ABSTRACT
Intravenous administration of N-(beta-alanyl-L-leucyl-L-alanyl-L-leucyl)doxorubicin (4) induces an acute toxic reaction, killing animals in a few minutes. This results from its positive charge at physiological pH combined with its propensity to form large aggregates in aqueous solutions. Negatively charged N-capped versions of 4 such as the succinyl derivative 5 can be administered by the iv route at more than 10 times the LD(50) of doxorubicin without inducing the acute toxic reaction, and they are active in vivo.
Subject(s)
Antineoplastic Agents/chemical synthesis , Doxorubicin/analogs & derivatives , Doxorubicin/chemistry , Doxorubicin/chemical synthesis , Oligopeptides/chemistry , Oligopeptides/chemical synthesis , Prodrugs/chemical synthesis , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/toxicity , Chromatography, High Pressure Liquid , Doxorubicin/administration & dosage , Doxorubicin/pharmacology , Doxorubicin/toxicity , Drug Stability , Female , Humans , Injections, Intraperitoneal , Injections, Intravenous , Lethal Dose 50 , Male , Mice , Mice, Inbred BALB C , Oligopeptides/administration & dosage , Oligopeptides/pharmacology , Oligopeptides/toxicity , Prodrugs/chemistry , Prodrugs/pharmacology , Prodrugs/toxicity , Solutions , Toxicity Tests, Acute , Tumor Cells, Cultured , Ultrafiltration , Xenograft Model Antitumor AssaysABSTRACT
Oligopeptidic derivatives of anthracyclines unable to penetrate cells were prepared and screened for their stability in human blood and their reactivation by peptidases secreted by cancer cells. N-beta-alanyl-L-leucyl-L-alanyl-L-leucyl-doxorubicin was selected as a new candidate prodrug. The NH2-terminal beta-alanine allows a very good blood stability. A two-step activation by peptidases found in conditioned media of cancer cells ultimately yields N-L-leucyl-doxorubicin. In vitro, when MCF-7/6 cancer cells are exposed to the prodrug, they accumulate about 14 times more doxorubicin than MRC-5 normal fibroblasts, whereas when exposed to doxorubicin the uptake is slightly higher in fibroblasts than in MCF-7/6 cells. This increased specificity of the prodrug over doxorubicin was confirmed in cytotoxicity assays using the same cell types. In vivo, the prodrug proved about nine times less toxic than doxorubicin in the normal mouse and also much more efficient in two different experimental chemotherapy models of human breast tumors.
Subject(s)
Antibiotics, Antineoplastic/pharmacokinetics , Doxorubicin/analogs & derivatives , Doxorubicin/pharmacology , Oligopeptides/pharmacology , Prodrugs/pharmacokinetics , Animals , Antibiotics, Antineoplastic/pharmacology , Antibiotics, Antineoplastic/toxicity , Biotransformation , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Doxorubicin/pharmacokinetics , Doxorubicin/toxicity , Drug Stability , Female , Humans , Lethal Dose 50 , Male , Mice , Mice, Inbred BALB C , Oligopeptides/toxicity , Prodrugs/pharmacology , Prodrugs/toxicity , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
The monoclonal antibody A33 recognises a tumour-associated antigen on human colorectal carcinoma, and has undergone preliminary evaluation in the clinic where selective localisation to hepatic metastases has been demonstrated [Welt et al. (1994) J. Clin. Oncol. 12, 1561-1571]. A33 and an A33 tri-fab fragment (TFM) were labelled with 90Y via a stable macrocyclic ligand for biodistribution and therapy studies in nude mice bearing SW1222 colon carcinoma xenografts. Biodistribution studies demonstrated tumour localisation for both A33 IgG and TFM with low bone, liver and kidney levels. Clearance of TFM from the blood was much faster than IgG and this led to lower tumour accumulation for TFM but superior tumour-blood ratios. The maximum per cent injected dose per g localised to tumour was 35.9% +/- 5.3% for A33 IgG and 12.9% +/- 4.6% for A33 TFM with tumour-blood ratios at 48 h after administration of 5.6 +/- 1.8 and 29.2 +/- 9.8 respectively. Autoradiography studies with 125I-labelled A33 IgG and TFM demonstrated a homogeneous distribution within tumour tissue which was not observed with other anti-colorectal tumour antibodies. TFM penetrated into the tumour tissue more rapidly than IgG. In therapy studies, a single dose of 90Y-A33 IgG (250 microCi per mouse) or 90Y-A33 TFM (300 microCi per mouse) led to complete regression of 2-week-old tumour xenografts with long-term tumour-free survivors. A transient drop in white blood cell count was observed with both IgG and TFM but was significantly more pronounced with IgG. The cell count fell to 8.4% of control for IgG, whereas with TFM cell counts fell to 51% of control before recovery. These results indicate that the more rapid blood clearance of 90Y-TFM confers reduced toxicity compared with 90Y-IgG although similar therapeutic effects are achieved. When the dose of 90Y-IgG was adjusted to give the same dose to tumour achieved with 300 microCi 90Y-TFM, a lesser therapeutic effect was observed. This may be owing to more rapid tumour penetration achieved with TFM. Both A33 IgG and TFM demonstrated potent anti-tumour effects against human tumour xenografts in this mouse model system. The stability of these 90Y-labelled conjugates and their effective tumour penetration are promising for the development of humanised reagents for clinical studies.
Subject(s)
Colorectal Neoplasms/radiotherapy , Radioimmunotherapy , Yttrium Radioisotopes , Animals , Antibodies, Monoclonal/pharmacokinetics , Autoradiography , Cell Division , Colorectal Neoplasms/pathology , Female , Humans , Immunoglobulin Fab Fragments , Immunoglobulin G , Leukocyte Count , Metabolic Clearance Rate , Mice , Mice, Nude , Time Factors , Tissue Distribution , Transplantation, Heterologous , Weight Loss , Yttrium Radioisotopes/pharmacokineticsABSTRACT
Radiolabeled antibodies have produced encouraging remissions in patients with chemotherapy-resistant hematological malignancies; however, the selection of therapeutic radionuclides for clinical trials remains controversial. In this study, we compared the internalization, lysosomal targeting, metabolism, and cellular retention of radiolabeled murine and humanized monoclonal antibodies targeting the CD33 antigen (monoclonal antibodies mP67 and hP67, respectively) on myeloid leukemia cell lines (HEL and HL-60) and of anti-carcinoma antibodies (monoclonal antibodies hCTM01 and hA33) targeting breast cancer and colorectal carcinoma cell lines (MCF7 and Colo 205, respectively). Each antibody was labeled with 125I (by the IodoGen method) and with 111In and 90Y using macrocyclic chelation technology. Targeted tumor cells were analyzed for retention and metabolism of radioimmunoconjugates using cellular-radioimmunoassays, Percoll gradient fractionation of cell organelles, SDS-PAGE, and TLC of cell lysates and culture supernatants. Our results suggest that antibodies are routed to lysosomes after endocytosis, where they are proteolytically degraded. [125I]monoiodotyrosine is rapidly excreted from cells after lysosomal catabolism of antibodies radioiodinated by conventional methods, whereas small molecular weight 111In and 90Y catabolites remain trapped in lysosomes. As a consequence of the differential disposition of small molecular weight catabolites, 111In and 90Y conjugates displayed superior retention of radioactivity compared with 125I conjugates when tumor cells were targeted using rapidly internalizing antibody-antigen systems (e.g., hP67 with HEL cells and hCTM01 with MCF7 cells). When tumor cells were targeted using antibody-antigen systems exhibiting slow rates of endocytosis (e.g., hP67 on HL-60 cells and hA33 on Colo 205 cells), little differences in cellular retention of radioactivity was observed, regardless of whether 125I, 111In, or 90Y was used.
Subject(s)
Antibodies, Monoclonal/metabolism , Breast Neoplasms/metabolism , Carcinoma/metabolism , Colorectal Neoplasms/metabolism , Immunoconjugates/metabolism , Indium Radioisotopes/metabolism , Iodine Radioisotopes/metabolism , Radioimmunotherapy , Yttrium Radioisotopes/metabolism , Antibodies, Monoclonal/therapeutic use , Antigens, CD/immunology , Antigens, Differentiation, Myelomonocytic/immunology , Antigens, Neoplasm/immunology , Breast Neoplasms/radiotherapy , Carcinoma/radiotherapy , Colorectal Neoplasms/radiotherapy , Drug Carriers , Female , HL-60 Cells , Humans , Immunoconjugates/therapeutic use , Indium Radioisotopes/therapeutic use , Iodine Radioisotopes/therapeutic use , Liposomes , Sialic Acid Binding Ig-like Lectin 3 , Yttrium Radioisotopes/therapeutic useABSTRACT
UNLABELLED: Radiolabeled MOC-31 retains its immunoreactivity and shows good in vivo immunolocalization to human SCLC xenografted in nude rats. METHODS: We evaluated the immunotargeting properties and safety of 111In-labeled monoclonal antibody (MAb) MOC-31 (125 MBq, 5 mg) in six patients with histologically proven small-cell lung cancer (SCLC). Scintigraphy and pharmacokinetics were performed up to 3 days after injection. RESULTS: No adverse reactions were found after injection of MAb MOC-31. Pharmacokinetics obtained from plasma radioactivity showed plasma disappearance described most properly by a monoexponential model with a mean half-life value of 17.0 +/- 1.4 hr. HPLC analysis documented the monomeric MOC-31 without evidence of immune complexes or radioactive lower molecular weight fractions. Mean 24-hr urinary excretion of radioactivity was 4.3% of the injected dose. Scintigraphy detected primary tumor or metastases in five of six patients. Localization of radioactivity in normal tissue was restricted, but additional experiments need to be performed to elucidate possible cross-reactivity of MOC-31 with normal tissue in vivo. CONCLUSION: Preliminary results justify further studies to reveal the possible usefullness of radiolabeled MOC-31 in the therapeutic and diagnostic management of SCLC.
Subject(s)
Antibodies, Monoclonal , Carcinoma, Small Cell/diagnostic imaging , Indium Radioisotopes , Lung Neoplasms/diagnostic imaging , Radioimmunodetection , Antibodies, Monoclonal/pharmacokinetics , Carcinoma, Small Cell/secondary , Humans , Indium Radioisotopes/pharmacokinetics , Pentetic Acid/pharmacokineticsABSTRACT
A type I ribosome inactivating protein, gelonin, was linked to Lym-1, a murine monoclonal antibody reactive with a polymorphic determinant of class II HLA-DR histocompatibility leukocyte antigen (HLA) on human lymphoma cells, via a disulfide linkage using the heterobifunctional cross-linking agent, N-succinimidyl-3-(2-pyridyldithio) propionate. This immunotoxin was purified from unreacted gelonin and unconjugated Lym-1 by fast protein liquid chromatography using sephacryl S-300 gel filtration and blue sepharose affinity gradient separation. Binding of Lym-1-gelonin immunoconjugate to human Raji Burkitt's lymphoma cells was demonstrated by indirect immunofluorescence using flow cytometry. Lym-1-gelonin was very active in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium salt and sulforhodamine B in vitro cytotoxicity assays against the Raji lymphoma cell line and confirmed the fact that monoclonal antibody Lym-1 internalizes into human lymphoma cells. A weaker cytostatic antiproliferative effect was also noted for unconjugated Lym-1. gamma-interferon augmented the antiproliferative effects of Lym-1-gelonin conjugate and unconjugated Lym-1, by having a direct cytotoxic effect on the Raji cells. Tumor necrosis factor-alpha also enhanced the antiproliferative effect of unconjugated Lym-1, but did not significantly augment the cytotoxic activity of the Lym-1-gelonin conjugate. These results suggest that anti-HLA class II monoclonal antibodies may be useful in constructing immunotoxins for the treatment of human lymphomas and leukemias expressing HLA class II antigens, and that unconjugated anti-HLA class II monoclonal antibodies may be therapeutically useful in conjunction with recombinant cytokines, especially gamma-interferon.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Burkitt Lymphoma/drug therapy , Growth Inhibitors/pharmacology , Immunoconjugates/pharmacology , Interferon-gamma/pharmacology , Plant Proteins/pharmacology , Tumor Necrosis Factor-alpha/pharmacology , Animals , Antibodies, Monoclonal/metabolism , Antineoplastic Agents, Phytogenic/metabolism , Binding Sites, Antibody , Burkitt Lymphoma/metabolism , Burkitt Lymphoma/pathology , Cells , Drug Synergism , Growth Inhibitors/toxicity , Humans , Immunoconjugates/isolation & purification , Immunoconjugates/metabolism , Mice , Plant Proteins/metabolism , Ribosome Inactivating Proteins, Type 1 , Tumor Cells, CulturedABSTRACT
Thirteen patients with relapsed or refractory Non-Hodgkin's Lymphoma were treated with 131I-Lym-1 during the course of a dose escalation trial. Principal aims were to establish the maximum tolerated single dose (MTD), as well as to assess clinical and dosimetric effects of the MTD. Patients were eligible if > 25% of tumor cells bound Lym-1 on immunohistochemistry, stain intensity was +2/4 or greater and human anti-mouse antibody (HAMA) assay was negative. Radioimmunotherapy was performed with escalating doses at levels of 50 mCi, 65 mCi/m2 and 80 mCi/m2 (50-139 mCi total). Patients were eligible for retreatment after 6-10 weeks if there was no severe toxicity, their disease was at least stable and HAMA remained negative. Three were retreated. Four have achieved partial responses which lasted 11, 11, 18 and 22 weeks. Acute toxicities included rigors (69%), fever (62%), nausea (46%), vomiting (46%), pruritus (23%), urticaria (23%), chest pain (23%) and bronchospasm (15%). HAMA developed in 3 patients. Myelosuppression, manifested as thrombocytopenia and neutropenia, was dose-limiting and defined the single dose MTD at 65 mCi/m2. Plasma radioactivity clearance was biphasic, with a 0.9 hr alpha-T1/2 and a 19.8 hr beta-T1/2. At completion of Lym-1 infusion, a mean of 45% of the injected dose was recoverable in the circulation. Images obtained within the first 2 hours indicated mean hepatic and splenic uptake was 29% and 11%, respectively. Radiation absorbed doses to tumor ranged from 18-61 rads; mean doses to whole body ranged from 17 to 71 rads.
Subject(s)
Immunotoxins/adverse effects , Immunotoxins/therapeutic use , Iodine Radioisotopes/therapeutic use , Lymphoma, B-Cell/radiotherapy , Adult , Aged , Animals , Antibodies, Monoclonal/adverse effects , Antibodies, Monoclonal/metabolism , Antibodies, Monoclonal/therapeutic use , Antigens, Neoplasm/analysis , Antigens, Neoplasm/immunology , Dose-Response Relationship, Drug , Dose-Response Relationship, Immunologic , Dose-Response Relationship, Radiation , Drug Administration Schedule , Female , Humans , Immunohistochemistry , Immunotoxins/metabolism , Infusions, Intravenous , Iodine Radioisotopes/blood , Iodine Radioisotopes/pharmacokinetics , Lymphoma, B-Cell/immunology , Male , Mice , Mice, Inbred BALB C , Middle Aged , Radiotherapy DosageABSTRACT
Twelve patients with metastatic colon cancer were treated with 131I-chimeric B72.3 (IgG-4) at total doses of 28 or 36 mCi/m2 in two or three weekly fractions. Bone marrow suppression was the only significant side effect. The degree of bone marrow suppression adjusted for whole-body dose was modestly but statistically significantly (p = 0.04) less than that seen with identical doses given as a single infusion for the total dose of 36 mCi/m2. Nine of twelve patients developed an antibody response to ch B72.3, which altered the kinetics of radiolabeled antibody in four patients given a second course of therapy. One patient had a minor response that lasted 4 mo. Fractionation of this particular radiolabeled antibody at the dose schedule used produced a modest increase in the therapeutic window in regard to administered dose.
Subject(s)
Colonic Neoplasms/radiotherapy , Radioimmunotherapy , Adult , Aged , Antibodies, Monoclonal/immunology , Antibody Formation , Bone Marrow Diseases/etiology , Colonic Neoplasms/blood , Colonic Neoplasms/immunology , Drug Evaluation , Female , Humans , Iodine Radioisotopes/administration & dosage , Leukocyte Count , Male , Middle Aged , Neoplasm Metastasis , Platelet Count , Radioimmunotherapy/adverse effects , Radiotherapy DosageABSTRACT
We had previously shown that the human colon produces at least two immunochemically distinct mucins, one neutral and the other a sialomucin [Gold et al. J. biol. Chem. 256, 6354-6358 (1981)]. In addition, the sialomucin was shown to contain an immunodeterminant restricted to colonic epithelium and may thus prove useful as a tissue-specific marker. In the current study we have shown that a specific linkage of sialic acid to the oligosaccharide backbone has a major role in the organ-specific immunodeterminant structure. Treatment of intact colonic mucin with sialidase (Cl. perfringens) cleaved 20-80% of the sialic acid as measured colorimetrically. Immunoreactivity was decreased by 0-42% with respect to the untreated material. Saponification (0.1 N KOH, 20 min at room temp) caused an approximate 90% decrease in immunoreactivity for each mucin. Subsequent to saponification, neuraminidase cleaved most of the sialic acid from the mucins. The majority of sialic acid was observed to be O-acetylated, thus making it sialidase-insensitive. Gas chromatography-mass spectrometric analyses of the trimethylsilyl sialic acid derivatives indicated the presence of NeuNAc; NeuNAc, 9-OAc; and NeuNAc, 7,9 diOAc as the major sialyl derivatives. The radioimmunoassay data appeared to indicate that O-acetylated sialic acid was necessary for immunoreactivity. It should be noted that jejunal mucin and bovine submaxillary mucin also contain O-acetylated sialic acid, but did not inhibit in our radioimmunoassay. This may have been due to differences in the O-acetylation pattern or the linkage of sialic acid to the core carbohydrate. Analyses of the partially methylated alditol acetate derivatives by gas chromatography-mass spectrometry of the untreated, as well as the saponified and neuraminidase treated, mucins revealed that sialic acid was attached to the carbohydrate core either to galactose, N-acetylglucosamine, and/or N-acetylgalactosamine. Linear regression analyses comparing immunoreactivity with specific epitope concns, in conjunction with RIA analyses of known structures, suggested that the organ-specific immunodeterminant was (or was dependent upon the presence of) the structure GlcNAc (1,3)[O-acetylated Neu5Ac(2,6)] GalNAc.
Subject(s)
Colon/immunology , Epitopes/analysis , Mucins/immunology , Chemical Phenomena , Chemistry , Gas Chromatography-Mass Spectrometry , Humans , RadioimmunoassayABSTRACT
We have evaluated 4 radioiodinated mouse monoclonal anticarcinoembryonic antigen antibodies (MAbs) by using the GW-39 human colorectal tumor xenograft transplanted i.m. in immunocompetent hamsters to determine whether there were any differences in their tumor localization properties. Additional comparisons were made to affinity-purified goat anticarcinoembryonic antigen antibody. Statistically significant differences were found in the percentage/g of tumor uptake and tumor/nontumor ratios among the antibodies, so that the antibodies could be ranked according to their tumor localization properties (NP-2 greater than NP-4 = goat antibody greater than NP-1 greater than NP-3). Although statistical differences were found, tumor/nontumor values generally were not distinguished by a factor of more than 1.5, suggesting that these differences may not be biologically significant. F(ab')2 fragments of NP-2 were found to be superior to NP-4 F(ab')2 fragments, giving tumor/liver and tumor/blood ratios of 16 and 11.5, respectively, within 3 days, in comparison to 5.4 and 3.8 for NP-4 F(ab')2 fragments. Mixtures of all of the MAbs or a mixture of NP-2 and NP-4 did not improve tumor localization, in comparison to NP-2 alone. These studies suggest that mixtures of these anticarcinoembryonic antigen MAbs may not afford better tumor imaging than the use of a certain single antitumor MAb.
Subject(s)
Antibodies, Monoclonal , Carcinoembryonic Antigen/immunology , Neoplasms, Experimental/diagnostic imaging , Animals , Antibody Specificity , Colonic Neoplasms/diagnostic imaging , Cricetinae , Humans , Radionuclide Imaging , Time Factors , Tissue DistributionABSTRACT
Carcinoembryonic antigen (CEA) was purified from GW-39 human tumor xenografts in hamsters by immunoaffinity chromatography. Binding of the antigen to immobilized monoclonal antibody provided a high degree of purification of CEA in a single step. A recovery of 79% and a 750-fold purification were obtained. The purified CEA has a molecular size of 180 kilodaltons, an isoelectric point of 4.4, and a specific activity of 0.94. About 73% of the radiolabeled GW-39 CEA reacted with goat anti-CEA serum. The amino acid and carbohydrate compositions of the GW-39 CEA were comparable to those of CEA preparations from other sources.
Subject(s)
Carcinoembryonic Antigen/isolation & purification , Colonic Neoplasms/immunology , Amino Acids/analysis , Animals , Antibodies, Monoclonal/immunology , Carbohydrates/analysis , Carcinoembryonic Antigen/analysis , Carcinoembryonic Antigen/immunology , Chromatography, Affinity , Cricetinae , Humans , Immunoelectrophoresis , Neoplasm Transplantation , Transplantation, HeterologousABSTRACT
Thirteen patients with a history of confirmed liver carcinoma were given either I131 goat polyclonal or murine monoclonal antibodies against alpha-fetoprotein (AFP), and then scanned with a gamma camera. In order to reduce background, nontarget activity, especially in the liver, blood pool, and reticuloendothelial system, 99mTc imaging agents were used for tumor image enhancement by computer-assisted subtraction. A sensitivity of 91% for the primary site, 50% for the lungs (33% for the chest area), and 75% for the abdomen and pelvis was achieved, with a specificity of 100%, 94%, and 100% for these sites, respectively. The accuracy was determined to be 93% for the liver, 86% for the lungs (77% for the chest), and 85% for the abdominal and pelvic area, resulting in an overall accuracy rate for imaging primary and metastatic hepatocellular cancer of 84% (90% if bone metastases are excluded). In two of the 13 patients, lesions that had been missed by conventional liver scintigraphy and transmission computed tomography (CT) were first shown by radioimmunodetection (RAID).
Subject(s)
Antibodies, Monoclonal , Antibodies , Carcinoma, Hepatocellular/diagnostic imaging , Hemangiosarcoma/diagnostic imaging , Liver Neoplasms/diagnostic imaging , alpha-Fetoproteins/immunology , Abdominal Neoplasms/diagnostic imaging , Abdominal Neoplasms/secondary , Adult , Carcinoma, Hepatocellular/secondary , Child, Preschool , Female , Hemangiosarcoma/secondary , Humans , Image Enhancement , Image Processing, Computer-Assisted , Male , Pelvic Neoplasms/diagnostic imaging , Pelvic Neoplasms/secondary , Radionuclide Imaging , Sodium Pertechnetate Tc 99m , Technetium Tc 99m Aggregated Albumin , Technetium Tc 99m Sulfur Colloid , Thoracic Neoplasms/diagnostic imaging , Thoracic Neoplasms/secondaryABSTRACT
Colon-specific antigen-p (CSAp) is a large molecular-sized protein restricted to normal and neoplastic gastrointestinal tissues and to some mucinous ovarian tumors. Murine monoclonal antibodies (MAbs) were raised against CSAp that was affinity purified with goat polyclonal antibodies from GW-39 human colonic carcinoma xenografts or against the CSAp-producing colon cancer cell line SW-948. Two of the MAbs, designated Mu-2 and Mu-4, recognized a CSAp determinant containing sialic acid, and this epitope was also expressed on bovine submaxillary mucin (BSM). Blocking experiments demonstrated that the Mu-2 and Mu-4 MAbs recognized different determinants. A third MAb, Mu-3, did not cross-react with BSM, but unlike Mu-2 and Mu-4, it did react with human saliva. Reactivity of Mu-3 with saliva did not correlate with major blood group and Lewis-related secretory blood group substances in saliva. This reactivity was not related to sialylated Lewis activity. The fourth antibody, Mu-1, appeared to react with a conformational determinant since its epitope was destroyed by heat treatment or thiol reduction. Enzyme immunoassays have demonstrated that all four epitopes may be expressed on one molecular species.
Subject(s)
Antibodies, Monoclonal/immunology , Antigens, Neoplasm/immunology , Epitopes/analysis , Animals , Colonic Neoplasms/immunology , Cross Reactions , Humans , Lewis Blood Group Antigens/immunology , Mice , Mice, Inbred BALB CABSTRACT
Human alpha 1-proteinase inhibitor (alpha 1-PI) yielded nine fragments on cleavage with CNBr. The amino acid sequences of these fragments were determined. Three of these CNBr-cleavage fragments, namely fragment I (residues 64-220), fragment II (residues 243-351) and fragment III (residues 1-63), were found to bind rabbit polyclonal antibodies against chemically oxidized alpha 1-PI and mouse polyclonal antibodies against native alpha 1-PI by the Bio-Dot method (enzyme-linked immunosorbent assay on nitrocellulose). These fragments, I, II and III, inhibited by 60%, 25% and 5% respectively the binding between alpha 1-PI and the rabbit antibodies. Fragments I, II and III were subjected to proteolytic digestion, and 15, ten and five peptides were obtained from these fragments respectively. Only four of these peptides showed binding to the mouse antibodies against native alpha 1-PI. These were residues 40-63, 79-86, 176-206 and 299-323. A panel of monoclonal antibodies was prepared by conventional hybridoma technology, with chemically oxidized alpha 1-PI as the antigen. The ability of the monoclonal antibodies to bind native alpha 1-PI and CNBr-cleavage fragments I-III was determined. The monoclonal antibodies fell into three categories. Most (over 90%) belonged to group I, which was capable of binding alpha 1-PI and only fragment I. Antibodies in groups II and III bound alpha 1-PI and either fragment II or fragment III respectively. The ability of the peptides derived from proteolytic digestion of fragments I, II and III to bind three monoclonal antibodies representing each of the three groups was determined. Among all the peptides tested, only one (residues 176-206) derived from fragment I showed binding to the antibodies from group I, one (residues 299-323) derived from fragment II showed binding to the antibodies from group II, and one (residues 40-63) from fragment III showed binding to the antibodies from group III. Each of these three peptides also inhibited the binding between alpha 1-PI and the corresponding monoclonal antibodies. From these data we concluded that at least four epitopic regions (residues 40-63, 79-86, 176-206 and 299-323) were present in alpha 1-PI. Specific monoclonal antibodies to three of these sites were obtained.
Subject(s)
Blood Proteins/immunology , Epitopes/analysis , Protease Inhibitors/immunology , Amino Acid Sequence , Amino Acids/analysis , Animals , Antibodies, Monoclonal , Antigen-Antibody Reactions , Binding, Competitive , Chromatography, Gel , Enzyme-Linked Immunosorbent Assay , Humans , Peptide Fragments/isolation & purification , alpha 1-AntitrypsinABSTRACT
We previously reported the characterization of a normal adult colonic mucin antigen which contained an organ specific immunodeterminant [Tissue Antigen 11, 362 (1978)]. In the present study we have investigated mucins produced at other levels of the gastrointestinal tract in order to determine if regional specificities exist. Mucins were isolated from normal adult stomach, jejunum, ileum and colon and used to prepare antisera in rabbits. By radioimmunoassay at least four distinct specificities were observed. Gastric, ileal and colonic mucins were shown to contain immunodeterminants which were organ specific. Antiserum directed toward jejunal mucin determinants was reactive with the entire gastrointestinal tract. However, by heterologous inhibition analyses employing purified mucins as inhibiting antigens, the anti-jejunum antiserum was shown to be capable of discriminating a determinant present in much higher epitope density within small intestinal mucins as compared to mucins of the stomach and colon. Thus, it appeared that immunologic determinants present within mucin type glycoproteins of the gastrointestinal tissues exhibit anatomic specificity. In each case the structure of the immunodeterminant was, or was dependent upon the presence of a sialic acid derivative.
Subject(s)
Digestive System/immunology , Mucins/immunology , Organ Specificity , Antigens/isolation & purification , Epitopes , Radioimmunoassay , Sialic Acids/immunology , Tissue DistributionABSTRACT
Two separate cell lines originating from distinct metastatic deposits in a patient with a primary colonic carcinoma have been established both in vivo and in vitro. One metastasis, OM-1, was found in the omentum, and the other, HOT-3, was located on the ovary. These two metastases differ in several significant characteristics, including growth kinetics and the production of carcinoembryonic antigen by the cultured cells. OM-1 xenograft tumors contain about 8-fold more colonic mucin antigen than do HOT-3 heterografts. Similarities also exist. Both cell lines contain extra chromosomes in the A group, and both are missing chromosomes in Pair 14 of the D group and in Pair 18 of the E group. Xenograft tumors from these two metastases contain equivalent amounts of carcinoembryonic antigen and nonspecific cross-reacting antigen. These metastases developed through a process of progression and natural selection that occurred during the course of the patient's disease. Thus, the cell lines established from them provide material which may be used to study functional intraneoplastic diversity.
Subject(s)
Colonic Neoplasms/pathology , Antigens, Neoplasm/analysis , Carcinoembryonic Antigen/analysis , Cell Line , Female , Humans , Karyotyping , Middle Aged , Neoplasm Metastasis , Neoplasm Transplantation , Transplantation, HeterologousABSTRACT
The tumor-associated antigen profile of a number of different human colon carcinomas xenografted into inbred Swiss nude mice was examined to determine whether the tumors could serve as useful models for antigen purification, radioimmunodetection, and immunotherapy. Extreme heterogeneity was observed both by radioimmunoassay and immunohistochemical procedures for the expression of carcinoembryonic antigen (CEA), colon-specific antigens (CSAp), and colonic mucin antigen (CMA) within the tumors. Four of 10 tumors (DLD-2, DLD-3, DLD-5, and HCT-10) were high producers of CEA (greater than 75 micrograms/g wet tissue wt). Two of these (DLD-2 and HCT-10) correlated with a high production of CSAp and CMA, whereas the other 2 produced low quantities of these antigens. Another tumor, HCT-14-OM1, produced large amounts of CMA yet produced moderate amounts of CEA and low quantities of CSAp. Immunohistochemical analyses of CEA gave a mostly diffuse cellular and cell surface staining for all of the tumors. Staining for CSAp was very focal, as in DLD-5 where only a few of the tumor cells were stained. Staining of CMA was limited; however, DLD-2, HCT-10, and HCT-14-OM1 showed intense cystoplasmic and intraluminal staining. A determination of the tumor antigen profile may be useful in characterization and classification of the tumor as well as enabling the selection of the proper antibody or antibodies for immunodetection and immunotherapy.
Subject(s)
Antigens, Neoplasm/analysis , Colonic Neoplasms/immunology , Animals , Antigens, Surface/analysis , Carcinoembryonic Antigen/analysis , Cell Line , Cytoplasm/immunology , Histocytochemistry , Mice , Mice, Inbred Strains , Mice, Nude , Neoplasm Transplantation , Neoplasms, Experimental/immunology , RadioimmunoassayABSTRACT
The physicochemical and immunological characteristics of colon-specific antigen-p (CSAp) in plasma and in colorectal and pancreatic tumors were investigated. CSAp in the plasma of a rectal cancer patient and in a colonic carcinoma xenografted in hamsters (GW-39 tumor) appeared to have similar chromatographic properties, being of a molecular size of 4 million or more. The activities of CSAp in both plasma and tumor were similarly destroyed by treatment with a thiol reagent. Finally, identical immunological reactions in radioimmunoassay and gel diffusion tests were obtained between the CSAp's in patient plasma and in GW-39 tumor tissue. Also CSAp in human pancreatic cancers xenografted in nude mice showed a precipitin line of complete identity with CSAp extracted from GW-39 human colonic carcinoma transplants. Thus, the CSAp's found in colorectal cancer patient plasma, in colonic carcinoma, and in pancreatic cancer tissues appear to be immunologically identical.
Subject(s)
Antigens, Neoplasm/analysis , Colonic Neoplasms/immunology , Pancreatic Neoplasms/immunology , Rectal Neoplasms/immunology , Animals , Cell Line , Chromatography, Gel , Cricetinae , Humans , Immunodiffusion , Mice , Mice, Nude , Neoplasm Transplantation , Neoplasms, Experimental/immunology , RadioimmunoassayABSTRACT
A radiometric immunoassay for detecting colon-specific antigen-p (CSAp) in the blood of patients suspected of having colorectal cancer has been developed and evaluated in 272 subjects of various disease entities. Using 10 units/ml as the cutoff value for normalcy, the results indicate that the highest number of elevated CSAp titers occurred in patients with advanced colorectal cancer (61%). Only one of 12 colonic adenoma patients had an elevated CSAp titer, and this was slightly above the 10 units/ml cutoff. Other nonneoplastic gastrointestinal disorders showed an 18% abnormal CSAp titer frequency, of which more than half bordered the upper limit of normalcy. CSAp elevations were also found in gastric cancer (20%), pancreatic cancer (20%), breast cancer (5%), and normal individuals (3%). CSAp was compared to carcinoembryonic antigen (CEA) in 44 colorectal cancer patients, in 12 patients with colonic adenomas, and in 62 patients with diverse gastrointestinal disorders. Using a CEA cutoff of 5 ng/ml, CSAp could increase the diagnostic accuracy of the CEA plasma test in colorectal cancer patients by 14%. In patients with colonic adenomas, the CSAp titer was normal when the CEA value was elevated in 25% of the cases. However, both were simultaneously elevated in only 3% of the patients with benign gastrointestinal disorders. Since the CSAp test was less frequently elevated (0-7%) in patients with breast, ovarian, lung, or cervical cancer than was found for CEA (39-75% elevated), it seems that the CSAp blood assay detects colorectal cancer more specifically than the more generally distributed CEA. These results suggest that the combined use of blood CEA and CSAp could enhance the discrimination of colorectal cancers from other nonmalignant gastrointestinal diseases, and could also serve to enhance the colorectal cancer-specificity of the CEA assay. Furthermore, serial monitoring of both markers in four advanced colorectal cancer patients indicated that they reflect disease activity, falling after successful treatment and rising again with recurrence and disease progression.
Subject(s)
Antigens, Neoplasm/analysis , Colonic Neoplasms/immunology , Rectal Neoplasms/immunology , Adenocarcinoma/immunology , Adenoma/immunology , Carcinoembryonic Antigen/analysis , Colonic Neoplasms/surgery , Epitopes , Fluorouracil/therapeutic use , Gastrointestinal Diseases/immunology , Humans , Male , Middle Aged , Neoplasm Metastasis , Radioimmunoassay , Rectal Neoplasms/drug therapy , Rectal Neoplasms/surgery , Time FactorsABSTRACT
Human colon adenocarcinomas xenotransplanted into BALB/c nude mice were examined for the ectopic synthesis of alpha 1-proteinase inhibitor(s) (alpha 1Pi). Tumor extracts were prepared by homogenization followed by molecular sieve chromatography on Sepharose 4B. Fractions with immunoreactive alpha 1Pi were concentrated and adsorbed onto a concanavalin A-Sepharose 4B affinity column, then eluted, concentrated, and analyzed. All 4 colon tumors examined produced alpha 1Pi with full antigenic identity to normal human plasma alpha 1Pi. Electrophoretic mobilities of 3 of the tumor-derived alpha 1Pi were different from the electrophoretic mobility of the normal human plasma alpha 1Pi. Activities of the inhibitors were determined by the reaction of each tumor extract with pancreatic elastase (PE) followed by immunoelectrophoresis of the reaction mixture against both anti-human alpha 1Pi and anti-PE antisera. Two of the tumor-derived alpha 1Pi examined were active in inhibiting PE.