Radioimaging of human malignant gliomas using indium-labelled monoclonal antibodies

A monoclonal antibody (MUC 2-63) was raised against a neuroectodermal antigen expressed on human malignant gliomas, neuroblastomas and melanomas. The mouse monoclonal antibody MUC 2-63 was of IgG1 isotype, it binds the antigenic determinants with the molecular weight of 32,000 Dalton present on the cell surface of native glioma cells. Seven patients with brain tumours, five with biopsy proven malignant gliomas, received an intravenous injection of the 111In-DTPA coupled monoclonal antibody MUC 2-63. Six patients had an uptake of 0.01-0.04% of the injected dose at the site of the tumour, which correlated to the computerized tomography (CT) images. The half-lives in the tumours of 111In were 38-259 h. The maximal uptake in the tumours were noticed between 40 and 67 h. One patient failed to demonstrate any intracranial uptake of the radioactivity. This patient had been treated with surgery and radiotherapy for a stage 2 testicular seminoma four years ago. He recently developed clinical signs of a primary brain tumour and the CT diagnosis was malignant glioma. All patients had nonspecific uptake in the liver, half-lives were between 60 and 84 h, the corresponding maximal uptake was detected between 22 and 45 h. No side effects were observed by administration of the mouse monoclonal antibody MUC 2-63.     

A rat model for imaging the effect of anti mouse antibody responses on the biodistribution of radiolabelled mouse monoclonal antibodies

Rats were injected both intradermally and intravenously with an IgG2b mouse monoclonal antibody (791T/36) and subsequently the biodistribution of intravenously injected 111In labelled antibody was examined by gamma camera imaging in these and control rats. The majority of pretreated rats showed a marked perturbation of the biodistribution of the radiolabelled antibody with a marked increase of the tracer in the liver. There was similar perturbation of the biodistribution of subsequently administered 111In labelled Fab fragment of the 791T/36 antibody and of another IgG2b monoclonal antibody. In contrast, there was little influence on the biodistribution of IgG2a or IgG1 monoclonal antibodies, indicating that the response in the rats was predominantly against the IgG2b isotype. This rat model system is amenable to examination of a number of aspects of the biological consequences of immune responses to foreign immunoglobulins relevant to their use for clinical imaging.     

A rapid method for the determination of human CEA/mouse anti-CEA immune complexes in patients undergoing immunoscintigraphy

We report here on a new and quick procedure to isolate human (hu) CEA mouse anti CEA immune complexes of sera from patients admitted for immunoscintigraphy with radiolabeled monoclonal anti CEA antibody. This method employs rabbit anti mouse IgG immune affinity chromatography together with a commercial CEA-IRMA kit for the specific isolation of immune complexes. By applying this procedure we were able to show that immune complex formation increased in parallel to CEA serum concentration. The formation of immune complexes did not significantly affect tumor detection by immunoscintigraphy.     

Falsely elevated results of radioimmunoassays using double antibody method: arguments for a third anti-rabbit IgG antibody present in certain human sera

Nine sera with falsely elevated results in a TSH radioimmunoassay were studied. Sera dilution and gel filtration showed that the immunoreactive TSH was different from the standard and labeled TSH. It coeluted with the immunoglobulin fraction of the serum. Rabbit serum decreased or emphasized the artifact according to the dose. Falsely elevated results appeared only in radioimmunoassays using a double antibody method for the separation of free and bound labeled hormone. FSH, LH, and beta HCG radioimmunoassays using the double antibody method underwent the same disturbance as the TSH assay in these patients. Our results suggest that the artifact may be due to the presence in certain human sera of anti-rabbit IgG antibodies, interfering in the radioimmunoassay by inhibition of the binding of first and second antibodies.     

间接血凝法检测抗Sm抗体和抗RNP

应用间接血凝法测定抗Sm抗体和抗RNP抗体。对不同疾病的检测结果表明,我们建立的这种方法与免疫电泳法结果无显著性差异(P〉0.05)。对SLE病45例检测抗Sm抗体阳性率为100％,抗RNP抗体阳性率为91％。抗Sm抗体可作为SLE的特异性标志抗体,抗RNP抗体在RA,PSS,DM,MCTD病人中检出,其中MCTD病23例,抗RNP抗体阳性率高达96％,且凝集效价高,可作为此病诊断的重要依据。     

日本人群中HEV感染的血清学调查

应用纯化的ＨＥＶ（８７Ａ株）热灭活抗原进行ＥＬＩＳＡ试验，测定日本大阪地区收集的１４９例各种肝病患者急性血清中的抗ＨＥＶ抗体、获得１９份阳性（阳性率为１２．７％），其中单纯抗ＨＥＶ ＩｇＭ阳性１１份和抗ＨＥＶ ＩｇＧ阳性６份。两者都阳性的２份。     

A rapid method for the determination of human CEA/mouse anti-CEA immune complexes in patients undergoing immunoscintigraphy

We report here on a new and quick procedure to isolate human (hu) CEA mouse anti CEA immune complexes of sera from patients admitted for immunoscintigraphy with radiolabeled monoclonal anti CEA antibody. This method employs rabbit anti mouse IgG immune affinity chromatography together with a commercial CEA-IRMA kit for the specific isolation of immune complexes. By applying this procedure we were able to show that immune complex formation increased in parallel to CEA serum concentration. The formation of immune complexes did not significantly affect tumor detection by immunoscintigraphy.Abbreviations CEA Carcinoembryonic antigen - DTE Dithioerythritol - EDTE Ethylenediaminetetraacetate - PMSF phenylmethylsulfonyl flouride - IRMA immunoradiometric assay - p.i. post injectionem Deceased     

Isotype switch variant anti-idiotype monoclonal antibodies: comparative radiolabeling and in vitro binding

The influence of antibody isotype on radiolabeling efficacy and immunoreactivity has been difficult to systematically examine as antibodies of different isotypes generally vary in both constant and variable regions. The recent ability to isolate class or isotype switch monoclonal antibodies that have identical binding regions but different constant regions allows for such a comparison to be undertaken. Two isotype switch variant families of murine anti-idiotypic monoclonal antibodies were studied for radioiodination efficacy and immunoreactivity following labeling. In one of the families (S3H5), the IgG2a isotype switch variant derived from an IgG2b parent had nearly 70% greater iodine incorporation and over 50% greater immunoreactivity than the IgG2b parent. In the other family (S5A8), the IgG2a isotype switch variant had virtually identical efficacy of iodine incorporation and binding to antigen after labeling as did its IgG2b parent. Differences in relative heavy and light chain iodine incorporation were seen among isotype switch variants and their parents regardless of alterations in quantitative iodine incorporation or immunoreactivity. We conclude that in certain instances, cloning of an isotype switch variant antibody can result in an antibody offspring that has superior radiolabeling characteristics to its parent antibody. This isotype switching approach may find utility in converting highly-specific, but difficult to label antibodies, to more useful agents.     

纤维连接蛋白降解片段（MAD2）单抗的制备及肝细胞癌患者血浆含量检测

应用杂交瘤技术制备了纤维连接蛋白（FN）降解片段MAD2的单克隆抗体（McAb)；建立了ＭＡＤ２检测的双抗体夹心ＥＬＩＳＡ法；对２２７例肝细胞癌（ＨＣＣ）、７６例肝转移癌（ＨＭＣ）、９８例消化道癌（ＡＣＣ）、１５６例慢性肝病（ＣＬＤ）患者和４８例健康人体血浆ＭＡＤ２含量进行测定。结果显示，制备的ＭＡＤ２ ＭcAb属ＩgG1，与ＦＮ无交叉反应；ＨＣＣ组血浆ＭＡＤ２含量均值与ＣＬＤ组、ＨＭＣ组、ＡＣＣ组和正常对照组比较差异有显著性意义（分别Ｐ＜0.01）,以HCC患者的最低MAD2值作临界值时，仅13.5%CLD、6.6%HMC和4.1?C超过此值，而健康人体血浆MAD2含量均低于临界值；65例血清AFP正常的HCC患者其血浆平均MAD2值仍明显高于非HCC肿瘤、CLD患者和正常对照。结合以前的结果，进一步表明，血浆MAD2检测可作为HCC诊断的新标志物，并可与AFP相互补充；ＭＡＤ２ ＭcAb的制备使MAD2的检测变得易行。     

Phase I trial of iodine-131-chimeric B72.3 (human IgG4) in metastatic colorectal cancer.

Twelve patients with metastatic colorectal cancer participated in a Phase I trial of 131I-labeled chimeric B72.3 (human IgG4). Consecutive groups of patients received 18 mCi/m2, 27 mCi/m2 and 36 mCi/m2. No acute side effects related to antibody administration were noted. Bone marrow suppression was the only side effect; it was dose-dependent and correlated with whole-body radiation dose estimates. The lowest dose level produced no marrow suppression, whereas 27 mCi/m2 resulted in Grade 1 and 2 marrow suppression in two of three patients. The maximum tolerated dose was 36 mCi/m2 with all six patients at this dose level having at least Grade 1 and two patients with Grade 3 and 4 marrow suppression. Eight of 12 patients had radioimmune imaging of tumor sites at 5-22 days. Seven patients had an antibody response to initial infusion. On retreatment, whole-body kinetics and imaging were altered for patients with a high anti-ch-B72.3 response. Thus, chimeric B72.3 (IgG4) has limited utility as a means of delivering multiple therapeutic doses of 131I in the majority of patients; alternative strategies including second generation anti-TAG-72 monoclonal antibodies, other radioisotopes and other chimeric human isotypes will need to be pursued.     

Gm typing by enzyme-linked immunosorbent assay (ELISA)

Summary A solid-phase ELISA for Gm typing is described. A mixture of anti-Gm serum (or monoclonal anti-Gm antibody) and test serum was incubated in microtiter wells coated with IgG or its fragments of appropriate Gm type. After washing of the wells, the bound antibody was detected with peroxidase-labeled second antibody. The Glm(3), G3m(16), and G3m(21) antigens could be identified by this technique. Since some of the human anti-Gm sera and anti-Rh₀ sera required for the conventional hemagglutination-inhibition method are hard to obtain, the ELISA system using anti-Gm antibodies and no anti-Rh₀ sera may serve as an alternative to the conventional method.Supported in part by grants-in-aid for Scientific Research, nos. 58480206 and 59570267, from the Ministry of Education, Science and Culture     

阿尔茨海默病多肽表位疫苗免疫原性研究

目的以淀粉样蛋白Aβ作为免疫靶标,优化阿尔茨海默病多肽B细胞表位疫苗。方法化学合成人Aβ1-42多肽、含B细胞表位的Aβ1-15多肽、含两个B细胞表位及一个辅助T细胞表位PADRE的多肽Aβ（1-15）2-PADRE及含13个氨基酸的PADRE。以上4种多肽免疫普通BALB/c小鼠,ELISA试验比较免疫后的小鼠血清抗体水平和亚型,点杂交分析其与不同状态Aβ的结合免疫特性。结果 Aβ（1-15）2-PADRE组免疫后诱导产生了良好的免疫效果,100μg组4次免疫后其抗体滴度可以达到1∶3500;抗体亚型分析后得知,其主要产生IgG1型抗体,免疫反应完全偏向于Th2型,其免疫血清可与不同状态Aβ的结合免疫特性不同。结论辅助T细胞表位PADRE增强了B细胞表位Aβ1-15的免疫效果,并且其免疫血清抗体与Aβ寡聚体结合效果更好。     

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.     

The humoral immune response to macrocyclic chelating agent DOTA depends on the carrier molecule.

The chelating agent 1,4,7,10-tetraazacyclododecane-N,N', N",N"'-tetraacetic acid (DOTA) is used to label monoclonal antibodies (mAbs) and peptides with (90)Y. DOTA allows the generation of clinically useful stable metallic radioconjugates for the treatment of a variety of tumors, but its immunogenicity has remained controversial. In this study, we evaluated the immune response to DOTA in a preclinical mouse model and in patients entered in a clinical trial. METHODS: Sera were obtained from BALB/c mice injected intraperitoneally or subcutaneously with different doses and formulations of syngeneic and xenogeneic mAbs or peptide (murine mAb Mov19 [mM19]; its chimeric version; murine V/human C ChiMov19 [cM19]; or Tyr(3)-octreotide)-DOTA conjugates. Sera from patients with neuroendocrine tumors, enrolled in a protocol for somatostatin receptor-mediated radionuclide therapy with (90)Y-DOTA-D-Phe(1)-Tyr(3)-octreotide (DOTATOC), were also collected before and after each treatment. Levels and specificity of antibody response to relevant (Mov19, ChiMov19, or Tyr(3)-octreotide) and nonrelevant (human serum albumin) DOTA targets were tested by enzyme-linked immunosorbent assay and competition assays. An anti-DOTA mAb (IgG1) derived from a ChiMov19-DOTA immunized mouse was used, in a competitive radioimmunoassay, to determine the efficiency of DOTA presentation on the different carriers. RESULTS: Depending on the immunogenicity and dosage of the mAb, a specific anti-DOTA response was revealed in the preclinical system. However, DOTA-peptide conjugate induced no immune-detectable response against either chelator or carrier. DOTA was poorly presented on small peptides, as determined using the anti-DOTA mAb. CONCLUSION: A humoral response against DOTA is possible, but only as a consequence of the response elicited against the carrier. Octreotide was not immunogenic. Thus, (90)Y-DOTATOC can be considered a safe and useful tool for receptor-mediated radionuclide therapy of somatostatin receptor-overexpressing tumors.     

Falsely elevated results of radioimmunoassays using double antibody method: Arguments for a third anti-rabbit IgG antibody present in certain human sera

Nine sera with falsely elevated results in a TSH radioimmunoassay were studied. Sera dilution and gel filtration showed that the immunoreactive TSH was different from the standard and labeled TSH. It coeluted with the immunoglobulin fraction of the seru. Rabbit serum decreased or emphasized the artifact according to the dose. Falsely elevated results appeared only in radioimmunoassays using a double antibody method for the separation of free and bound labeled hormone. FSH, LH, and HCG radioimmunoassays using the double antibody method underwent the same disturbance as the TSH assay in these patients. Our results suggest that the artifact may be due to the presence in certain human sera of anti-rabbit lgG antibodies, interfering in the radioimmunoassay by inhibition of the binding of first and second antibodies.     

Comparative biodistributions and rates of catabolism of radiolabelled anti-CEA monoclonal antibody and control immunoglobulin in nude mice with human tumour xenografts showing specific antibody localization

The rate of catabolism of a radiolabelled monoclonal anti-CEA antibody has been compared with that of control IgG1 in control nude mice and in mice with CEA producing xenografts and antigen negative xenografts. The rate of catabolism of antibody, but not of IgG, was increased 3-4 fold in mice with xenografts localizing the antibody, but not in mice with antigen negative tumours. There was no evidence of immune complex formation and/or clearance of antibody from the serum of xenografted mice and the current interpretation of these findings is that following tumour immune directed fixation, antibody is subsequently catabolized faster than in the general metabolic pool. The present data indicates that this is about six times as rapid as in the animal as a whole on a weight to weight tissue basis.     

Radioimaging of light chain amyloid with a fibril-reactive monoclonal antibody.

Currently, there are no available means in the United States to document objectively the location and extent of amyloid deposits in patients with systemic forms of amyloidosis. To address this limitation, we have developed a novel diagnostic strategy, namely, the use of a radiolabeled fibril-reactive murine monoclonal antibody (mAb) as an amyloid-specific imaging agent. The goal of this study was to determine the pharmacokinetics, biodistribution, and ability of this reagent to target the type of amyloid that is formed from immunoglobulin light chains, that is, AL. METHODS: Subcutaneous tumors (amyloidomas) were induced in BALB/c mice by injection of human AL fibrils. The IgG1 mAb designated 11-1F4 and an isotype-matched control antibody were radioiodinated, and the pharmacokinetics and localization of these reagents were determined from blood and tissue samples. Amyloidoma-bearing animals that received (125)I- or (124)I-labeled antibodies were imaged by whole-body small-animal SPECT/CT or small-animal PET/CT technology, respectively. RESULTS: Radioiodinated mAb 11-1F4 retained immunoreactivity, as evidenced by its subnanomolar affinity for light chains immobilized on 96-well microtiter plates and for beads conjugated with a light chain-related peptide. Additionally, after intravenous administration, the labeled reagents had the expected biologic half-life of murine IgG1, with monoexponential whole-body clearance kinetics. In the amyloidoma mouse model, (125)I-11-1F4 was predominately localized in the tumors, as demonstrated in biodistribution and autoradiographic analyses. The mean uptake of this reagent, that is, the percentage injected dose per gram of tissue, 72 h after injection was significantly higher for amyloid than for skeletal muscle, spleen, kidney, heart, liver, or other tissue samples. Notably, the accumulation within the amyloidomas of (125)I- or (124)I-11-1F4 was readily visible in the fused small-animal SPECT/CT or small-animal PET/CT images, respectively. CONCLUSION: Our studies demonstrate the amyloid-imaging capability of a radiolabeled fibril-reactive mAb and provide the basis for a clinical trial designed to determine its diagnostic potential in patients with AL amyloidosis and other systemic amyloidoses.     

Comparative tumour localization properties of radiolabelled monoclonal antibody preparations of defined immunoreactivities

The immunoreactive fraction of an anti-CEA monoclonal antibody preparation has been progressively decreased by the addition of increasing proportions of impurity in the form of immunologically inert mouse immunoglobulin. Following radioiodination, the immunoreactive fractions of the preparations were determined and their localization in a human tumour xenograft in nude mice was assessed. There was a progressive decline in tumour localization, from tumour to blood ratios of 2:1 with unadulterated antibody to 0.6:1 with preparations only 15% with respect to the initial antibody. These findings demonstrate that the immunoreactive fraction of monoclonal antibody preparations is a major limiting factor in tumour localization and this has implications for experimental and clinical applications of monoclonal antibodies.     

A phase I trial combining high-dose 90Y-labeled humanized anti-CEA monoclonal antibody with doxorubicin and peripheral blood stem cell rescue in advanced medullary thyroid cancer.

This trial determined the pharmacokinetics, dosimetry, and dose-limiting toxicity of 90Y-hMN-14 IgG (humanized anticarcinoembryonic antigen [CEA, or CEACAM5] monoclonal antibody; labetuzumab), combined with doxorubicin and peripheral blood stem cell (PBSC) support in advanced medullary thyroid cancer (MTC) patients. METHODS: Fifteen patients received an infusion of 111In-hMN-14 IgG. One to 2 wk later, 14 patients received 90Y-hMN-14 IgG, starting at 740 MBq/m2, followed 24 h later with a fixed intravenous bolus dose of doxorubicin (60 mg/m2). Preharvested PBSCs were reinfused when the 90Y activity in the body was < or =111 MBq/m2. RESULTS: The mean red marrow dose estimated for the 90Y-hMN-14 IgG was 1.65 +/- 0.59 mGy/MBq (n = 11), with normal organs ranging from approximately 2.3 to 4.4 mGy/MBq. Eighty percent of all known lesions (125/156), including 78 of 79 bone and 16 putatively occult lesions, were targeted. The average radiation dose to the tumor was 15.1 +/- 10.8 mGy/MBq (55.8 +/- 39.8 cGy/mCi) 90Y-hMN-4 IgG (n = 29 tumors in 8 patients), with a majority of the lesions receiving >2,000 cGy at an administered dose of < or =1,480 MBq/m2. The average tumor-to-red marrow, tumor-to-liver, tumor-to-lungs, and tumor-to-kidneys ratios were 15.0 +/- 11.0, 5.1 +/- 3.6, 6.9 +/- 6.1, and 9.0 +/- 8.7, respectively. Cardiopulmonary toxicity was dose limiting at 1,850 MBq/m2. Minor responses were noted in 2 patients and 1 patient had a partial response (68% reduction in local and hepatic metastatic disease). CONCLUSION: This treatment combination was well tolerated with complete recovery of blood counts and reversible nonhematologic toxicities at the maximum tolerated dose of 1,480 MBq/m2. Evidence of antitumor response in these patients with advanced cancer was modest, but encouraging; this type of treatment may be more successful if applied to more limited, earlier-stage disease.     

Comparative tumour localization properties of radiolabelled monoclonal antibody preparations of defined immunoreactivities

The immunoreactive fraction of an anti-CEA monoclonal antibody preparation has been progressively decreased by the addition of increasing proportions of impurity in the form of immunologically inert mouse immunoglobulin. Following radioiodination, the immunoreactive fractions of the preparations were determined and their localization in a human tumour xenograft in nude mice was assessed. There was a progressive decline in tumour localization, from tumour to blood ratios of 2:1 with unadulterated antibody to 0.6:1 with preparations only 15% with respect to the initial antibody. These findings demonstrate that the immunoreactive fraction of monoclonal antibody preparations is a major limiting factor in tumour localization and this has implications for experimental and clinical applications of monoclonal antibodies.