Purification and analysis of a human sarcoma associated antigen.

S1, a heterophile antigen present on human sarcoma cell lines in culture, has been previously defined by this laboratory [1,2]. This antigen is also present in guinea-pig kidney. Purification of the antigen to homogeneity has now been achieved by a combination of ammonium sulfate fractionation, DEAE-cellulose, sephadex, high pressure liquid chromotography and affinity chromotography. S1 is a monomeric protein of 70,000 Da, as indicated by the presence of a single band on SDS-PAGE. Amino acid analysis demonstrates the prevalence of glycine, lysine and glutamic acid. Aspartic acid was found to be the N-terminal residue with further sequence of glycine-valine-alanine-glutamic acid (gly-val-ala-glut).

Labeling of sarcoma associated monoclonal antibody with 111In, 67Ga and 125I.

Labeling of human sarcoma-associated murine monoclonal antibody (MAb) 23H7 with 67Ga and 111In by the bifunctional ligand method is reported. 67Ga was chelated to the MAb via desferrioxamine B and 111In via the cyclic anhydride of DTPA. Higher specific activity was obtained with 67Ga (4-5 microCi/micrograms) as compared with 111In (2 microCi/micrograms). The binding capacity of the MAb was confirmed by repeated indirect immuno-fluorescence assays performed before and after labeling. A fast blood clearance was observed: 33% recovered dose (R.D.) blood level 3 h post-injection as compared with 56% after injection of control polyclonal IgG. Preliminary results on chemically induced sarcoma bearing mice showed a relatively high tumor uptake of the labeled antibody.

Human sarcoma-associated murine monoclonal antibody labeled with indium-111, gallium-67, and iodine-125.

Monoclonal antibody (MAb) 23H7 is a hybridoma-derived IgG that is generated following fusion of mouse myeloma cell line P3U1 and spleen cells from BALB/c mice hyperimmunized with a human fibrosarcoma. It detects a mesenchymal antigen of 23KD expressed on human sarcoma tissues and other neoplasms, including myeloid leukemias, but it rarely binds to normal tissues. The MAb 23H7 was labeled with 67Ga and 111In using the bifunctional ligand method. The 67Ga was chelated to the MAb via desferrioxamine B, while 111In was chelated via the cyclic anhydride of diethylenetriamine pentaacetic acid. Higher specific activity was obtained with 67Ga than with 111In (4.5 and 2 muCi/microgram, respectively); both gave stable complexes. When 23H7 was labeled with 125I, considerable breakdown was observed. This, together with the physical shortcomings of this isotope, emphasizes the advantages of labeling with 111In and 67Ga. The rapid blood clearance of the labeled sarcoma-associated MAb may be beneficial for early tumor uptake and for imaging shortly after injection.

The effect of insulin on human-mouse hybridoma formation.

To evaluate the effect of insulin on the formation of human-mouse hybridoma clones, P3U1 mouse plasmacytoma cells were fused with human lymph-node lymphocytes in the presence of polyethylene glycol. After fusion, cells were grown for 2 weeks in HAT medium supplemented with insulin (H1AT, 10(-1)-10(-5) units/ml) or in HAT medium alone. The addition of 10(-3) units/ml of insulin to HAT medium resulted in an over two fold increase in the number of clones formed and in the average colony size compared to growing the fused cells in HAT medium alone. In view of the recent increasing interest in human-mouse hybridoma fusions it is suggested that selective medium HAT should be supplemented by insulin (H1AT) to enhance the number of colonies formed and provide a more efficient way for stabilizing the newly formed hybrids.

Monoclonal antibody defining fibroblasts appearing in fetal and neoplastic tissues.

VIF3 is a hybridoma-derived mouse IgG monoclonal antibody (MoAb) generated in a fusion with the use of a malignant fibrous histiocytoma as the immunizing agent and shown to recognize a 70-kilodalton antigen expressed within connective tissues. Of 55 human tissue culture cell lines tested by indirect immunofluorescence, VIF3 was shown to bind to 20 of 35 (57%) sarcomas, 4 of 9 (44%) normal fibroblasts, and none of 11 carcinomas and other neoplasm-derived cell lines. A panel of over 259 human frozen tissue sections obtained from surgical pathology specimens, postmortem studies, and elective abortions was used to further determine the histopathologic specificity of VIF3. MoAb VIF3 was found to bind to 15 of 19 (79%) human sarcoma tissue sections tested. It was also shown to recognize an antigen expressed on a subset of fibroblasts dispersed within the stroma of carcinomas obtained from all 46 patients tested, as well as a subset of cells within 3 of 10 benign hyperplastic tissues (30%). VIF3-positive cells were detected in all 60 fetal tissues tested including amniotic membranes, placentas, and umbilical cords. In contrast, fibroblasts of normal adult tissues tested stained infrequently (22/97 or 23%) with this reagent. The results confirm that this MoAb is directed against a human connective tissue-specific marker. VIF3 detects a marker appearing on fetal fibroblasts that is typically not present in normal adult tissues, but reappears in association with neoplastic diseases. MoAb VIF3 therefore defines a fibroblast-oncofetal antigen and as such may serve as a probe for immunopathologic studies.

Detection of retroviral particles in hybridomas secreting monoclonal antibodies.

Electronmicroscopy of hybridoma clones derived by fusing BALB/c mouse spleen cells with P3U1 mouse plasmacytoma cells to generate monoclonal antibodies against human sarcoma antigens, revealed the presence of large number of viral particles. These particles were also seen budding from the cell surfaces. The intracytoplasmic particles were intracisternal and resembled type-A oncornavirus, while the budding and extracellular forms, with a centrally located nucleoid, resembled mature type-C oncornaviruses. Cells of the parental P3U1 palsmacytoma cell line and of the NS-1 myeloma cell line contained morphologically identical viral structures. The scientific and medical communities engaged in hybridoma research should be alert to the possible presence of viruses in hybridomas and their products. The question is raised as to whether it is safe to use mouse monoclonal antibodies for clinical purposes, both diagnostic and therapeutic.

Modulation of hybridoma formation by dexamethasone.

In an effort to determine the effect of dexamethasone on hybridoma formation, spleen cells from BALB/c mice hyperimmunized with sheep red blood cells (SRBC) were fused with mouse plasmacytoma cells (P3U1) in the presence of polyethylene glycol (PEG). Dexamethasone was added in decreasing doses (10(-3) to 10(-9) mM) to the hypoxanthine-aminopterin-thymide (HAT) medium immediately after the PEG-mediated cell fusion. 10(-3) mM of this steroid was found to inhibit markedly the number and size of hybridoma clones generated, while 10(-5) mM dexamethasone was shown to enhance hybridoma formation. The effect of 10(-3) mM dexamethasone was most pronounced when added immediately after fusion. When this dose was given 48 or 120 h after cell fusion, the extent of the inhibitory effect was less pronounced. High concentration of dexamethasone may also inhibit monoclonal antibody production by hybridomas once generated. An increase in the number of clones formed was observed when 10(-5) mM dexamethasone was added to HAT medium as well as an increase in the average colony size. Large clones were also observed with lower dexamethasone doses ranging from 10(-7) to 10(-9) mM. Possible mechanisms on the effect of dexamethasone on hybridoma formation are discussed.

The use of insulin-enriched culture medium and human-human hybridoma formation: a preliminary study.

Human lymphoblastoid cell line WI-L2-729-HFZ was fused with human lymph-node lymphocytes in one fusion and with human spleen cells in another fusion to generate human-human hybridomas. In both, increasing doses of insulin were added to the HAT medium immediately after the PEG-mediated cell fusion (10(-1)-10(-5) IU/ml) and the number of clones formed was determined 3 weeks later. 10(-3) IU/ml of insulin resulted in a 2- to 5-fold increase in the number of clones generated compared to the control plates. In view of the known difficulties in generating high-yield human-human hybridoma fusions, it is suggested that the use of insulin-supplemented HAT medium may provide a more efficient way in obtaining such clones.

A high-affinity monoclonal antibody (GIF-1) to human gamma-interferon: neutralization of interferon mediated inhibition of retrovirus production and 2'-5' (A) synthetase induction.

An hybridoma clone secreting an IgG1 monoclonal antibody (GIF-1) specific for human gamma-interferon (HuIFN-gamma) has been generated using HAT medium supplemented with insulin (HIAT) at the initial stage of cell fusion. This antibody is capable of neutralizing the antiviral activity of HuIFN-gamma, the ability of HuIFN-gamma to inhibit retroviral replication in RD-114 cells, and the ability of HuIFN-gamma to induce the 2'-5' oligoadenylate (A) synthetase in RD-114 and HeLa cells. Eluate from an immunoaffinity column containing GIF-1 yielded two protein bands of molecular weight of 20 and 25 kd when subjected to SDS-PAGE.

The interaction of Kaposi's sarcoma with monoclonal antibodies to human sarcoma and connective tissue differentiation antigens.

Four monoclonal antibodies (McAbs) previously generated against human soft tissue sarcomas and reacting with connective tissue differentiation antigens were evaluated for their interaction with tissues obtained from patients with classic Kaposi's sarcoma. Biopsy was performed on active neoplastic lesions from the skin of 26 patients, frozen sections were prepared, and the binding of the McAbs was tested using the indirect immunofluorescence assay. Clinically uninvolved skin from the same patients as well as skin and muscle from eight non-cancer patients were treated similarly and served as controls. McAbs IXG11, 23H7, IIIE5, and 15G5 interacted strongly with the Kaposi's sarcoma lesions and weakly with the uninvolved skin in 22 of 26 (84%), 23 of 26 (88%), 12 of 14 (85%), and 1 of 6 (16%) of the patients, respectively. IXG11, 23H7, and IIIE5 interacted weakly with the skin of seven of eight non-cancer patients. McAb 15G5 was found to bind strongly to tumor lesions, to the respective uninvolved skin in four of five Kaposi's sarcoma patients, and also to skin and connective tissues of muscle from non-cancer patients. The mode of interaction was morphologically different for each McAb. It is suggested that McAbs IXG11, 23H7 and IIIE5 identify markers whose expression is markedly increased in Kaposi's sarcoma lesions as compared with uninvolved skin of the same patients. These markers may serve as immunologic probes for the investigation of this neoplastic process.

Harnessing T-lymphocytes for human cancer immunotherapy.

After nearly a decade of controversy, the concept of adoptive immunotherapy in humans is gaining greater acceptance. More recently, investigators have made use immunotherapeutically of T-lymphocytes nonspecifically activated in vitro by a number of agents, including lymphokines, lectins, and autologous and allogeneic tumor cells. The limitations for the investigational use of these highly specialized and "educated" lymphocytes have been the inability to generate sufficient numbers of cells in vitro for adoptive transfer experiments and to sustain their growth over long periods of time. While marked success has been demonstrated over the years in tumor-bearing animal models, the feasibility of such work in humans has been greatly improved by the experimental expansion and maintenance of immune lymphocytes (those exposed to antigenic challenge) in vitro using either highly purified or recombinant, interleukin 2. As a result, large numbers of lymphocytes can successfully be infused into patients, and whole body scans can show migration of these labeled cells to the lung, liver, and spleen. The use of nontoxic, nonspecific activated "killer" lymphocytes is an innovative approach with enormous potential. This report presents discussion of these findings and addresses the issue of an alternative approach to cancer treatment therapy, the in vivo use of cloned cytotoxic T-lymphocytes sensitized to the autologous tumor.

Clonal stability and heterogeneity of hybridomas: analysis by multiparameter flow cytometry.

Flow cytometry of cellular DNA and RNA content was employed to determine DNA ploidy, proliferation, and RNA content of hybridoma cultures shortly after cell fusion and sequentially thereafter. The parental mouse myeloma cell line P3UI was characterized by tetraploid DNA content, S-phase 50%, and high RNA, the parental mouse spleen cells by diploid DNA content, low proliferation, and low RNA content. Hybridoma cultures studied as early as 21 days after fusion were found to contain the sum of the parental cells' DNA content (hexaploid), or if less, more than that of the myeloma parental cells. Only one clone of 35 tested was shown to be hexaploid and the rest hypertetraploid or hyperpentaploid. Hybrid cell cultures were frequently found to contain a variable mixture of unfused parental cells. The high proliferation of hybridoma cells determined by flow cytometry indicates that these cells would eventually overgrow the parental cells. Flow cytometry also enabled an accurate estimation of the effect of various doses of dexamethasone added to HAT medium immediately after cell fusion on hybridoma formation. Cultures treated with 10(-5) mM of the hormone had a higher DNA ploidy than cultures grown in the presence of 10(-3) mM dexamethasone. No parental cells were observed in the hybridoma cultures studied with this hormone. Sequential DNA/RNA measurements of hybridoma clones showed a decrease in DNA ploidy over time with high dexamethasone doses and a minimal increase or no change with low hormone dose. Flow cytometry is suggested to be a useful technique for evaluating the effects of various agents on DNA ploidy and proliferation and on stability of fused cells.

Monoclonal antibodies to human sarcoma and connective tissue differentiation antigens.

The use of monoclonal antibodies to distinguish human sarcoma from carcinoma cells has been explored. Spleen cells from a BALB/c mouse immunized with a human malignant fibrohistiocytoma were fused with cells of the mouse P3U1 plasmacytoma cell line. Antibodies were then screened for reactivity against human sarcoma and carcinoma cells growing in culture. This work has yielded 2 immunoglobulin G monoclonal antibodies VIE4 and VIF3 which, respectively, reacted with 85% (17 of 20) and 90% (18 of 20) of sarcoma lines tested but with none of eight carcinoma cell line preparations. Reactivity against normal fibroblasts was also demonstrated. By immunofluorescence, the antigens detected by the two antibodies appear to have distinctive intracellular distributions. Immunoprecipitation with VIF3 has shown that it is detecting a protein with a molecular weight of 70,000. When tested against pathological frozen tissue sections, VIF3 reacted with four of 11 and VIE4 with three of 11 human sarcomas but with none of ten carcinomas tested. VIF3 occasionally bound to normal adult connective tissues, whereas no such reactivity was seen with VIE4. These antibodies appear to be directed to fibroblastic markers associated with sarcomas and connective tissue differentiation antigens.

Corynebacterium parvum toxicity in patients with limited and advanced malignancy.

The toxicity of intravenously administered Corynebacterium parvum was observed in 14 patients with stage II melanoma and in 14 patients with advanced ovarian carcinoma. Those with melanoma were rendered disease-free by surgery prior to treatment. The ovarian cancer patients had failed chemotherapy with alkylating agents and were receiving C. parvum prior to chemotherapy as part of an immunochemotherapy trial. Both clinical and laboratory parameters were observed. The mean daily C. parvum dose for melanoma patients was 2.03 mg/m2 and for ovarian carcinoma patients 2.02 mg/m2. The most important clinical toxic effects noted were fever, chills, blood pressure changes, headache, nausea, vomiting and diaphoresis. Laboratory toxicity was mild, with small decreases in hemoglobin levels, white blood cell counts and uric acid and albumin concentrations occurring in some patients. Serum bilirubin and SGOT levels tended to rise. In addition to determining the frequency of clinical toxic effects by treatment course, consideration was also given to frequency per treatment day, correlation of the occurrence of different toxicities in the same patient, time of onset of each toxicity and, for vital signs, to intensity of change and duration. In this analysis no major differences in toxicity were observed when C. parvum was given to the two patient groups.

The addition of insulin to HAT medium (HIAT) enhances hybridoma formation.

To assess the effects of insulin on the formation of hybridomas, sheep red blood cell (SRBC) immunized spleen cells from BALB/c mice were fused with P3U1 mouse myeloma cells. After fusion, cells were grown for 2 weeks in HAT medium containing insulin (HIAT) (doses ranging between 10(-1) to 10(-9) units/ml) or HAT medium alone. The number of hybridoma colonies was found to be significantly increased in the presence of HIAT medium compared to HAT alone. In addition, the average size of the hybridoma clones was at least doubled and the cumulative colony size index per plate increased several folds. A significant rise in the number of wells containing clones secreting anti-SRBC monoclonal antibodies was again shown in the presence of HIAT compared to HAT medium alone. The maximal effect of insulin on the above biological parameters ranged between 10(-1) and 10(-4) units/ml. It is concluded that the addition of insulin to HAT medium (HIAT) enhances hybridoma formation and thus, its more frequent use may considerably expediate ongoing research efforts on the production of monoclonal antibodies.

Enhancement of hybridoma formation by addition of insulin to HAT medium (HIAT).

The effects of insulin on the formation of hybridoma clones following fusion experiments with SRBC immunized BALB/c mouse spleen cells and P3U1 mouse plasmacytoma cells were evaluated. The addition of insulin to HAT medium (HIAT) resulted in significant increases in the number and size of hybridoma colonies generated. The total number of anti-SRBC antibody-secreting clones also increased as much as sevenfold using insulin-supplemented medium compared to HAT alone. In view of the increasing interest in hybridoma technology for monoclonal antibody production, the use of insulin-supplemented medium (HIAT) may significantly expedite ongoing work by providing a more efficient method for the establishment of stable clones.

Prolonged remissions of metastatic breast cancer achieved with a six-drug regimen of relatively low toxicity.

A combination of six chemotherapeutic agents was used to treat 30 women with unresectable metastatic carcinoma of the breast. In the first year five drugs (Cytoxan, methotrexate, 5-fluorouracil, vincristine, and prednisolone [CMFVP]) were given using a weekly schedule for administration of intravenous drugs. During the next year, a seven-week treatment cycle was introduced, with CMFVP given for four weeks, followed by an Adriamycin combination (Adriamycin, cyclophosphamide, and prednisone [ACP]) for three weeks and then the cycle repeated. Treatment was continued for three years or to time of relapse. Overall response rate was 66.7% (20/30). The median duration of response was 40 months and the median survival 39 months. Premenopausal women fared better than postmenopausal women with comparable response rates, duration of response and survival being 81.5%, 41 months, 56 months versus 50%, 20 months and 27 months. Of 16 premenopausal patients treated 7 achieved a complete response (CR) and, of these, 5 remained free of disease at 3 years. For these five individuals all treatment was then stopped. Disease recurred in two patients by five months but three remain disease-free after 43, 40 and 34 months, respectively without therapy. Toxicity was generally limited to heartburn and modest hair loss. This regimen appears to be more effective than those previously employed for metastatic breast cancer. However, comparative trials will be necessary to confirm its advantages.

An enzyme-linked immunosorbent assay (ELISA) for the detection of monoclonal antibodies recognizing surface antigens expressed on viable cells.

A solid-phase enzyme-linked immunosorbent assay (ELISA) has been developed for detecting monoclonal antibodies binding to surface antigens expressed on viable adherent cells of tumor cell lines. This assay utilizes a sheep anti-mouse IgG to which a beta-galactosidase is linked. It is highly sensitive and permits quantification of IgG monoclonal antibody levels. In studies of monoclonal antibodies prepared against human tumors, the ELISA assay revealed the presence of antigens which were not seen using acetone-fixed cell immunofluorescence methods. This assay is safe, rapid, low cost, and gives reproducible quantitative results. As such it should prove useful to laboratories engaged in the study of antigens expressed on cell membranes.