抗登革病毒单抗的独特型抗体的制备与鉴定

从19种抗登革病毒单克隆抗体(简称单抗)中筛选出4种具有中和活性的单抗。经过纯化免疫新西兰大白兔制备多克隆抗独特型抗体(Ab2)。制备的4种抗独特型抗体血清有很高的特异性,其中具有全交叉反应的3种单抗的抗血青中Ab2的水平较高。采用简便的去除抗血清中的同种型和同种异型抗体的方法,用夹心ELISA法检测Ab2的水平取得了满意的结果。     

Labeling of monoclonal antibodies with radionuclides

Antibodies, specifically monoclonal antibodies, are potentially very useful and powerful carriers of therapeutic agents to target tissues and diagnostic agents. The loading or charging of antibodies with agents, especially radiotracers, is reviewed here. The choice of radioisotope for immunodetection and/or immunotherapy is based on its availability, half-life, nature of the radiation emitted, and the metabolic pathways of the radionuclide in the body. Most important of all are the derivatization techniques available for labeling the antibody with the given radionuclide. Isotopes of iodine and divalent metal ions are the most commonly used radionuclides. Antibodies labeled with iodine at tyrosine residues are metabolized rapidly in vivo. This leads to the incorporation of metabolized radioactive iodine into various tissues, mainly the thyroid gland and stomach, and to the accumulation of high levels of circulating iodine in the blood, which masks tumor uptake considerably. To overcome these limitations, the use of iodohippurate as an iodine-anchoring molecule to the protein should be considered. When divalent or multivalent metal ions are used as the preferred radionuclide, bifunctional chelating reagents such as ethylenediaminepentaacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA) are first coupled to the protein or antibody. These chelating molecules are attached to the protein by formation of an isopeptide linkage between the carboxylate of the chelating reagent and the amino group of the protein. Several procedures are available to generate the isopeptide linkage. When the anchoring of the chelating agent through isopeptide linkage results in the inactivation of the antibody, periodate oxidation of the carbohydrate moiety of the antibody, followed by reductive coupling of chelator, could be considered as an alternative. There is still a need for better, simpler, and more direct methods for labeling antibodies with radionuclides.     

Labelling monoclonal antibodies with yttrium-90

Using the cyclic DTPA derivatisation procedure developed by Hnatowich, conditions have been optimised for labelling three tumour-associated monoclonal antibodies with 90Y. High labelling efficiencies (greater than 95%) at modest specific activities (1 mCi/mg) could be routinely obtained. Radiochemical stability of the radiolabelled preparations in vitro was good, but radiolysis resulted in early losses of antibody immunoreactivity.     

Quantitative SPECT of uptake of monoclonal antibodies

Absolute quantitation of the distribution of radiolabeled antibodies is important to the efficient conduct of research with these agents and their ultimate use for imaging and treatment, but is formidable because of the unrestricted nature of their distribution within the patient. Planar imaging methods have been developed and provide an adequate approximation of the distribution of radionuclide for many purposes, particularly when there is considerable specificity of targeting. This is not currently the case for antibodies and is unlikely in the future. Single photon emission computed tomography (SPECT) provides potential for greater accuracy because it reduces problems caused by superimposition of tissues and non-target contributions to target counts. SPECT measurement of radionuclide content requires: (1) accurate determination of camera sensitivity; (2) accurate determination of the number of counts in a defined region of interest; (3) correction for attenuation; (4) correction for scatter and septal penetration; (5) accurate measurement of the administered dose; (6) adequate statistics; and (7) accurate definition of tissue mass or volume. The major impediment to each of these requirements is scatter of many types. The magnitude of this problem can be diminished by improvements in tomographic camera design, computer algorithms, and methodological approaches.     

Labelling monoclonal antibodies with Yttrium-90

Using the cyclic DTPA derivatisation procedure developed by Hnatowich, conditions have been optimised for labelling three tumour-associated monoclonal antibodies with ⁹⁰Y. High labelling efficiencies (>95%) at modest specific activities (1 mCi/mg) could be routinely obtained. Radiochemical stability of the radiolabelled preparations in vitro was good, but radiolysis resulted in early losses of antibody immunoreactivity.Abbreviations PBS Phosphate buffered saline - BSA Bovine Serum Albumin - HSA Human Serum Albumin - ITLC Instant thin layer chromatography - HPLC High pressure liquid chromatography - CAE Cellulose acetate electrophoresis - ELISA Enzyme linked immunosorbent assay - RIA Radioimmune assay - DTPA Diethylenetriaminepentaacetic acid     

Considerations for tomographic imaging of monoclonal antibodies

Monoclonal antibodies have begun to assume a significant role in clinical research. The ability to label these agents has initiated research in the areas of radioimmunodetection and radioimmunotherapy. In the case of antibodies directed against tumor antigens, imaging has been employed to help assess location and extent of disease, and to provide information and extent of disease, and to provide information concerning biodistribution to be used in subsequent dosimetric calculations. Because of the low counting statistics characteristic of such images, the use of single photon emission computed tomography (SPECT) is suggested as a potential method of improving the diagnostic yield from image data. Careful attention to acquisition parameters and image processing options is needed if these goals are to be achieved.     

Radiolabeled monoclonal anti-tumor antibodies in diagnosis and therapy

Clinical work with radiolabeled anti-tumor antibodies has made remarkable progress in the past few years. Still, there is much to be done before these new reagents can have a substantial impact on the practical management of patients. In this discussion, the properties of an "ideal" radiolabeled antibody and important factors for in vivo localization in tumors are reviewed. Potential approaches to improving the localization of currently available "tumor specific" monoclonal antibodies are discussed and examples of patients examined and treated with this method are presented. Experience to date suggests that within the foreseeable future, radiolabeled antibody techniques will become a "genuinely decisive technology".     

破伤风毒素单克隆抗体的制备及鉴定

目的 一株抗破伤风毒素鼠源单克隆抗体的制备与鉴定.方法 从实验室前期以破伤风类毒素为抗原筛选得到的6株稳定细胞株中选取4E12进行制备及鉴定,首先将4E12细胞株扩大培养后腹腔注射BALB/c小鼠制备腹水,经Protein G柱和PD10脱盐柱两步纯化鼠源单抗;其次通过SDS-PAGE电泳检测抗体纯度并通过分型试剂盒鉴定抗体轻重链亚型,免疫印迹法鉴定抗体结合区域及表位类型,非竞争ELISA法测定抗体亲和力常数以及小鼠中和实验评价抗体中和活性.结果 获得能稳定分泌抗体的杂交瘤细胞株4E12,其细胞培养上清效价为4×103,抗体轻重链分别为κ型和IgG1型,CDR3序列分别为SQSTHVPPT和ARKGTGTGFNY;4E12腹水抗体效价为3.2×104,抗体经纯化后纯度＞95％,以线性表位与TeNT/Hc结构域特异性结合,两者的亲和力常数为3.6×10-10 mol/L,小鼠中和实验结果显示,4E12抗体可部分中和破伤风毒素.结论 筛选到一株高亲和力的鼠源单抗,为破伤风毒素检测及中和抗体研发奠定了基础.     

Myelotoxicity and RBE of 211At-conjugated monoclonal antibodies compared with 99mTc-conjugated monoclonal antibodies and 60Co irradiation in nude mice.

The rationale of this study was to determine the myelotoxicity in nude mice of the alpha-emitter 211At conjugated to monoclonal antibodies (mAbs) and to compare the effect with an electron emitter, (99m)Tc, and external irradiation from a 60Co source, for estimation of the relative biological effectiveness (RBE). METHODS: 211At and (99m)Tc were conjugated to the IgG1 mAbs MX35 and 88BV59. Nude female BALB/c mice, 8- to 12-wk old, were injected intraperitoneally or intravenously. The biodistribution was determined 3, 6, and 18 h after injection. The bone-to-blood and bone marrow-to-blood activity concentration ratios (BBLR and BMBLR, respectively) were determined for simultaneously injected 211At- and (99m)Tc-mAbs. Bone marrow samples were taken from the femur. For each mouse, the whole-body retention was measured as well as the blood activity by repeated blood samples from the tail vein (0), 1, 3, 6, 12, and 18 h after injection. External-beam irradiation from a 60Co source was also performed at 3 different dose levels. White blood cell (WBC) counts, red blood cell counts, platelet counts, and hemoglobin were determined for each mouse initially and on days 1, 4, 5, 7, 15, 22, and 27 after injection. The calculations of the absorbed dose to the bone marrow were based on the BBLR, BMBLR, the cumulated activities, and the absorbed fractions. The absorbed fractions, phi, for alpha-particles and electrons in the bone marrow were calculated using Monte Carlo simulations based on a bone marrow dosimetry model. RESULTS: The BMBLR was 0.58 +/- 0.06 and 0.56 +/- 0.06 for the 211At- and (99m)Tc-mAbs, respectively. No significant variation in BMBLR with time was found. The absorbed fractions for alpha-particles and electrons in the bone marrow were 0.88 and 0.75, respectively. The mean absorbed fractions of the photons from (99m)Tc were 0.033 and 0.52 for 140 and 18.3 keV, respectively. When different amounts of 211At- and (99m)Tc-mAbs (0.09-1.3 and 250-1,300 MBq, respectively) were administered intraperitoneally or intravenously, corresponding to absorbed doses to the bone marrow of 0.01-0.60 and 0.39-1.92 Gy, respectively, the WBC counts was suppressed by 1%-90% and 23%-89%, respectively. When external-beam irradiation with a 60Co source was performed to absorbed doses of 1.4, 1.9, and 2.4 Gy, the WBC counts was suppressed by 47%-90%. These results indicate a myelotoxic in vivo RBE of 3.4 +/- 0.6 for alpha-particles compared with (99m)Tc and 5.0 +/- 0.9 compared with 60Co irradiation. CONCLUSION: The effect on the WBC counts from bone marrow irradiation with 211At-mAbs indicates an in vivo RBE of 3.4 +/- 0.6 in comparison with (99m)Tc-mAbs. The RBE value compared with external irradiation is 5.0 +/- 0.9.     

Site-specifically 89Zr-labeled monoclonal antibodies for ImmunoPET

Three thiol reactive reagents were developed for the chemoselective conjugation of desferrioxamine (Df) to a monoclonal antibody via engineered cysteine residues (thio-trastuzumab). The in vitro stability and in vivo imaging properties of site-specifically radiolabeled ⁸⁹Zr-Df-thio-trastuzumab conjugates were investigated.MethodsThe amino group of desferrioxamine B was acylated by bromoacetyl bromide, N-hydroxysuccinimidyl iodoacetate, or N-hydroxysuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate to obtain thiol reactive reagents bromoacetyl-desferrioxamine (Df-Bac), iodoacetyl-desferrioxamine (Df-Iac) and maleimidocyclohexyl-desferrioxamine (Df-Chx-Mal), respectively. Df-Bac and Df-Iac alkylated the free thiol groups of thio-trastuzumab by nucleophilic substitution forming Df-Ac-thio-trastuzumab, while the maleimide reagent Df-Chx-Mal reacted via Michael addition to provide Df-Chx-Mal-thio-trastuzumab. The conjugates were radiolabeled with ⁸⁹Zr and evaluated for serum stability, and their positron emission tomography (PET) imaging properties were investigated in a BT474M1 (HER2-positive) breast tumor mouse model.ResultsThe chemoselective reagents were obtained in 14% (Df-Bac), 53% (Df-Iac) and 45% (Df-Chx-Mal) yields. Site-specific conjugation of Df-Chx-Mal to thio-trastuzumab was complete within 1 h at pH 7.5, while Df-Iac and Df-Bac respectively required 2 and 5 h at pH 9. Each Df modified thio-trastuzumab was chelated with ⁸⁹Zr in yields exceeding 75%. ⁸⁹Zr-Df-Ac-thio-trastuzumab and ⁸⁹Zr-Df-Chx-Mal-thio-trastuzumab were stable in mouse serum and exhibited comparable PET imaging capabilities in a BT474M1 (HER2-positive) breast cancer model reaching 20–25 %ID/g of tumor uptake and a tumor to blood ratio of 6.1–7.1.ConclusionsThe new reagents demonstrated good reactivity with engineered thiol groups of trastuzumab and very good chelation properties with ⁸⁹Zr. The site-specifically ⁸⁹Zr-labeled thio-antibodies were stable in serum and showed PET imaging properties comparable to lysine conjugates.     

Immunoreactivity assay for labeled anti-melanoma monoclonal antibodies

A convenient, rapid, and reproducible assay was developed to evaluate the immunoreactivity of radiolabeled monoclonal antibodies against three different human melanoma-associated antigens, p97, a proteoglycan and a GD3 ganglioside. A cloned melanoma cell line (M 2669 CL 13) was selected as the target and, when fixed with paraformaldehyde, showed binding as good as or better than that obtained with live cells for the three antigens. Fixed cells retained good binding properties stored at 4 degrees C for over 6 mo. This assay has general applicability to other antigen-antibody systems for testing chemically modified monoclonal antibodies or fragments during the development of a radiopharmaceutical or as a routine quality control measure for clinical agents.     

Breast cancer imaging with mouse monoclonal antibodies

The localization of 111In-labelled MA5 monoclonal antibody, reactive with a breast tumor associated antigen, was studied in 17 patients. MA5 was selected because 1) it reacts with greater than 95% of primary and metastatic lesions, 2) the recognized antigen is present on the cell surface in vivo and 3) MA5 gives excellent localization in human breast tumor xenografts. Each patient received 2 mg antibody labeled with 5 mCi 111In and in some cases, 3 mg or 18 mg unlabeled carrier antibody. No serious allergic reactions were noted. There was a large uptake in the liver, less significant uptake in the spleen and bone, and minimal accumulation in the bowel. Bone lesions, primary tumors, soft tissue recurrences and lung metastases larger than 3 cm diameter were imaged, while only 1 lesion smaller than 3 cm was detected. Non specific accumulation of tracer was noted at the site of a port-a-cath, in a hematoma, in fibrocystic lesions, and at sites of previous radiation treatment. Extensive fibrosis and poor vascularization characteristic of breast tumors may explain in part the limited sensitivity of the imaging.