Immunologic and biochemical analysis of TNT-1 and TNT-2 monoclonal antibody binding to histones.

Two novel murine monoclonal antibodies that bind to intracellular antigens, designated TNT-1 and TNT-2, have been generated by immunizing mice with nuclear extracts from human lymphoma cells. The monoclonal antibodies were initially identified by indirect immunofluorescence on lymphoma cell lines and subsequently were found to stain the nucleus of all cell types from several species including plants by indirect immunofluorescence techniques. Immunoblot analysis demonstrated that TNT-1 bound to a protein of 22 kD and TNT-2 bound to two proteins of 15 and 22 kD, consistent with the known molecular weight of histones. To characterize their immunoreactivity, competition radioimmunoassays were performed using purified histone fractions H1, H2a, H2b and H3. By these assays, TNT-1 was found to bind to histone fraction H1 and TNT-2 to an epitope common to histone fractions H1 and H3. Histones are found in abundance in the nucleus of the cell and are known to play a major role in chromosome structure and gene expression. Upon cell death, histones remain tightly bound to DNA and consequently provide an abundant intracellular antigen that can be exploited in targeting necrotic cells, such as those found in tumors.

Chromatographic methods for large-scale preparation of immunoglobulin G2a monoclonal antibodies Lym-1 and TNT-1 F(ab')2 fragments.

Lym-1 and TNT-1 are two murine immunoglobulin G2a monoclonal antibodies (MAbs) which have been used for clinical trials in cancer patients. This paper describes methods for large-scale preparation of F(ab')2 fragments from 50 mg to 4 g of MAbs Lym-1 and TNT-1. Digestion of MAbs with pepsin was optimized and performed at pH 3.8, a pepsin/antibody ratio of 1:250, and 3-4 h of incubation at 37 degrees C. The F(ab')2 fragments were purified by tandem column procedures using fast protein liquid chromatography. Quality control analyses of the products included protein purity, isoelectric point, immunoreactivity, and endotoxin level. The results revealed that the chromatographic procedures are practical, simple, and effective, and can be used to produce gram quantities of clinical-grade F(ab')2 fragments for the diagnosis of cancer in patients.

Cell based radioimmunoassays to quantitate the immunoreactivity of TNT monoclonal antibodies directed against intracellular antigens.

Direct and indirect radioimmunoassay (RIA) procedures to determine the amount of binding of a mouse monoclonal antibody (MoAb) reactive with an intracellular antigen present in human cells are described. In these RIAs, mouse IgG2a MoAb, designated as Tumor Necrosis Treatment (TNT-1) antibody, paraformaldehyde/acetone fixed cells, and Sephadex beads were used to standardize the assay conditions. In the direct RIA, 83% of the 125I-labeled TNT-1 MoAb was bound to the target cells within 30 min after the addition of reagents. The amount of binding of the MoAb was directly proportional to the amount of antigen present in the assay. When the direct RIA was carried out using different types of target cells, 125I-labeled TNT-1 MoAb showed greater than 70% binding. In the indirect RIA, the amount of binding of secondary 125I-labeled goat anti-mouse IgG antibody to the target cells was linear. These results suggest that the indirect RIA can be used to estimate the immunoreactivity of the unlabeled TNT-1 MoAb present in crude protein preparations. Based on the results of RIAs the following two conclusions are drawn: 1) the direct RIA can be used to estimate rapidly the amount of immunoreactive TNT-1 MoAb present in 125I-labeled antibody preparations and 2) the indirect RIA which estimates the amount of immunoreactivity of unlabeled TNT-1 MoAb can be used to monitor the purification and study the characteristics of the MoAb present in crude protein preparations. These methods enable the quantitative measurement of MoAbs reactive against intracellular antigens using standard RIA procedures.

A live cell enzyme-linked immunosorbent assay for detecting human hepatoma membrane antigens.

Live cell enzyme-linked immunosorbent (ELISA) and fixed cell indirect immunofluorescence (IF) assays were compared to screen mouse hybridomas producing immunoreactive monoclonal antibodies against cell membrane antigens expressed on Ha22T, a human hepatoma cell line. While performing live cell ELISA, two parameters were tested to improve the viability of the target cells. The first parameter was the inclusion of growth medium in the assay buffers, and the second was performing the assay incubations at 37 degrees C in an incubator containing 5% CO2 in the air. Fixed cell IF detected and classified 46% of the hybridomas secreting monoclonal antibodies reactive with membrane, cytoplasm, cytoskeleton, and nuclear antigens of Ha22T cells. Fixed cell IF was able to reveal mixtures of two or more hybridomas growing in the same well secreting antibodies to different cell organelles. The live cell ELISA, on the other hand, identified 12 additional membrane reactive monoclonal antibodies from the hybridoma supernatants that were not reactive by IF. These results disclose that cell fixation procedures used for IF either completely or partially inactivated some of the cell membrane antigens. We, therefore, propose the use of a combination of immunoassays to select the maximum number of hybridomas secreting useful monoclonal antibodies from somatic cell fusions.

Variations in the secretion of monoclonal antibodies by human-human hybridomas.

A series of human-human hybridomas derived from a single fusion of UC 729-6 with lymph node lymphocytes were examined for the type and nature of macromolecules synthesized and secreted. One hybrid, VLN3G2, secreted fourfold higher IgG than that present in the cytoplasm over 4 days of growth, while the IgM distribution was opposite to that of IgG. VLN5C7, contrary to VLN3G2, contained several-fold more cytoplasmic IgG as well as IgM than the amounts secreted over the same period of time. Of the secreted IgG and IgM by both of these hybridomas, only the IgG showed immunoreactivity against target A431 cell surface antigen(s). Another hybridoma, termed VLN1H12, secreted immunoreactive IgM against target A431 cells, but no detectable IgG. Cytoplasmic proteins prepared by repeated freeze-thaw of the hybridoma cells, membrane proteins obtained by NP-40 extraction of the cell membrane, and secreted proteins present in the supernates of the various hybridomas were assayed by an enzyme-linked immunoassay (ELISA), to understand the discrepancy observed in the immunoglobulins of the cellular and extracellular compartments. The parental UC 729-6 cell line used in these cell fusions produced only trace amounts of immunologically inactive IgM and no detectable IgG. Molecular sieving column chromatography of these hybridoma supernates suggested the presence of intact IgG and IgM molecules and the absence of free heavy chains or hybrid antibodies containing both mu and gamma heavy chains. Intrinsic labeling of VLN3G2 hybridoma cells with 35S-methionine demonstrated the presence of not only a nonimmunoglobulin protein but also a small molecular weight protein-A-binding polypeptide in the culture supernatant. 35S-methionine-incorporated IgG and IgM antibodies, isolated from spent media, cytoplasm, and cell membranes of VLN3G2, also showed binding to protein-A-bearing bacteria. In conclusion, the differences observed in the amounts of secreted MAbs by the human-human hybridomas were not due to the decreased synthesis of these molecules.

Experimental radioimmunotherapy of a xenografted human colonic tumor (GW-39) producing carcinoembryonic antigen.

Experiments were undertaken to evaluate the antitumor effects of 131I-labeled goat antibody immunoglobulin G prepared against carcinoembryonic antigen in hamsters bearing the carcinoembryonic antigen-producing GW-39 human colonic carcinoma. At a single injection of 1 mCi 131I and higher, a marked growth inhibition of GW-39 tumors, as well as a considerable increase in the survival time of the tumor-bearing hamsters, could be achieved. At a dose of 1 mCi, the radioactive affinity-purified antibody appeared to be superior to radioactive normal goat immunoglobulin G in influencing tumor growth and survival time, but no significant difference could be seen at the higher dose of 2 mCi given. Radiobiological calculations indicated that the tumors received, at up to 20 days after therapy, 1325 rads for the specific antibody and only 411 rads for the normal immunoglobulin G preparation. These findings encourage the further evaluation of antibodies to tumor markers for isotopic cancer therapy.

Experimental studies of tumor radioimmunodetection using antibody mixtures against carcinoembryonic antigen (CEA) and colon-specific antigen-p (CSAp).

Experiments with the GW-39 human colonic carcinoma growing in hamsters showed that injection of radioactive antibody to a colorectal-specific, tumor-associated antigen, CSAp, results in better tumor radiolocalization than was seen previously with radioantibodies to carcinoembryonic antigen (CEA). However, a mixture of both radioactive antibodies resulted in potentiation of CEA-tumor radioimmunodetection without affecting CSAp-tumor radiolocalization. Hence, multi-marker antibody mixtures may be the method of choice in cancer radioimmunodetection.

Further studies on a human lung tumor-associated antigen. Comparison of antigens from different tumors.

A human lung tumor-associated antigen was purified to homogeneity from a crude cell-free extract of a human lung adenocarcinoma using standard biochemical procedures. In order to facilitate monitoring the recovery of antigen, trace amounts of previously purified and radioiodinated antigen from another lung tumor were added to the crude extract. The purified antigen was a glycoprotein and contained sialic acid. The antigen had a molecular weight of 76,000 and appeared to contain three subunits, each with a molecular weight of 25,000. The antigen had the following physical properties: Stokes radius, 39.4 A; S20,w, 4.24 S; D20,w, 5.15 x 10(-7) cm2 S-1; and a frictional ratio of 1.40. In addition, the purified, radioiodinated antigen retained complete immune reactivity since it could be quantitatively precipitated with specific immune serum. All of these properties were in close agreement with the properties of another antigen which was purified from a separate human lung tumor. Thus, it appeared from the biochemical and immunochemical criteria presented in this report that a common and identical antigen was isolated from two distinct human lung tumor extracts.

Purification and Some Biological Properties of Asparaginase from Azotobacter vinelandii.

Asparaginase was found in the soluble fraction of cells of Azotobacter vinelandii, and its activity remained the same during growth of the organism in a nitrogen-free medium. The specific activity and the yield of A. vinelandii increased twofold in the presence of ammonium sulfate. Within limits, the temperature (30 to 37 degrees C) and pH (6.5 to 8.0) of the medium showed little effect on the levels of enzyme activity. The enzyme was purified to near homogeneity by standard methods of enzyme purification, including affinity chromatography, and had optimum activity at pH 8.6 and 48 degrees C. The approximate molecular weight was 84,000. The apparent K(m) value for the substrate was 1.1 x 10 M. Metal ions or sulfhydryl reagents were not required for enzyme activity. Cu, Zn, and Hg showed concentration-dependent inhibition, whereas amino and keto acids had no effect on the enzyme activity. Asparaginase was stable when incubated with rat serum and ascites fluid. The enzyme had no effect on the membrane of sheep erythrocytes and did not inhibit the incorporation of radioactive precursors into deoxyribonucleic acid, ribonucleic acid, and protein in Yoshida ascites sarcoma cells. Asparaginase activity was not detected in the tumor cells.