7/alpha H7 3.2 x 10(9) M-1). By in vivo injection of the antiidiotype, a rapid dose dependent clearance of circulating radiolabeled idiotypes was demonstrated and a decrease in total body radioactivity was recorded with a concomitant dramatic increase in non-protein-bound 125I excreted in the urine. It is concluded that idiotypic-antiidiotypic interactions offer advantages in the regulation of antibody levels in vivo.
One way of selectively improving the efficiency of radioimmunolocalization and radioimmunotherapy is to eliminate redundant, circulating, non-targeting radiolabeled antibodies after saturation of the target sites. Secondary antibodies of different types have been proposed as clearing agents for such purposes. The conceptually different approaches of the 'secondary antibody' strategy including its advantages and limitations are discussed. This mini-review also presents a model describing the kinetics of the components (the antigen, the primary and secondary antibodies) and approaches required to improve the efficacy of both radioimmunolocalization and radioimmunotherapy.
In this antigen target system, a single injection of a large amount of antibody was found to be more efficient than the same antibody dose subdivided into three or ten fractions. It was concluded that not only the radioactivity but also the amount of antibody per fraction should be considered when determining optimal fractionated radioimmunotherapy.