In vivo antitumor effect of methotrexate conjugated to a monoclonal IgM antibody specific for stage-specific embryonic antigen-1, on MH-15 mouse teratocarcinoma

Summary Methotrexate (MTX) was coupled to an IgM monoclonal antibody specific for stage-specific embryonic antigen-1 (SSEA-1), and the resulting immunoconjugate (MTX-anti-SSEA-1) was used for in vivo drug targeting in mice bearing MH-15 teratocarcinoma. Immunoconjugates having an average of 65 mol MTX/mol antibody retained full antigen-binding capacity. Mice bearing well-established tumors (approx. 1 g) were treated i.v. using the immunoconjugate. MTX-anti-SSEA-1 at 15 mg/kg of drug had significant antitumor activity with no significant systemic toxicity. Neither an irrelevant isotype-matched conjugate, MTX-MOPC-104E, prepared from the MOPC 104E myeloma protein, nor free MTX injected alone or with either antibody had any signficant antitumor effect. These results indicate that IgMs can be effective drug carriers for tumor targeting in spite of their high molecular mass, and that antigens that are selectively accessible in tumors, even though present in normal tissues, can be suitable targets for in vivo chemoimmunotherapy.Supported by intramural research funds from the Veteran's Administration, Pittsburgh Cancer Institute, and NIH grants CA34798 and CA 14551     

Tumor radioimmunolocation: differential antibody retention by antigenic normal tissue and tumor

On the assumption that a high specificity would be required for in vivo tumor location and drug targeting, much labor has been expended to produce monoclonal antibodies directed to antigens found primarily on tumors. However, for at least one tumor-associated antigen, stage-specific embryonic antigen 1 (SSEA-1), the concentration of the target antigen need not be higher in the tumor than that found in normal tissues for successful use of the corresponding monoclonal antibody in in vivo tumor location; rather, accessibility and antibody metabolism are more important.     

Distribution of murine stage-specific embryonic antigens in the kidneys of three rodent species

The distribution of two mouse stage-specific embryonic antigens (SSEA-1 and SSEA-3), recognized by monoclonal antibodies, was investigated in the kidneys of mice, rats and gerbils. Both antigens, with the exception of SSEA-3, which was not found in the kidney of the gerbil, were detectable in at least some portions of the kidney in all three species. These data suggest that while unique antigenic determinants may be evolutionarily conserved in homologous tissues of different species, their distribution may vary anatomically.     

Developmentally regulated cell surface structures on mouse and human embryonal carcinoma cell lines

Three monoclonal antibodies 5.1.H, 8.7.D and 13.7.A raised against semi-purified Tera 1 membrane fractions recognize distinct onco-foetal antigens which are developmentally regulated on cells such as Tera 2 clone 13 and appear to be restricted in their expression to undifferentiated ectoblastic cells and certain organized cystic structures mimicking the foetal intestine. These antigens, absent from normal adult tissues, differ markedly from glycosidic stage-specific antigens such as 75.12 which, while functioning as embryonal carcinoma differentiation markers, are also expressed on certain adult tissues. No evidence for a role of fucosyltransferases in regulating either 75.12 or SSEA-1 antigen expression on embryonal carcinoma cells or for the presence of lectin-like structures recognizing these antigens on such cells was found.     

Density comparisons of heavy chains of membrane and secreted immunoglobulins of mouse.

In order to explore structural differences between membrane and secreted immunoglobulins the buoyant densities of mouse immunoglobulin (Ig) heavy (H) chains were compared by isopycnic centrifugation in CsCl containing guanidine hydrochloride. The buoyant densities, under denaturing conditions, of mouse myeloma protein MOPC 21 IgG, MOPC 315 IgA and MOPC 104E IgM H chains were consistent with their carbohydrate contents. Mouse membrane IgM and MOPC 104E-secreted IgM H chains were of equal density. The buoyant densities of MOPC 104E-secreted IgM and spleen-cell-secreted IgM H chains were indistinguishable. The IgD-like membrane H chain was denser than membrane IgM H chain, and its carbohydrate content was calculated to be 15.5%. The resolution of the technique was sufficient to conclude that the apparent 1500 mol.wt. difference, as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, between membrane and secreted IgM H chains was due to peptide rather than to carbohydrate. The results also imply that intact membrane IgM and IgD bind detergent and are thus integral membrane proteins.     

Inhibition of growth of MOPC 104E cells in immunosuppressed mice

In this report we provide evidence that suggests that MOPC 104E may come under regulation in highly immunosuppressed hosts depleted of T cells. Mice that are adult thymectomized, total body irradiated, and transplanted with bone marrow cells were able to resist the growth of MOPC 104E cells. Spleen cells from such animals had low NK activity and no cytotoxicity against MOPC 104E, and poor response to Con A, PHA, and LPS. The animals were deficient in Lyt-1+ and Lyt-2+ cells. The growth of MOPC 104E cells was measured by using the circulating level of MOPC 104E IgM in vivo in mice treated by different modalities. We observed that inhibition of tumor growth in vivo varied with the treatment of the host. Growth was inhibited in the host in the following order: ATXBM greater than XBM greater than NORMAL greater than ATx mice.     

Carbohydrate-derivatized immunoconjugate of the anti-(carcinoembryonic antigen) monoclonal antibody C46: Immunohistological reactivity and pharmacokinetic comparison with a randomly derivatized C46 immunoconjugate

Summary In this study, a site-specific glycyl-tyrosyl-(N--diethylenetriaminepentaacetic acid)-lysine (GYK-DTPA) immunoconjugate of the anti-carcinoembryonic antigen monoclonal antibody C46 (C46-GYK-DTPA) was characterized by immunohistological and immunofluorescence methods for reactivity with normal and neoplastic human tissues. In addition, pharmacokinetic studies assessed the ability of C46-GYK-DTPA labeled with¹¹¹ In to localize to and image human tumor xenografts in nude mice. The native antibody and the site-specific immunoconjugate exhibited similar patterns of reactivity with normal human tissues. C46 did not bind to the surface of normal human granulocytes, which indicates lack of reactivity with normal cross-reacting antigen. C46-GYK-DTPA reacted with 100% of the colon, breast and renal carcinomas examined and with two of three lung carcinomas, but did not react with any sarcomas, melanomas or lymphomas examined. Intravenously administered, C46-GYK-DTPA-¹¹¹In rapidly localized to and imaged LS174T human colon adenocarcinoma xenografts in nude mice, reaching maximal levels of about 25% of injected dose/g tumor within 1 day. No unusual localization to any non-tumor tissue or organ was seen; the level of radioactivity in the normal tissues and organs was at or below that in the blood. The accessible binding sites in 1 g tumors appeared to be saturated at an antibody dose between 100 µg and 1000 µg/mouse. Further, in a direct in vivo comparison, the site-specific conjugate C46-GYK-DTPA had more favorable pharmacokinetics and better tumor localization than a randomly derivatized C46 immunoconjugate (C46-DTPA). These findings suggest that the site-specific immunoconjugate C46-GYK-DTPA may be useful in the diagnosis and therapy of colon cancer and other adenocarcinomas expressing carcinoembryonic antigen.     

Characterization of the glycosylation of a human IgM produced by a human-mouse hybridoma

We analysed the oligosaccharides of a human IgM produced bya human-human-mouse hybridoma at each of its five conservedheavy chain glycosylation sites. Consistent with previous reports,this IgM possesses sialylated oligosaccharides at Asn171, Asn332and Asn395, and high-mannose-type oligosaccharides at Asn402.In contrast to previous reports for human IgMs, we find thatAsn563 is not occupied by oligosaccharide on perhaps 25% ofIgM heavy chains, while occupied Asn563 sites contain both high-mannose-typeand sialylated oligosaccharides. These latter results are consistentwith the glycosylation at Asn563 previously reported for themouse MOPC 104E IgM. We demonstrate that both the human hybridomaIgM and the mouse MOPC 104E IgM are mixtures of pentamers andhexamers, raising the possibility that the unique findings concerningthe glycosylation at Asn563 in this study and the previous studyof the MOPC 104E IgM could be related, at least in part, tothe different packing requirements of the hexameric geometryand the accessibility of oligosaccharides in the hexameric geometryfor processing to complex type. In addition, we used high-pHanion-exchange (HPAE) chromatography, neutral anion-exchangechromatography, fluorophore-assisted carbohydrate electrophoresisand Western blots to compare the oligosaccharide compositionsof the human hybridoma IgM, pooled human serum IgM and two mousemonoclonal IgMs (MOPC 104E and TEPC 183). Of note is the presenceof N-glycolylneuraminic acid (NeuGc) and N-acetymeuraminic acid(NeuAc) at a 2:1 ratio in the oligosaccharides of the humanhybridoma IgM. The presence of both NeuGc and NeuAc complicatesthe interpretation of HPAE chromato-graphs. glycosylation high-pH anion-exchange chromatography human IgM human—mouse hybridoma oligosaccharide     

Murine embryonic antigen (SSEA-1) is expressed on human cells and structurally related human blood group antigen I is expressed on mouse embryos

The stage-specific embryonic antigen (SSEA-1), present on embryonal carcinoma cells and on murine preimplantation embryos, is defined by a monoclonal antibody. The antigenic determinant of SSEA-1 is a carbohydrate structurally related to the human blood group antigen I. Since it has been suggested that the I antigen might represent a precursor or SSEA-1, we used antibodies to SSEA-1 and to I to analyze their expression on mouse preimplantation embryos. Both are expressed on mouse embryos; moreover, I is expressed on earlier embryos than SSEA-1. The I antigen is defined by its expression on human erythrocytes; accordingly, we examined expression of I and SSEA-1 on human peripheral blood elements. We find SSEA-1 to be expressed exclusively on human granulocytes while I is found only on erythrocytes. These results suggest that these closely related antigens can be independently expressed. Analysis of the expression of I and SSEA-1 was then extended to a series of mouse and human cell lines; some express both, some express only one, and some express neither of these antigens. The activation of specific glycosyltransferases and/or glycosidases during development and differentiation appears to be the biochemical mechanism regulating expression of these antigens.     

Heterogeneous expression of two oncodevelopmental antigens, CEA and SSEA-1, in colorectal cancer

Summary Colorectal carcinomas are composed of heterogeneous cell subpopulations which may be instrumental in conferring metastatic potential and therapeutic refractoriness to these tumours. To assess cellular heterogeneity, the expression has been examined of two oncodevelopmental antigens, carcinoembryonic antigen (CEA) and stage-specific embryonic antigen 1 (SSEA-1), by double immunofluorescence microscopy on 11 human colorectal carcinomas. Although both antigens were expressed in each tumour, their regional and cellular locations differed considerably. SSEA-1 expression was rarely expressed in poorly differentiated cancers but was enhanced with increasing degrees of differentiation. CEA expression was independent of histological differentiation. SSEA-1 was expressed with similar frequency in cell membranes, cytoplasm, and glandular contents regardless of degree of differentiation. Cytoplasmic staining with CEA however, was limited to more poorly differentiated tumours. In normal mucosa remote from the tumours and transitional mucosa adjacent to them, SSEA-1 stained only a few lower crypts whereas CEA stained a majority of both upper and lower crypts. Although biochemical studies have indicated that the SSEA-1 epitope may reside on CEA molecules, the fact that colon cancer tissues express these two antigens quite heterogeneously suggests differences in antigenic processing which may be dependent upon the degree of cellular differentiation.     

A pluripotent human stem-cell clone isolated from the TERA-2 teratocarcinoma line lacks antigens SSEA-3 and SSEA-4 in vitro, but expresses these antigens when grown as a xenograft tumor

Human embryonal carcinoma (EC) cells generally express the cell-surface, stage-specific embryonic antigens 3 and 4 (SSEA-3 and SSEA-4), the epitopes of which are defined by two monoclonal antibodies that recognize different portions of an extended globoseries oligosaccharide. To examine further the relationship between these epitopes and the human EC phenotype, we investigated the properties of two newly isolated clones from the human teratocarcinoma cell line, TERA-2. One clone expresses SSEA-3 and SSEA-4; the other does not. Nevertheless, these clones otherwise resemble one another, and based upon their morphology, their expression of other cell-surface antigens, and their ability to form xenograft tumors containing a variety of cell types, we conclude that both clones are composed of pluripotent human EC cells. When exposed to retinoic acid in vitro, neither clone differentiates as extensively as other clones that we have previously derived from TERA-2. These observations indicate heterogeneity among stem cells derived from a single human teratocarcinoma, and suggest that SSEA-3 and SSEA-4 are not necessarily integral features of the human EC phenotype. On the other hand, EC cells in xenograft tumors derived from the SSEA-3- and SSEA-4-negative clone re-express these epitopes. Further, this re-expression is stable, since EC cell lines that are SSEA-3- and SSEA-4-positive grow out when the tumors are explanted in vitro. We conclude that the expression of these globoseries epitopes can be modulated by environmental influences.     

Expression of murine early embryonic antigens, SSEA-1 and antigenic determinant of EMA-1, in embryos and ovarian follicles of a teleost medaka (Oryzias latipes)

Stage-specific embryonic antigen-1 (SSEA-1) and the antigenic determinant of monoclonal antibody EMA-1 are expressed in a stage-specific manner in mouse early embryos. To study whether these antigens generally exist in fish, expression of the antigens was examined in embryos, ovarian follicles, and adult tissues of a teleost medaka (Oryzias latipes), using immunohistochemical techniques. In 1-cell-stage embryos, these carbohydrate antigens were found in numerous cytoplasmic granules in the blastodisc and the cortical cytoplasm. These granules gradually decreased in number as the embryos developed. In 4-cell-stage embryos, the antigens appeared on the cleavage planes and were located on the cleavage planes within the blastoderm in the following cleavage stages. In blastula-stage embryos, the expression was ubiquitously found on the cell surface of blastomeres. At the mid-gastrula stage, the antigens were restricted to the enveloping layer, yolk syncytial layer, and cortical cytoplasm, but were rarely found in deep cells that contribute to formation of the embryonic body. In later-stage embryos and adult fish, the antigens were located in various tissues. In ovarian follicles, the antigens were found in granules of oocytes and granulosa cells. These observations were basically consistent with those in mice; however, expression in 1-cell-stage embryos and ovarian follicles has not been observed in mice. This unexpected finding suggests that the antigens are produced in granulosa cells and transferred to 1-cell-stage embryos via oocytes, and that the antigens involved in the early developmental process are maternally prepared in teleosts.     

Monoclonal antibodies to murine thrombospondin-1 and thrombospondin-2 reveal differential expression patterns in cancer and low antigen expression in normal tissues

There is a considerable interest for the discovery and characterization of tumor-associated antigens, which may facilitate antibody-based pharmacodelivery strategies. Thrombospondin-1 and thrombospondin-2 are homologous secreted proteins, which have previously been reported to be overexpressed during remodeling typical for wound healing and tumor progression and to possibly play a functional role in cell proliferation, migration and apoptosis. To our knowledge, a complete immunohistochemical characterization of thrombospondins levels in normal rodent tissues has not been reported so far. Using antibody phage technology, we have generated and characterized monoclonal antibodies specific to murine thrombospondin-1 and thrombospondin-2, two antigens which share 62% aminoacid identity. An immunofluorescence analysis revealed that both antigens are virtually undetectable in normal mouse tissues, except for a weak staining of heart tissue by antibodies specific to thrombospondin-1. The analysis also showed that thrombospondin-1 was strongly expressed in 5/7 human tumors xenografted in nude mice, while it was only barely detectable in 3/8 murine tumors grafted in immunocompetent mice. By contrast, a high-affinity antibody to thrombospondin-2 revealed a much lower level of expression of this antigen in cancer specimens. Our analysis resolves ambiguities related to conflicting reports on thrombosponding expression in health and disease. Based on our findings, thrombospondin-1 (and not thrombospondin-2) may be considered as a target for antibody-based pharmacodelivery strategies, in consideration of its low expression in normal tissues and its upregulation in cancer.     

Improved immunohistochemical method for detecting hypoxia gradients in mouse tissues and tumors.

We describe an improved immunohistochemical procedure for detecting regions of hypoxia in normal organs and tumors in mice. The method employs a primary fluorescein-conjugated mouse monoclonal antibody directed against pimonidazole protein adducts that are created in hypoxic tissues and a secondary mouse anti-fluorescein antibody that is conjugated to horseradish peroxidase. Using these reagents, we clearly visualized the regions of relative hypoxia in implanted tumors in mice as well as in normal organs such as liver and kidney. Significantly, the resulting tissue sections were remarkably free of the background staining that is characteristically observed when rodent antibodies are used to detect antigens in rodent tissues.     

Defective immune adherence and elimination of hepatitis B surface antigen/antibody complexes in patients with mixed essential cryoglobulinemia type II

Mixed essential cryoglobulinemia type II (monoclonal Ig/polyclonal IgG) is characterized by systemic vasculitis caused by the deposition of circulating immune reactants that include the monoclonal component. Such reactants may include immune complexes (IC) formed from exogenous Ag. IC binding to E C receptor type 1 appears to play a role in transport and buffering of such IC (immune adherence: IA). To define the mechanisms responsible for immune deposition, 7 patients with cryoglobulinemia type II (IgM kappa/polyclonal IgG) and 14 normal volunteers were injected i.v. with hepatitis B surface Ag/antibody complexes. Two minutes after injection, only 19.4% (mean) of the circulating complexes were bound to E in patients as compared with 63.1% in normal subjects. This IA correlated directly with C4 and inversely with the IgM rheumatoid factor (RF) titer. Disappearance of IC was faster in patients (mean elimination rate: 15.7%/min) than in normal subjects (9.3%). In vitro experiments demonstrated that C depletion, interference with IC opsonization by monoclonal IgM RF, and decreased binding of opsonized IC in the presence of monoclonal RF are each associated with decreased IA. These observations suggest that, in patients with cryoglobulinemia type II, monoclonal IgM RF and low C contribute to reducing IA of circulating IC that might be rapidly trapped in tissues, resulting in injury.     

Reactivity of a Monoclonal Antibody Raised against Human Leukemic Cells to Embryonic and Adult Tissues of the Mouse and Teratocarcinomas

C-9-1, a monoclonal IgM antibody raised against human null cell acute lymphocytic leukemia cells reacted with restricted regions of embryonic and adult tissues of the mouse. The antigen positive sites in the embryos included embryonic ectoderm, visceral endoderm, trophoblastic cells invading the maternal decidua of 5∼7-day embryos, primordial germ cells of 10∼12-day embryos, epithelium of nasal chamber, the bronchus, Mullerian duct, epididymis and bladder of 12∼17-day embryos. In the adult mice, C-9-1 antigen was detected in renal tubules, a part of stomach, bladder, endometrium and epididymal sperm. Embryonal carcinoma cells, but not endodermal cells of teratocarcinoma expressed the antigen. Thus, C-9-1 antigen showed distribution similar to SSEA-1. However, C-9-1 antigen was not detected in preimplantation embryos, nor in oviduct, both of which are positive for SSEA-1.     

Immunoregulatory activity of human bone marrow. Identification of suppressor cells possessing OKM1, SSEA-1, and HNK-1 antigens

Human bone marrow contains natural regulatory cells capable of suppressing the in vitro primary IgM response of normal tonsillar cells. The suppression is mediated by non-T cells possessing Fc receptors, OKM1, SSEA-1, and HNK-1 antigens on their surface. The suppression was abrogated by treatment of bone marrow cells (BMC) with anti-HNK-1 or anti-SSEA-1 antisera and complement. Furthermore, BMC depleted of HNK-1+ cells could respond in a primary in vitro antibody response when provided with accessory T cells and macrophages from tonsillar cells. Our findings support the idea that HNK-1+ and HNK-1- BMC populations act antagonistically in the regulation of antibody synthesis. Further, the finding of HNK-1+, SSEA-1+, and OKM1+ suppressor cells in human bone marrow may represent a precursor phenotype of mature natural killer cells with potent immunoregulatory activity.     

Expression of the HNK-1/NC-1 epitope in early vertebrate neurogenesis

Summary A family of glycoconjugates has recently been shown to share a common carbohydrate epitope recognized by the mouse monoclonal antibody HNK-1. The specificity of HNK-1 was found to be similar to that of another monoclonal antibody, NC-1. These two IgM monoclonal antibodies were raised after immunization of mice with a human T-cell line and avian neural crest-derived ganglia, respectively. The antigens recognized by these antibodies include the myelin-associated glycoprotein, MAG, a glycolipid of defined structure, and a set of molecules involved in cell adhesion. The timing and pattern of appearance of these antigens are distinct. Moreover, the epitope may be absent on an antigen at a given stage or in a given tissue. Therefore, although the molecules able to carry the NC-1/ HNK-1 epitope are numerous and expressed in various tissues, the use of the monoclonal antibodies on tissue sections has proven adequate for following the migration of avian neural crest cells, the major cell lineage recognized by NC-1 and HNK-1 during early embryogenesis. Analogies in several other species have been found on the basis of HNK-1 reactivity. In this study we show that NC-1 and HNK-1 can be used successfully to label migrating neural crest cells in dog, pig and human. On the other hand, the NC-l/HNK-1 epitope was not present on migrating crest cells in amphibians or mice and was found only transiently on the neural crest of rats.     

IgA anti-dextran B1355 responses.

Injection of mice bearing the Ig-1a allotype with dextran B1355 results in an IgM antibody response that is generally regarded as thymus independent. Moreover, the antibody is directed to alpha[1,3] determinants on dextran B1355 and shares cross-reacting idiotypic determinants with a lambda 1 IgA (J558) myeloma protein as well as a lambda 1 IgM (MOPC 104E) myeloma protein. In this study, we show that BALB/c (Ig-1a) mice injected with dextran B1355 produced highly significant IgA anti-dextran responses with specificity directed to the alpha[1,3] epitope. Kinetics of the IgA anti-dextran response in BALB/c mice paralleled kinetics of the IgM response. However, the magnitude of the IgA response was markedly T cell dependent and age dependent.     

Synthesis, surface deposition, and secretion of immunoglobulins by Abelson virus-transformed lymphosarcoma cell lines.

Three Abelson virus-transformed lymphoma cell lines were established in tissue culture and the immunoglobulin biosynthesis by these cell lines was studied. Two of the cell lines (ABLS-1 and ABLS-5) were found to synthesize monomeric IgM molecules which were deposited in the cell membrane, probably to serve as an antigen receptor. The third cell line (ABLS-8) was found to synthesize membrane-associated IgM as well as cellular IgG molecules. In addition, these cell lines were found to synthesize a protein of 35,000 molecular weight which is also membrane-associated and which has the capability to bind the immunoglobulin (MAID). It is speculated that this protein might play a role in adapting the receptor immunoglobulin molecule to the hydrophobic environment of the cell membrane. The kinetics of amino acid incorporation into immunoglobulins by these cell lines show that they produce immunoglobulins at a rate which is two orders of magnitude smaller than plasmacytoma cells (MOPC 104E). These results suggest that Abelson virus transforms thymus-independent lymphocytes in various stages of maturation and these lymphocytes might be of B cell origin. The T lymphoma (P1798) used as a control cell line was found occasionally to produce minute amounts of immunoglobulin.