Distinct effects on splicing of two monoclonal antibodies directed against the amino-terminal domain of galectin-3

Previous experiments had established that galectin-3 (Gal3) is a factor involved in cell-free splicing of pre-mRNA. Addition of monoclonal antibody NCL-GAL3, whose epitope maps to the NH₂-terminal 14 amino acids of Gal3, to a splicing-competent nuclear extract inhibited the splicing reaction. In contrast, monoclonal antibody anti-Mac-2, whose epitope maps to residues 48-100 containing multiple repeats of a 9-residue motif PGAYPGXXX, had no effect on splicing. Consistent with the notion that this region bearing the PGAYPGXXX repeats is sequestered through interaction with the splicing machinery and is inaccessible to the anti-Mac-2 antibody, a synthetic peptide containing three perfect repeats of the sequence PGAYPGQAP (27-mer) inhibited the splicing reaction, mimicking a dominant-negative mutant. Addition of a peptide corresponding to a scrambled sequence of the same composition (27-mer-S) failed to yield the same effect. Finally, GST-hGal3(1-100), a fusion protein containing glutathione-S-transferase and a portion of the Gal3 polypeptide including the PGAYPGXXX repeats, also exhibited a dominant-negative effect on splicing.     

鼠单克隆抗-HBs的单克隆抗独特型抗体的制备及其免疫原性的研究 Ⅱ.Ab_2—1的免疫原性研究

用一株单克隆抗独特型抗体细胞株,Ab_2-1免疫4只BALB/c小鼠和2只家兔。每只小鼠每次经腹腔接种100#gAb_2-1,共接种4次;每只家兔每次经皮下注射1mgAb_2-1,共注射4次。经免疫的小鼠和家兔都产生了抗-HBs,经两种竞争性抑制试验证实,这些抗-HBs是特异性的。小鼠抗-HBs滴度为2~(-6)至2~(-9);家兔抗-HBs滴度从2~(-10)至2~(-12)。这些资料提示:单克隆抗独特型抗体能模拟HBsAg,具有类似HBsAg的免疫原性。     

变速上样在MabSelect填料上分离WLB303

随着抗体表达量的提升和生产规模的扩大,Protein A亲和层析不仅需要高载量填料,也需要提高工艺效率。变速上样的方法可以在满足载量要求的同时大大缩短工艺耗时。通过测定WLB303单克隆抗体在GE MabSelect填料多个保留时间的动态载量,建立一元三次方程拟合载量和保留时间的关系。以该方程计算获得填料在快、中、慢速下不同保留时间的动态载量表,并以此作为变速上样组合的参考依据。在2.7ml层析柱上使用精纯样品测试最快的变速上样组合的可行性;然后用常规纯化工艺在7ml层析柱上使用细胞培养澄清液,对最快的变速上样组合和恒速上样的两种工艺周期进行了比较,证实变速上样的方式能明显提升整体工艺效率。     

抗酸土脂环酸芽孢杆菌(Alicyclobacillus acidoterrestris)单克隆抗体制备

【背景】基于本实验室已经建立的脂环酸芽孢杆菌检测和鉴定方法工作基础之上,以期建立具有很好的经济价值和实用价值,且更为方便、快捷、准确、特异、灵敏的检测方法。【目的】实现对果汁生产中脂环酸芽孢杆菌从原料到成品的快速检测和鉴定。【方法】采用杂交瘤细胞技术,以Alicyclobacillus acidoterrestris(ATCC49025)免疫BALB/c小鼠,用建立的间接ELISA方法筛选杂交瘤细胞,得到了3株能稳定分泌A.acidocaldarius抗体的杂交瘤细胞株,其中两株为Ig G1亚类,并对其进行生物学特性的鉴定。【结果】得到的两株单抗3F7和9C4是针对不同的抗原位点,且多次传代后稳定性基本保持不变;特异性实验表明两株单抗均不与A.acidocaldarius(NCIMB11725)、Bacillus cereus(ATCC11778)、Bacillus subtilis(ATCC11774)、A.cycloheptanicus(ATCC49029)等发生交叉反应。【结论】3F7和9C4这两株单抗可以进一步用于检测胶体金试纸条的研制。     

Antitumor effects of a novel monoclonal antibody with high binding affinity to ganglioside GD3

Ganglioside GD3, which is one of the major gangliosides expressed on the cell surface human tumors of neuroectodermal origin, has been studied as a target molecule for passive immunotherapy. We established ten kinds of anti-GD3 monoclonal antibodies (mAb) of the mouse IgG3 subclass by immunization with purified GD3 and melanoma cells. One of the established mAb, KM641, showed major reactivity with GD3 and minor reactivity with GQ1b out of 11 common gangliosides in an enzymelinked immunosorbent assay. Immunostaining of gangliosides, separated on thin-layer chromatography plates, using KM641 revealed that most of the melanoma cell lines contained immunoreactive GD3 and GD3-lactone at a high level, but only the adrenal gland and the urinary bladder out of 21 human normal tissues had immunoreactive GD3. In immunofluorescence, KM641 bound to a variety of living tumor cell lines especially melanoma cells, including some cell lines to which another anti-GD3 mAb R24, established previously, failed to bind. High-affinity binding of KM641 to a tumor cell line was quantified by Scatchard analysis (K _d = 1.9×10^–8 M). KM641 exerted tumor-killing activity in the presence of effector cells or complement against melanoma cells expressing GD3 at a high level. Not only natural killer cells but also polymorphonuclear cells were effective as the effector cells in antibody-dependent cellular cytotoxicity. Intravenous injection of KM641 markedly suppressed the tumor growth of a slightly positive cell line, C24.22 (7.2×10⁵ binding sites/cell), as well as a very GD3-positive cell line, G361 (1.9×10⁷ binding sites/cell), inoculated intradermally in nude mice. KM641, characterized by a high binding affinity for GD3, has the potential to be a useful agent for passive immunotherapy of human cancer.     

Tumor labeling in vivo using cyanine-conjugated monoclonal antibodies

Far-red-emitting cyanine fluorochromes have many properties desirable for in vivo imaging: absorption and emission at wavelengths where blood and tissue are relatively transparent, high quantum yields, and good solubility even at high molar ratios of fluorochrome to antibody. Potentially, conjugation by multiple linkages should minimize hydrolysis in vivo. We conjugated two tumor-targeting monoclonal antibodies: anti-SSEA-1 (IgM, ) at ratios of 1.2–35 mol dye/mol antibody and 9.2.27 (IgG_2a, ) at 0.6–6 mol dye/mol antibody, using the cyanine fluorochromes Cy3.18, Cy5.18, and Cy5.5.18. Nude mice were inoculated using the SSEA-1-expressing MH-15 teratocarcinoma or the 9.2.27 antigen-expressing SK-MEL-2 melanoma to give tumors at several sites. Conjugated antibody was injected, and mice were imaged immediately after injection and at appropriate intervals thereafter using a standard camera lens, dissecting microscope, or endoscopes. Images were acquired using either an image-intensified video camera or cooled CCD cameras. Immediately after injection, major blood vessels and the heart, liver, and kidneys were readily visualized. After 1 day, tumor-targeting antibody conjugates were concentrated in tumors and there was little circulating conjugate; however, the bladder and kidneys were still visible. Tumors labeled by specific antibody were the most fluorescent tissues at 2 days after injection, but non-specific antibody conjugates did not concentrate in the tumors. The small intestine was weakly visualized by both specific and non-specific antibody conjugates. These data support the possibility of visualizing tumor metastasis by optical means, including currently available endoscopes.This work was supported by funds of the Division of Urologic Surgery, Department of Surgery, University of Pittsburgh; by an NSF Center Grant (MCB-8920118) to the Center for Light Microscope Imaging and Biotechnology, Carnegie-Mellon University; and by intramural funds of the VAMC     

Murjne Anti-Idiotypic Monoclonal Antibodies to Syngeneic Antihuman High Molecular Weight-Melanoma Associated Antigen Monoclonal Antibodies: Development,Characterization, and Clinical Applications

Following a discussion of the rationale underlying the selection of human melanoma to test the usefulness of anti-idiotypic monoclonal antibodies in the therapy of solid tumors, the development of the anti-idiotypic monoclonal antibody MoAb) MF11–30 is described. This antibody recognizes a private idiotope within the antigen-combining site of the immunizing antihuman high molecular weight melanoma-associated antigen MoAb 225.28. The results of a phase I clinical trial with the MoAb MF11–30 in patients with advanced melanoma are described. The lack of toxic effects and the minor responses in six patients suggest that these studies should be extended to a larger number of patients with an emphasis on the analysis of the mechanisms underlying the clinical response.     

Expanded bed adsorption processing of mammalian cell culture fluid: comparison with packed bed affinity chromatography

A comparison between expanded bed adsorption and conventional packed bed Protein A Fast Flow to purify the anti-rHBsAg mAbs from feedstock is presented in this work. Direct capture by STREAMLINE expanded bed adsorption chromatography resulted in 92% product recovery and sevenfold more concentrated product with similar purity levels compared to that obtained by the standard packed method. The process time and buffer consumption were reduced in the expanded bed adsorption method not only with the binding-elution conditions but also with the use of NaOH during the cleaning-in-place step. The latter is the most widely accepted agent in downstream processing, being a cost effective technique that provides not only efficient cleaning but also sanitizes complete column systems and destroys pirogens.     

Adriamycin(hydrazone)-antibody conjugates require internalization and intracellular acid hydrolysis for antitumor activity

Summary Adriamycin hydrazone (ADM-Hzn) immunoconjugates have previously been shown to exhibit antibody-directed antitumor activity in vitro and in vivo. In this report, the biological and biochemical properties of the mAb and linker were investigated. Conjugates prepared with two antibodies 5E9 [anti-(transferrin receptor)] and G28.1 (anti-CD37), (which internalize from the surface of target cells following binding) were more cytotoxic in vitro and had greater antitumor activity against Daudi B lymphoma tumor xenografts than a non-internalizing immunoconjugate prepared with mAb 2H7 (anti-CD20). In addition, the 13-acylhydrazone bond linking the drug to the mAb was labile at pH 5 and released unmodified ADM at a rapid rate (t1/2 = 2.5 h). Immunoconjugates prepared with an oxime linkage at the C-13 position were stable to acid and were not cytotoxic. These findings suggest that internalization of ADM-Hzn immunoconjugates and release of free ADM from the mAb in acidic intracellular compartments were important steps in the mechanism of action of ADM-Hzn immunoconjugates.     

Radioimmunotherapy of small-cell lung cancer xenografts using131I-labelled anti-NCAM monoclonal antibody 123C3

We have studied the therapeutic efficacy of¹³¹I-labelled monoclonal antibody 123C3 in human small-cell lung carcinoma xenografts established from the NCI-H69 cell line in nude mice. Several radiation does were administered intraperitoneally and different treatment schedules were tested. The maximal tolerated dose, 2×500 Ci, resulted in complete remission of tumours smaller than 200 mm³ and long-lasting remission (more than 135 days) of the larger tumours. In control experiments, treatment with unlabelled monoclonal antibody 123C3 did not affect the tumour growth rate, while the effect of radiolabelled non-relevant, isotype-matched, monoclonal antibody M6/1 was minor and transient. Regrowth of the tumours occurred in all cases and could not be attributed to loss of neural cell adhesion molecule (NCAM) expression. Tumour recurrence is probably caused by insufficient radiation dosage. Radiation-induced toxicity was monitored by assessment of weight and bone marrow examination. Weight loss was observed in all treatment groups, but the mice regained their initial weight within 14 days, except for the group receiving the highest radiation dose (3×600 Ci). In this group all mice died as a result of radiotoxicity. Of the mice injected with 600 Ci radiolabelled control antibody, 50% died within 2 weeks after administration. Apparently the higher uptake of the radiolabelled monoclonal antibody in the tumour reduced systemic radiation toxicity.     

Domain structure of neurofilament subunits as revealed by monoclonal antibodies

Monoclonal antibodies have been prepared against purified neurofilament (NF) subunits (NF68, NF150, and NF200). From 25 fusions, several hundred strongly positive antibodies have been obtained. Among them are antibodies against the specific subunits as well as antibodies recognizing common antigenic determinants. These have all been characterized according to the following properties: ELISA (enzyme-linked immunosorbant assay) testing against each subunit, immunoblots against enriched neurofilament preparation, immunoblots of cyanogen bromide or chymotrypsin-treated neurofilaments, immunofluorescence with PC12 cells, and immunohistochemistry of cerebellum. Whereas the antibodies against the NF68 and NF150 appear to react with single cyanogen bromide fragments, the antibodies against the NF200 react with multiple cyanogen bromide fragments. These data are consistent with the hypothesis that the NF200 is partially composed of several repeated structural determinants. Furthermore, all of the antibodies that react with the NF200 recognize the solubilized "sidearm" domain from limited chymotryptic digestions. The locations of the common and variable domains of the three subunits are discussed in light of these results.