Biomedical subjects

L B Shih

Publications and source records attributed to L B Shih.

At least 19 recordsLinked to original sources

PubMed1999

Targeting human cancer xenografts with monoclonal antibodies labeled using radioiodinated, diethylenetriaminepentaacetic acid-appended peptides.

A new nonmetabolizable peptide approach to the production of residualizing radioiodine was evaluated in nude mice bearing xenografts of human lung adenocarcinoma (Calu-3) and B-cell lymphoma (Ramos). Monoclonal antibodies (MAbs) RS7 (anti-epithelial glycoprotein-1) and LL2 (anti-CD22) were radioiodinated using the thiol-reactive diethylenetriaminepentaacetic acid-D-peptide adducts IMP-R1 and IMP-R2. 125I-IMP-R1- and 125I-IMP-R2-labeled MAbs were compared to the MAbs iodinated by the conventional chloramine-T approach, (111)In, and 131I-dilactitoltyramine (DLT). In vivo biodistribution studies demonstrated a significant improvement in the tumor accretion of radiolabel using the 125I-IMP-R1 labeled MAbs compared with the conventionally iodinated antibodies. For example, at day 7, the percentage of injected dose per gram of tissue in Calu-3 was 7.9 +/- 4.1% and 18.1 +/- 7.9% (P < 0.05) for the conventional 131I- and 125I-IMP-R1-RS7, respectively, and tumor:nontumor ratios were 2.6-4.5-fold higher with the 125I-IMP-R1-RS7. It is estimated that 131I-IMP-R1-RS7 would deliver a dose to tumor (at the estimated maximum tolerated dose) 3.9 times greater than conventional 131I-labeled RS7, 1.4 times greater than 90Y-labeled RS7, and 0.7 times that of 131I-DLT-labeled RS7. Tumor accretion of 125I-IMP-R2-RS7 was also improved compared with conventionally iodinated antibody. However, this label also caused a large increase in kidney accretion. Similar improvements in tumor accretion and tumor:nontumor ratios were observed when 125I-IMP-R1-LL2 was used in the Ramos model. IMP-R1 offers a practical and useful residualizing radioiodine label because labeling efficiency is at least 10 times greater than that of the residualizing label DLT, without MAb aggregation. Structural modifications can be envisioned for further improvements in radioiodine incorporation, specific activity, and tumor dosimetry, and efforts along these lines are under way.

Animals↗

PubMed1999

The effects of domain deletion, glycosylation, and long IgG3 hinge on the biodistribution and serum stability properties of a humanized IgG1 immunoglobulin, hLL2, and its fragments.

Antibody (Ab) fragments are preferred agents for imaging applications because of their rapid clearance from the blood, thereby providing high tumor:blood ratios within a few hours. Several preclinical studies have also suggested that Ab fragments might be preferred for therapeutic applications over an intact IgG. The purpose of this project was to develop engineered Ab fragments using a humanized anti-carcinoembryonic antigen and anti-CD22 Ab as the parent. Three types of variants were prepared: a deltaCH2 (deletion mutant missing the CH2), a gamma3 F(ab')2 containing the human IgG3 hinge, and three glycosylated variants. The gamma3 F(ab')2 and glycosylated variants were developed because of the potential for site-specific linkage to the Ab in its divalent or monovalent fragment. The gamma3 F(ab')2 variant contains 10 cysteine residues that could be used for direct coupling using thiol chemistry, whereas the glycosylated variants have N-linked glycosylation sites engineered in the CH1 domain (two variants) as well as the VK domain (one variant). All of these variants were successfully prepared and shown to react with the target antigen. All Abs could be purified to a single peak by size-exclusion HPLC, but the deltaCH2 variant showed two distinct peaks, which were believed to be both the divalent and monovalent forms of this fragment. The two CH1 glycosylated variants showed differences in the extent of glycosylation. Modeling studies suggest that one variant would be better suited for site-specific coupling than the other because the carbohydrate chain is extended further away from the antigen-binding site. The Abs were radioiodinated to determine their pharmacokinetic behavior in mice. All of the humanized Ab divalent fragments cleared nearly 20 times faster from the blood than the murine parent F(ab')2 over a 24-h period. The glycosylated fragments showed some added stability compared to the other fragments over 4 h, but by 24 h, they had cleared to the same extent. Size-exclusion high-performance liquid chromatography of blood samples indicated that the humanized Ab fragments were quickly degraded in the blood. Thus, there is an inherent instability of the divalent fragments from these humanized IgG1 constructs that may affect their utility in imaging or therapy applications.

Animals↗

PubMed1998

Carbohydrates engineered at antibody constant domains can be used for site-specific conjugation of drugs and chelates.

To improve the efficiency of site-specific conjugation of chelates and drugs to antibodies, and to minimize the incidence of immunoreactivity perturbation to the resultant immunoconjugates, Asn-linked oligosaccharide moieties were designed and engineered into the constant domains of a humanized anti-CD22 monoclonal antibody, hLL2. From 10 potential glycosylation mutants, two CH1 domain glycosylation sites, HCN1 and HCN5, were identified that were positioned favorably for glycosylation. The carbohydrate (CHO) chains attached at these sites were differentially processed so that HCN5-CHOs were physically larger than HCN1-CHOs. Although both the CH1-appended CHOs, and the LL2 Vkappa-appended CHOs conjugated efficiently with small chelates, the HCN5-CHOs, due to the structural and positional superiority, appear to be a better conjugation site for large drug complexes, such as 18 kDa doxorubicin (DOX)-dextran.

Amino Acid Sequence↗

PubMed1997

Generation of a high-producing clone of a humanized anti-B-cell lymphoma monoclonal antibody (hLL2).

BACKGROUND: LL2 is a murine immunoglobulin (Ig)G2a-kappa anti-B-cell monoclonal antibody with proven targeting and therapeutic efficacy in the management of non-Hodgkin's lymphoma (NHL). The authors had previously generated a humanized LL2 (hLL2) that demonstrated binding properties identical to those of LL2. Nevertheless, the productivity of the cell line was insufficient for large-scale production of the antibody for clinical studies. Therefore, the authors chose an amplifiable system for the generation of hLL2. METHODS: The hLL2 sequences were ligated into the expression vector pdHL2, which has a dhfr amplifiable gene, and were incorporated into the SP2/0 cells by electroporation. A methotrexate (MTX) resistant clone producing hLL2 was identified. Stepwise increases in MTX concentrations, from 0.1 to 5 microM, and subcloning of the cells by limiting dilution were performed. RESULTS: By amplifying the dhfr and hLL2 genes with stepwise increases in the MTX concentration, the antibody production was enhanced from its original 1.4 to 70 +/- 5 mg per liter of culture media. Subsequent subcloning further improved the productivity. Immunoreactivity of the antibody was conserved, as proven by enzyme-linked immunosorbent assay and cell-binding assays. By isoelectrofocusing, the isoelectric point (pI) of the antibody was measured at approximately 9.6. The productivity of the clone was not affected by culture conditions or storage of the cells in liquid nitrogen. CONCLUSIONS: By means of gene amplification, the authors have generated a high-producing hLL2-IgG clone suitable for production of the quantity of antibody necessary for clinical diagnostic and therapeutic trials of NHL patients.

Antibodies, Monoclonal↗

PubMed1997

The advantage of residualizing radiolabels for targeting B-cell lymphomas with a radiolabeled anti-CD22 monoclonal antibody.

CD22 antibodies (Abs) bound to B-cell lymphomas are known to be internalized and catabolized rapidly. Therefore, it would be expected that use of CD22 as a target for radioimmunotherapy should be enhanced by the use of "residualizing" radiolabels, which are trapped within the cell after catabolism of the Ab to which they had been conjugated. Our study was intended to evaluate this hypothesis using Ab LL2. In initial experiments, we found that LL2 binding was strongly temperature dependent, with approximately 15-fold greater binding at 37 degrees C than at 0 degrees C. A series of experiments suggested that this difference is due to a conformational change in the antigen at low temperature, so that the LL2 epitope is partially blocked. In vitro, residualizing labels-including 125I-dilactitol tyramine and 111In-DTPA-were retained by cells much longer than a conventional iodine label. In vivo, residualizing labels also showed a marked advantage in terms of uptake by Ramos B-cell lymphoma xenografts in nude mice. However, the absolute Ab uptake by xenografts was quite low, in comparison with results obtained with many carcinoma xenografts, which appears to be due in part to vascular properties of the B-cell lymphoma xenografts.

Animals↗

PubMed1997

Advantage of residualizing radiolabels for an internalizing antibody against the B-cell lymphoma antigen, CD22.

LL2 is an anti-CD22 pan-B-cell monoclonal antibody which, when radiolabeled, has a high sensitivity for detecting B-cell, non-Hodgkin's lymphoma (NHL), as well as an antitumor efficacy in therapeutic applications. The aim of this study was to determine whether intracellularly retained radiolabels have an advantage in the diagnosis and therapy of lymphoma with LL2. In vitro studies showed that iodinated LL2 is intracellularly catabolized, with a rapid release of the radioiodine from the cell. In contrast, residualizing radiolabels, such as radioactive metals, are retained intracellularly for substantially longer. In vivo studies were performed using LL2-labeled with radioiodine by a non-residualizing (chloramine-T) or a residualizing method (dilactitol-tyramine, DLT), or with a radioactive metal (111In). The biodistribution of a mixture of 125I (non-residualizing chloramine-T compared to residualizing DLT), 111In-labeled LL2 murine IgG2a or its fragments [F(ab')2, Fab'], as well as its humanized, CDR-grafted form, was studied in nude mice bearing the RL human B-cell NHL cell line. Radiation doses were calculated from the biodistribution data according to the Medical International Radiation Dose scheme to assess the potential advantage for therapeutic applications. At all assay times, tumor uptake was higher with the residualizing labels (i.e., 111In and DLT-125I) than with the non-residualizing iodine label. For example, tumor/blood ratios of 111In-labeled IgG were 3.2-, 3.5- and 2.8-fold higher than for non-residualizing iodinated IgG on days 3, 7 and 14, respectively. Similar results were obtained for DLT-labeled IgG and fragments with residualized radiolabels. Tumor/organ ratios also were higher with residualizing labels. No significant differences in tumor, blood and organ uptake were observed between murine and humanized LL2. The conventionally iodinated anti-CD20 antibody, 1F5, had tumor uptake values comparable to those of iodinated LL2, the uptake of both antibodies being strongly dependent on tumor size. These data suggest that, with internalizing antibodies such as LL2, labeling with intracellularly retained isotopes has an advantage over released ones, which justifies further clinical trials with residualizing 111In-labeled LL2 for diagnosis, and residualizing 131I and 90Y labels for therapy.

Animals↗

PubMed1995

In vitro and in vivo reactivity of an internalizing antibody, RS7, with human breast cancer.

RS7, a murine IgG1 antibody raised against human lung carcinoma, possesses pancarcinoma reactivity. The antigen defined by this antibody is present in tumors of the lung, stomach, bladder, breast, ovary, uterus, and prostate. Efficient targeting and therapy by radiolabeled RS7 has been demonstrated previously in animals bearing Calu-3 (an adenocarcinoma of the lung) xenografts. In this study, the efficiency of tumor targeting and the efficacy of therapy of this antibody in nude mice bearing the MDA-MB-468 human breast carcinoma were evaluated. The tumor:nontumor ratios of RS7 were 1.9-2.1 times higher than those for Ag8 (the control antibody) on day 14, except for the heart. These values were similar to that of RS7 in the Calu-3 xenograft model. Radioimmunotherapy using 250 microCi of 131I-labeled RS7 in animals bearing approximately 0.1 cm3 tumors (approximately 10 days old) caused the disappearance of tumors in 6 of 10 animals at 2 weeks postinjection. Tumors eventually disappeared from all animals, and animals remained tumor-free until the termination of the study (11 weeks of duration), except for one animal that developed a transient reappearance of tumor. The tumors in animals that received an equal dose of 131I-labeled Ag8, or unlabeled RS7 or Ag8, either were unchanged or continued to grow. Treatment that used 275 microCi of 131I-labeled RS7 in animals carrying established tumors (1 month old, approximately 0.2-0.3 cm3) showed that this antibody is effective in controlling the growth of this tumor. Tumors in the treatment group began to disappear between the second and third weeks after the injection of the radiolabeled antibody. Seven of 10 animals remained tumor free at 15 weeks after the injection. Tumors in animals that received an equal dose of control antibody persisted but grew at a slower pace compared to the untreated group. No systemic toxicity was observed.

Adenocarcinoma↗

PubMed1995

Development and evaluation of the specificity of a rat monoclonal anti-idiotype antibody, WN, to an anti-B-cell lymphoma monoclonal antibody, LL2.

Anti-idiotype monoclonal antibodies (Mabs) to mLL2, an anti-B-cell lymphoma and CD22-specific murine IgG2a-kappa Mab, were generated by hybridoma technology from splenocytes of Copenhagen rats immunized with mLL2 F(ab')2. Mab WN, an IgG2a-kappa, was selected based on its specific binding to mLL2 and not other IgG isotypes or anti-B-cell Mabs. In a radioimmunoassay, WN was found to inhibit the binding of 125I-labeled mLL2 to Raji cells and to have no effect on the binding of other B-cell-reactive antibodies. Using high performance liquid chromatography analysis, WN was shown to complex specifically with both mLL2 and mLL2 Fab'. Meanwhile, we have constructed chimeric (cLL2) and humanized (hLL2) versions of LL2. Both cLL2 and hLL2 were demonstrated to retain the original antigen specificity and affinity of mLL2 [S.O. Leung et al., Proc. Am. Assoc. Cancer Res., 2872 (abstract), 34: 481, 1993]. The specific binding of WN to either radioiodinated or peroxidase-conjugated mLL2 was inhibited in a dose-response manner, and to a similar extent by mLL2, cLL2, and hLL2. Since the mLL2 complementarity-determining regions are the only sequences common to mLL2, cLL2, and hLL2, the result confirms that WN is specific to the antigen-binding complementarity-determining regions. A WN binding assay is currently being evaluated as a substitute for the tedious, and sometimes inconsistent, Raji cell-binding assay for the determination of LL2 immunoreactivity. In conclusion, we have developed an anti-idiotype Mab, WN, to mLL2. Its potential use as a surrogate antigen for B-cell lymphoma is under investigation.

Animals↗

PubMed1995

Construction and characterization of a humanized, internalizing, B-cell (CD22)-specific, leukemia/lymphoma antibody, LL2.

The murine monoclonal antibody, LL2, is a B-cell (CD22)-specific IgG2a which has been demonstrated to be clinically significant in the radioimmunodetection of non-Hodgkin's B-cell lymphoma. The antibody carries a variable region-appended glycosylation site in the light chain and is rapidly internalized upon binding to Raji target cells. Humanization of LL2 was carried out in order to develop LL2 as a diagnostic and immunotherapeutic suitable for repeated administration. Based on the extent of sequence homology, and with the aid of computer modeling, we selected the EU framework regions (FR) 1, 2 and 3, and the NEWM FR4 as the scaffold for grafting the heavy chain complementarity determining regions (CDRs), and REI FRs for that of light chains. The light chain glycosylation site, however, was not included. Construction of the CDR-grafted variable regions was accomplished by a rapid and simplified method that involved long DNA oligonucleotide synthesis and the polymerase chain reaction (PCR). The humanized LL2 (hLL2), lacking light chain variable region glycosylation, exhibited immunoreactivities that were comparable to that of chimeric LL2 (cLL2), which was shown previously to have antigen-binding properties similar to its murine counterpart, suggesting that the VK-appended oligosaccharides found in mLL2 are not necessary for antigen binding. Moreover, the hLL2 retained its ability to be internalized into Raji cells at a rate similar to its murine and chimeric counterparts.

Amino Acid Sequence↗

PubMed1994

Internalization and intracellular processing of an anti-B-cell lymphoma monoclonal antibody, LL2.

The successful clinical experience with antibody LL2 (an IgG2a, anti-B-cell lymphoma antibody) in radioimmunodetection and radioimmunotherapy suggests that this antibody may have potential as a carrier of cytotoxic agents. The internalization, cellular trafficking, and catabolism of this antibody in target human Burkitt lymphoma cells (Raji) were investigated. Internalization of intact antibody as well as of the F(ab')2 and Fab' fragments was detected by an FITC-labeled anti-mouse second antibody probe, and evaluated by fluorescence microscopy. Internalization of intact IgG (or the fragments) was observed as early as 5 min after incubation at 37 degrees C. Initially, the internalized antibodies were present as micro-particles inside the cell membrane, and were translocated to the lysosomal compartment within 2 hr. The anatomic location of the internalized antibody, before translocation to the lysosomal compartment, was deduced by comparing the fluorescence images obtained with the antibody to those obtained with fluorescent probes with known cellular distribution in a co-internalization study. A Golgi-like compartment was found to be involved in the translocation of the antibody. Cellular catabolism of the bound antibody was studied by using 125I-labeled antibody on the target cells. At 21 h, 40% of the radioactivity was released into the supernatant as degraded fragments. The observation suggested that the antibody was degraded mainly in the lysosomes, since the degradation was significantly inhibited in the presence of lysosomal inhibitors such as ammonium chloride or leupeptin. Subcellular fractionation of Raji cells after the binding of 125I-labeled LL2 indicated that the antibody was translocated to lysosomes as evidenced by SDS-PAGE. The rate of internalization (Ke) of LL2, and the re-expression of the antigen were determined. The rapid internalization of LL2 and the re-expression of the antigen suggest that this antibody may have potential as a therapeutic immunoconjugate, since it could deliver a higher accumulation of cytotoxic agents into lymphoma cells.

4-Chloro-7-nitrobenzofurazan↗

PubMed1994

Processing of antibody-radioisotope conjugates after binding to the surface of tumor cells.

BACKGROUND: Previous experiments indicated that most antibodies binding to cell surface antigens are internalized gradually and degraded within lysosomes, with a half-life of degradation of approximately 1 day, for most antibodies. The research discussed in this article extended our studies to eight additional antibodies reacting with six different antigens, including three antigens anchored in the membrane by glycosyl-phosphatidylinositol. The authors also tested antibodies labeled with 111indium, as well as 125iodine, to determine whether different radiolabels would be processed differently. METHODS: Antibodies were radiolabeled with 125I or with 111In bound to benzyl-DTPA. After binding to the surface of tumor cells in vitro, excess antibody was washed away, and the fate of the radiolabel was investigated over periods of 3-7 days. Radiolabel released into the supernatant or retained by the cells was analyzed to determine whether it was still on intact antibody. RESULTS: In 13 of the 15 antibodies that were tested, a similar pattern of irreversible binding and gradual catabolism was observed. Iodine conjugated to antibodies was released rapidly from the cell after antibody catabolism. In contrast, the 111In was retained within the cell much longer than 125I, with the rate of degradation and release into the medium being at least fivefold slower. More than 50% of the bound 111In was still present on the cells after 7 days. Biochemical analysis of the retained 111In extracted cells after 4-6 days demonstrated that it was no longer associated with antibodies and was in a low molecular weight form, probably still associated with the chelator benzyl-DTPA. CONCLUSIONS: Different radiolabels are processed by tumor cells differently, after catabolism of the antibody to which they originally were conjugated. The data suggest that the prolonged retention of 111In, relative to that of 125I, is due not to deiodination of iodine conjugates, but rather to intracellular retention of catabolic products containing 111In, perhaps within lysosomes. The use of radioisotopes that are retained within cells after antibody internalization and degradation may improve both radioimmunodetection and radioimmunotherapy of cancer.

Antibodies, Monoclonal↗

PubMed1994

Internalization of an intact doxorubicin immunoconjugate.

An immunoconjugate between doxorubicin and anti-(carcinoembryonic antigen) (CEA) was prepared by using aminodextran (M(r) = 40,000) as the intermediate carrier, and the carbohydrate moiety of the antibody as the linking site. The resulting immunoconjugate was subjected to an in vitro evaluation for the internalization on the target cells (Lo Vo), and compared to that of unconjugated antibody, as well as the cellular uptake of unconjugated doxorubicin. The internalization was evaluated microscopically by following the translocation of the red fluorescence of doxorubicin and the green fluorescence of the fluorescein-isothiocyanate-labeled goat anti-(mouse Ig) antibody, which visualizes the location of the primary mouse antibody. Anti-CEA monoclonal antibody (NP-4) was found to internalize into Lo Vo cells. The immunoconjugate made with this antibody was similarly internalized, and the doxorubicin was found to distribute with the primary antibody. The cell surface and cytoplasm were the major compartments of their distribution. These results indicate that the drug molecules were indeed delivered into the cells by the antibody as an intact conjugate. Unconjugated doxorubicin, on the contrary, was quickly absorbed by the cells and concentrated in the nucleus within 30 min, and never showed a distribution in the cytoplasm or cell membrane as in the nucleus by this procedure. The intermediate drug conjugate, doxorubicin-dextran, did not show internalization. The internalization of NP-4 antibody (or the doxorubicin conjugate) was also confirmed by studying the intracellular catabolism of the cell-bound antibody (or conjugate). The release of the degraded antibody by the cells, as differentiated by trichloroacetic acid precipitation techniques, was considered an indication of internalization. Lysosomes were involved in the degradation, since the process was markedly inhibited in the presence of the lysosomal enzyme inhibitor, ammonium chloride.

Antibodies, Monoclonal↗

PubMed1994

Chimerization of LL2, a rapidly internalizing antibody specific for B cell lymphoma.

LL2 is a murine monoclonal antibody (MAb) that has been shown to be effective for the diagnosis and treatment of patients with non-Hodgkin's B cell lymphoma. Studies have also shown that radiolabeled murine LL2 (mLL2) or mLL2 and fragments thereof coupled to Pseudomonas exotoxin (PE) can effectively target human B cell lymphoma in mice. We have obtained the DNA sequences encoding the VK and VH domains of mLL2, an IgG2a MAb, which were combined with their respective human kappa and IgG1 constant region domains and expressed in SP2/0 cells. Like its murine counterpart, the chimeric LL2 (cLL2) antibody is glycosylated in the light chain variable region. Chimerization did not interfere with the immunoreactivity of the antibody, as determined by a competitive binding assay, where either antibody shows equivalent inhibition of the binding of its counterpart to the Raji cell membrane surface antigen, CD22. Both antibodies bind and are rapidly internalized by Raji cells, whereas an irrelevant humanized antibody did not bind and was not internalized under similar conditions. The internalization rates of the bound murine or chimeric antibodies were nearly identical, with Ke values of 0.106 and 0.118 min-1 for mLL2 and cLL2, respectively. The observed close equivalence between the murine and chimeric antibodies suggests potential advantages of the latter as a less immunogenic agent. Studies are currently underway to evaluate the chimeric antibody as a potential therapeutic immunoconjugate.

Amino Acid Sequence↗

PubMed1994

The processing and fate of antibodies and their radiolabels bound to the surface of tumor cells in vitro: a comparison of nine radiolabels.

UNLABELLED: Processing radiolabeled degradation products is the key factor affecting retention of antibodies within the cell. In this study, we have analyzed the processing of antibodies labeled in nine different ways. METHODS: Antibodies were labeled with three different radioisotopes and seven different forms of 125I. Eight of the radiolabels (except 188Re) were conjugated to the same antibody, MA103, and tested on the renal carcinoma cell line SK-RC-18 and/or the ovarian carcinoma cell line SK-OV-6. Rhenium conjugation utilized the antibody RS7, the target cell line ME180 and three of the other radiolabels were also tested with this antibody-target cell combination for comparison. RESULTS: Iodine conjugated to antibodies by conventional methods was rapidly released from the cell after antibody catabolism. In contrast, iodinated moieties, such as dilactitol-tyramine and inulin-tyramine were retained within cells four to five times longer. CONCLUSIONS: The use of radiolabels that are trapped within cells after antibody catabolism can potentially increase the dose of radiation delivered to the tumor, from the same amount of radioactivity deposited by a factor of four or five. The prolonged retention of 111In relative to 125I is not due to deiodination of iodine conjugates, but rather to intracellular retention of catabolic products containing 111In, perhaps within lysosomes.

Acetylgalactosamine↗

PubMed1993

Specificity and properties of MAb RS7-3G11 and the antigen defined by this pancarcinoma monoclonal antibody.

The murine monoclonal antibody (MAb) RS7-3G11 is an IgG1 with pancarcinoma reactivity, which has been raised against human squamous-cell carcinoma of the lung. Immunoperoxidase staining of frozen tissue sections demonstrated that the antigen defined by RS7-3G11 is present in tumors of the lung, stomach, bladder, breast, ovary, uterus and prostate. The rate and extent of internalization of RS7-3G11 into Calu-3, an adenocarcinoma of the lung cell line, was investigated using unconjugated MAb, followed by fluorescence labelling, and by binding 125I-RS7-3G11 followed by acid removal of surface-bound antibody. Rapid internalization of MAb RS7-3G11 into target cells was observed. Antibody internalization was noted at 30 min, and by 2 hr virtually all MAb RS7-3G11 was internal. Although MAb RS7-3G11 was raised against non-small-cell carcinoma of the lung, ME-180, a cervical-carcinoma cell line, expresses higher quantities of the antigen than the lung-carcinoma cell lines. Due to the higher antigen density in ME-180 cells, this line was used for immunoprecipitation studies and antigen purification. Immunoprecipitation studies using the ME-180 cervical-carcinoma cell line metabolically labeled with [3H]leucine or [3H]glucosamine demonstrated that the antigen defined by RS7-3G11 is a glycoprotein of M(r) 46 kDa. Deglycosylation by treatment with endoglycosidase-F resulted in a protein with a M(r) of 35 kDa. RS7-3G11-antigen was purified from ME-180 tissue-culture cells using affinity-column chromatography. By SDS-PAGE it was seen that the antigen was highly purified. The major band appeared at M(r) of 45 to 48 kDa. This result is in agreement with the immunoprecipitation studies. The broad band observed in the SDS-PAGE is typical of many glycoproteins, and suggests heterogeneity of glycosylation. Chemical and enzymatic treatments of the antigen, followed by Western blot analyses, suggest that the RS7-3G11 antigenic determinant is composed of a conformation-dependent peptide.

Animals↗

PubMed1992

The fate of antibodies bound to the surface of tumor cells in vitro.

The fate of monoclonal antibodies binding to the surface of human tumor cells in vitro was investigated. Seven antibodies, labeled with 125I, were tested on four cell lines, which included a melanoma and carcinomas of the ovary, kidney, and lung. The antibodies were selected only by the criterion that they not be rapidly internalized via coated pits, so that they would be representative of most antibodies reacting with cell surface antigens. After allowing binding during a 2-h incubation, unbound antibody was removed, and the release of intact or degraded antibody in the supernatant was monitored. The data demonstrate that most bound antibody was gradually degraded and released from the cell over a 2-3-day period, probably via internalization, while only a small fraction, less than 20% for most antibodies, appeared to dissociate intact. One exceptional antibody, MW207, dissociated largely intact. The release of intact antibody was virtually complete within 4 h, and radioactivity released after this time was predominantly in degraded form. These results demonstrate that antibody binding to the surface of viable cells must in general be considered irreversible, and hence the concept of affinity is not applicable. Since an Fab fragment of one of the antibodies dissociated rapidly, such irreversible binding appears to require bivalent attachment. Another conclusion of this study is that most antibodies binding to the cell surface are gradually internalized, which we suggest is due to the normal turnover of cell surface constituents via non-clathrin-dependent endocytosis. Several experimental approaches indicated that a large fraction of antibody retained by the cells, for at least 2 days after binding, was present at the cell surface.

Ammonium Chloride↗

PubMed1991

Anthracycline immunoconjugates prepared by a site-specific linkage via an amino-dextran intermediate carrier.

Anthracycline, either daunomycin or doxorubicin, was site specifically attached to the carbohydrate moiety of a monoclonal anticarcinoembryonic antigen antibody by using amino-dextran as the intermediate carrier. The reaction resulted in an immunoconjugate that contains approximately 20 to 25 molecules of drug per molecule of immunoglobulin G. Flow-cytometric studies revealed the retention of the antibody-binding activity. The immunoconjugate was cytotoxic to the target cells, as examined by the 75selenomethionine incorporation studies, and remained efficient for targeting a human colonic tumor (GW-39) in the nude mouse model. The conjugate possessed a greater antitumor activity against the subcutaneous tumor than either the free drug or an irrelevant antibody conjugate, and it was well tolerated by the animals at a much higher dose level than was the unconjugated drug.

Animals↗

PubMed1990

A fluorouridine-anti-CEA immunoconjugate is therapeutically effective in a human colonic cancer xenograft model.

5-fluorouridine (FUR), an antineoplastic agent, was site-specifically conjugated to the carbohydrate moiety of a anticarcinoembryonic antigen (CEA) monoclonal antibody (MAb) by using amino-dextran as the intermediate carrier. The final immunoconjugate contains approximately 30-35 molecules of FUR per molecule of immunoglobulin, has immunoreactivity retained as examined by flow cytometry, and is cytotoxic to the target cells as examined by 75selenomethionine incorporation studies. In the GW-39/nude mouse model, the conjugate remained efficient in targeting the human colonic tumor and possessed greater inhibitory growth effects on the subcutaneous tumor than free FUR or an irrelevant antibody conjugate. In addition, the reduced host toxicity of the conjugate may permit the use of this agent in a high-dose therapy of this tumor system.

Animals↗