Activated human lymphocytes and aggressive non-Hodgkin's lymphomas express a homologue of the rat metastasis-associated variant of CD44.

A recently described splice variant of CD44 expressed in metastasizing cell lines of rat tumors, has been shown to confer metastatic potential to nonmetastasizing rat pancreatic carcinoma and sarcoma cell lines. Using antibodies raised against a bacterial fusion protein encoded by variant CD44 sequences, we have explored the expression of variant CD44 glycoproteins on human lymphoid cells and tissues and on non-Hodgkin's lymphomas. Normal lymphohematopoietic cells express barely detectable low levels of variant CD44 glycoproteins, whereas T lymphocytes, upon activation by mitogen or antigen, transiently upregulate expression of specific CD44 variant glycoproteins. The reaction pattern of various antibodies indicates that these CD44 variants contain the domain encoded by exon v6, which is part of the variant that in the rat confers metastatic capability. It is interesting that overexpression of v6 was also found in several aggressive, but not low-grade, non-Hodgkin's lymphomas.

Expression of CD44 variant proteins in human colorectal cancer is related to tumor progression.

Specific CD44 variant glycoproteins are overexpressed at particular stages of colorectal tumor progression. Some variants of the CD44 glycoprotein without exon v6 sequences appear at the earliest stage of tumorigenesis, i.e., in early adenomas. Expression of variants containing exon v6 sequences is largely restricted to the advanced stages of tumor development and in addition is more prevalent and intense in metastatic (Dukes C/D) than in nonmetastatic (Dukes A/B) carcinomas. The observation that CD44 variants containing a protein domain of CD44 that confers full metastatic potential to rat carcinoma and sarcoma cell lines is increasingly expressed during colorectal tumor progression indicates that this domain may have an important role in tumor progression and metastasis in humans. Information on v6 expression, which can be obtained by routine immunohistochemistry, may prove of important prognostic value, particularly in carcinomas (Dukes A and B) that have not yet given rise to detectable metastases.

Poly(A) polymerase activity during cell cycle and erythropoietic differentiation in erythroleukemic mouse spleen cells.

Poly(A) polymerase activity was studied in lysates of cultured murine erythroleukemic cells (Friend cells). Incorporation of ATP into acid-precipitable products is dependendent on the presence of Mn2+ or Mg2+ and of an RNA primer. The reaction is specific for ATP as the substrate (KM=290 290 micron, it is not inhibited by actinomycin D and only slightly interferred with by ethidium bromide. Cordycepin 5'-triphosphate and sodium pyrophosphate inhibit the enzyme activity. The chain length of the products of the reaction is dependent on the primer concentration and reaches up to 30 nucleotides. Poly(A) polymerase activity is low in resting (G1 phase) cells 75 nmol ATP incorporated/h per 10(6) cells) and increases to a level about twice as high in early S phase of the cell cycle. A possible model for regulation of enzyme activity is discussed. Polymerase activity in the early phase of erythropoietic differentiation of the cells induced by butyric acid does not show any difference in comparison to untreated controls. A decrease in enzyme activity to levels characteristic for cells in G1 phase accompanies shutdown of cell growth in the course of the ongoing differentiation. Analysis of the DNA content of the cells revealed that erythropoietic differentiation of Friend cells induced by butyric acid is characterized by arrest of the cells in G1 phase of the cell cycle. Poly(A) polymerase activity in erythroleukemic cells is thus controlled only by the phase of the cell cycle; it is not affected by changes in gene expression during erythroid differentiation.

Isolation and partial characterization of the human erythrocyte band 7 integral membrane protein.

Monoclonal antibodies to the Mr 31,000 major integral membrane protein of the human erythrocyte band 7 region were used to identify the corresponding polypeptide chain and epitope-carrying fragments on immunoblots. Analysis of the erythrocyte membrane, membrane fractions, and cytosol revealed that the Mr 31,000 band 7 integral membrane protein is unique and not related to any of the other water-soluble or membrane-bound band 7 components. Cross-reacting proteins were identified in the membranes of other mammalian erythrocytes and in cell lines of epithelial and lymphoid origin. Proteolytic digestion of intact human erythrocytes or erythrocyte membranes demonstrated that the band 7 integral membrane protein has an intracellular domain larger than Mr 12,000; it does not have an extracellular one. One of the monoclonal antibodies was employed for the isolation of band 7 integral membrane protein by immunoaffinity chromatography; subsequent Edman degradation revealed a blocked N-terminus.

Human interferon omega 1: isolation of the gene, expression in Chinese hamster ovary cells and characterization of the recombinant protein.

A gene encoding human interferon omega-1 (IFN-omega 1) was isolated from a cosmid library, sequenced and expressed in Chinese hamster ovary (CHO) cells under the control of an SV40-derived promoter/enhancer sequence. Culture supernatants of stably transfected cell clones contained biologically active IFN-omega 1 at concentrations up to 10 micrograms/l. Amplification of the expression vector containing a dhfr gene under methotrexate selection pressure resulted in yields up to 200 micrograms/l. Production of IFN-omega 1 was further enhanced 2- to 3-fold by propagation of the cells in the presence of n-butyrate. IFN-omega 1 was purified from culture supernatants by monoclonal antibody affinity chromatography. The resulting protein was at least 95% pure as determined by reverse-phase HPLC and size-exclusion HPLC. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed two bands of about the same intensity with apparent molecular masses of 24.5 and 22.5 kDa. Upon treatment with peptide:N-glycosidase F, both bands were shifted to lower molecular masses (20.5 and 18.5 kDa), indicating that CHO cell-derived IFN-omega 1 is glycosylated; Asn-78 was identified as the glycosylation site. Analysis of the carbohydrate moiety using glycosidases and lectins revealed the presence of biantennary complex oligosaccharides containing neuraminic acid. Amino acid sequencing showed that only about 40% of the molecules have the expected N-terminus, whereas the others carry two additional amino acids derived from the signal sequence. C-terminal amino acid sequencing using carboxypeptidase P demonstrated that the smaller form of the protein lacks nine amino acids. Disulfide bridges were shown to connect Cys residues 1 and 99 as well as 29 and 139, respectively, as in IFN-alpha. The specific antiviral activity of recombinant, glycosylated human IFN-omega 1 on human cells was 2.6 x 10(8) IU/mg, not significantly different from that of the authentic, human leukocyte-derived protein.

Safety and biodistribution of 99mTechnetium-labeled anti-CD44v6 monoclonal antibody BIWA 1 in head and neck cancer patients.

The CD44 protein family consists of isoforms, encoded by standard exons and up to nine alternatively spliced variant exons (v2-v10), which are expressed in a tissue-specific way. Expression of v6-containing variants (CD44v6) has been related to aggressive behavior of various tumor types and was shown to be particularly high in squamous cell carcinoma (SCC). Therefore, CD44v6 might be a suitable target for radioimmunoscintigraphy (RIS) and therapy. The present study evaluates the novel high-affinity murine anti-CD44v6 monoclonal antibody (MAb) BIWA 1 for its safety and targeting potential in patients with SCC of the head and neck (HNSCC). Twelve HNSCC patients, who had planned to undergo resection of the primary tumor and neck dissection, were included. Preoperatively, 2, 12, or 52 mg of 99nTc-labeled MAb BIWA 1 was administered. RIS results obtained 21 h after injection were compared with palpation, computed tomography, and magnetic resonance imaging, with histopathology as the gold standard. Moreover, biodistribution of BIWA 1 was evaluated by radioactivity measurement in blood and bone marrow and in biopsies from the surgical specimen obtained 40 h after injection. The distribution of BIWA 1 in tumor biopsies was analyzed by immunohistochemistry. BIWA 1 integrity in the blood was assessed by high-performance liquid chromatography and related to soluble CD44v6 levels in serum samples. No drug-related adverse events were observed. Human antimouse antibody responses were observed in 11 patients. The diagnostic efficacy of RIS appeared to be comparable for the three BIWA 1 dose levels and for the four diagnostic methods. Besides activity uptake in tumor tissue, minimal accumulation of activity was observed in mouth, lungs, spleen, kidney, bone marrow, and scrotal area. Analysis of tissue biopsies revealed high uptake in tumors, with a mean value of 14.2+/-8.4% of the injected dose/kg tumor tissue and a mean tumor:blood ratio of 2.0+/-1.4 at 40 h after injection. Differences among the three dose groups were not statistically significant, although a trend toward lower uptake in the highest dose group was noted. Distribution of BIWA 1 throughout the tumor was heterogeneous for all dose groups, which might be related to the high affinity of the MAb. The mean biological half-life in blood (34.5+/-6.1 h) was not dose dependent. Extensive complex formation of BIWA 1 was observed in the 2-mg group, most probably with soluble CD44v6 present in the blood, and complex formation relatively diminished upon increase of the MAb dose. BIWA 1 is a promising MAb for targeting HNSCC because it can be safely administered to HNSCC patients, while it shows high and selective tumor uptake. However, BIWA 1 is immunogenic, and therefore a chimerized or humanized derivative of BIWA 1 with intermediate affinity will be used in future clinical trials.

Cloning of human lysozyme gene and expression in the yeast Saccharomyces cerevisiae.

cDNA clones encoding human lysozyme were isolated from a human histiocytic cell line (U-937) and a human placenta cDNA library. The clones, ranging in size from 0.5 to 0.75 kb, were identified by direct hybridization with synthetic oligodeoxynucleotides. The nucleotide sequence coding for the entire protein was determined. The derived amino acid sequence has 100% homology with the published amino acid (aa) sequence; the leader sequence codes for 18 aa. Expression and secretion of human lysozyme in Saccharomyces cerevisiae was achieved by placing the cloned cDNA under the control of a yeast gene promoter (ADH1) and the alpha-factor peptide leader sequence.

Natural human tumor necrosis factor beta (lymphotoxin). Variable O-glycosylation at Thr7, proteolytic processing, and allelic variation.

Natural human tumor necrosis factor beta (TNF-beta) purified from supernatants of a human B-lymphoblastoid cell line was found to be heterogeneous in molecular mass, with seven components resolved by gel electrophoresis. All components are N-glycosylated at Asn62; N-glycosylation does not contribute to heterogeneity. In addition, part of the molecules are O-glycosylated at Thr7; O-glycosylation is heterogeneous due to variable decoration with neuraminic acid. The four lower molecular mass forms are derived from the full-length protein by trypsin-like proteolytic cleavage in the N-proximal region; these clipped molecules lack O-linked carbohydrates. Two allelic variants differing in amino acid position 26 (threonine/asparagine) were identified.

Expression of CD44 isoforms in human skin cancer.

In animal models, isoforms of CD44 (CD44v) containing sequences encoded by one or several of ten different exons (v1-v10) contribute to tumour metastasis. In certain human cancers, CD44v6 expression is associated with poor prognosis. This paper examines CD44v expression in skin carcinogenesis and skin cancer metastasis. CD44v expression was studied in basal cell carcinoma (BCC), squamous cell carcinoma (SCC), primary malignant melanoma (PMM), metastases of MM (MMM), benign melanocytic naevi (BMN) and normal skin (NS) by immunohistochemistry and reverse transcript polymerase chain reaction (RT-PCR). BCC, SCC and NS expressed several CD44v, including v6, albeit in different distributions and intensities. PMM, MMM and BMN expressed isoforms containing v7/8 and v10, but failed to express epitopes encoded by v5 or v6. Thus, different CD44 isoforms are found in human skin cancers and are modulated during carcinogenesis. However, we did not observe a correlation of CD44v6 expression with metastatic potential.

Splice variants of the cell surface glycoprotein CD44 associated with metastatic tumour cells are expressed in normal tissues of humans and cynomolgus monkeys.

Certain isoforms of the CD44 glycoprotein family play an essential role in the metastatic spread of tumour cells. Protein expression of such CD44 isoforms has also been observed in a variety of human malignancies. In this study, we compared the expression of exon v5- and v6-containing CD44 isoforms in normal human and cynomolgus monkey (Macacca fasciculata) tissues. Cloning and sequencing of cynomolgus CD44 exons v5 and v6 revealed a homology of 97% and 95%, respectively, between man and monkey. Two monoclonal antibodies (MAbs) directed against an epitope encoded by human exon v5 (VFF8) and an epitope encoded by exon v6 (VFF18) were used to determine expression of CD44 isoforms in man and monkey. Immunohistochemical screening of a representative profile of normal human and cynomolgus tissues revealed that expression of exon v5- and v6-containing CD44 isoforms was almost identical in the two species. Exon v6 staining was observed only in a subset of epithelial tissues, whereas v5 staining was additionally detected on certain non-epithelial tissues. These data suggest that cynomolgus monkey could serve as a system to test the usefulness of antivariant CD44 MAbs with regard to antibody-based tumour therapy.

Highly sensitive enzyme immunoassay for human lymphotoxin (tumor necrosis factor beta) in serum.

We have developed a rapid, simple and highly sensitive 'sandwich' enzyme immunoassay (ELISA) for the detection and quantification of human lymphotoxin (= tumor necrosis factor beta) in serum. The assay, performed in microtiter plates, employs two monoclonal murine antibodies able to neutralize the cytotoxic activity of lymphotoxin. In a one-step procedure, antibody LTX-21 (IgG2b) coated on to the solid phase captures antigen present in the sample; subsequently antibody LTX-22 (IgG1), covalently coupled to horseradish peroxidase, labels the bound antigen. The assay is able to detect lymphotoxin spiked into human serum in concentrations as low as 7 pg/ml, whereas human tumor necrosis factor alpha does not cross-react even at 10(7)-fold higher concentrations. Only biologically active protein is recognized by the antibodies, since inactivation of lymphotoxin measured by bioassay results in a parallel decrease in immunoreactivity. Natural, glycosylated lymphotoxin shows the same reactivity as recombinant, unglycosylated protein. The assay will be useful for the quantification of endogenous human lymphotoxin in serum, other body fluids, and culture supernatants of human cells, and can also be used to monitor levels of recombinant human lymphotoxin in animal studies and clinical trials.

Highly sensitive enzyme immunoassays for antibodies to human tumor necrosis factor (TNF-alpha) and lymphotoxin (TNF-beta).

Two 'inverse sandwich' enzyme immunoassays (ELISAs) were developed for the detection and quantification of antibodies to human tumor necrosis factor (TNF-alpha) and lymphotoxin (TNF-beta), respectively. In these one-step assays, antibodies present in the sample linked antigen which had been covalently coupled to horseradish peroxidase to antigen bound to a solid phase (microtiter plates). The limits of detection of the assays were lower than those of neutralization bioassays; antibodies to TNF-alpha and TNF-beta being detected at concentrations as low as 2 ng/ml and 0.5 ng/ml, respectively, and no cross-reactivity was observed. The advantages of these ELISAs over other assay methods currently in use for the detection of antibodies include: (i) the convenience of the microtiter plate format and its suitability for testing large numbers of samples; (ii) the absence of radioactive tracers and precipitation steps; (iii) the high stability of the reagents; (iv) the avoidance of second antibodies and, thus, the possibility of testing samples from various species without modification of the assay and (v) the ability to detect low-affinity antibodies due to the absence of competitive reactions. The assays may be used without modification for the detection of antibodies in serum samples from both man and laboratory animals as well as in other samples such as hybridoma supernatants.

A monoclonal antibody-based enzyme immunoassay for quantitation of human tumor necrosis factor binding protein I, a soluble fragment of the 60 kDa TNF receptor, in biological fluids.

Three hybridoma cell lines secreting monoclonal IgG antibodies specific for human tumor necrosis factor-binding protein I (TNF-BP I), the extracellular domain of the 60 kDa TNF receptor, were developed by fusion of spleen cells from mice immunized with TNF-BP I purified from urine. The antibodies recognize three different epitopes on TNF-BP I. Two of the antibodies were used to develop a two-site ('sandwich') enzyme immunoassay with horseradish peroxidase as the marker enzyme. The assay was able to measure TNF-BP I in serum, urine and cell culture supernatants with a sensitivity of about 200 ng/l and a precision better than 10%. TNF-BP I was detected in the serum of healthy individuals at a mean concentration of 2.1 +/- 1.0 micrograms/l (mean +/- standard deviation; range, 0.52-5.4 microgram/l, n = 42); no significant difference was seen in patients with chronic polyarthritis (2.3 +/- 0.79 micrograms/l; n = 15). Serum TNF-BP I was significantly elevated in patients with burns (6.5 +/- 1.7 micrograms/l; n = 10) and markedly increased in patients with renal failure (49 +/- 17 micrograms/l; n = 6). TNF-BP I was also detectable in urine from normal individuals (2.2 +/- 1.2 micrograms/l; range 0.78-4.3 micrograms/l; n = 16). Culture supernatants of several human tumor cell lines also contained TNF-BP I. The assay will be a useful tool to detect activation of the TNF receptor by the physiological ligands, TNF-alpha and TNF-beta, as well as transmodulation by other mediators in various pathological conditions.

Characterization of the human erythrocyte complement receptor CR1 (C3b receptor) by epitope mapping.

Monoclonal antibodies and an anti-idiotypic serum against human complement receptor CR1 (C3b receptor, immune adherence receptor) were used to identify CR1 and some of its proteolytic fragments by an immunoblotting technique. The anti-idiotypic serum had a specificity for the C3b-binding site, as could be shown by its cross-reactivity with complement factor H. The monoclonal antibodies GARP-4 and GARP-37 were specific for epitopes located nearby the ligand-binding site, because they blocked the immune adherence reaction. For the immunoblotting technique, it was essential to use non-reducing conditions, since reduction of CR1 destroyed the epitopes. Therefore, mainly large (disulphide-linked) fragments of CR1 were obtained. A chymotryptic fragment of Mr 56,000 identified by GARP-4, was the smallest cleavage product to be associated with the C3b-binding domain. Different proteases gave CR1 degradation products of similar Mr, indicating the presence of distinct domains, three of which had a Mr approximately 38,000. A schematic model of CR1 substructure was deduced from the epitope mapping data.

Molecular cloning and expression of human and rat tumor necrosis factor receptor chain (p60) and its soluble derivative, tumor necrosis factor-binding protein.

Tumor necrosis factor-alpha (TNF-alpha), a protein released by activated macrophages, is involved in a wide variety of human diseases including septic shock, cachexia, and chronic inflammation. TNF binding protein (TNF-BP), a glycoprotein with high affinity to TNF-alpha isolated from urine, acts as an inhibitor of TNF-alpha by competing with the cell-surface TNF receptor. We report here the partial amino acid sequencing of human TNF-BP as well as the isolation, sequence, and expression of cDNA clones encoding a human and rat TNF receptor. The calculated Mr of the mature human and rat TNF receptor chains is 47,526 and 48,072, respectively. The extracellular ligand binding domain represents the soluble TNF-BP which is released by proteolytic cleavage. TNF-BP contains 24 cysteine residues and three potential N-glycosylation sites and shows sequence homology to the extracellular portions of TNF-R p80 chain and nerve growth factor receptor. Transfection of the human TNF receptor cDNA into mammalian cells resulted in increased binding capacity for TNF-alpha and increased reactivity with a monoclonal antibody directed against the human TNF receptor chain p60. When a stop codon was introduced into the cDNA at the site corresponding to the carboxyl terminus of TNF-BP, transfected cells secreted a protein that reacted with antibodies raised against natural TNF-BP.

Expression of CD44 isoforms in human renal cell carcinomas.

A series of 27 renal cell carcinomas 4 oncocytomas and 7 samples of tumour free kidney parenchyma were analysed immunohistochemically using eight different CD44 isoform-specific monoclonal antibodies. In normal kidney expression of CD44 isoforms (containing variant exons v6, v7/8 and v10) was found predominantly at the distal tubules. The majority of clear cell carcinomas investigated showed expression of variant exons v5, v7/8 and v10, but not v6. Lack of CD44v6 expression was confirmed by reverse transcription-polymerase chain reaction analysis. Carcinomas of the chromophilic cell type were almost completely devoid of CD44 expression, including the standard form CD44s. This study shows that there are statistically significant differences in the CD44 expression pattern of the two major histological subtypes of renal cell carcinomas (clear cell and chromophilic carcinomas). Moreover, the almost complete lack of CD44 expression in chromophilic carcinomas contrasts with carcinomas of other histogenetic origin investigated including stomach, breast and lung which express various CD44 isoforms abundantly.

Expression of CD44 isoforms on isolated bone marrow plasma cells and peripheral CD19+ B cells of patients with multiple myeloma and healthy individuals.

The expression of certain isoforms of CD44 was shown to correlate with aggressiveness and metastatic potential of various tumour types. We analysed the expression of the adhesion molecule CD44 and its variant domains (v6, v7, v7/8, v10) on isolated bone marrow (BM) plasma cells and peripheral blood (PBL) CD19+ B cells of 21 patients with MM and 15 healthy donors. B cells and plasma cells were isolated by immunomagnetic sorting and analysed by two-colour flow cytometry. The expression of CD44 isoforms was significantly higher on PBL B cells of patients with MM than in healthy controls. The elevated expression of CD44 isoforms (v6, v7/8, v10) on PBL B cells correlated with reduced overall survival in MM. CD44 isoforms were more strongly expressed on "larger", activated B cells. Furthermore, CD44 isoforms were found to be simultaneously expressed with CD38hi and CD56 on both, B lymphocytes and plasma cells of patients with MM. The determination of CD44 isoforms on circulating B cells may be helpful in defining prognostically unfavourable subgroups in MM.

Proteins induced by recombinant equine interferon-beta 1 within equine peripheral blood mononuclear cells and polymorphonuclear neutrophilic granulocytes.

Peripheral blood mononuclear cells (PBMC) and polymorphonuclear neutrophilic granulocytes (PMN) as well as embryonic equine dermal fibroblasts and the equine fibroblast line E. Derm which were used as controls, were treated with recombinant equine interferon-beta 1 (rEqIFN-beta 1) in vitro which induced the expression of different proteins in these cells. A 74 kDa protein was induced in PBMC and an 82 kDa protein was additionally found in the equine fibroblast E. Derm cell line following treatment with rEqFN-beta 1. Both proteins reacted with anti-mouse and anti-human Mx protein antisera in immunoblot tests. The 74 kDa and perhaps the 82 kDa components may thus represent equine 'Mxanalogous proteins'. The 74 kDa protein was only detected in PBMC of ten out of 20 horses examined. The induction of Mx protein in the horse by Type 1 interferon may therefore resemble that in the mouse, where Mx protein is involved in selective resistance to influenza virus. The influence of rEqIFN-beta 1 on protein expression in equine PBMC and PMN was monitored by metabolic labeling and 2-D gel electrophoresis. Proteins of 82, 74, 58 and 40 kDa were induced in PBMC following exposure to rEqIFN-beta 1. A constitutively expressed 35 kDa protein, however, was no longer demonstrable upon treatment with interferon. None of the proteins induced within PBMC was found in highly purified PMN treated with interferon. PMN exposed to rEqIFN-beta 1 synthesized four proteins in the range of 25 to 27 kDa. These proteins have not been described in interferon-treated PMN of any other species.

Kinetics of inhibition of replication of vesicular stomatitis virus in blood mononuclear cells of horses after in vitro and in vivo treatment with recombinant equine interferon-beta 1.

Recombinant equine interferon-beta 1 (reqIFN-beta 1) induces an antiviral state in blood mononuclear cells (BMC) of horses. Maximal protection against replication of vesicular stomatitis virus is achieved 6 hours after treatment with IFN in vitro and in vivo. Duration of the protective effect depends on the dose of IFN in vitro and in vivo. Availability of reqIFN-beta 1 in cultures of BMC for up to 48 hours does not prolong the antiviral state. The protective effect on BMC after treatment with IFN has similar duration in vivo and in vitro. Monitoring of the effect of IFN in vivo is, thus, simplified because the antiviral state may be recorded by testing cells twice (ie, before and 6 hours after application of interferon). All further tests may be performed in vitro. Multiple administration of reqIFN-beta 1 do not prolong duration of the protective phases after each administration. Duration of the antiviral state depends only on the dose of reqIFN-beta 1.

[Use of monoclonal antibodies in the purification and analysis of human interferon]. / Anwendung monoklonaler Antikörper bei der Reinigung und Analyse von Humaninterferon.

This paper describes the development of hybridoma cell lines secreting monoclonal antibodies to human IFN-alpha and the implementation of these antibodies in the purification of recombinant IFN-alpha 2 and the quantification of this protein by means of immunological assay procedures.