Biomedical subjects

J C Roder

Publications and source records attributed to J C Roder.

At least 91 records · Page 5Linked to original sources

PubMed1985

Spontaneous and induced primary oncogenesis in natural killer (NK)-cell-deficient beige mutant mice.

Spontaneous tumor development and primary oncogenesis were compared in a large number of NK4-deficient, homozygous C57Bl/6-bg/bg mice and their NK normal, heterozygous +/bg littermate controls. In a group of 167 retired breeders followed for spontaneous tumors, the probability of survival for mice eventually dying with a tumor was greater for the NK-competent, +/bg than for the NK-deficient, homozygous C57BL/6-bg/bg mice (p = 0.0019), although the higher overall incidence of tumors in the bg/bg group (48%) was not significantly different from that in the +/bg group (37%). In the bg/bg group the incidence of tumor death appeared to increase relatively sharply in the 25- to 29-month age bracket compared to the fairly regular increase in incidence observed in the +/bg group. All the spontaneous tumors except 2 (discovered in +/bg mice) were classified histologically as widely disseminated malignant lymphomas. The other two were one squamous-cell carcinoma and one sarcoma. A total of 73 bg/bg mice injected s.c. with benzo[alpha]pyrene (BP) had a higher overall incidence of tumors (81%) (rhabdomyosarcomas) than 138 +/bg mice (64%) and in the largest group (0.3 mg, n = 85) the bg/bg group developed tumors, at a higher incidence (p = 0.01) and with a shorter latency (p = 0.025) than the +/bg group. On the other hand, mice injected with dimethylbenzanthracene or given 4 weekly doses of 160 rads of gamma irradiation showed no difference in overall tumor incidence. In addition, mice injected with various doses of DMBA-induced murine leukemia virus (DMBA-LV) also showed no significant difference in tumor incidence. Others have reported that some of these treatments (DMBA, split-dose irradiation) cause profound NK suppression, thereby reducing NK differences between the two groups of mice. These results suggest that a partial NK impairment in beige mutant mice early in life may lead to significantly greater rates of death with spontaneous malignant tumors late in life. Some primary oncogenesis treatments (BP) but not others (DMBA, split-dose irradiation, leukemia viruses) cause tumors with a greater incidence and shorter latency in beige mice. The results suggest, but do not prove, that NK cells play a role in surveillance against spontaneously arising, and possibly some types of carcinogen-induced, tumors.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1985

Amplified sequences from chromosome 15, including centromeres, nucleolar organizer regions, and centromeric heterochromatin, in homogeneously staining regions in the human melanoma cell line MeWo.

Homogeneously staining regions (HSRs) in the human melanoma cell line, MeWo, are located on an X and a der(15) chromosome. These regions are homogeneously stained with quinacrine fluorescence, but stain differentially with conventional Giemsa, G-banding, C-banding, and distamycin A/4',6-diamidino-2-phenylindole. There are five and six blocks of positively staining material on the X and der(15) HSRs, respectively. Hybridization in situ with a cloned repetitive Kpn I family member has confirmed the amplification of this sequence along the HSRs. With silver staining of the nucleolar organizer regions (NORs) there appear to be three very strong and two weaker pairs of NORs along the HSR of the X chromosome and four strong and one weaker pair on that of the der(15) chromosome. There was little NOR staining on the normal acrocentric chromosomes in these cells, suggesting preferential transcription of the NORs in the HSRs. Centromere-dot staining revealed a distribution of multiple centromeres similar to the NORs along the two HSRs. These data suggest that a unit composed of the short arm and centromere of chromosome #15 has been amplified and that the HSR present on the X chromosome probably arose by a translocation from chromosome #15.

Cell Line↗

PubMed1985

Organization of a repetitive human 1.8 kb KpnI sequence localized in the heterochromatin of chromosome 15.

We have isolated a repetitive 1.8 kb KpnI DNA sequence which is amplified in the homogeneously staining regions of a human melanoma cell line. Under low stringency conditions this sequence (D15Z1) hybridized in situ to the centromeric heterochromatin of chromosomes 1, 9, 15p, 16, and distal Yq as well as to the short arms of the other acrocentric chromosomes. Under conditions of high stringency, labelling was predominantly on the short arm of chromosome 15. D15Z1 was shown to be present at approximately 3,000 copies per haploid genome and organized in long tandem arrays showing restriction site heterogeneity. Sequences homologous to D15Z1 were highly enriched in the less dense shoulder region of a Ag+-Cs2SO4 gradient. Analysis of D15Z1 indicated that this sequence is composed of tandemly arranged imperfect repeats of the consensus 5' AATGG 3' similar to previously identified satellite III sequences. Digestion of D15Z1 with HinfI resulted in a series of restriction fragments making up a subset of the HinfI ladder components of satellites III and IV. These data suggest that D15Z1 represents a chromosome 15 specific domain of human satellites III or IV and that it makes up the major fraction of the heterochromatin of this chromosome. Possible relationships between this sequence and the cytochemical staining properties of human chromosomes with distamycin A/DAPI, D280/170, and antiserum to 5-methylcytosine are discussed.

Chromosomes, Human, 13-15↗

PubMed1985

Segregation of the NK-sensitive phenotype in human x mouse somatic cell hybrids reveals separate genetic control of recognition and postrecognition determinants.

In an attempt to investigate the nature of tumor cell-derived membrane surface determinants involved in natural killer cell (NK) recognition or postrecognition events, we have constructed human X mouse interspecies somatic cell hybrids. Highly NK-sensitive (NKs) human tumor cells were fused with NK resistant (NKr) mouse fibroblasts (LMTK-) in polyethylene glycol and selected in hypoxanthine/aminopterin/thymidine medium and ouabain. Hybrids generated from NKs erythroleukemia cells (K-562) or NKs retinoblastoma cells (Y-79) with LMTK- displayed an intermediate NK-sensitive phenotype. One Y-79 X LMTK- hybrid (YL-22) retained a high level of susceptibility to NK binding and cytolysis, as determined by 51Cr release and in cold-target inhibition assays. On the other hand, human NKr RAJI cells generated NK-resistant hybrids when fused with LMTK- fibroblasts. Four hybrids (KL-12, YL-2, YL-22, and YL-43) displaying consistent NK sensitivity were subsequently cloned by limiting dilution. Various hybrid clones derived from the KL-12 hybrid (K-562 X LMTK-) demonstrated a range of NK-sensitive phenotypes. However, the uncloned KL-12 and most cloned lines derived from this hybrid competed against 51Cr-labeled K-562 targets as well as unlabeled K-562 parental cells, regardless of their NK-sensitive phenotype. These findings raise the possibility that chromosomal segregation may be affecting a postbinding step in this hybrid system. The NK-sensitive hybrids exhibited a limited number of human chromosomes as assessed by quinacrine banding. Furthermore, human transferrin receptor (TfR) expression, as monitored by flow cytometry using the B3/25 monoclonal antibody, demonstrated no clear correlation with NK sensitivity or competitive ability in either KL or YL hybrid clones, thus arguing against the involvement of the TfR in human NK recognition. These results suggest that the NK-sensitive phenotype in human tumor cells may be regulated by genes encoded by a limited number of human chromosomes.

Animals↗

PubMed1985

The characterization and cellular distribution of a family of antigens related to myelin associated glycoprotein in the developing nervous system.

The antigenic epitope detected on myelin associated glycoprotein (MAG) by the monoclonal antibody HNK-1 (Leu 7) was sensitive to degradation by trifluoromethane-sulfonic acid (TFMS) and is therefore probably carbohydrate in nature. This antigen was found to be widely distributed within the rat and chicken embryonic nervous system and was present on cultured central and peripheral neurons (100%), oligodendrocytes (100%) and astrocytes (70-80%) as detected by double marker immunofluorescence. The antigen could be removed from cultured neurons by trypsinization and its resynthesis was blocked by cycloheximide, suggesting that the carbohydrate epitope detected by HNK-1 was attached to a de novo synthesized protein. Several molecular species were detected on Western blots of detergent extracts from 13-15d rat embryonic brain and neuron-enriched cultures from chick spinal cord and dorsal root ganglia. Protein components with molecular weights in the ranges of 90-100 kd to 280 kd were observed and comprise a family of glycoproteins containing the HNK-1 reactive carbohydrate epitope present on MAG. These glycoproteins could play a role in intercellular interactions within the developing nervous system.

Animals↗

PubMed1985

Human monoclonal antibodies against Mycobacterium leprae.

Human hybridomas were constructed which produce antibodies against three different extracts of Mycobacterium leprae. A thioguanine-resistant (Thgr), ouabain-resistant (Ouar), human lymphoblastoid cell line, KR-4, was hybridized with Epstein-Barr virus-transformed cell lines from lepromatous leprosy patients with fusion frequencies of greater than 10(-5). Non-Epstein-Barr virus-transformed donor cells fused at much lower rates (less than 2 X 10(-7]. Hybrids were selected in medium containing hypoxanthine aminopterin thymidine and 10(-5) M ouabain. An enzyme-linked immunosorbent assay was used to screen for antibodies against three crude extracts of armadillo-derived M. leprae, including (i) a soluble sonic extract preparation, (ii) sodium dodecyl sulfate extract of insoluble sonicated M. leprae, and (iii) a purified phenolic glycolipid antigen. Of a total of 2,200 final clones screened, 359 were found to secrete antibody which bound to soluble sonic extracts and the sodium dodecyl sulfate extract (6.7 and 9.6%, respectively), whereas 12.5% (21 out of 168) showed positivity to the glycolipid antigen. Four selected hybridomas also reacted with the deacylated derivative of M. leprae phenolic-glycolipid antigen. The specificity of these monoclonal antibodies was partially determined by screening on a panel of crude extracts from four other mycobacteria. Nine clones of 122 showed reactivity to M. leprae only. The predominant immunoglobulin was immunoglobulin M, and quantities up to 10 micrograms/ml were produced. Antibody production by hybrid clones was stable in more than 75% of the clones grown in continuous culture. By comparison, 10,000 Epstein-Barr virus-transformed lymphocyte clones from lepromatous leprosy patients were screened for anti-M. leprae antibody production, and all of the 42 clones that were initially positive in the enzyme-linked immunosorbent assay lost their antibody-producing capabilities within 6 weeks in culture. These results suggest that a combination of Epstein-Barr virus transformation and hybridization may be an optimal method in producing human monoclonal antibodies from leprosy patients.

Antibodies, Bacterial↗

PubMed1985

Natural killer cell-mediated lysis involves an hydroxyl radical-dependent step.

The role of oxygen radicals in lysis of K562 target cells by human natural killer (NK) cells was determined by addition of scavengers of these free radicals. Lysis was greatly reduced under hypoxic conditions. Superoxide dismutase and cytochrome c, scavengers of superoxide anions, and catalase and scavengers of hypochlorite had no effect on lysis. Of 15 hydroxyl radical scavengers tested, 13 inhibited lysis. These were not toxic, because cell morphology and spontaneous chromium release were not affected and preculture with scavengers was not inhibitory. These scavengers differed widely in structure, but degree of inhibition of lysis correlated with their rate constants (k) for reaction with hydroxyl radical (k vs log inhibitor concentration required to decrease lysis by 50%: r = -0.9202, p less than 0.001), showing that inhibition was due to inactivation of the hydroxyl radical. Target cell binding was not reduced at concentrations that inhibited lysis. Inhibitors of the lipoxygenase pathway also decreased lysis, suggesting this pathway to be the source of hydroxyl radicals. In view of the reported requirements for hydroxyl radical-mediated lipid peroxidation for optimal secretory activity in a number of cell types, it appears that the generation of hydroxyl radicals by NK cells is required for delivery of cytotoxic factors.

Arachidonic Acid↗

PubMed1985

An exoglycosidase-sensitive triggering site on NK cells which is coupled to transmethylation of membrane phospholipids.

Glycosidic enzymes were used as probes to analyze the mechanism of NK cell-mediated cytotoxicity. Pretreatment of nylon wool-enriched CBA/J spleen cells, a murine NK clone, or human peripheral blood lymphocytes (PBL) with alpha-mannosidase, an exoglycosidase, led to a marked dose-dependent inhibition of NK lytic activity against YAC-1.2 or K562 tumor cells. Maximal inhibition occurred after a 60-min pretreatment of murine effectors at 37 degrees C, and the kinetics of NK inhibition by alpha-mannosidase was similar to the reported kinetics for enzymatic activity. Released hexose was detected chemically in the supernatant of mouse spleen cells treated with NK inhibitory dose of alpha-mannosidase, and inactivation of enzymatic function with EDTA reversed the NK inhibitory effect. These results suggest that alpha-mannosidase inhibited NK function by virtue of its enzymatic action. Culture of human PBL for 20-hr after treatment with this enzyme led to a greater than 70% recovery in NK lytic function. Recovery was blocked by incorporating tunicamycin, a glycosylation inhibitor of asparagine-linked glycoproteins, into the culture medium. These results suggest that the alpha-mannosidase-sensitive site may be de novo synthesized glycoprotein. Neuraminidase, beta-galactosidase, endo-beta-N-acetylglucosaminidase-D and H, and peptide-N-glycosidase treatments did not inhibit human NK cell lysis of K562 cells. Pretreatment of nylon wool-enriched CBA/J spleen cells or Percoll-enriched human LGL with alpha-mannosidase did not influence their capacity to bind YAC 1.2 target cells or K562 target cells, respectively, Ca++ pulse experiments revealed that the alpha-mannosidase-sensitive site on the NK cells was involved after target-effector binding but before the Ca++ influx. Pretreatment of effector cells with this enzyme which normally occurs after effector-target cell interaction. These results suggest that the phospholipid methylation reaction is coupled to the alpha-mannosidase-sensitive site on the NK cells. By analogy to other physiologic systems, such as histamine release in mast cells, the triggering of phospholipid methylation in the NK cells may serve as a mechanism for signal transduction across the plasma membrane.

Animals↗

PubMed1985

Differential expression of the Leu-7 antigen on human lung tumor cells.

The HNK-1 monoclonal antibody detects an antigen (Leu-7) on a subpopulation of large granular lymphocytes which have natural killer cell function. Recently this antigen has been found on nonhemopoietic tissues. In the present study human lung tumor cells were examined for the presence of Leu-7 antigen using an enzyme-linked immunosorbent assay, immunoperoxidase staining, and fluorescence-activated cell sorter analysis. All small cell lung tumor cells tested were Leu-7 positive. In contrast only two of seven biopsy specimens from small cell lung cancer patients were Leu-7 positive. Several large cell lung tumor lines were Leu-7 positive while an adenocarcinoma and squamous cell carcinoma were negative. These results indicate that expression of Leu-7 antigen on lung tumor cells is heterogeneous both in vitro and in vivo. Small cell lung tumor lines have been reported to undergo histological conversion in vitro accompanied by the loss of a number of biochemical markers. In our study histologically converted cells exhibited much less reactivity with HNK-1 than did the parent cells. These results indicate that the degree of expression of Leu-7 antigen may be under the control of differentiation-related events. Thus monoclonal antibody HNK-1 has been very useful in studying heterogeneity within and among lung tumor cells.

Antibodies, Monoclonal↗

PubMed1985

DNA amplification and tumorigenicity of the human melanoma cell line MeWo.

Homogeneously staining regions (HSRs) were found in hypodiploid cells (40%) of a subline of the human melanoma cell line, MeWo, (MeWo-C) but were absent from the hypotetraploid cells (60%). Another subline (MeWo-B) was also shown to contain two populations of cells, 70% hypodiploid and 30% hypotetraploid. None of the MeWo-B cells contained HSRs, but all four cell types from both sublines shared marker chromosomes indicating their common origin. The hypodiploid MeWo-B cells were karyotypically similar to the hypodiploid MeWo-C cells except for the presence of the HSRs in the latter. Both MeWo-C and MeWo-B sublines were injected into BALB/c nude mice. The MeWo-C cells were markedly more tumorigenic than MeWo-B cells as judged by tumor incidence, latency, average tumor size, and tumor take values. Cytogenetic and flow cytofluorometric analyses of the tumors induced by MeWo-C cells revealed a shift in the tumor cell population from 40% to greater than 90% HSR-containing hypodiploid cells during tumor growth. Hybridization of tumor DNA to a probe (D15Z1), the sequence of which is amplified in the HSRs, also indicated an increase in the proportion of HSR-bearing cells during tumor growth. No such selective advantage was found with the hypodiploid, HSR-lacking MeWo-B cells. The results suggest that HSRs found in the human melanoma line MeWo may confer enhanced tumorigenicity to the cells containing them.

Animals↗

PubMed1984

In vitro stimulated lymphocytes as a source of human hybridomas.

Stimulation of peripheral blood lymphocyte (PBL) cultures from tetanus toxoid (TT)-immunized donor with Epstein-Barr virus (EBV) yielded cells with much higher frequencies of hybrid formation (36 X 10(-7) compared to unstimulated PBL or cells cultured with pokeweed mitogen or TT antigen. The proportion of hybridomas (approximately 1%) producing anti-TT antibody was similar in EBV- and TT-stimulated cultures. A marked increase in immunoglobulin secretion was observed after hybridization and preselection of EBV subcultures for high anti-TT production prior to fusion resulted in a fivefold increase in TT-specific hybridomas (p less than 0.001). Most (20/21) specific hybrids produced IgM anti-TT, whereas one (1/21) produced IgG anti-TT, possibly due to the immature stage of differentiation in EBV-stimulated parental cells. The ability to choose an antigen, immunize a human subject and expand the rare antigen-specific B cells from PBL, in vitro, with EBV, prior to fusion, should yield an increasing spectrum of human monoclonal antibodies for diagnostic, therapeutic or basic studies.

Adult↗

PubMed1984

Human monoclonal antibodies.

The technology for the production of murine monoclonal antibodies has been refined enormously since its introduction in 1975. However, the technology for generating human monoclonal antibodies has only recently come into its own. In this review, three currently available approaches to the production of human monoclonal antibodies are described. These include the hybridoma technique, based on the fusion of antibody-producing human B lymphocytes with either mouse or human myeloma or lymphoblastoid cells; the EBV immortalization technique, based on the use of Epstein-Barr virus (EBV) to 'immortalize' antigen-specific human B lymphocytes; and the EBV-hybridoma technique, based on a combination of the first two methods. The EBV-hybridoma system retains the advantageous features of the other two systems while overcoming their pitfalls and may be the current method of choice for producing human monoclonal antibodies with a defined specificity.

Antibodies, Monoclonal↗

PubMed1984

The cooperative effect of the satin and beige mutations in the suppression of NK and CTL activities in mice.

The functional activity of natural killer (NK) cells has been found to be modulated by several point mutations associated with coat color. The most commonly studied gene, beige (Bg), has been found to block a postrecognition event in the lytic cycle. Four other coat color mutations in the mouse (satin, leaden, fuzzy, pale ears) were studied for their effect on NK cell function, and only one, satin (Sa), was found to be suppressive. When both the Sa and Bg mutations were present in the same animal, their effects were synergistic in the suppression of NK levels. Normal numbers of NK cells were present in these double mutants, as determined by the frequency of IgG2b binding cells and by antiasialo GM1 staining. The ability of Sa/Bg NK cells to recognize and bind targets suggests that the defect is localized in the postbinding cytolytic pathway. These genes were not specific for NK cells and also suppressed alloimmune cytolytic T lymphocyte function. Since Sa/Bg mice are much more suppressed in NK function than Bg mice, we suggest that this double mutant may be a better model for NK deficiency in vivo.

Animals↗

PubMed1984

The inhibitory effect of phenothiazines on NK-mediated cytolysis of tumor cells.

Non-toxic concentrations of fluphenazine caused a marked (90%) inhibition of NK-mediated cytolysis of YAC-1 tumor cells. The biologically inactive sulphoxide derivative was not inhibitory and the efficacy of inhibition of other compounds was directly correlated (r = -0.96, p less than 0.02) with their reported affinities for calmodulin. Fluphenazine may act on the earliest stages of the target-effector interaction since conjugate formation between CBA effectors and YAC target cells decreased from 20% to 6% (p less than 0.02) upon pre-treatment with fluphenazine. However, fluphenazine was not selective for NK cells since cytotoxic T lymphocytes, derived from both mixed lymphocyte culture and by concanavalin A stimulation, revealed depressed cytolytic activity against P815 tumor targets after fluphenazine treatment. Tumoricidal activity by activated macrophages and effectors of antibody-dependent cell-mediated cytotoxicity was also blocked. Fluphenazine inhibition was reversible, since addition of 1.25-5 micrograms/ml of the calcium ionophore A23187 to fluphenazine-treated effectors restored NK binding and cytolytic functions to normal levels. Calmodulin was isolated from NK-enriched populations by affinity chromatography on sepharose-fluphenazine columns. Pre-treatment of effector cells with [3H]fluphenazine and isolation of calmodulin by immunoprecipitation and SDS-polyacrylamide gel electrophoresis showed that fluphenazine entered the cells and bound a calmodulin-like molecule. These data are compatible with the suggestion that fluphenazine inhibits NK function by inactivating the calcium-calmodulin complex and thereby altering binding events in the target-effector interaction. Other actions of the phenothiazines are also possible.

Animals↗

PubMed1984

Involvement of hydroxyl free radical, but not superoxide, in the cytolytic pathway of natural killer cells. Revision of an earlier hypothesis.

Our earlier hypothesis suggested that NK effectors, in response to tumor cells, generate superoxide anion (O2.-) which was necessary for NK cytolysis to proceed. This event could be detected by chemiluminescence. More recent data suggests that O2.- is not necessary for NK cytolysis and that chemiluminescence is the result of an NK-tumor cell-monocyte interaction. Hydroxyl radicals however do contribute to the NK-mediated cytolytic process. The source of OH. is unknown but does not appear to be generated by the NADPH oxidase system. We suggest that hydroxyl radical generation is a necessary event in NK cytolysis but we do not yet know if it acts at the level of NK activation, delivery of the lethal hit or in autolysis by the tumor cells.

Cytotoxicity, Immunologic↗

PubMed1984

A human hybrid myeloma for production of human monoclonal antibodies.

We produced somatic cell hybrids between human myeloma cells and a lymphoblastoid cell line that is hypoxanthine phosphoribosyl transferase-deficient and ouabain-resistant. These hybrids were phenotypically similar to the human myeloma parental cells and grew as well as the human lymphoblastoid parental cells. After counterselection in 6-thioguanine, mutants that were 6-thioguanine-and ouabain-resistant were obtained, one of which was used as a fusion partner with lymphoblastoid B cells that produce anti-tetanus toxoid (TT) antibodies. These hybrids secreted human anti-TT monoclonal antibodies in much larger amounts than the parental lymphoblastoid cells, and were stable for a period of over 10 mo until the present time. Thus, by hybridizing plasmacytomas with lymphoblastoid cells, we constructed a fusion partner that secretes large amounts of immunoglobulin (Ig), grows at a fast rate, has a high fusion frequency, and supports the production of monoclonal antibodies over long periods of time. Moreover, anti-TT antibody-producing hybrids have been grown as solid tumors in irradiated BALB/c nude mice and then adopted to ascites growth, producing 1 to 8 mg of human immunoglobulin per 1 ml of ascites fluid.

Animals↗

PubMed1984

Amplified KpnL repetitive DNA sequences in homogeneously staining regions of a human melanoma cell line.

The human melanoma cell line MeWo was found to contain two populations of cells, one containing 83 chromosomes (hypotetraploid) and the other containing 43 chromosomes (hypodiploid). All of the hypodiploid cells, but none of the hypotetraploid cells, contained chromosomes with long homogeneously staining regions (HSR's) when examined with quinacrine fluorescence. These long HSR's on an X-chromosome and derivative chromosome 15, produced characteristic patterns of positive- and negative-staining areas along the HSR's with both conventional Giemsa (G) staining and C-banding. The C- and G-positive regions stained with distamycin A-DAPI, which is specific for the centromeric heterochromatin of chromosomes 1, 9, 15p, 16, and Y. DNA extracted from MeWo cells and digested with the restriction enzymes KpnL or Sau3A exhibited marked amplification of a 1.8-kilobase fragment. The amplified Sau3A fragment (D15Z1) was cloned, mapped, and partially sequenced. The sequenced region contained a five-base-pair repeat unit (adenine-adenine-thymine-guanine-guanine) that has undergone considerable divergence. Estimates of the size of the HSR's and the amount of amplified DNA suggested that each HSR contained at least 30,000 copies of the 1.8-kb KpnL fragment, representing about 50% of each HSR. The amplified sequence was identified as one member of the previously described KpnL family of repeated sequences. In situ hybridization of D15Z1 to MeWo metaphase chromosomes resulted in heavy labeling over both HSR's. These data suggested that centromeric heterochromatin and neighboring sequences on chromosome 15 have been amplified and some of this material translocated to the X-chromosome.

DNA Restriction Enzymes↗

PubMed1984

A strategy for the production of human monoclonal antibodies reactive with lung tumor cell lines.

Epstein-Barr virus (EBV)-immortalized cell lines were established from lymphocytes derived from peripheral blood, draining lymph nodes, bone marrow aspirates, tumors, and pericardial effusions from lung cancer patients. Ten of these lines were cloned and screened against glutaraldehyde-fixed lung tumor cells for tumor-specific antibody production using an enzyme-linked immunosorbent assay. None of the 140 clones tested secreted specific antibody, suggesting that B-lymphocytes specific for tumor antigens are rare in lung cancer patients. The EBV lines from the lung cancer patients were then hybridized with a thioguanine-resistant, ouabain-resistant human B-lymphoblastoid fusion partner KR-4, in an attempt to rescue low frequency B-cell precursors. Supernatants from more than 4500 hybridomas surviving hypoxanthine-aminopterin-thymidine:ouabain selection were screened against human lung tumor cells in an enzyme-linked immunosorbent assay. Over 360 hybrids showed significant levels of activity, although most were not tumor cell specific since they also reacted with EBV-infected cells from the lymphocyte donor. Two hybridomas showed apparent specific binding early after fusion, but this activity was lost upon continued growth although, in general, hybrids continued to secrete high levels of immunoglobulin M (up to 50 micrograms/ml), in some cases, beyond 1 year in culture. The human EBV-hybridoma system described here may be useful for rescuing low-frequency tumor-reactive B-cell precursors in lung cancer patients.

Adenocarcinoma↗