Preparation and functional evaluation of new doxorubicin immunoconjugates containing an acid-sensitive linker on small-cell lung cancer cells

 The anthracycline doxorubicin (DOX) is one of the most effective drugs for the treatment of small-cell lung cancer (SCLC), but its clinical application is limited by unspecific side-effects like cardiotoxicity. In the present study doxorubicin was conjugated to the monoclonal antibodies (mAb) SEN7, MOC31, and SWA11 via a novel acid-sensitive hydrazone linker. These mAb recognize SCLC-associated antigens of cluster 1 (NCAM), cluster 2 (EGP-2/GA733-2), and cluster 4 (CD24) respectively. To assess their potential therapeutic use against SCLC, the antigen-binding activities, the rates of internalization and the cytotoxic effects of the immunoconjugates were examined on tumour cell lines. The preparation procedure preserved the antigen-binding activities of the mAb and yielded immunoconjugates with average drug : mAb ratios of 7 : 1. The hydrazone linker was found to be stable at neutral pH but to release doxorubicin under acidic conditions. In contrast to SEN7-DOX, MOC31-DOX and SWA11-DOX were rapidly internalized into SCLC target cells upon binding to their specific cell-surface antigens. Accordingly, both immunoconjugates proved to be highly cytotoxic agents, inhibiting thymidine incorporation by 50% at concentrations between 0.5 μM and 1 μM and were 100-fold more selective than free doxorubicin. The results suggest that binding to selective cell-surface antigens, rapid internalization and efficient release of doxorubicin from the mAb by acid hydrolysis are required for the selective and potent function of the immunoconjugates. In particular, the use of MOC31-DOX for targeted cytotoxic therapy might be promising because of the limited cross-reactivity of the mAb with normal human tissues and its recently demonstrated tumour localization potential in SCLC patients. Received: 22 September 1995 / Accepted: 23 November 1995     

Methods for production of monoclonal antibodies with specificity for human lung cancer cells

Summary We have developed a screening strategy and technology to produce monoclonal antibodies with specificity for human lung cancer cells. Mice and rats were immunized with well-characterized tissue culture lines of human small cell lung cancer (SCLC), mouse myeloma x spleen hybrids formed by the technique of Kohler and Milstein, and the resulting culture fluids were screened for antibody binding phenotype using a radioimmunoassay. To facilitate testing large numbers of culture fluids, a 96-well, microtiter based, resuable, replicating device was designed. Using this, many hybridoma culture fluids were replica plated for antibody binding tests on a series of human target cell plates. Hybrids producing antibodies that reacted with the immunizing SCLC line and another independent SCLC line, but not with autologous B-lymphoblastoid cells derived from one of the patients, were identified, selected, and then repeatedly recloned using the same screening strategy. With this technology, hybridomas representing less than 0.5% of all hybrids generated could be isolated and stable antibody producing cultures derived. Such antibodies reacted with a panel of well-characterized SCLC lines and SCLC samples taken directly from patients but not with a variety of normal tissues. Using these antibodies we can demonstrate: tumor cell contamination of bone marrow specimens, marked heterogeneity of antigen expression on cells within individual SCLC lines and individual patients, and inhibition of clonal growth of SCLC lines in soft agarose assays. All of these findings have potential clinical and cell biologic application. Presented in the symposium on The Biology of Hybridomas at the 32nd Annual Meeting of the Tissue Culture Association, Washington, D.C., June 7–11, 1981. This symposium was supported in part by the following organizations: Bethesda Research Laboratories, Cetus Corporation, Hybritech Incorporated, MAB-Monoclonal Antibodies, Inc., National Capital Area Branch of the Tissue Culture Association, New England Nuclear Corporation, and Ortho Pharmaceutical Corporation.     

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.     

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。     

BW1023U90: A new GRP receptor antagonist for small-cell lung cancer cells

Gastrin-releasing peptide (GRP) receptor antagonists were synthesized and their ability to interact with small-cell lung cancer (SCLC) cells determined. [¹²⁵I]BW1023U90, bound with high affinity (K_d = 2 nM) to a single class of sites (Bmax = 55 fmol/mg protein) using SCLC cell line NCI-H345. [¹²⁵I]BW1023U90 binding was time dependent and reversible even at 37°C as the ligand was minimally internalized. Specific [¹²⁵I]BW1023U90 binding was inhibited with high affinity by GRP as well as bombesin (BB) but not neuromedin B (NMB). BW1023U90 inhibited the ability of BB to elevate cytosolic Ca²⁺ and increase the growth of SCLC cells. A BW1023U90 analogue, BW2258U89 (10 μg/day, SC) slowed SCLC xenograft formation in nude mice and [¹²⁵I]BW1023U90 localized to SCLC tumors 1 h after injection into nude mice. BW2258U89 (4% by weight) was placed in microspheres and slowly released over a 3-week period in nude mice bearing SCLC xenografts. The microspheres containing BW2258U89 strongly inhibited SCLC growth in vivo. A radioimmunoassay was developed for the GRP receptor antagonists and the rabbit antiserum cross-reacted totally with BW2258U89 or BW1023U90. BW2258U89 immunoreactivity (5 nM) was detected in the plasma of nude mice containing the microspheres after 1 week. These data suggest that GRP receptor antagonists bind to receptors on SCLC tumors.     

Specificity of monoclonal antibodies generated against arabinoxylans of cereal grains

Xylooligosaccharides substituted by arabinose have been produced by degradation of wheat flour arabinoxylans with an endoxylanase. These oligosaccharides were coupled to carrier proteins (KLH and BSA) and three monoclonal antibodies were isolated. The specificity of antibody recognition was studied using arabino-xylo-oligosaccharides exhibiting different pattern of substitution by arabinose.

ELISA competition tests and molecular modelling suggest that the conformation adopted by beta-(1→4) linked xylose residues is an antigenic determinant recognized by the different antibodies. Arabinose was not specifically involved in the interaction of antibody and epitope.     

Functional properties of FC-2.15, a monoclonal antibody that mediates human complement cytotoxicity against breast cancer cells

FC-2.15 is a murine IgM monoclonal antibody (mAb) that recognizes a cell-surface antigen (Ag2.15) expressed in most tumor-proliferating cells of human breast carcinomas and other neoplasias. In this study the cytotoxic ability of mAb FC-2.15, its cell-surface binding properties and endocytosis in Ag2.15-expressing (Ag2.15⁺) cells were investigated. A⁵¹Cr-release assay was used to test the FC-2.15-mediated cytotoxicity. When human serum was used as source of complement, FC-2.15 exerted a strong cytotoxic effect against human Ag2.15⁺ cells such as MCF-7 (breast cancer cell line), primary breast carcinoma cells, polymorphonuclear leukocytes and chronic myeloid leukemia cells. The mAb concentration range was 1–50 g/ml. Cytotoxicity was completely abolished when complement was inactivated. Only 3.8±2.9% of MCF-7 cells survived the treatment with FC-2.15 in the presence of human serum. A flow-cytometry assay was performed to study the Ag2.15 expression of the surviving cells and they were found to be Ag2.15^–. FC-2.15 did not mediate antibody-dependent cell cytotoxicity when different effector cells were used. Scatchard analysis with¹²⁵I-FC-2.15 on MCF-7 cells demonstrated an affinity constant of 6.9×10⁷ M^–1 and 2.8×10⁶ antigenic sites/cell.¹²⁵I-FC-2.15 was internalized to cytoplasmic vesicles reaching a maximum of 27% after 6 h incubation, followed by the release of labeled degradation products to the supernatant. FC-2.15 appears to exert its cytotoxic effect mainly in the presence of human complement, it reacts with intermediate affinity with a high-density surface antigen, and it is slowly internalized by Ag2.15⁺ cells.     

Cytotoxic properties of a ricin A chain immunotoxin recognising the cluster-5A antigen associated with human small-cell lung cancer

Summary The cytotoxic properties of a ricin A chain immunotoxin made with the mouse monoclonal antibody SWA20, recognising a family of sialoglycoprotein antigens selectively expressed by human small-cell lung cancer (SCLC), were examined using a panel of tumour cell lines in tissue culture. SWA20—ricin-A-chain was selectively toxic to the SW2, NCI-H69 and GLC-8 SCLC cell lines, inhibiting the incorporation of [³H]leucine by 50% at a concentration of 0.2–2 nM, but had no selective activity against the NCI-H23 and NCI-H125 lung adenocarcinoma or the control CEM T-lymphoblastoid cell lines. The SWA20 immunotoxin intoxicated the SW2 cell line rapidly, inhibiting [³H]leucine incorporation by 50% within 2 h compared with 0.5 h for ricin. Analysis of the effects of SWA20—ricin-A-chain on the growth of SW2 cells using a limiting-dilution clonogenic assay revealed that the immunotoxin could eliminate 95% of clonogenic malignant cells. Although SWA20—ricin-A-chain was found to be rapidly active against the majority of tumour cells, its action was limited by the presence of insensitive cells expressing low levels of the target antigen.     

Generation of monoclonal antibodies against non-structural protein 3AB of foot-and-mouth disease virus

To identify linear epitopes on the non-structural protein 3AB of foot-and-mouth disease virus (FMDV), BABL/c mice were immunized with the 3AB protein and splenocytes of BALB/c mice were fused with myeloma Sp2/0 cells. Two hybridoma monoclonal antibodies (mAbs) cell lines against the 3AB protein of foot-and-mouth disease virus (FMDV) were obtained, named C6 and E7 respectively. The microneutralization titer was 1:1024 for mAb C6, and 1:512 for E7. Both mAbs contain kappa light chains, and were of subclass IgG2b. In order to define the mAbs binding epitopes, the reactivity of these mAbs against FMDV were examined by indirect ELISA. The results showed that both mAbs can react with FMDV, but had no cross-reactivity with Swine Vesicular Disease (SVD) antigens. The titers in abdomen liquor were 1:5×10⁶ for C6 and 1:2×10⁶ for E7. In conclusion, the mAbs obtained from this study are specific for the detection of FMDV, can be used for etiological and immunological researches on FMDV, and have potential use in diagnosis and future vaccine designs.     

High-throughput screening of monoclonal antibodies against plant cell wall glycans by hierarchical clustering of their carbohydrate microarray binding profiles

Antibody-producing hybridoma cell lines were created following immunisation with a crude extract of cell wall polymers from the plant Arabidopsis thaliana. In order to rapidly screen the specificities of individual monoclonal antibodies (mAbs), their binding to microarrays containing 50 cell wall glycans immobilized on nitrocellulose was assessed. Hierarchical clustering of microarray binding profiles from newly produced mAbs, together with the profiles for mAbs with previously defined specificities allowed the rapid assignments of mAb binding to antigen classes. mAb specificities were further investigated using subsequent immunochemical and biochemical analyses and two novel mAbs are described in detail. mAb LM13 binds to an arabinanase-sensitive pectic epitope and mAb LM14, binds to an epitope occurring on arabinogalactan-proteins. Both mAbs display novel patterns of recognition of cell walls in plant materials.     

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     

Voltage-dependent sodium channels in human small-cell lung cancer cells: Role in action potentials and inhibition by lambert-eaton syndrome IgG

Sodium channels of human small-cell lung cancer (SCLC) cells were examined with whole-cell and single-channel patch clamp methods. In the tumor cells from SCLC cell line NCI-H146, the majority of the voltage-gated Na⁺ channels are only weakly tetrodotoxin (TTX)-sensitive (K _d =215 mm). With the membrane potential maintained at –60 to –80 mV, these cells produced all-or-nothing action potentials in response to depolarizing current injection (>20 pA). Similar all-ornothing spikes were also observed with anodal break excitation. Removal of external Ca²⁺ did not affect the action potential production, whereas 5 m TTX or substitution of Na⁺ with choline abolished it. Action potentials elicited in the Ca²⁺-free condition were reversibly blocked by 4 mm MnCl₂ due to the Mn²⁺-induced inhibition of voltage-dependent sodium currents (I _Na). Therefore, Na⁺ channels, not Ca²⁺ channels, underlie the excitability of SCLC cells. Whole-cell I _Na was maximal with step-depolarizing stimulations to 0 mV, and reversed at +45.2 mV, in accord with the predicted Nernst equilibrium potential for a Na⁺-selective channel. I _Na evoked by depolarizing test potentials (–60 to +40 mV) exhibited a transient time course and activation/ inactivation kinetics typical of neuronal excitable membranes; the plot of the Hodgkin-Huxley parameters, m and h, also revealed biophysical similarity between SCLC and neuronal Na⁺ channels. The single channel current amplitude, as measured with the inside-out patch configuration, was 1.0 pA at –20 mV with a slope conductance of 12.1 pS. The autoantibodies implicated in the Lambert-Eaton myasthenic syndrome (LES), which are known to inhibit I _Ca and I _Na in bovine adrenal chromaffin cells, also significantly inhibited I _Na in SCLC cells. These results indicate that (i) action potentials in human SCLC cells result from the regenerative increase in voltage-gated Na⁺ channel conductance; (ii) fundamental characteristics of SCLC Na⁺ channels are the same as the classical sodium channels found in a variety of excitable cells; and (iii) in some LES patients, SCLC Na⁺ channels are an additional target of the pathological IgG present in the patients' sera.Department of Biomedical EngineeringThis study was supported by National Institutes of Health grant NS18607 and a research grant from the Muscular Dystrophy Association. Dr. Y.I. Kim is the recipient of a Javits Neuroscience Investigator Award from the National Institute of Neurological Disorder and Stroke.     

Immunotoxins to human small-cell lung cancer

Ricin A chain ITs directed against a variety of the common cell-surface antigens associated with SCLC exerted selective toxic effects on SCLC cell lines. The potency of the cytotoxic effects matched or exceeded that previously reported for ricin A chain ITs directed against identical or similar antigens on other types of carcinoma, suggesting that SCLC may be uniquely sensitive to this type of IT.     

Development of monoclonal antibodies specific to urochordate intracellular epitopes

Sixteen monoclonal antibodies (MAbs) specific to 2 urochordate genera (Botryllus schlosseri and Botrylloides) intracellular epitopes were generated in mice immunized with a mixture of fresh and paraformaldehyde-fixed cells obtained from animal's blood and cells from dissociated organs. Hybridoma clones were selected by ELISA tests and immunohistochemistry assays on paraffin-embedded animal tissues. Five MAbs were tested for reactions with different zooidal organs and cell compartments; 7 MAbs were tested, separately, on 5 different botryllid colonies (3 Botryllus and 2 Botrylloides). The results revealed high polymorphism. Whereas some of the MAbs recognized, specifically, only part of the botryllid genotypes tested, others recognized only part of the cellular compartments. These MAbs will be used as an important tool in the study of botryllid ascidian immunology and developmental biology, revealing the first wide panel of MAbs specific to urochordate intracellular antigens.     

Carbohydrate vaccines that induce antibodies against cancer. 1. Rationale

  Resistance to chemotherapy is a major cause for failure in the treatment of lung cancer. Compared to conventional cytotoxic drugs, immunotoxins act by different mechanisms and thus might be promising for the treatment of chemoresistant cancer. The monoclonal antibody MOC31 recognises the epithelial glycoprotein-2 (EGP-2), a cell-surface antigen associated with small-cell lung cancer (SCLC) and a major fraction of lung adenocarcinomas. An immunotoxin composed of MOC31 and a recombinant form of Pseudomonas exotoxin A lacking the cell-binding domain (ETA_{252 – 613}) was prepared, and its effect on lung cancer cell lines examined. MOC31-ETA_{252 – 613} was selectively cytotoxic to EGP-2-positive SCLC and adenocarcinoma cell lines inhibiting proliferation by 50% at concentrations ranging from 0.01 nM to 0.3 nM. Moreover, the immunotoxin reduced the numberof clonogenic tumour cells from cultures by factors of 10⁴ and 10⁵ during a 24-h and a 3-week exposure respectively. In athymic mice, the immunotoxin, which revealed a serum half-life of approximately 4 h, caused substantial regression of small (40 mm³) chemoresistant tumour xenografts and significantly delayed the growth of larger tumours (120 mm³). This finding indicates that MOC31-ETA_{252 – 613} may be useful for the treatment of lung cancer in the setting of chemoresistant minimal residual disease. Received: 31 October 1996 / Accepted: 5 December 1996     

Establishment of internal-image anti-idiotype monoclonal antibodies to a human antibody to lung cancer

 Internal-image anti-idiotype antibodies are expected to enhance anticancer effector mechanisms in vivo. The objective of this study was to establish hybridomas producing anti-idiotype monoclonal antibodies against a human monoclonal antibody (hmAb) 4G12 that reacts strongly with lung squamous cell carcinomas. BALB/c female mice 6 weeks old were immunized with 4G12. Splenocytes were hybridized with P3U1 cells and hybrid cells secreting anti-4G12 hmAb were cloned. Two clones reacted with 4G12 hmAb but not with 3H12 IgM hmAb, human IgM, human serum or fetal calf serum. These two Ab2 antibodies (IgG1κ) 2B12 and 2H1 demonstrated 91.5% and 90.3% inhibition in their reactivity with radiolabelled 4G12 on PC10 cells, indicating that 2B12 and 2H1 antibodies were of the Ab2β type. In criss-cross inhibition assays, the binding of 2B12 or 2H1 to 4G12 was not inhibited by 2H1 or 2B12. Thus 2B12 and 2H1 were thought to recognize the different epitopes on the antigen-binding sites. Antisera against 2B12 and 2H1 demonstrated specific reactivity to PC10 cells. The two Ab2β antibodies, 2B12 and 2H1, express internal images of lung squamous cell carcinoma recognized by the 4G12 antibody and may be useful for cancer immunotherapy. Received: 20 September 1996 / Accepted: 2 January 1997     

Production of monoclonal antibodies against 3α,20β-hydroxysteroid dehydrogenase from Streptomyces hydrogenans

Abstract A procedure is described for the production of monoclonal antibodies (mAbs) against 3α,20β-hydroxysteroid dehydrogenase (3α,20β-HSD) from the actinomycete Streptomyces hydrogenans ATCC 19631. Clones which were obtained after fusion of immune cells were screened by solid-phase ELISA and immunoblotting. About 5.2% of the clones secreted immunoglobulins with specificity for 3α,20β-HSD. The purified mAbs were found to belong to subclass IgG₁ and to recognize both the native enzyme as well as its identical subunits which were obtained by SDS denaturation. However, the activity of the tetrameric holoenzyme was only weakly diminished in the presence of these mAbs.