Production of antibodies in plants: approaches and perspectives.

Advances in molecular biology, immunology, and plant biotechnology have changed the paradigm of plant as a food source to so-called plant bioreactor to produce valuable recombinant proteins. These include therapeutic or diagnostic monoclonal antibodies, vaccines, and other biopharmaceutical proteins. The plant as a bioreactor for the production of therapeutic proteins has several advantages, which include the lack of animal pathogenic contaminants, low cost of production, and ease of agricultural scale-up compared to other currently available systems. Thus, plants are considered to be a potential alternative to compete with other systems such as bacteria, yeast, or insect and mammalian cell culture. Plant production systems, particularly therapeutic antibodies, are very attractive to pharmaceutical companies to produce the antibodies in demand. Currently, we have successfully developed a plant system for production of anti-rabies monoclonal antibody and anti-colorectal cancer monoclonal antibody. The effective plant production system for recombinant antibodies requires the appropriate plant expression machinery with optimal combination of transgene expression regulatory elements, control of posttranslational protein processing, and efficient purification methods for product recovery. However, there are several limitations that have to be resolved to establish the efficient plant system for antibody production. Here, we discuss the approaches and perspectives in plant systems to produce monoclonal antibody.

Application of liquid separation techniques to the determination of the main urinary nicotine metabolites.

A rapid procedure for the analysis of the main nicotine metabolites (cotinine, trans-3'-hydroxycotinine) in urine has been worked out. The procedure includes isolation of nicotine and its metabolites from urine by means solid-liquid extraction technique using resin Amberlite XAD-2 and then quantitation by the use of thin-layer chromatography with densitometry (in reflection mode). GC-MS was applied to confirm the results obtained by TLC. The procedure was applied to the analysis of cotinine concentrations in urine samples taken from children living in Upper Silesia region (Poland). Among 444 investigated children we did not find cotinine almost in 60% but in 15% of this population, there were children who could have been exposed to cigarette smoke.

[Alice Hamilton (1869-1970): a pioneer of occupational medicine and public health]. / Alice Hamilton (1869-1970) -- pionier medycyny pracy i zdrowia publicznego.

Dr. Alice Hamilton (1869-1970) was the mother of occupational health a pioneer in public health in the United States. She worked as a doctor in Hull House, the first settlement house, and she was an advocate of the birth-control movement. She led pioneering studies of occupational head, mercury, carbon monoxide poisoning and many other chemical intoxications of workers. She was an assistant professor of industrial medicine at the Harvard Medical School (1919-1935). During the years 1924-1930 she worked for the Health Organization of the League of Nations. From 1943 she acted as a vice-president of the American Health Association. Alice Hamilton was an expert in the field of occupational lead poisoning.

Vaccination with carbohydrate peptide mimotopes promotes anti-tumor responses.

Tumor-associated carbohydrate (TAC) antigens are important targets in cancer vaccine efforts. Carbohydrates are, however, frequently poor immunogens, in that they are T-cell-independent antigens. Molecular mimicry of TAC by peptides is an alternative approach to generating anti-carbohydrate immune responses. Here we demonstrate that peptide mimotopes can elicit antibody responses that cross-react with representative human TAC antigens. Primary immunization with such a multiple antigenic peptide, along with QS-21 as adjuvant, elicits cytotoxic antibodies reactive with naturally occurring forms of TAC expressed on tumor cells, and vaccination of mice with peptide mimotopes reduced tumor growth and prolonged host survival in a murine tumor model.

Towards the development of peptide mimotopes of carbohydrate antigens as cancer vaccines.

Tumor-associated carbohydrate antigens are considered important targets in efforts to develop cancer vaccines. To further enhance vaccine efforts, we are developing peptide mimotopes of tumor-associated carbohydrate antigens that can elicit functional immune responses. Mapping peptide epitopes with anticarbohydrate antibodies can lend to defining structural relationships that can go undetected by screening of carbohydrate antigens alone. Here we contrast reactivity patterns for peptides using monoclonal antibodies (MAbs) directed to the neolactoseries related Lewis Y (LeY) and sialyl-Lewis X (sLeX) antigen and the GD3/GD2 ganglioside antigen. We observe that representative MAbs cross-react with a WRY-containing peptide and that this motif type is isolated by the respective monoclonal in peptide phage display screening. Primary immunization with multiple antigen peptide preparations with QS-21 adjuvant efficiently elicited cytotoxic IgM antibodies for a murine Meth A fibrosarcoma line expressing sLeX. The cytotoxicity of IgG polyclonal response was found to be as effective as IgM in mediating complement-dependent cytotoxicity against the Meth A line. These experiments suggest that peptide mimotopes of the LeY and sLeX tumor-associated carbohydrate antigen and QS-21 adjuvant could be considered as an immunogenic therapeutic vaccine in carcinoma and melanoma patients in the minimal residual disease setting.

Antigenic and immunological mimicry of peptide mimotopes of Lewis carbohydrate antigens.

Peptides may substitute for carbohydrates in reactions with carbohydrate-specific molecules. Recently, we found that peptides containing aromatic residues mimic mucin and histo-blood group related carbohydrate epitopes, eliciting polyclonal responses cross-reactive with bacterial and viral antigens that express these carbohydrate forms. These results demonstrate that peptides can function in in vivo and in vitro models as carbohydrate surrogate antigens. To further explore the nature of the antigenic and immunogenic properties of such mimotopes, synthetic peptides with aromatic amino acids were tested to delineate reactivity patterns with several anti-neolactoseries monoclonal antibodies (MAbs). These MAbs recognize biologically important conformations of the histo-blood group related Lewis antigens expressed on the surface of a variety of human cancers. Results by ELISA demonstrate that the MAbs can distinguish particular peptide motifs that include the sequences GGIYYPYDIYYPYDIYYPYD, GGIYWRYDIYWRYDIYWRYD and GGIYYRYDIYYRYDIYYRYD. Substitution of Arg by Pro diminished the reactivity of the anti-Lewis Y (LeY) MAb BR55-2. Binding of LeY to BR55-2 was inhibitable by the Arg containing peptides. Serum against all three peptides displayed reactivity with synthetic histo-blood group related antigen probes. Immunologic presentation of the peptides as multiple antigen peptides (MAPs) improved peptide ability to induce LeY specific immune responses. Serum bound to human tumor cells that preferentially expressed neolactoseries antigens, but not to normal tissues. Immunoprecipitation of human breast tumor cell lysates before and after treatment with tunicamycin confirmed serum carbohydrate binding. The anti-peptide sera mediated tumor cell killing by complement mediated cytotoxicity. These results indicate that mapping peptide epitopes with anti-carbohydrate antibodies can lend to defining antibody fine specificities that can go undetected by screening of carbohydrate antigens alone. In addition, these results confirm that peptides and carbohydrates can bind to the same antibody binding site and that peptides can structurally mimic salient features of carbohydrate epitopes.

[Level of stress during pregnancy estimated by mothers and average weight of newborns and frequency of low birth weight]. / Poziom stresu w czasie ciazy w ocenie matek a sredni ciezar ciala noworodków i czestosc wystepowania malej urodzeniowej masy ciala.

Low birth weight is still important health problem in many countries. Children's low birth weight increases mortality, injures central nervous system, somatic, interferes with intellectual and emotional development. Low birth weight is frequently occurring in Poland--between 7-9% of live births. There are many risk factors, among them behavioural and environmental. In Poland an attention was put on chemical and physical environmental factors. Behavioural factors (stress) are disregarded. In the present paper it was decided to check the relationship between stress during pregnancy (estimated by pregnant), child birth weight and frequency of low birth weight. The research was carried out by use of a questionnaire using the "case-control study". In the research were involved 450 mothers of new-born children (the group of cases: untimely, premature delivery or child birth weight below 2500 g) and 450 mothers of new-born children (control group-physiologically delivered). Mothers were asked about their relations to the pregnancy; professional and personal stress during pregnancy was estimated. The results were analysed by counting risk ratio coefficient (RR) and correlation coefficient. The research showed, that there is no relation between acceptation of pregnancy, stress and frequency of low birth weight or the average child birth weight. The researches didn't prove unfavourable influence of stress reaction caused by professional and personal stressors on intrauterine foetus development.

Peptide mimicry of carbohydrate epitopes on human immunodeficiency virus.

Cancer-related, mucin-type carbohydrate epitopes, principally mannose and sialo-syl residues, are expressed on the envelope protein gp 160 of the human immunodeficiency virus (HIV). Anticarbohydrate antibodies directed toward these and other carbohydrate epitopes are known to neutralize HIV-1 infection by cell-free virus. Carbohydrates, however, being T cell-independent antigens, typically elicit diminished immune responses. To overcome this potential draw back, we have examined the ability of peptides that mimic such epitopes to elicit immune responses that cross-react with carbohydrate structures. We report that mouse polyclonal antisera generated against peptides that mimic mucin-related carbohydrate epitopes have anti-HIV-1 activity. Generation of antibodies was not lr-gene restricted, as at least two different strains of mice. Balb/c (H-2d) and C57Bl/6 (H-2b), responded equally to the peptides. The antipeptide sera displayed neutralizing activity against HIV-I/MN and HIV-I/3B viral strains. This neutralization was as good as human anti-HIV sera. These results indicate that peptide mimics of carbohydrates provide a novel strategy for the further development of reagents that elicit immune responses to carbohydrate epitopes associated with many infectious organisms and tumor cells.

External beam radiation enhances antibody mediated radiocytotoxicity in human glioma cells in vitro.

Enhanced accumulation of monoclonal antibodies in tumor tissue has been observed as a result of external beam irradiation (EBR). This effect was mainly attributed to increased vascular leakage due to unspecific radiation damage of vascular endothelial cells. The aim of this study was to investigate the effects of EBR on expression and antibody-binding of epidermal growth-factor receptor (EGF-R) in human glioma cells in-vitro. High-grade glioma cells were irradiated with conventional x-rays (0-3600 Rad) and surface binding, internalization and radiocytotoxicity of 125I-labeled monoclonal antibody (MAb) 425, specific for human EGF-R, was tested. EBR showed a short-term dose and time dependent increase of specific MAb 425 binding and internalization in receptor positive cell lines U87-MG and A1207. This effect was probably due to a mitotic block and an increase in cellular volume. Combination of EBR and 125I-425 showed additive effects on cell vitality/survival and was more pronounced in contact inhibited cells as compared to cells growing in a log-phase. We assume that cells exposed to 125I-labeled MAb 425 are only able to accumulate a critical number of DNA double-strand breaks when the doubling-time is prolonged e.g. under contact-inhibition or radiation induced mitotic blockade. We conclude that EBR has no negative effects on EGF-R expression, MAb-binding and internalization. The combination of EBR and 125I-MAb 425 enhances cytotoxic efficacy and thus supports adjuvant use in the clinical management of high-grade glioma.

Peptide mimicry of adenocarcinoma-associated carbohydrate antigens.

Carbohydrate antigens have been identified as significant antigens in many human tumors either by analyzing antibodies in patients' sera or by using monoclonal antibodies of either mouse or human origin. Three carbohydrate epitopes present on cancer-associated mucins [sialyl-Lewis A (SLA), sialyl-Lewis X (SLX), and sialyl-Tn (STn)] may have functional significance in metastasis. Subsequently, these antigens are considered as targets for active specific immunotherapy. Carbohydrates, as T-cell-independent antigens, often elicit diminished immune responses. To overcome this drawback, carbohydrates are typically coupled to protein carriers to elicit immunoglobulin G (IgG) responses as opposed to low-affinity IgM responses, which often times accompanies carbohydrate-based immunizations. In addition, some complex carbohydrates are difficult to synthesize. This latter aspect is further magnified if one considers that clustering of epitopes on neoglycoproteins must be emulated in the synthesis process, leading to multiple presentation or tandem repeats of the synthetic carbohydrate immunogen. Here, we examine the hypothesis that peptides that mimic carbohydrates might be developed to induce immune responses that target and mediate the killing of tumor cells, particularly breast cancer cells in an adjuvant-type setting. We have found that carbohydrate-mimicking peptides retain carbohydrate-like conformations, inducing anti-carbohydrate immune responses against breast tumor cells and mediating their killing by a complement-dependent mechanism.

Molecular recognition of the Lewis Y antigen by monoclonal antibodies.

The murine monoclonal antibody BR55-2 is directed against the tumor-associated antigen Lewis Y oligosaccharide. The Lewis Y core antigen is a difucosylated structure consisting of four hexose units. Analysis of binding profiles of lactoseries isomeric structures by BR55-2 suggest that the binding epitope includes the OH-4 and OH-3 groups of the beta-D-galactose unit, the 6-CH3 groups of the two fucose units and the N-acetyl group of the subterminal beta-D-N-acetylglucosamine (beta DGlcNAc). To elucidate the molecular recognition properties of BR55-2 for the Y antigen, BR55-2 was cloned, sequenced and its three-dimensional structure was examined by molecular modeling. The crystal structure of BR96, another anti-Lewis Y antibody, solved in complex with a nonoate methyl ester Lewis Y tetrasaccharide, and the lectin IV protein in complex with a Lewis b tetrasaccharide core were used as a guide to probe the molecular basis for BR55-2 antigen recognition and specificity. Our modeling study shows that BR55-2 shares similar recognition features for the difucosylated type 2 lactoseries Lewis Y structure observed in the BR96-sugar complex. We observe that a major source of specificity for the Lewis Y structure by anti-Y antibodies emanates from interaction with the beta-D-N-acetylglucosamine residue and the nature of the structures extended at the reducing site of the fucosylated lactosoamine.

HLA-B-restricted, CD8+ cytolytic human T cell clones derived from a melanoma-invaded lymph node.

We have reported that medium containing recombinant human IL-1 (rIL-1), rIL-2, rIL-4, and rIL-6 (MB-1,2,4,6 medium) efficiently expanded autologous tumor specific CTLs in vitro. For further examination of the CTLs cultured in MB-1,2,4,6, the therapeutic activity on tumor growth inhibition in vivo and established CTL clones were studied. In vivo therapy with the CTLs in combination with rIL-2 was highly effective. To investigate CTL clones, 19 CD8+ T cell clones were obtained by limiting dilution method, each clone retained autologous-melanoma-specific, HLA-class I-restricted cytolytic activity. Four T cell clones were analyzed in detail. These T cell clones displayed a CD3+, CD4-, CD8+, CD11b-, CD16-, CD56-, CD45RA-, TcR alpha/beta + phenotype and monoclonal antibodies to HLA class I, CD3, and CD8 antigens inhibited their cytolytic activity. Moreover, these CTL clones recognize one of common melanoma antigens associated with HLA-B (B49). Analysis of the effect of different cytokines on the proliferation and cytotoxicity of cloned CTL revealed the highest growth rate in MB-1,2,4,6 but no dependence on particular cytokine combinations for autologous tumor-specific cytolytic activity. These results suggest (1) the usefulness of MB-1,2,4,6 medium in expanding autologous tumor-specific CTLs, which can be used in adoptive immunotherapy, (2) HLA-B molecules can present one of common melanoma antigens, and (3) the independence of CTLs from any cytokine combination once they become target-specific.

Elevated expression of H type GDP-L-fucose:beta-D-galactoside alpha-2-L-fucosyltransferase is associated with human colon adenocarcinoma progression.

GDP-L-fucose:beta-D-galactoside alpha-2-L-fucosyltransferase (EC 2.4.1.69) is a key enzyme in the biosynthesis of fucosylated type 1 and 2 lactoseries structures, such as Lewis b and the H type 2 and Lewis Y, respectively, that are accumulated in colon adenocarcinoma. Analysis of the mRNA transcript level for the human H gene-encoded beta-D-galactoside alpha-2-L-fucosyltransferase revealed 40- and 340-fold increases in the mRNA levels in all adenocarcinomas and tumor cell lines, respectively, compared to normal colon mucosa where a low level of mRNA transcript was detected. A variable increase in mRNA transcript levels was observed in 50% of adenomatous polyps. Nucleotide sequence analysis of the protein coding region of the cDNAs derived from normal colon, adenoma, and colon adenocarcinoma revealed 100% homology, suggesting that there are no tumor-associated allelic variations within the H beta-D-galactoside alpha-2-L-fucosyltransferase cDNA. These results suggest that beta-D-galactoside alpha-2-L-fucosyltransferase expression highly correlates with malignant progression of colon adenocarcinoma.

Cytokine combinations for induction of antigen-specific cytolytic T lymphocytes from peripheral blood lymphocytes.

We studied effects of varied cytokine combinations on allogeneic cytotoxic T lymphocyte (CTL) generation in a mixed lymphocyte-tumor cell culture. Sensitized with allogeneic melanoma cell line (MM-8.1 or MM-28), peripheral blood mononuclear cells (PBMC) were cultured in medium containing various combinations of cytokines that included human recombinant interleukin-2 (rIL-2) alone (MB-2), rIL-2 and rIL-4 (MB-2,4) and rIL-1, rIL-2, rIL-4 and rIL-6 (MB-1,2,4,6). Lymphocytes proliferated for more than 50 days and manifested stimulating cell-specific cytotoxicities in 1 of 5 cultures with MB-2, in 4 of 5 with MB-2,4 and in 5 of 5 with MB-1,2,4,6. Lymphocytes grew up to 10(6) fold in culture with MB-2,4 or MB-1,2,4,6 at day 90-100. Stimulating cell MM-8.1 (HLAABC+,DR-) induced CD3+, CD8+, CD56- CTLs and MM-28 (HLA-ABC+, DR+) mainly generated CD3+, CD4+, CD56- CTLs. Monoclonal antibodies against HLA-ABC and HLA-DR molecules suppressed the stimulating cell-specific cytolytic activity of CD8+ and CD4+ CTLs, respectively. These data indicate that combination of rIL-1, rIL-2, rIL-4 and rIL-6 offer an efficient system to generate allogeneic, tumor-specific CTLs from PBMCs and that HLA molecules expressed on stimulating cells play an important role in CTL generation.

Epidermal growth factor receptor 425 monoclonal antibodies radiolabeled with iodine-125 in the adjuvant treatment of high-grade astrocytomas.

Fifty-nine patients with primary presentation of high-grade gliomas of the brain, 13 with astrocytomas with anaplastic foci and 46 with glioblastoma multiforme, were treated with surgical intervention and definitive postoperative radiation therapy followed by multiple intravenous administration of iodine-125-labeled monoclonal antibody-425, which binds specifically to human epidermal growth factor receptor. The total cumulative labeled antibody doses ranged from 40 to 296 mCi. The administration of the radiolabeled antibody was performed in most instances within 3 months following completion of the primary surgery and radiation therapy. No significant life-threatening toxicities were observed during the trial. At one year, 34 (58%) of the 59 patients in the trial were alive. The median overall survival for both groups was 13.5 months.

Enhancement of monoclonal antibody efficacy: the effect of external beam radiation.

External beam irradiation has been shown to enhance accumulation of monoclonal antibodies (MAb) in tumors in vivo. This effect is mainly attributed to an unspecific damage of vascular endothelial cells resulting in an increased vascular leakage. The aim of our studies was to determine the effects of external beam radiation on the expression and function of the epidermal growth factor receptor (EGF-R) in vivo. Expression and internalization of EGF-R was tested in vivo, employing 125I-MAb 425 that binds specifically to the human EGF-R. Irradiation of human high-grade glioma cell lines U87-MG and A1207 with increasing doses (0-3600 Rad) of 240 kVp X-rays, markedly enhanced the binding of 125I-MAb 425 to the cell surface. This effect could only be observed for a few days following irradiation. No correlation of the radiation dose and overexpression of EGF-R were found. At the same time, irradiation stimulated significant and dose-dependent internalization of 125I-MAb. Internalization and intranuclear accumulation of 125I-MAb are necessary to explain the radiocytotoxic effects of 125I. The combination of external beam irradiation and labeled MAb 425 showed at least additive effects on tumor cell survival, when the interval between irradiation and MAb treatment was short. Our data support the clinical observations in the adjuvant treatment of high grade gliomas with 125I-MAb 425 following surgery and external beam radiation.

Proliferation, phenotype, and cytotoxicity of human lymphocytes isolated from lymph nodes invaded by melanoma cells.

The effects of human recombinant interleukin-4 (rIL-4) on metastatic melanoma (lymph node)-derived T lymphocytes cultured with human recombinant interleukin-2 (rIL-2) were studied. Lymphocytes isolated from melanoma-invaded lymph nodes were cultured in media with rIL-2 in the presence (MB-2,4) or absence (MB-2) or rIL-4 for up to 48 days. A majority of lymphocytes grown in both cultures were CD3+ T lymphocytes. Addition of rIL-4 to the rIL-2 culture abrogated the growth of the CD3-, CD56+ cell population [natural killer (NK) cell], which were present in culture with rIL-2 alone. Lymph-node-derived T lymphocytes that had expanded under MB-2,4 conditions lysed only autologous melanoma cells and they maintained the autologous-specific cytolytic activity during the entire culture period. They did not lyse K562, Daudi, or allogeneic target cells. Monoclonal antibodies (MAbs) against CD3 molecules and MHC class I molecules but not MHC class II molecules inhibited the specific lytic functions of T lymphocytes under MB-2,4 culture conditions. Collectively, these data indicate that in lymphocyte culture derived from melanoma-invaded lymph nodes, rIL-4 inhibits the rIL-2-dependent proliferation of NK cells and antigen nonspecific killer T lymphocytes and also effectively abrogates the rIL-2-induced NK and lymphokine-activated killer (LAK) activities.

In vitro expansion of tumor-specific, HLA-restricted human CD8+ cytolytic T lymphocytes.

The effect of cytokines with human recombinant interleukin-2 (rIL-2) on cytolytic T cell (CTL) generation was studied. Lymphocytes were isolated from involved lymph nodes of melanoma patients and expanded in medium containing rIL-2 alone or in combination with other human cytokines (rIL-1, rIL-4, rIL-6, and recombinant human tumor necrosis factor-alpha (rTNF alpha)). Lymphocytes incubated with rIL-2 alone did not grow, whereas addition of the other cytokines augmented IL-2-mediated lymphocyte proliferation. In all cultures, the majority of expanded lymphocytes were CD3+, CD56- T cells. Lymphocytes cultured with rIL-1, rIL-2, rIL-4, and rIL-6 exhibited cytolytic activity specific for autologous melanoma, which increased during the culture period (24.08 and 58.18% at 16 and 30 days in culture, respectively) without detectable changes in cell surface phenotype and remained high even after 100 days in culture. Moreover, the cytolytic activity was inhibited by monoclonal antibodies (mAbs) against HLA-class I, CD3, and CD8 molecules but not by mAbs against HLA-class II or CD4 molecules. Lymphokine-activated killer (LAK) activity was detected in lymphocytes cultured with rIL-1, rIL-2, and rIL-6 in the presence or absence of rTNF alpha. These data indicate that lymphocytes derived from melanoma-invaded lymph nodes and cultured in the presence of rIL-1, rIL-2, rIL-4, and rIL-6 offers an efficient system to expand CD8+ CTLs with HLA-restricted cytolytic specificity against autologous tumor cells.

Oncogene-targeted antisense oligodeoxynucleotides combined with chemotherapy or immunotherapy: a new approach for tumor treatment?

Synthetic oligodeoxynucleotides (antisenses) complementary to bcr/abl breakpoint junction transcript on Philadelphia chromosome, or c-myb protooncogene inhibit partially the proliferation of Philadelphia positive leukemic cells (antisenses against bcr/abl and c-myb) and other tumor cells (antisenses against c-myb). This phenomenon is accompanied by specific downregulation of mRNA level of the particular gene. To develop a more effective procedure of tumor treatment the combination of low dose of cytostatic and bcr/abl or c-myb antisenses against Philadelphia chromosome positive cell line BV173, and the combination of anti-tumor cytotoxic T lymphocytes (CTL) and c-myb antisenses against melanoma cell line MM-28, were tested in vitro. Our results indicate that the combinations of conventional chemotherapeutic agent and antisense against bcr/abl or c-myb or tumor specific CTL and antisense against c-myb, are highly effective in killing of tumor cells and sparing normal cells. This creates the possibility to develop a more selective and effective treatment of neoplasia.