ovalbumin and Multiple-Myeloma

The use of monoclonal antibodies is ubiquitous in science and biomedicine but the generation and validation process of antibodies is nevertheless complicated and time-consuming. To address these issues we developed a novel selective technology based on an artificial cell surface construct by which secreted antibodies were connected to the corresponding hybridoma cell when they possess the desired antigen-specificity. Further the system enables the selection of desired isotypes and the screening for potential cross-reactivities in the same context. For the design of the construct we combined the transmembrane domain of the EGF-receptor with a hemagglutinin epitope and a biotin acceptor peptide and performed a transposon-mediated transfection of myeloma cell lines. The stably transfected myeloma cell line was used for the generation of hybridoma cells and an antigen- and isotype-specific screening method was established. The system has been validated for globular protein antigens as well as for haptens and enables a fast and early stage selection and validation of monoclonal antibodies in one step.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Antibody-Producing Cells; Antigens; Biotinylation; Cell Fusion; Cell Line, Tumor; Epitopes; ErbB Receptors; Hemagglutinins; Humans; Hybridomas; Mice; Multiple Myeloma; Ovalbumin; Recombinant Fusion Proteins; Transfection

Ionizing radiation induces direct and indirect killing of cancer cells and for long has been considered as immunosuppressive. However, this concept has evolved over the past few years with the demonstration that irradiation can increase tumor immunogenicity and can actually favor the implementation of an immune response against tumor cells. Adoptive T-cell transfer (ACT) is also used to treat cancer and several studies have shown that the efficacy of this immunotherapy was enhanced when combined with radiation therapy. α-Radioimmunotherapy (α-RIT) is a type of internal radiotherapy which is currently under development to treat disseminated tumors. α-particles are indeed highly efficient to destroy small cluster of cancer cells with minimal impact on surrounding healthy tissues. We thus hypothesized that, in the setting of α-RIT, an immunotherapy like ACT, could benefit from the immune context induced by irradiation. Hence, we decided to further investigate the possibilities to promote an efficient and long-lasting anti-tumor response by combining α-RIT and ACT. To perform such study we set up a multiple myeloma murine model which express the tumor antigen CD138 and ovalbumine (OVA). Then we evaluated the therapeutic efficacy in the mice treated with α-RIT, using an anti-CD138 antibody coupled to bismuth-213, followed by an adoptive transfer of OVA-specific CD8+ T cells (OT-I CD8+ T cells). We observed a significant tumor growth control and an improved survival in the animals treated with the combined treatment. These results demonstrate the efficacy of combining α-RIT and ACT in the MM model we established.

Topics: Adoptive Transfer; Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Bismuth; CD8-Positive T-Lymphocytes; Cell- and Tissue-Based Therapy; Combined Modality Therapy; Disease Models, Animal; Female; Mice; Mice, Inbred C57BL; Multiple Myeloma; Ovalbumin; Radioimmunotherapy; Syndecan-1

IFN-tau, a type I IFN, is an antiviral, immunomodulating, and antiproliferative agent similar to IFN-alpha and IFN-beta, but IFN-tau lacks the toxicity associated with high concentrations of these IFNs in tissue culture and in animal studies. We have previously shown that IFN-tau inhibits antibody production in a murine model of an autoimmune disease.. We investigate the effectiveness of ovine IFN-tau and other type I IFNs in suppressing the development of allergic sensitization in a murine model of allergy by using ovalbumin (OVA) antigen as an allergen and in suppressing IgE production by using a human IgE-producing myeloma cell line.. Mice that were treated with IFN-tau in vivo before and after intraperitoneal immunization with aluminum hydroxide-precipitated OVA had significantly lower OVA-specific IgE levels than the PBS-treated group. IFN-tau-treated mice had reduced inflammatory cell infiltration into the lung tissue. Furthermore, in vitro IFN-tau treatment of splenocytes taken from OVA-immunized mice suppressed OVA-induced proliferation. Also, treatment of the IgE-producing human myeloma cell line U266BL with IFN-tau-reduced IgE production and inhibited cell proliferation compared with media controls. Similar suppression of proliferation and inhibition of IgE production was seen with other type I IFNs, as well as a humanized IFN-tau/IFN-alphaD chimeric that consists of residues 1 to 27 of the ovine IFN-tau and residues 28 to 166 of the human IFN-alphaD. The chimeric was not toxic to human peripheral white blood cells at concentrations as high as 10(5) U/mL, whereas human IFN-alphaD was toxic at 10(3) U/mL.. These data suggest that IFNs may be useful in preventing allergic sensitization by suppressing the production of allergen-specific IgE antibodies without toxic side effects.

Topics: Animals; Antibody Formation; Cattle; Cell Division; Disease Models, Animal; Humans; Immunoglobulin E; Interferon Type I; Interferon-alpha; Interleukin-4; Leukocytes, Mononuclear; Lung; Mice; Mice, Inbred BALB C; Multiple Myeloma; Ovalbumin; Pregnancy Proteins; Recombinant Fusion Proteins; Respiratory Hypersensitivity; Sheep; Spleen; Tumor Cells, Cultured

Six fluorescent antihuman Ig preparations were tested for their Ig class specificity by reacting them with highly purified IgG, IgM, IgA, and OVA coupled covalently to Sepharose beads. OVA was used as a measure for nonimmunologic binding. Bead fluorescence was determined by microfluorometry. The amounts of USS and NSS were expressed quantitatively. These data were compared with the performance of these particular conjugates in a biologic system, namely, monoclonal bone marrow cells. Five of the six conjugates satisfied the requirement of monospecific activity; one did not. At a dilution of 1 : 8, the five monospecific conjugates reacted between five and 50 times stronger with their appropriate antigens than with OVA-coupled beads. Cross reactivity with other Ig classes, after correction for OVA staining was maximally 6%. The conjugate that was nonspecific in the bone marrow system gave very high cross reactivity with the Ig-coupled beads. A good correlation was found between OVA bead staining and nonimmunologic binding of conjugates in bone marrow slides. In this respect, conjugates prepared from antibody preparations isolated by solid immunoadsorbents proved to be superior to globulin or whole IgG fractions. Ig coupled to Sepharose beads seems to represent a very promising substrate for conjugate specificity testing.

Topics: Adsorption; Animals; Bone Marrow; Bone Marrow Cells; Epitopes; Fluorescent Antibody Technique; Goats; Humans; Immunodiffusion; Immunoelectrophoresis; Immunoglobulin A; Immunoglobulin G; Immunoglobulin M; Multiple Myeloma; Ovalbumin; Polysaccharides; Rabbits; Sepharose; Sheep; Staining and Labeling; Swine; Waldenstrom Macroglobulinemia

Guinea pigs were immunized with liposomal model membranes actively sensitized with 2,4-dinitrophenyl-aminocaproylphosphatidyl-ethanolamine. The immune response was characterized by the formation of both IgM and IgG anti-DNP plaque-forming cells (PFC) which paralleled the appearance of anti-DNP antibodies in the serum. Plaque inhibition by DNP-lysine indicated that the PFC produced after liposomal immunization fell in a much narrower range of avidity groups than the PFC obtained after immunization with DNP-albumin and, in this regard, resembled MOPC 315. The restricted nature of the serum IgG anti-DNP antibodies was confirmed by isoelectric focusing and revealed the expression of a limited number of clones in all cases.

Topics: Animals; Antibody Formation; Antibody Specificity; Antibody-Producing Cells; Dinitrophenols; Erythrocytes; Female; Guinea Pigs; Hemagglutination Tests; Hemolytic Plaque Technique; Immunoglobulin G; Immunoglobulin M; Isoelectric Focusing; Liposomes; Lysine; Multiple Myeloma; Myeloma Proteins; Ovalbumin; Plasmacytoma; Serum Albumin; Sheep

MPC-11 myeloma cells synthesizing IgG were labeled in vitro with 3H-uridine and the cell lysates, or purified polysomes treated with rabbit antiserum against the purified MPC-11 protein. The specificity of the immune precipitation was tested by rabbit anti-egg albumin and by precipitation of polysomes from "non-producer" myeloma and HeLa cells. The specific precipitation was 10 to 15% of the total cytoplasmic RNA for the IgG-producing clone of MPC-11 and about 2% for the non-producer clone or HeLa cells. Up to 20% of the total polysomal and ribosomal RNA were precipitated from the IgG producing clone. This value correlated with the IgG synthesis of these cells which is also about 20% of the total protein produced. The results suggest that myeloma cells produce a relatively large quantity of IgG due to the presence of a large amount of specific m-RNA molecules.

Topics: Animals; Antibodies; Clone Cells; Immunoglobulin G; Mice; Multiple Myeloma; Ovalbumin; Polyribosomes; Precipitin Tests; RNA, Messenger; RNA, Neoplasm; RNA, Ribosomal

Topics: Animals; Antigen-Antibody Reactions; Antigens; Binding Sites; Blood Protein Electrophoresis; Cell Membrane; Complement System Proteins; Dinitrophenols; Erythrocytes; Female; gamma-Globulins; Guinea Pigs; Haptens; Hemagglutination Tests; Hemocyanins; Hemolysis; Histamine Release; Immunization; Immunoglobulins; Mast Cells; Mice; Multiple Myeloma; Neoplasm Proteins; Nitrobenzenes; Ovalbumin; Passive Cutaneous Anaphylaxis; Rabbits; Rats; Serum Albumin, Bovine; Sheep

The ability of passively administered antibody to suppress the immune response against homologous antigenic determinants while concomitantly enhancing the response against other unrelated determinants of the same antigen molecule has been established in two distinct antigen-antibody systems: (a) guinea pig gamma(2)-immunoglobulin + passive anti-F(ab')(2) antibody, where suppression of anti-F(ab')(2) antibody synthesis is accompanied by enhancement of the anti-Fc response; and (b) human secretory IgA + passive anti-serum IgA antibody, where suppression of antibody production against the alpha and L chains accompanies augmentation of the response to the secretory component. The mechanisms of the suppressive and enhancing effects are probably unrelated for the following reasons: (a) Enhancement of the response to certain determinants may be obtained without discernible suppression of the response to the homologous determinants; and (b) the F(ab')(2) fragments of passive antibody can mediate immune suppression but were not observed to enhance the response against the unrelated determinants of the same antigen molecule. Also, the timing for achieving maximum suppression or enhancement of antibody formation is not the same; enhancement was obtained only at a later time. Both the enhancement and suppressive effects were obtained with the purified gammaG fraction of antisera. This finding rules out an exclusive role of gammaM antibody in the enhancement phenomenon.

Topics: Animals; Antibodies; Antibody Formation; Antibody Specificity; Antigens; Chickens; Colostrum; Epitopes; Freund's Adjuvant; Guinea Pigs; Humans; Immune Sera; Immunity, Maternally-Acquired; Immunochemistry; Immunoglobulin A; Immunoglobulin G; Immunosuppression Therapy; Iodine Isotopes; Multiple Myeloma; Neoplasm Proteins; Ovalbumin; Precipitin Tests; Rabbits

Topics: Adsorption; Animals; Antibodies; Antibody Formation; Azo Compounds; Benzoates; Binding Sites; Cattle; Complement System Proteins; Haptens; Immune Sera; Immunochemistry; Immunoglobulin G; Iodine Isotopes; Isoelectric Focusing; Multiple Myeloma; Neoplasm Proteins; Ovalbumin; Polymers; Rabbits; Serum Albumin

Topics: Agammaglobulinemia; Animals; Freund's Adjuvant; gamma-Globulins; Immune Sera; Immunization; Immunoelectrophoresis; Injections, Intraperitoneal; Mice; Multiple Myeloma; Oils; Ovalbumin; Plasmacytoma; Research; Vaccination

Topics: Bence Jones Protein; Beta-Globulins; Carbohydrates; Chemical Phenomena; Chemistry; Chromatography; Electrophoresis; gamma-Globulins; Hemoglobins; Hot Temperature; Immunochemistry; Immunodiffusion; Immunoelectrophoresis; Multiple Myeloma; Muramidase; Ovalbumin; Pepsin A; Polymers; Ribonucleases; Serum Albumin; Serum Globulins; Transferrin; Trypsin