Human lung tumor-associated antigen identified as an extracellular matrix adhesion molecule.

A single chain glycoprotein with an estimated molecular mass of 160 kD (gp160) was previously identified as a human lung tumor-associated antigen. This tumor marker is shown here to be associated noncovalently with a second 130-kD protein. Sequential immunoprecipitation studies of surface iodinated lung tumor cell lysates reveal that this heterodimeric complex is indistinguishable serologically and structurally from the integrin VLA-2, found originally on activated T lymphocytes and platelets. The VLA-2-like complex expressed on the lung tumors possesses similar characteristic Mg2+ dependent binding of collagen and laminin as observed with VLA-2 on normal cells. RNA analysis indicates that human lung tumors express at least 20 times more VLA-2 alpha chain message than normal adult human lung tissue. The results presented here raise the possibility that the overproduction of VLA-2 may be involved in the pathogenesis of human lung tumors by modulating the invasive and metastatic potential of the tumor.

COOH terminus of membrane IgM is essential for an antigen-specific induction of some but not all early activation events in mature B cells.

Transfectants of mature B cell lines that bind phosphorylcholine were made in order to understand the role of the COOH terminus of the mu chain of membrane IgM (mIgM) in generation of antigen-specific signals. A chimeric receptor (I-A alpha tail) was constructed by replacing 40 amino acids from the mu COOH terminus with that of major histocompatibility complex class II I-A alpha chain. The effect of wild-type and chimeric tails were studied on representative immediate-early antigen-specific signals. The I-A alpha tail hybrid, but not the wild-type receptor, was defective in antigen-driven Ca2+ mobilization, although it could effectively endocytose ligand-receptor complexes. Signal(s) transduced through the wild-type receptor led to transient induction of selected immediate-early gene messages (Egr-1, c-fos, Jun) above basal levels. However, the signal(s) generated after crosslinking of the I-A alpha tail receptor either showed no effect (c-fos) or actually repressed basal level expression of Egr-1 and Jun. Thus, we have established that receptor-mediated endocytosis can be distinguished from other early events associated with B cell activation, based on their differential dependence upon the structural fidelity of the COOH-terminal sequence of mIgM.

Spectrin immunofluorescence distinguishes a population of naturally capped lymphocytes in situ.

Immunofluorescence analysis of mammalian lymphocytes using antiserum directed against chicken erythrocyte alpha-spectrin revealed a lymphocyte population in which spectrin antigen was arranged in the form of a discrete cap (hereafter referred to as capped lymphocytes). This subset could be easily distinguished from other lymphocytes in which the spectrin antigen was diffusely distributed near the plasma membrane (noncapped lymphocytes). The subset of capped lymphocytes could be visualized in situ and in isolated cells in the absence of added ligand. Using frozen sections of lymphoid organs that were fixed in formaldehyde prior to the immunofluorescence procedure, capped lymphocytes were found in characteristic locations depending on the tissue examined. In the thymus, the major population of medullary lymphocytes were capped whereas cortical lymphocytes were mostly noncapped. In Peyer's patches, capped lymphocytes were interspersed with noncapped lymphocytes throughout the tissue. In the spleen, capped lymphocytes were concentrated in the periarterial lymphoid sheath of the white pulp and in lymph nodes they were found predominantly in the paracortical and cortical regions. Capped lymphocytes were not visible in the thymus until just before birth and did not appear in the spleen until 3 d after birth. When lymphocytes were isolated from lymphoid organs, fixed in formaldehyde and prepared for immunofluorescence, capped and noncapped lymphocytes were still identifiable and present in the same relative proportions as seen in situ. Results identical to those described above are obtained using antisera directed against guinea pig fodrin. Natural capping of proteins previously shown to co-migrate with a variety of cell surface macromolecules after cross-linking may be a new means of identifying various stages of lymphocyte activation or differentiation.

Heterogeneity in lymphocyte spectrin distribution: ultrastructural identification of a new spectrin-rich cytoplasmic structure.

Spectrin-like proteins are found in a wide variety of non-erythroid cells where they generally occur in the cell cortex near the plasma membrane. To determine the intracellular distribution of alpha-spectrin (alpha-fodrin) in lymphocytes, we have developed an immunoperoxidase method to localize this protein at the ultrastructural level. Of considerable interest, particularly with regard to our efforts to determine the function of spectrin in this cell type, was the finding that its subcellular localization and its relationship with the plasma membrane can vary dramatically. Based on its position in the cell, alpha-spectrin can occur in two forms in lymphocytes: one that associates closely with the plasma membrane and another that occurs at some distance from the cell periphery, either as a single large aggregate or as several smaller ones. The single large aggregate of spectrin is a stable feature in a number of lymphocyte cell lines and hybrids which were used to examine its ultrastructural characteristics. A previously undescribed cellular structure, consisting of a meshwork of spectrin filaments and membranous vesicles, was identified in these cells. This structure could be induced to dissipate in response to membrane perturbants (e.g., hyperthermia and phorbol esters, known effectors of lymphocyte function and differentiation) and the patterns resulting from the redistribution of spectrin were a reflection of those observed routinely in lymphocytes in situ. The correlation between naturally occurring spectrin localization patterns and those seen after membrane perturbation suggested the possibility that spectrin distribution is indicative of particular maturation stages or functional states in lymphocytes. The implications of these findings with regard to the role of spectrin in lymphocyte function are discussed.

Integrin alpha 2 beta 1 is upregulated in fibroblasts and highly aggressive melanoma cells in three-dimensional collagen lattices and mediates the reorganization of collagen I fibrils.

The ability of cultured human fibroblasts to reorganize and contract three dimensional collagen I gels is regarded as an in vitro model for the reorganization of connective tissue during wound healing. We investigated whether adhesion receptors of the integrin family are involved. It was found that synthesis and transcription of the alpha 2 beta 1 integrin (but not of alpha 1 beta 1 or alpha 3 beta 1) is selectively upregulated when fibroblasts are seeded into type I collagen gels. Time course experiments revealed that high synthetic levels of alpha 2 beta 1 parallel the gel contraction process and return to "baseline" levels after the contraction has subsided. Furthermore, function-blocking mAbs directed to the alpha 2 and beta 1 chain of integrins inhibited gel contraction. Remodelling of connective tissue can be important for tumor cells during invasion and formation of metastases. Therefore, we tested human melanoma cell lines for this function. Five out of nine melanoma lines contracted collagen gels in vitro. Among these, two highly aggressive melanoma cell lines (MV3 and BLM) most efficiently contracted gels almost reaching the rate of normal adult fibroblasts. In these cells, synthesis of alpha 2 beta 1 was also significantly upregulated when seeded into collagen I gels. Moreover, function blocking anti-alpha 2 in conjunction with anti-beta 1 chain mAbs completely inhibited gel contraction for several days. Other melanoma cells (530) with lower metastatic potential which were not able to contract gels, showed no induction of alpha 2 beta 1 synthesis in gel culture. Our results suggest an important role of integrin alpha 2 beta 1 in the contraction of collagen I by normal diploid fibroblasts during wound healing and in the reorganization of collagen matrices by highly aggressive human melanoma cells.

Human villous adenomas engrafted into scid mice survive for prolonged period without malignant transformation.

Human villous adenomas are thought to represent premalignancies that subsequently give rise to colorectal adenocarcinomas. Currently there is no in vivo model in which to study the dedifferentiation and malignant transformation of these tumors. We establish here that human villous adenomas can be successfully engrafted into severe combined immunodeficient (scid) mice. Furthermore, these xenografts remain viable for up to 18 mo after either a subcutaneous or intraperitoneal inoculation of the human tissue. Tumors grew slowly and secreted a clear mucinous fluid. Examination of the tumors histologically at 1, 4, and 12 mo after implantation revealed that the villous polypoid structure was maintained and islands of atypical cells were observed within pockets of mucin surrounding the adenomatous tissue. No gross or histologic evidence of malignancy was detected throughout the 20-mo observation period. The human identity of the cells in the graft was confirmed by DNA in situ hybridization with a human-specific probe. We conclude that the human-scid xenograft described here represents a viable animal model with which to study the potential malignant dedifferentiation of villous adenomas over a prolonged period of time and to evaluate the possible contribution of selected oncogenic vectors on the malignant transformation of these adenomas.

Human lung tumor growth established in the lung and subcutaneous tissue of mice with severe combined immunodeficiency.

We report here that a mouse mutant (C.B-17 scid) which lacks functional B- and T-lymphocytes can be used to propagate a human lung tumor. The heterotransplanted tumor cells generated palpable s.c. tumors by 18 days in 100% of the mice inoculated s.c. with greater than 4 X 10(6) cells. All tumors grew progressively with no sign of regression. A portion of the scid mice given injections i.v. of the human lung tumor cells developed multiple tumor nodules in the lung by 15 weeks after the inoculation of tumor cells. The tumor nodules were shown by karyotype analysis to be human cells, and the histopathology of the tumor nodules revealed a pattern of growth that was consistent with that of the original tumor. The human lung tumor used in the study expresses an Mr 160,000 cell surface glycoprotein that has been shown to occur on a large proportion of human lung tumors and tumor cell lines. A monoclonal antibody specific for Mr 160,000 glycoprotein was used to demonstrate that this tumor-associated antigen is stably expressed by the s.c. tumors and the lung tumor nodules in the scid mice. The mutant mice with this severe combined immunodeficiency represent a new and viable model for propagating human tumors and for evaluating the efficacy of novel drug delivery protocols in the treatment of human cancer.

Selective growth inhibition of human lung cancer cell lines bearing a surface glycoprotein gp160 by 125I-labeled anti-gp160 monoclonal antibody.

Monoclonal antibody 5E8 which is specific for a Mr 160,000 glycoprotein (gp160) on the surface of human lung cancer was radiolabeled with 125I. Radiolabeled 5E8 antibody is shown here to suppress the growth of gp160 positive human lung tumor cell lines in a dose-dependent fashion, but this same radiolabeled antibody does not alter the growth of gp160 negative lung tumor cell lines. Neither the unlabeled 5E8 nor a control radiolabeled monoclonal antibody has any effect upon the growth of gp160 positive tumors. The specificity of radiolabeled antibody mediated tumor killing is further demonstrated by the ability of unlabeled 5E8 to inhibit tumor killing by 125I-5E8. The efficiency with which the labeled tumor specific antibody suppressed tumor colony formation is enhanced by increasing the molar ratio of 125I to 5E8. This ratio could be increased to a level of two without affecting the capacity of the antibody to bind to the cell surface antigen. An attempt to increase the efficiency of tumor killing by the addition of a second antibody subsequent to incubation with 125I-5E8 was unsuccessful. These results indicate that 125I is a viable isotope and gp160 represents an appropriate target for radioimmunotherapy of human lung cancer.

Cell surface glycoprotein associated with human lung tumors that is similar to but distinct from the epidermal growth factor receptor.

A cell surface glycoprotein (gp160) present on the surface of non-small cell human lung tumors is characterized and compared with the epidermal growth factor receptor (EGFR). The epitope on gp160 recognized by monoclonal antibody 5E8 is shown to be part of the protein moiety of the molecule and is found to be relatively stable. The epitope is stable over a wide pH range and after treatment with urea as well as most ionic and non-ionic detergents. We have observed that gp160 is similar in several respects to the EGFR. However, despite the similarities, several independent lines of experimental evidence presented here suggest that gp160 and the EGFR are distinct molecules. The first evidence suggesting that these two molecules are different is that the EGFR, but not gp160, is constitutively detectable in the A431 cell tissue culture supernatant, and that a pulse of these cells with epidermal growth factor (under conditions which permit the internalization of the receptor-ligand complexes) significantly reduces the expression of the EGFR without noticeably affecting the level of gp160 on the cell surface. Two very different immunofluorescent patterns marking the position of gp160 and EGFR are observed using monoclonal antibodies specific for each molecule. Using an enzyme-linked immunosorbent assay, it was determined that these same monoclonal antibodies do not cross-inhibit one another, and it was established that gp160, but not EGFR, was retained on an affinity column containing anti-gp160 antibodies immobilized to the solid matrix. An additional finding that supports the notion that gp160 and the EGFR are distinct molecules is that one human lung tumor cell line (Calu-3) has been identified which expresses gp160 but not the EGFR on its surface. These results indicate that there are characteristics which distinguish gp160 from the EFGR, and we establish here that these distinguishing features reflect differences at the protein moiety and not simply differential glycosylation. We conclude from these studies that we have identified and characterized a cell surface molecule that resembles in several respects the epidermal growth factor receptor. This cell surface molecule represents a potentially useful target for the immunotherapy and diagnosis of human non-small cell lung cancer.

In situ tumor vaccination with interleukin-12-encapsulated biodegradable microspheres: induction of tumor regression and potent antitumor immunity.

An alternative technology for the local and sustained delivery of cytokines to tumors for cancer immunotherapy was evaluated and shown here to induce tumor regression, suppression of metastasis, and development of systemic antitumor immunity. Treatment of tumor-bearing BALB/c mice with a single intratumoral injection of biodegradable polylactic acid microspheres loaded with recombinant interleukin-12 (IL-12) promoted complete regression of the primary tumor and prevented the metastatic spread to the lung. Mice that experienced tumor regression after being treated rejected a subsequent challenge with live tumor cells, which indicated the development of systemic antitumor immunity. In situ tumor vaccination, ie., injection of IL-12 microspheres into existing tumors, was superior to vaccination of mice with mixtures of tumor cells (live or irradiated) and IL-12 microspheres in inducing systemic antitumor immunity. The sustained release of IL-12 from the microspheres was superior to bolus injection of free IL-12, and intratumoral delivery of microspheres was more effective than other routes of administration. These studies establish the utility of biodegradable polymer microspheres as a clinically feasible alternative to systemic cytokine therapy and cytokine gene-modified cell vaccines for the treatment of neoplastic disease.

Antitumor immune response of human peripheral blood lymphocytes coengrafted with tumor into severe combined immunodeficient mice.

Here, it is established that human peripheral blood lymphocytes (HuPBLs), injected s.c. with a human lung tumor into severe combined immunodeficient (SCID) mice, engraft and display antitumor cytotoxic activity. Initial studies used HuPBLs from normal donors and an allogeneic tumor cell line derived from biopsy tissue of a patient with a squamous cell carcinoma of the lung. Evidence of HuPBL antitumor activity was revealed by a cell dose-dependent suppression of the tumor xenograft. Tumor suppression was shown to be dependent upon both CD8+ T cells and CD56+ natural killer cells in the donor HuPBLs. By titrating the antitumor activity of HuPBLs in SCID mice with and without cytokines, it was established that interleukin (IL)-12 enhanced the HuPBL-mediated tumor suppression and that IL-2 had a synergistic effect upon the IL-12 enhancement of cytotoxicity. Subsequent studies revealed that a lung cancer patient's PBLs also suppress the growth of the patient's (autologous) tumor when coinjected s.c. with the tumor cells into SCID mice. The patient's antitumor immunity was shown to be mediated by CD8+ T cells and CD56+ natural killer cells. The data presented here indicate that the s.c. coengraftment of HuPBLs and tumor into SCID mice represents a viable model with which to study (and to periodically monitor) patients' immune responses to their tumors for extended periods of time and suggest that this SCID/Winn assay could be used to evaluate novel immunotherapeutic approaches, such as bolus injections of cytokines, cytokine gene therapy, or vaccination strategies for the treatment of human cancer.

Immunospecific targeting of cytosine arabinonucleoside-containing liposomes to the idiotype on the surface of a murine B-cell tumor in vitro and in vivo.

A new tumor model is described that is suitable for the evaluation of antibody-directed drug-delivery protocols and a modification in the procedure for covalently coupling antibody to the surface of drug-containing liposomes is presented. These immunospecific liposomes containing cytosine arabinonucleoside (Ara-C) have been tested in vitro and in vivo for their ability to kill a B-cell tumor. The target of the immunospecific-Ara-C liposomes is the idiotype associated with an antigen-specific immunoglobulin receptor on the cell surface of a murine B-cell hybrid (2C3). Affinity-purified antibodies specific for the idiotype were covalently coupled to modified lipid on the surface of the large unilamelar liposomes containing drug. These liposomes were shown to kill idiotype-positive 2C3 cells in vitro, but not idiotype-negative variants of this same cell line. It was also established in vitro that the drug-containing liposomes were at least 40 times more efficient than free Ara-C in the killing of the tumor cells. The 2C3 tumor was also propagated in vivo following the i.p. administration of tumor cells. The tumor grew initially as multiple foci within the peritoneum and subsequently spread to the spleen. Tumor-bearing mice were treated either with free Ara-C or with immunospecific liposomes containing Ara-C. Tumor growth in the primary tumor nodules and in the spleen was monitored by the administration of bromodeoxyuridine to the tumor-bearing animals followed by the immunofluorescent staining of cells with a monoclonal anti-bromodeoxyuridine antibody to estimate the proportion of cells in S phase. Our data from five out of seven animal experiments shows that the immunospecific-Ara-C liposomes, but not free drug, reduced tumor growth in the spleen. However, neither the liposomes containing drug nor the free drug were able to alter the growth of the primary tumor nodules growing in the peritoneal cavity. These results suggest that immunospecific-Ara-C containing liposomes may be useful in conjunction with other cytoreductive protocols in controlling tumor growth or preventing the spread of the tumor to other sites, but that immunospecific-Ara-C containing liposomes by themselves are not likely to eliminate an established tumor in vivo. We also demonstrate here that the administration of immunospecific-Ara-C containing liposomes in an animal having high levels of circulating tumor-associated antigen (i.e., IgG containing the idiotype) represents a potential clinically relevant hazard which must be considered when designing antibody-directed drug-delivery protocols.

Arrest of human lung tumor xenograft growth in severe combined immunodeficient mice using doxorubicin encapsulated in sterically stabilized liposomes.

Incorporation of polyethylene glycol-derivatized phospholipids into liposomes results in carriers that can enhance the therapeutic efficacy of encapsulated drugs by imparting the ability to evade the reticuloendothelial system and remain in the circulation for prolonged periods. In this study, doxorubicin encapsulated in these sterically stabilized liposomes (S-DOX) is shown to completely arrest the growth of human lung tumor xenografts in severe combined immunodeficient (scid) mice. Doxorubicin administered at equivalent doses as free drug or encapsulated into conventional liposomes was ineffective at completely arresting the growth of this human tumor, although a decrease in tumor growth rate compared to untreated controls was observed. Scid mice were found to be significantly more susceptible to the toxic effects of doxorubicin than were immunocompetent C.B-17 control mice, a characteristic that is likely to result from the deficit in DNA repair mechanisms previously identified in scid mice. However, doxorubicin toxicity in scid mice could be minimized while maintaining the antitumor activity of doxorubicin encapsulated in sterically stabilized liposomes by administering the drug in multiple weekly injections at low doses. This report provides the first evidence that antitumor drugs delivered in sterically stabilized liposomes are more effective at arresting the growth of human tumors than are conventional delivery systems. In addition, the scid mouse is presented as a viable model in which to study novel chemotherapeutic approaches to the treatment of human cancer.

Antibody targeting of doxorubicin-loaded liposomes suppresses the growth and metastatic spread of established human lung tumor xenografts in severe combined immunodeficient mice.

Beta1 integrins, expressed on the cell surface of human non-small cell lung carcinomas, are used here as a target for the selective delivery of anti-cancer drug-loaded liposomes. Fab' fragments of a monoclonal antibody specific for human beta1 integrins were conjugated to sterically stabilized liposomes. Confocal microscopy of beta1 integrin-positive lung tumor cells incubated with fluorescently labeled anti-beta1 Fab immunoliposomes revealed a tumor-specific binding and efficient internalization of the liposomes into the tumor cells. The ability of these liposomes to deliver cytotoxic drugs to the tumor and kill these cells was demonstrated in vitro by incubating tumor cells with doxorubicin-loaded anti-beta1 Fab' immunoliposomes. The drug-loaded immunoliposomes were >30-fold more cytotoxic to the tumor cells than drug-loaded liposomes without antibody, nonspecific Fab' control immunoliposomes with drug or immunoliposomes without drug. The therapeutic efficacy of doxorubicin-loaded immunoliposomes was also evaluated in a metastatic human lung tumor xenograft/severe combined immunodeficient (SCID) mouse model. SCID mice that received i.v. injections of human lung tumor cells developed primary tumor nodules in the lung that subsequently metastasized to the liver and adrenal gland. Treatment of SCID mice bearing established lung tumor xenografts with doxorubicin-loaded anti-beta1 Fab immunoliposomes resulted in a significant suppression of tumor growth (monitored periodically by quantifying serum levels of a tumor marker), whereas tumors grew progressively in mice treated with control formulations. In addition to suppressing the growth of the primary lung tumor nodules, the immunoliposomes prevented the metastatic spread of the tumor to the liver and adrenal glands and increased the median survival time of the tumor-bearing mice. We conclude that Fab' immunoliposomes directed to tumor-associated integrins represent a potentially viable approach clinically for the selective delivery of drugs to solid tumors and may be useful in preventing the metastatic spread of lung cancer.

Doxorubicin encapsulated in sterically stabilized liposomes is superior to free drug or drug-containing conventional liposomes at suppressing growth and metastases of human lung tumor xenografts.

Liposomes containing polyethylene glycol-derivatized phospholipids are able to evade the reticuloendothelial system and thereby remain in circulation for prolonged periods. We report here that doxorubicin encapsulated in these sterically stabilized liposomes (S-DOX) suppresses the growth of established human lung tumor xenografts in severe combined immunodeficient (SCID) mice and inhibits the spontaneous metastases of these tumors. The enhanced therapeutic efficacy of S-DOX compared to free doxorubicin was demonstrated in two independent human/mouse models. In the first model, S-DOX inhibited the growth of a human non-small cell lung tumor xenograft established orthotopically in the lungs of SCID mice. Treatment of these mice with S-DOX, but not with free drug, suppressed the growth of the tumor in the lung, prevented metastasis from the lung, and enhanced survival percentage. In another model, the human lung tumor is engrafted into gonadal fat pad of SCID mice. Human tumor xenografts grow floridly in this site of engraftment, and the tumor spreads from this primary site into the peritoneal cavity and subsequently reaches the liver and lung. In this model, free drug suppressed the growth of the primary tumor but had no effect upon the subsequent spread of the tumor into the peritoneal cavity, liver, and lung. In contrast, treatment of the tumor-bearing mice with S-DOX (but not with doxorubicin in conventional liposomes) suppressed the tumor spread to the peritoneal cavity, completely arrested metastasis to the liver and lung, and suppressed the growth of the primary tumor xenograft. This report provides the first evidence that antitumor drugs delivered by sterically stabilized liposomes can arrest the metastasis of human tumor xenografts.

Membrane-associated glycoprotein (gp 160) identified on human lung tumors by a monoclonal antibody.

A monoclonal antibody (5E8) has been used to identify and structurally characterize a previously unreported macromolecule present on the surface of human lung tumors. This antibody was derived from a hybrid clone that was produced using spleen cells of mice immunized with a surgically excised squamous cell carcinoma. Using immunofluorescence, the 5E8 antibody was observed to stain many different human lung tumor cell lines and surgically excised human lung tumors including squamous cell carcinomas, adenocarcinomas, alveolar carcinomas, and a portion of the large cell tumors tested. With few exceptions, notably the basal layer of the skin, little or no detectable staining of 5E8 to normal human tissues (lung, brain, kidney, heart, stomach, breast, erythrocytes, or lymphocytes) was observed. The 5E8 antibody was used to immunoprecipitate detergent lysates of biosynthetically labeled or surface radioiodinated lung tumors. Analysis of the immunoprecipitates by sodium dodecyl sulfate gel electrophoresis revealed a major band and a faster migrating second minor band. The molecular weights of these two proteins were estimated to be 160,000 and 120,000, respectively. The addition of a reducing agent to the gels did not alter the migration pattern of the immunoprecipitated macromolecules. The removal of a terminal carbohydrate, sialic acid, did not restrict the binding of 5E8 to the tumor-associated antigen. However, labeling studies using galactose oxidase and tritiated borohydride revealed the presence of galactose on the immunoprecipitated protein. This major Mr 160,000 glycoprotein that was identified on two different human lung tumor cell lines was also found on a human large cell tumor tissue obtained by surgical biopsy. The 5E8 antibody and the Mr 160,000 glycoprotein that it recognizes represent two very useful components with which to test several new antibody-mediated drug delivery systems in the treatment of human lung tumors. The tumor-associated glycoprotein also represents a potential analyte for a diagnostic or prognostic immunoassay for lung cancer.

Expression of mu and gamma 1 membrane forms of immunoglobulin segregate in somatic cell hybrids.

In the present investigation, we have utilized the somatic cell hybridization technique to generate an experimental model for studying the differential expression of membrane (mIg) and secreted (sIg) forms of immunoglobulin that characterize different stages of B cell development. We describe here that fusion of the dextran-binding myeloma, MOPC 104E (mu, lambda 1) and the phthalate-binding B cell hybridoma, 2C3E1 (gamma 1, kappa) results in the formation of antigen-specific, double hybrids (tribrids) that coexpress both parental secreted forms of Ig but express only one of the two possible membrane forms of immunoglobulin (Ig). This segregated expression of membrane Ig is a new and unexpected finding that has been substantiated here by both immunological and biochemical methods. Analysis by SDS-containing polyacrylamide gels (SDS-PAGE) reveals distinct and characteristic migration patterns for each of the four Ig heavy chains in the tribrids (mu membrane, mu secreted, gamma 1 membrane and gamma 1 secreted). Immunochemical analysis of the immunoglobulin from the tribrids confirms the coexpression of both secreted forms of immunoglobulin in most of the tribrid lines tested and indicates that about 30% of the tribrids express only phthalate-specific gamma 1 membrane Ig, while 38% express only dextran-binding mu membrane Ig. About 30% of the tribrids secrete both antibodies but express no membrane form and less than 1% are non-secretors. Approximately 2% initially express both membrane forms of Ig, as determined by immunocytoadherence assay using appropriate target cells but subsequently express only one membrane form during propagation in vitro. SDS-PAGE analysis of surface labeled tribrids confirms that in tribrids expressing membrane Ig, only a single mIg is synthesized. These results suggest that the expression of the secreted and membrane forms of immunoglobulin are separately regulated and the tribrids represent a model with which to study the mechanisms involved in the regulation of each structurally distinct immunoglobulin form.

Antitumor efficacy of a human interleukin-12 expression plasmid demonstrated in a human peripheral blood leukocyte/human lung tumor xenograft SCID mouse model.

Genes encoding the p35 and p40 subunits of human interleukin-12 (IL-12) and the bacterial aminoglycoside phosphotransferase were cloned into a mammalian expression plasmid. The resultant plasmid, pCMVIL-12neo, was used to transfect human lung tumor cell lines in vitro. Stably transfected subclones were generated and found to secrete human IL-12 for at least 10 days following a lethal dose of gamma-radiation. The ability of the IL-12--producing tumor cells to promote an antitumor response in vivo was evaluated in SCID mice co-engrafted subcutaneously with human peripheral blood lymphocytes (PBLs) and viable human lung tumor cells (SCID-Winn assay). Using this model system, it was established that IL-12 released locally into tumors by irradiated IL-12--transfected cells activated the human PBL and promoted their ability to suppress tumor development in a dose-dependent fashion. PBL subset depletion studies revealed that the antitumor effect promoted by the IL-12--modified cells was dependent on the presence of human CD8(+) T cells and, to a lesser extent, human CD56(+) natural killer cells within the xenograft. We conclude that (a) irradiated human lung tumor cells genetically modified with pCMVIL-12neo secrete bioactive human IL-12 at concentrations sufficient to promote a human lymphocyte-mediated antitumor response in the microenvironment of the xenograft, and (b) that the SCID-Winn assay provides a useful model for the preclinical evaluation of cytokine-based human immunotherapy protocols.

Screening and replica plating of anti-hapten hybridomas with a transfer template hemolytic spot assay.

A localized hemolysis in gel assay is described for screening microcultures of hybridomas for the production of anti-hapten antibody. The keys to the rapid screening assay reported here are a special fenestrated transfer template and an improved hapten conjugated target cell. The transfer template is a 96-well plate with a calibrated hole in the bottom center of each well. To assay for anti-hapten antibodies, the transfer template is positioned over a 96-well microculture plate containing the growing hybridomas. After making contact with the tissue culture supernatant each orifice of the transfer template retains approximately 2 microliter of tissue culture supernatant. The transfer template is then placed onto an assay slide containing a thin layer of hapten conjugated target erythrocytes incorporated into agarose. After incubation with an anti-immunoglobulin and complement, areas of localized hemolysis in the gel indicate hybridomas which are secreting anti-hapten antibodies. The assay detects as little as 10 pg of antibody. Since the transfer template can be used as a replica plate, one can repeatedly transfer samples to various slides which contain either different hapten target cells or different hapten analog inhibitors in the agarose layer. Therefore, in addition to rapidly screening microcultures for positive hybridomas this procedure permits the characterization of each monoclonal antibody's fine specificity.

A filtration double antibody radioimmunoassay that simplifies and semi-automates the isolation of immune precipitates.

A semi-automation of fluid phase double antibody radioimmunoassay has been developed. The immune precipitate that was formed in 96-well microtitration plates was harvested and washed on microfibre filters using a Titertek cell harvester. A disc transfer system originally designed for use with the harvester was used as a quick and easy method of transferring the filter discs containing immune precipitate into vials for counting. The results of radioimmunoassay using the microtitration plate-filtration and conventional tube-centrifugation method are essentially identical. The microtitration plate-filtration radioimmunoassay has the following advantages over the conventional tube-centrifugation method: (1) there is no centrifugation required; (2) handling of microtitration plate is easier than the tubes in racks; and (3) it requires much less time to perform the assay.