Mouse-specific antibody responses to a monoclonal antibody during repeated immunoscintigraphy investigations: comparison of antibody titres and imaging studies in a rat model

As a model for human mouse-specific antibody responses in patients undergoing immunoscintigraphy, we have investigated in rats the production of mouse-specific antibodies (MA) to the mouse monoclonal antibody 791T/36. At intervals of between 5 and 16 weeks the rats were given repeated cycles of intravenous (IV) injections of antibody with or without a simultaneous intradermal (ID) injection. The IV dose was 60 micrograms/kg, a dose similar to that used in many clinical immunoscintigraphy studies. The ID injection was 2 micrograms, which mimicks the skin test dose often given in clinical imaging protocols. The study was carried out with both 131I-labelled antibody and with antibody labelled with 111In by diethylenetriamine-penta-acetic acid (DTPA) chelation. MA was measured with a passive haemagglutination assay using sheep red blood cells coated with the monoclonal antibody. Of rats given ID injections of unlabelled antibody at the same time as the IV imaging doses, 9/20 produced MA during 4 cycles of injections. In contrast, only 2/16 rats given only the IV dose produced MA. Both 131I- and 111In-labelled antibody appeared equally immunogenic with 5/18 and 6/18 overall responders, respectively. The production of MA was associated with a significant perturbation in the biodistribution of the IV dose of labelled antibody as seen by gamma-camera imaging of the rats given 111In-labelled antibody. There was clearance of immune complexes to the liver, this organ accumulating up to 90% of the whole body count rate of radiolabel. MA titres of between 1/100 and 1/78,000 caused equal perturbation of biodistribution, although below 1/100 the effect was more variable.     

Does immunoscintigraphy serve clinical needs effectively? Is there a future for radioimmunotherapy?

Since 1980, immunoscintigraphy has been performed in thousands of patients, and its clinical value has been demonstrated for selective indications in malignant (early detection of recurrences of colorectal and ovarian carcinomas) and non-malignant (cardiovascular and inflammatory) pathology. However, many clinicians are not yet very convinced of its efficiency. Opinions range between favourable interest and marked scepticism. The causes of this inconclusive verdict include an often moderate target-to-background ratio in images, the immunogenicity of injected murine antibodies and the fact that a true benefit for the patient has not yet been clearly demonstrated in large series of patients. Future prospects could significantly improve this and involve the reduction of non-specific activity in normal tissues (to improve disease target contrast and thus make image interpretation easier) and the decreased immunogenicity of injected immunoconjugates (to permit repetition of examinations). Radioimmunotherapy, an innovative and promising approach, is still limited by numerous problems. The results of clinical studies are still inconclusive, being encouraging only for specific indications. In the future, pre-targetting techniques should allow the rapid elimination of radioactivity from normal tissues, resulting in a significant increase in tumour-to-normal tissue ratios. Progress is also required in the choice of radionuclides and labelling techniques and in methods for dosimetric estimations. The clinical indications of radioimmunotherapy after systemic injection will concern mainly radiosensitive tumours such as lymphomas, small-cell lung cancers and neuroblastomas. After endocavitary injection, radioimmunotherapy could prove efficient in the treatment of micrometastases of ovarian carcinomas. For all indications, this new approach should be combined with other therapeutic modalities. Offprint requests to: J.F. Chatal     

Radioimmunoimaging of subacute infective endocarditis using a technetium-99m monoclonal granulocyte-specific antibody

Immunoscintigraphy with a technetium-99m murine monoclonal IgG₁ antibody directed against non-specific cross-reacting antigen (NCA-95) and carcinoembryonic antigen was performed with 20 patients with suspected subacute infective endocarditis (SIE) and 6 controls with suspected inflammatory/infectious disease elsewhere in the body. Immunoscintigraphy and echocardiography localised SIE in 11 of 15 patients in whom the disease could be confirmed. In 4 patients with validated SIE, the immunoscan was abnormal, and the echocardiogram was normal. In another 4 patients, the result was exactly the opposite. These findings suggest that the combination of immunoscintigraphy and echocardiography improves diagnostic efficacy in patients with suspected SIE. Offprint requests to: D.L. MunzDedicated to Professor F. Wolf on the occasion of his sixtieth birthday     

Quantitative studies of monoclonal antibody targeting to disialoganglioside GD2 in human brain tumors

Iodine-131 3F8, a murine IgG₃ monoclonal antibody that targets to G_D2-bearing tumors, was administered intravenously to 12 patients with brain tumors. Six patients received 2 mCi (0.74 Bq) of¹³¹I-3F8, five patients 10 mCi (3.7 Bq)/1.73 m² of¹³¹I-3F8, and one patient 2.6 mCi (0.96 Bq) of¹²⁴I-3F8, with no side-effects. Nine of 11 malignant gliomas and the single metastatic melanoma showed antibody localization, with the best tumor delineation on single-photon emission tomography (SPET) following 10 mCi (3.7 Bq)/1.73 m² dose. No nonspecific uptake in the normal craniospinal axis was detected. There was no difference in the pharmacokinetics of low-dose versus the higher-dose antibody groups; plasma and total-body half-lives were 18 h and 49 h, respectively. Surgical sampling and time-activity curves based on quantitative imaging showed peak uptake in high-grade glioma at 39 h, with a half-life of 62 h. Tumor uptake at time of surgery averaged 3.5×10^–3 %ID/g and peak activity by the conjugate view method averaged 9.2×10^–3 %ID/g (3.5–17.8). Mean radiation absorption dose was 3.9 rad per mCi injected (range 0.7–9.6) or 10.5 cGy/Bq (range 1.9–26). There was agreement on positive sites when immunoscintigraphy was compared with technetium-99m glucoheptonate/diethylene triamine penta-acetic acid planar imaging, thallium-201 SPET, and fluorine-18 fluorodeoxyglucose positron emission tomography. Taken together, these data suggest that quantitative estimates of antibody targeting to intracranial tumors can be made using the modified conjugate view method.     

The preparation and characterisation of 111In-labelled 791T/36 monoclonal antibody for tumour immunoscintigraphy

The anti-human tumour monoclonal antibody 791T/36 was conjugated to the cyclic dianhydride of DTPA and radio-labelled with ¹¹¹In. The labelling method proved to be both simple and reliable and would be suitable for routine clinical use. Subsequent characterisation of this radio-pharmaceutical in vitro and in tumour-bearing hosts gave a strong indication as to its suitability for clinical tumour localisation studies.     

Volume rendering and bicolour scale in double isotope studies: application to immunoscintigraphy and bone landmarking

Combining the volume rendering and bicolour visualization tehniques is proposed as an aid in interpreting single photon emission tomography (SPET) immunoscintigraphy (IS) recorded simultaneously with SPET bone landmarking (BL). The combination helps in localizing abnormal monoclonal uptake and in differentiating it from a physiological radioactivity distribution. The so-called rendered images (RIs) are obtained in both IS and BL studies according to a depth- weighted maximum activity projection algorithm. Fused BL and IS RIs are constructed by a simple, pixel by pixel addition. They are displayed using a bicolour grey-red scale, which makes it possible to visualize both studies by a transparency effect. This method was applied to patients suspected of suffering colorectal or ovarian cancer recurrences, in whom monoclonal antibodies against carci-noembryonic antigen, B72-3 or OC125 labelled with indium-111 were used. Offprint requests to: A. Loboguerrero     

Inhibition of the hepatocyte uptake of radiolabelled monoclonal antibodies by chelating agents

The imaging of small abdominal tumours with indium 111 labelled monoclonal antibodies (MAbs) is often obscured by the uptake of activity into the hepto cytes of normal liver tissue. A model has therefore been developed to analyse reagents which may inhibit the he patocyte uptake of¹¹¹In-MAb whilst preserving tumour uptake. Primary rat hepatocyte cultures and an epithelial membrane antigen (EMA) expressing tumour cell line (MCF7), recognised by the EMA-specific MAb ICR2, were obtained in tissue culture. Monolayers of both cells were incubated with the¹¹¹In-MAb with or without the additional reagents and the cell uptake then measured and expressed per milligram of cell protein using a Lowry protein assay. No preferential reduction in hepatocyte uptake was noted by incubating cells with either saturated or unsaturated transferrin. The chelating agent, diethylene triamine penta-acetic acid (DTPA), however, significantly reduced the uptake of activity in hepatocytes but not the tumour cell line (TP<0.05). An optimum concentration and time period for incubating DTPA with labelled MAb was established. The mean hepatocyte uptake was reduced by 80% with a 1 h incubation with 1 mM DTPA. These results suggest that DTPA may have a role in reducing the liver uptake of radioactivity in patient studies using¹¹¹In-MAb.This work was presented at the Society of Nuclear Medicine annual meeting, St. Louis, USA, July 1989     

A new approach to image subtraction in immunoscintigraphy: Preliminary results

Recently proposed image comparison software is applied to immunoscintigraphy. The software performs geometric and gray level registration of two images and generates an image of the statistically significant differences. It permits the comparison of scintigraphic images recorded at different times. It is used to subtract ^113mIn and ¹¹¹In-phytate colloid liver scans and early (blood pool) images from ¹³¹I or ¹¹¹In-monoclonal Ab images and to compare Ab images recorded at different times. Using the procedures made possible by this software, only images recorded using the same radionuclide or using radionuclides of about the same energy are compared. Anti CEA, 19-9 and OC 125 F(ab)2 fragments labeled with ¹³¹I or ¹¹¹In are used in 32 patients with 47 demonstrated recurrences or metastases of colorectal or ovarian cancers. The overall sensitivities of the unprocessed and processed images are 25/47 and 41/47 respectively. The improvement in sensitivity is particularly high in the liver when In labelled Ab are used. This technique improves the contrast of the images, but the interpretation must take into account the components of the non target activity (kidney, bone marrow, colon...) which are not removed by the image subtraction method.     

European multicentre study on melanoma immunoscintigraphy by means of99mTc-labelled monoclonal antibody fragments

A total of 493 melanoma patients were investigated by 20 European nuclear medicine departments by means of the saine^99mTc-labelled immunoradiopharmaceutical and the same immunoscintigraphy (ISG) protocol. (i) No chemical or clinical toxicity was detected during or following the studies. (ii) Positive results were obtained in 287/363 (79%) patients (321 carrying known lesions and 42 carrying previously occult lesions): in 231 (80%) of them, 402/402 lesions were imaged, in the remaining 56 ISG-positive patients, 108/204 lesions were imaged; in 76 patients 0/122 lesions were ïmaged. (iii) The fraction of melanoma lesions visualized by ISG was 510/728 (70A%); 605 of these lesions were already documented at the time of the study, and 123 were previously occult. (iv) A total of 218 documented melanoma lesions (30%) were not visualized by ISG in 132 patients: about 70% of the ISG-negative lesions were of small size (less than 2 cm diameter). (v) The melanoma nature of 69/123 previously occult lesions was confirmed by clinical criteria and/or additional investigations in follow-up studies. The results obtained in this study are similar to those obtained in the Italian Multicentre Study which had previously been carried out with 258 melanoma patients.     

Human breast tumor imaging using 111In labeled monoclonal antibody: Athymic mouse model

The monoclonal antibody (MoAb) 323/A3, an IgG1, was raised against the human breast tumor cell line MCF-7 and recognized a 43 Kd membrane associated glycoprotein. Histochemical studies with the antibody detected 75% of metastatic lymph nodes, 59% of primary breast tumors, and showed some staining in 20% of benign breast lesions. For radionuclide imaging, the MoAb 323/A3 was labeled with both ¹²⁵I and ¹¹¹In, via covalently coupled diethylenetriaminepentaacetic acid (DTPA) by the mixed anhydride method. The antibody activity of the DTPA modified 323/A3 was assessed by an immunoassay using viable and fixed MCF-7 target cells. Male athymic nude mice bearing BT-20 human mammary tumors were injected with dual ¹²⁵I/¹¹¹In labeled DTPA 323/A3 via the tail veins. The animals were imaged with a gamma camera equipped with a pinhole collimator at 1–3 h, 1, 2, 3, 4 and 5 days after the tracer administration. On day 5 or 6, the animals were killed, and the biodistribution of the radiotracers was determined for the blood, thyroid, heart, lungs, liver, spleen, kidneys, gastrointestinal tract and tumor. Target to blood ratio at 6 days for the ¹¹¹In tracer was 24:1 in the group with a mean tumor weight of 0.492 g, and 13:1 in another group with a mean tumor weight of 0.1906 g (day 5). However, the ¹²⁵I activity showed only 3.6:1 and 5.4:1 target to blood ratios in the corresponding groups. The larger tumors localized less ¹¹¹In tracer (27.13%±7.57% injected dose/g, Mean±SD) than the smaller tumors (52.75%±22.25% ID/g). Analysis of the gamma images showed that the maximum tracer concentration occurred in the tumors at about 2 to 3 days after intravenous tracer administration. The excellent tumor resolution observed with BT-20 tumors may be due to increased 43 Kd glycoprotein antigen density in this tumor cell line.     

Comparative imaging and biodistribution studies with an anti-CEA monoclonal antibody and its F(ab)2 and Fab fragments in mice with colon carcinoma xenografts

An IgG₁ mouse monoclonal antibody directed against CEA has been digested with papain to yield F(ab)₂ and Fab fragments. Following radioiodination, intact antibody and fragments showed specific binding to cells of a CEA-producing tumour, although the immune reactivities of the fragments were lower than that of intact antibody. Gamma scintigraphy of nude mice bearing CEA-producing human tumour xenografts and injected with ¹³¹I-labelled fragments showed earlier and superior imaging of tumours than did ¹³¹I-intact antibody, and this was most marked with the Fab fragment. Sequential dissection analyses showed that this was due to earlier and higher tumour-to-blood ratios with fragments than with intact antibody, but in absolute terms the degree of localization of both fragment types was significantly lower than that of intact antibody.     

Uptake of 99mTc labelled (Fab')2 fragments of monoclonal antibody 225.28S by a benign ocular naevus

Malignant melanoma is one of the most common primary intraocular neoplasms. Recently, ^99mTc radiolabelled (Fab')₂ fragments of monoclonal antibody 225.28S raised against cutaneous melanomas have been used for imaging uveal melanomas. We report here a case where uptake of radiolabelled antibody was observed in a choroidal melanoma of the right eye and a benign choroidal naevus of the left.     

In vivo labelling of granulocytes with 99mTc anti-NCA monoclonal antibodies for imaging inflammation

We looked for inflammatory lesions in 45 patients using in vivo labelled granulocytes. For the purposes of cell labelling we used a murine, monoclonal antibody reacting with NCA and CEA (BW 250/183) (Bosslet et al. 1985) labelled with ^99mTc-pertechnetate. All abscesses and other inflammatory lesions were visualized with excellent quality scintigrams between 2 and 6 h after the injection. As the antibody can be stored in a freeze-dried form and labelled at any time in any Department of Nuclear Medicine with ^99mTc-pertechnetate, without cell isolation being necessary, the method appears to be suitable even for use in acute diagnosis. No side-effects have so far occurred, even in patients injected up to three times.     

抗河流弧菌单克隆抗体的建立及初步鉴定

目的 制备高效、特异的抗河流弧菌单克隆抗体(monoclonal antibody,mAb),为海洋致病细菌的快速免疫诊断提供技术支持.方法 选用雌性BALB/c小鼠(8周龄)制备成为灭活河流弧菌免疫小鼠,利用杂交瘤细胞融合技术,制备出抗河流弧菌杂交瘤细胞株,用Giemsa染色对其进行染色体鉴定,并用酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)方法筛选其与河流弧菌及其他重要海洋细菌的交叉反应特异性.结果 共获得4株抗河流弧菌mAb,均符合杂交瘤细胞染色体特点,具有良好的特异性和免疫反应性,分别命名为01H10,02F12,05F3和03G10.结论 本研究制备、筛选出抗河流弧菌的mAb,有助于建立抗河流弧菌快速免疫检测试剂盒.     

Differences in tumour and normal tissue concentrations of iodine- and indium-labelled monoclonal antibody.

The poor imaging characteristics of ¹³¹I have resulted in the use of alternative radionuclides for radiolabelling monoclonal antibodies. Clinical imaging studies have shown that, in addition to the more suitable energy of emission of ¹¹¹In over ¹³¹I for gamma camera detection, the ¹¹¹In-labelled antibody appears to clear from the blood-stream at a faster rate than that of ¹³¹I-labelled antibody, resulting in greater tumour-to-background image contrast.However, measurements of the activity in blood samples from patients demonstrate that both ¹³¹I- and ¹¹¹In-labelled anitbodies clear from the circulation at similar rates. This discrepancy is probably due to the different biological fates of the two radionuclides and warrants further scientific study.     

Detection of deep venous thrombosis with indium 111-labelled monoclonal antibody against tissue plasminogen activator

The administration of a radiolabelled monoclonal antibody against tissue plasminogen activator allows detection of areas with increased fibrinolytic activity, i. e. those with an active thrombotic lesion. Eight patients with phlebographically verified deep venous thrombosis were examined. At the time of immunoscintigraphy study they were examined receiving anticoagulant therapy. Some 75–85 MBq indium 111-labelled antibody were injected, and scintigrams were obtained after 30 min and after 24 h. The precise site of the thrombus could not be visualized after 30 min due to high background activity, whereas after 24 h it was detectable in all patients. The thrombus/background ratios achieved are twice as high as those observed in a human antifibrin antibody study. These preliminary data suggest a high sensitivity of our t-PA-specific antibody for the detection of active deep venous thrombosis in man, and our antibody seems to offer theoretical advantages over both platelet and fibrin-specific antibodies.     

Localization of melanoma with radiolabelled monoclonal antibody fragments and iodoamphetamine

In two melanoma patients, metastases accumulated both ^99mTc-labelled monoclonal anti-tumor F(ab)₂ fragments and N-isopropyl-p-(¹²³I)-iodoamphetamine. Small metastatic deposits were localized only by labelled antibody, for which a higher target-to-nontarget ratio was observed than for radioiodoamphetamine, indicating that immunoscintigraphy may be the more sensitive method. In these two patients positive immunohistochemical staining for the antibody used was observed, whereas in a third patient, with no concentration of labelled antibody, the staining result was negative showing the specificity of the immunoscintigraphy findings. It is possible that the accumulation of radio-iodoamphetamine is due to binding to melanin but this is not certain as tissue samples from one of the two patients with positive scintigrams did not contain stainable melanin.     

Detection of thyroid tumour using a monoclonal 123I anti-human thyroglobulin antibody

A monoclonal antibody to human thyroglobulin was radiolabelled with ¹²³I NaI and shown to be a stable and biologically active reagent in vivo. When injected intravenously into 12 patients with cancer of the thyroid on thyroxine-replacement therapy, 6 of the 12 patients had localization of the labelled antibody in tumour sites. These results were compared to ¹³¹I scans done on the same patients 1 month after stopping thyroxine. The biological half-life of the antibody in the blood was influenced by the levels of circulating thyroglobulin.     

In vivo imaging of rat lymphocytes with an indium 111-labelled anti-T cell monoclonal antibody: a comparison with indium 111-labelled lymphocytes

An indium 111-labelled mouse anti-rat T cell monoclonal antibody, MRC OX-19, was injected intravenously into rats to establish the usefulness of radiolabelled anti-lymphocyte antibodies in imaging lymphoid tissues. Antibody binding in vivo, measured by immunofluorescence analysis of cell suspensions made from lymphoid tissues, was detectable on lymphocytes in blood, spleen and lymph nodes. The extent of binding was time and antibody-dose dependent. Doses of antibody above 80 g/kg body weight resulted in modulation, i.e. loss of CD 5 (T 1) molecules from the cell surface, although the cells remained in the circulation. Modulation was demonstrable within 2 h and for at least 24 h after a single injection of antibody. Intravenous injection of¹¹¹In-MRC OX-19 resulted in levels of in vivo binding comparable with those seen with unlabelled antibody. Scintillation imaging showed early splenic localisation persisting over 48h, a more gradual localisation in the lymph nodes seen clearly at 24 h and a steady background. Comparison of the in vivo distribution of labelled antibody and¹¹¹In-tropolone-labelled lymphocytes showed that both could be used for external imaging of lymphocytes by scintillation camera.     

Immunoscintigraphic localization of inflammatory lesions: Clinical experience

This clinical study was based on the experimental results reported in the two preceding papers, showing that the highly selective affinity of the¹²³I-anti-CEA monoclonal antibody 47 (¹²³I-Mab_gc) for human granulocytes makes this compound suitable for the immunoscintigraphic detection of inflammatory lesions. Forty five patients with suspected infections have been studied after infusion of 4 mCi (148 MBq)¹²³I-Mab_gc corresponding to 120 μg labeled protein. No adverse reactions have been seen. Because of the high number of labeled cells, the quality of the images was excellent. SPECT was performed in 15 cases in order to define the extent of the lesion. Infectious foci were usually seen 3-5 h postinjection, but the unimpaired function of the granulocytes guarantees diagnostically relevant examinations over a much longer period of time. Scans were read as being negative if no pathological accumulation of activity was detected after 24 h. The new scanning method is technically easy to perform and provides distinct advantages over other techniques necessitating in vitro labeling of the white blood cells. Therefore, recommended indications are acute infections of unknown origin or extent, especially recurrent episodes of osteomyelitis and infections of joint prostheses.