Use of phage display to isolate specific human monoclonal antibody fragments against a potential target for multiple myeloma

<p><b>INTRODUCTION</b>Multiple myeloma (MM), a malignancy of plasma cells, accounts for 10% of all haematological malignancies and is currently incurable. Although it can be treated, the disease tends to relapse after several years and becomes increasingly resistant to conventional therapy. Investigations into using humoral therapy for MM are now underway with a view that novel therapeutic agents may provide a more targeted therapy for MM.</p><p><b>MATERIALS AND METHODS</b>Here, phage display, a faster and more efficient method compared to classical hybridoma fusion technology, was used as a proof-of-concept to isolate several single-chain Fragment variables (scFv) against Ku86.</p><p><b>RESULTS</b>Anti-Ku86 polyclonal scFvs biopanning was successful where third round scFvs (A(450)~1.1) showed a 1/3 increase in binding as compared to the fi rst round scFvs (A(450)~0.4) with 100 microg/mL of antigen (purified human Ku86). Subsequent selection and verification of monoclonal antibodies using third round biopanning revealed 4 good affinity binding clones ranging from A(450)~0.1 to A450~0.15 on 12.5 microg/mL of antigen as compared to low binders (A(450)~0.07) and these antibodies bind to Ku86 in a specific and dose-dependent manner. Comparative studies were also performed with commercially available murine antibodies and results suggest that 2 of the clones may bind close to the following epitopes aa506-541 and aa1-374.</p><p><b>CONCLUSIONS</b>These studies using phage display provide an alternative and viable method to screen for antibodies quickly and results show that good affinity antibodies against Ku86 have been successfully isolated and they can be used for further studies on MM and form the basis for further development as anti-cancer therapeutic agents.</p>

Antitumor immune responses induced by idiotype-pulsed dendritic cells with cell-penetrating peptide vaccination in vivo / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To confirm the therapeutic effect of dendritic cell (DC) vaccine on treatment for mice with lymphoma and the protective effect of DC vaccine loaded with different antigens on the tumor-bearing BAL B/c mice.</p><p><b>METHODS</b>Firstly, a mouse tumor model was set up by s. c. inoculation of 1 x 10(6)/mouse A20 tumor cells. Then different DC vaccines were injected, respectively, and the tumor size and survival time were observed. Secondly, the immunized mice with DC vaccines were challenged with A20 tumor cells, and observed whether a new tumor occurred in the mice and the time of survival.</p><p><b>RESULTS</b>The tumor of mice immunized with Id-DC vaccines grew slower than the controls (mean time of survival was 40.4 days vs. 33.4 days), but statistically not significantly different. The tumor of mice injected with CPP-Id-DC vaccines grew slower than that injected with Id-DC vaccines and controls, and one of 5 mice got CR and the tumor in another one mouse became stable. The median survival time was 70.8 days during a 90-days observation period. The difference was significant (P<0.01). The mice injected with Id-DC vaccines were challenged with A20 tumor cells showed new tumor occurred at 7 - 12 days, and 1 of the 5 mice survived for 60 days. The mice injected with CPP-Id-DC vaccines had no tumor.</p><p><b>CONCLUSION</b>The DC loaded with CPP-Id was better than that loaded with Id alone in treating B cell lymphoma, and It can enhance their antitumor responses and prolong the survival time of the A20 tumor animal models. The vaccine of DC loaded with CPP-Id can protect mice from A20 tumor cell challenge.</p>

In vitro anti-tumor effect of CTL induced by HSP70-Id complex-modified dendritic cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To prepare the tumor antigen peptide complex (HSP70-1d) of HSP70 and idiotype (Id) from SmIg ScFv fragment in patients with Chronic B cell leukemia (B-CLL), and to study the anti-tumor effect of cytotoxic T lymphocyte (CTL) induced by HSP70-Id complex-modified dendritic cell (DC) in vitro and explore their immune mechanism.</p><p><b>METHODS</b>Purified HSP70 was combined into peptide complex (HSP70-Id) with the prepared Id-ScFv from B-CLL cells in vitro by using biochemical technique. The plastic-adherent monocytes from human peripheral blood were cultured and induced into DC with rhGM-CSF and rhIL-4 using cell culture and separation technique. The cultured DC were harvested and pulsed by HSP70-Id complex. DC morphology was observed under converted phase microscope and its phenotype was characterized by FCM on 8th day as well as their secreting cytokines were measured. Host lymphocytes were stimulated by DC loaded with HSP70-Id complex and co-cultured in the medium containing IL-2. The activation and proliferation of lymphocytes were examined by MTr test, which was also used to assay cytotoxicity of CTL elicited by modified DC to Daudi, K562 and HepG2 tumor cells, and FCM analyzed the changes of T lymphocyte subsets.</p><p><b>RESULTS</b>Mature DCs were obtained successfully, showing typical morphology and phenotypic properties, the expression ratio of cellular surface molecules, CD1a was 20% - 30%, CD83 was more than 72% , both CD86 and HLA-DR over-expressed obviously in the complex-loaded DC group secreting cytokines of Thl type, IL-12 and TNF-alpha. The culturing lymphocytes that were activated by modified DC could more effectively and specifically kill Daudi (71. 24%), but not K562 and HepG2 tumor cells. Results of FCM assay demonstrated that percentage of CD4+ and CD8+ T lymphocytes cocultured with complex-modified DC increased notably to 56.51% and 70.21%, respectively. CD4+ T/ CD8+ T proportion was changed from 1.49 to 0.81. The dose of peptide would be reduced to 1/50 if specific CTL induced by complex-modified DC instead of directly by peptide complex.</p><p><b>CONCLUSION</b>DCs modified by HSP70-Id complex exhibit powerful biological activities, and could induce CTL to specific cytotoxicity against carcinoma cells. It might be produced by cooperation of CD4+ T, CD8+ T lymphocytes and DC. The results also suggested that DC modified by HSP70-Id complex can present antigen and induce CTL with high efficacy and specificity.</p>

Construction and expression of a prokaryotic expression plasmid of idiotypic vaccine against B cell lymphoma: encoding the fusion genes of single-chain variable fragment and MCP-3 / 中国实验血液学杂志

The aim was to construct a prokaryotic expression plasmid encoding the fusion gene of single-chain variable fragment and monocyte chemotactic protein-3 (MCP-3). The cDNAs of immunoglobulin (Ig) VH and Ig VL were amplified by RT-PCR and assembled into the single-chain variable fragment (scFv) by recombinant PCR method. The cDNAs of Ig VH and Ig VL were connected by a (Gly4Ser)3 linker. Then, the fragments of scFv and MCP-3 were connected with a NDAQAPKS spacer, using recombinant PCR method again. The results indicated that the fusion gene of scFv-MCP-3 were constructed correctly and cloned into the prokaryotic expression plasmid successfully identified by sequencing and restriction endonucleases examination. Finally, the fusion protein was expressed in E coli DH5alpha under induction by arabinose. And the fusion protein was 65 kD and account for 30% of the total protein of the bacteria. In conclusion, a prokaryotic plasmid, encoding the fusion gene of single-chain variable fragment with MCP-3 and expressing idiotype protein vaccination against B cell lymphoma, was constructed correctly.

Exploration of immunoblot profiles of Neospora caninum probed with different bovine immunoglobulin classes

The present study was attempted to compare the Neospora caninum (N. caninum) antigenic bands recognized by different bovine immunoglobulin classes. A total 10, 5, 2, and 6 antigenic bands were exhibited on immunoblot profiles against bovine IgM, IgE, IgA, and IgG, respectively. A 46 kDa band was probed as a common antigenic band except IgA; 69 kDa band was bovine IgM and IgE; 33, 37, 55, and 79 kDa bands were bovine IgM and IgG; 72 kDa band was found IgM and IgA profiles. Based on the analysis, it appeared that different immunoglobulin classes recognizing different antigenic molecules were cooperating to cope with neosporosis.

Immuno-therapeutic study of anti-idiotype minibody (single chain Fv-CH3) on ovarian carcinoma bearing mice / 中国医学科学院学报

<p><b>OBJECTIVE</b>An anti-idiotypic minibody with optimal antigenicity which mimicking ovarian cancer antigen was used for therapeutic research in mice model bearing ovarian cancer.</p><p><b>METHODS</b>Using gene engineering technique, prokaryotic expression vector was constructed by genetic fusion of 6B11scFv to human IgG1 hinge and CH3 region. The fusion protein named minibody was induced with IPTG in E. coli and analyzed with Western blot and inhibition ELISA tests respectively. Twenty human-PBL-SCID mice bearing i.p. Skov3.ip1 cells were divided into two groups (10 per-group), 10 mice were immunized repeatedly by minibody every two weeks for three times. Indirect ELISA test was employed for analyzing the characterization of anti-anti-idiotypic scFv (Ab3). The latent period of ascites growth and the mean survival time were observed respectively. CD4+ and CD8+ T cells from the spleen of immunized mice were assayed by flow cytometry.</p><p><b>RESULTS</b>SDS-PAGE gel electrophoresis showed that a protein band with molecular weight of 50,000 appeared as the expected size after transformation and induction the host bacteria BL21 (DE3). The expressed minibody could be reacted with COC166-9 (Ab1 of 6B11) and binding goat anti-human IgG1 antibody in Western blot. Inhibition ELISA showed minibody had the capacity of binding ovarian cancer monoclonal antibody COC166-9 instead of primal antigen. Ab3 could be detected in the sera of immunized mice with minibody by ELISA test. Ab3 reached the highest at the 14th day after last vaccination and lasted for 6 weeks. The ratio of CD4+/CD8+ was the highest at the 13th day after last vaccination. The latent period of ascites growth were (37.7 +/- 5.5) days and (48.6 +/- 14.3) days (P = 0.04) respectively; while the mean survival time were (42.5 +/- 1.8) days and (59.4 +/- 16.8) days (P = 0.011) in the control and minibody group respectively.</p><p><b>CONCLUSIONS</b>These results demonstrate the successful construction and expression minibody with good immune activities of 6B11scFv and human IgG1 molecules function. Antigenicity is increased without adjuvants and partial humanization is realized. Minibody can induce humoral anti-idiotypic immunity responses against ovarian carcinoma in vivo. When ascites formation was delayed or prevented and the survival was prolonged in minibody group. We expect that minibody may be used as tumor vaccine to ovarian carcinoma in the future clinical trails.</p>

Idiotipos y antiidiotipos: estructura y función en autoinmunidad / Anti-idiotypes and idiotypes: structure and autoimmunity function

Se presenta una revisión sobre la estructura de las inmunoglobulinas y los idiotipos que constituyen las regiones hipervariables que unen al antígeno; según la teoría de jerne, un idiotipo genera otro anticuerpo (antiidiotipo) que reconoce los epitopos de las regiones hipervariables, creando una red que en teoría regula la síntesis de anticuerpos. Funcionalmente existen dos clases: 1) Los idiotipos que constituyen imágenes internas del sitio de unión con el antígeno y 2) Los que reconocen sitios diferentes. Cuando los idiotipos son compartidos por individuos de una misma especie se denominan públicos y son generados vía línea germinal, y los que están presentes en un solo individuo corresponden a idiotipos privados y son producto de mutación somática. En autoinmunidad los idiotipos identifican autoanticuerpos patogénicos órgano-específicos como en pénfigo o penfigoide; éstos son capaces de inducir directamente lesión tisular, que es reproducible en animales. Los órgano-inespecíficos como los autoanticuerpos antinucleares de lupus, generalmente no inducen lesión directa. Finalmente, se revisan las evidencias y modelos experimentales que sugieren que un idiotipo autoinmune humano inyectado en un animal genera un antiidiotipo, que simula al antígeno, este genera un tercer anticuerpo idéntico al autoanticuerpo humano y los animales desarrollan síntomas similares a los observados en padecimientos autoinmunes como lupus

Anticorpo sérico e antigliadina no diagnóstico e seguimento da doença celíaca / Serum antigliadin antibodies in the diagnosis and follow-up of celiac disease

Foram estudadas prospectivamente, no período de 1985 a 1990, 236 crianças com suspeita de má-absorçäo, encaminahdas para biopsia de intestino delgado e prova de absorçäo do D-Xilose. Foram colhidas uma ou mais amostras de sangue para dosagem de anticorpo antigliadina. O objetivo do trabalho foi a avaliaçäo do comportamento dos anticorpos antigliadina classes IgG e IgA no diagnóstico diferencial entre doença celíaca e outras causas de má-absorçäo e no acompanhamento de pacientes celíacos, nas diferentes fases do preenchimento dos critérios diagnósticos. Foram estudadas 20 crianças com doença celíaca confirmada por três biopsias de intestino delgado; 12 crianças com suspeita de doença celíaca com duas biopsias intestinais, antes e um ano após retirada do glúten; 106 crianças com enteropatia ambiental; 45 crianças com diarréia protraída e 56 crianças com baixa estatura. Os anticorpos foram úteis no diagnóstico diferencial entre doença celíaca e outras enteropatias, sendo o da classe IgG mais sensível e o da classe IgA mais específico no diagnóstico. No seguimento dos pacientes celíacos os títulos de anticorpo foram significantemente mais altos na vigência de glúten na dieta do que na fase de restriçäo, demonstrando ser bom indicador da presença de lesäo intestinal nesses pacientes

Some aspects of idiopatyc regulation in Chagas disease and human schistosomiasis

We developed an approach for the examination of antiidiotypic cell-mediated reactivity during chronic human infections. Antibodies against Schistosoma mansoni egg antigens (anti-SEA) and against Trypanosoma cruzi epimastigotes (anti-EPI) prepared from pooled and individual sera from patients with either schistosomiasis mansoni or Chagas disease were immunoaffinity purified on appropriate columns and used to stimulate patients' T cells. These antibodies preparations and their F (ab)2 fragments stimulated in a dose-dependent assay the proliferations of peripheral blood mononuclear cells (PBMC) and T lymphocytes from some, but not all actively infected individuals. Anti-idiotypic T cells can recognize and respond to anti-SEA or anti-EPI idiotypes directly. We contend that the most likely explanation of this stimulations is that the idiotype expressed on these anti-SEA or anti-EPI antibodies are acting as immunogens to stimulate antiidiotypic T lymphocytes that develop during the course of the infection. Immunization of rabbits with these antibodies (anti-SEA or anti-EPI) preparations, followed by absorption of the rabbit antisera on absorbants of normal Ig, produced specific anti-id reagents. Use of these reagents in competitive ELISA distinguished the idiotypic expression on anti-SEA or anti-EPI preparations obtained from patients' sera with different clinical forms of schistosomiasis or Chagas disease, respectively. The anti-SEA Id-reactive T cells from S. mansoni infected patients were capable of regulating autologous in vitro granuloma formation.

Development of anti-idiotypic antibodies to HLA antigens during pregnancy.

Thirty one pregnant women whose sera were initially positive for anti HLA antibodies were retested for lymphocytotoxicity 7-9 months after delivery. In the second testing 10 women were found to have lost the cytotoxic antibodies. These sera were tested for presence of anti-idiotypic antibodies by complement dependent cytotoxicity (CDC). In all ten sera tested, CDC blocking antibody was detected by inhibition technique at different dilutions. These antibodies directed specifically against the HLA antigen of the spouse. This CDC blocking factor could be the anti-idiotypic antibody (Ab2) directed against the cytotoxic anti-HLA antibody (Ab1). In one woman who conceived a second time, repeat sample obtained at the 7th month of the second pregnancy showed reappearance of the specific anti-HLA antibody. This may suggest that the Ab1-Ab2 network is under dynamic regulation.

Immunoregulation by processed immunoglobulin on B-cells.

According to Jerne's network hypothesis, the unique amino acid sequences of Ig variable regions, that is, the idiotypic determinants can function in immunoregulatory mechanisms and cellular interactions. Indeed, Id-specific T-cells (mostly CD4+) have since been described, but the nature of Id-positive Ig on B-cells involved in recruiting T-cells is unclear. Studies from our ongoing investigation presented here clearly show that Id can evoke both CD4+ and CD8+ T cells, and exist not only as the integral components of a bona fide antigen-binding receptor Ig but also as distinct molecular entities in processed forms on the cell surface of B-lymphocytes. Using a B-cell hybridoma, 2C3, that expresses anti-hapten (phthalate) antibody receptors on the cell surface, we induced both Id-specific CD4+ and CD8+ T effector cells. The CD4+ T cells were suppressive and mediated generation of Id-loss 2C3 variants, whereas CD8+ T cells were highly cytotoxic and selectively eliminated 2C3 cells both in vitro and in vivo. These effector cells could be induced by cell membrane-associated Ig but not by its soluble form, secreted by 2C3 cells. Antibodies to MHC class I but not class II molecules were inhibitory to this induction. Furthermore, brefeldin A (BFA), an inhibitor of MHC class I mediated processing, blocked induction of CTL but had no effect on the expression of membrane Ig. Moreover, chloroquine, an inhibitor of class II-mediated processing, had no effect. A few reports have recently appeared indicating that an exogenous Ig can be processed by B-cells in the context of MHC class II proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for a relationship between the expression of dominant idiotypes in the offspring's of schistosoma mansoni infected rats and the immunopathology of the disease

In Schistosomiasis, there is increasing evidence, in both human and murine models, that there is association between the idiotypic regultion and the immunopathology of the disease. The presence of dominant shared idiotypes across species had been described by both our laboratory and others. It is not clear, if this dominant idiotypes is a consequence of the immunopathology of egg deposition, or the presence of such dominant idiotypes direct the immunopathology, and consequently the morbidity of the disease. In this study, the presence of dominant idiotypes in the offsprings of S. mansoni-infected rats and their effects on the morbidity in subsequent infections have been evaluated. In the offsprings, the idiotypes levels were found to be in three levels; high, intermediate and low levels in comparison to the maternal and control level. All the groups were infected with 100 cercaria. The infection were confirmed by the presence of ova in stool examination after 8 weeks of infections. After 11 weeks of infections, rats stopped passing ova in their stools. Four weeks later, the rats were sacrificed and examined for pathology. Our data indicated that rat offsprings with high level of idiotypes showed maximum granulomatous reaction and egg deposition, while rat offsprings with low level of idiotypes showed minimum granulomatous reaction and egg deposition. Significance of our studies in the predicion of the prognosis of human schistosoma infection will be discussed. Supported by USAID/MOH Egypt, 263-0140.2 Project, SRP grant No 01-03-34

Human schistosomiasis mansoni: studies on in vitro granuloma modulation

Infection with Schistosoma mansoni induces humoral and T cell mediated responses and leads to delayed hipersensitivity that results in granulomatous inflamatory disease around the parasite eggs. Regulation of these responses resulting in a reduction in this anti-egg inflamatory disease is appsrently determined by idiotypic repertoires of the patient, associated with genetic background and multiple external factors. We have previously reported on idiotype/anti-idiotype-receptor transactions in clinical human schistosomiasis. These findings support a hypothesis that anti-SEA cross-reactive idiotypes develop in some patients during the course of a chronic infection and participate in regulation of anti-SEA cellular immune responses. We repport here on experiments wich extend those observations to the regulation of granulomatous hypersensitivity measured by an in vitro granuloma model. T cells from chronic intestinal schistosomiasis patients were stimulated in vitro with anti-SEA idiotypes and assayed in an autologous in vitro granuloma assay for modulation of granuloma formation. These anti-SEA idiotype reactive T cells were capable of regulating autologous in vitro granuloma formation. This regulatory activity, initiated with stimulatory anti-SEA idiotypic antibodies, was antigenically specific and was dependent on the present of intact (F(ab')2 immunoglobulin molecules. The ability to elicit this regulatory activity appears to be dose dependent and is more easily demonstrated in chronically infected intestinal patients or SEA sensitized individuals. These data support the hypothesis that anti-SEA cross reactive idiotypes are important in regulating granulomatous hypersensitivy in chronic intestinal schistosomiasis patients and these cross-reactive idiotypes appear to play a major role in cell-cell interactions which result in the regulation of anti-SEA cellular immune responses

Inducción experimental de factores reumatoides de baja afinidad y reactividad cruzada con nucleoproteinas no histonas mediante anti-idiotipos dirigidos a factores reumatoides de alta afinidad y reactividad cruzada con histonas H1 y H2b / Experimental induction of low affinity and crossed reaction of rheumatoid factors with non-histone nucleoproteins by means of anti-idiopathic substances directed at affinity and crossed reactivity towards H1 and H2b histonic rheumatoid factors

El análisis de la reactividad cruzada de factores reumatoides contra antígenos nucleares ha demostrado diferencias entre pacientes con artritis reumatoide y sujetos sanos seropositivos. Este trabajo demuestra que en artritis reumatoide, pero no en sujetos sanos, existe una subpoblación de moléculas que amén de compartir un idotipo público, muestran alta afinidad por IgG y reactividad cruzada contra histonas. Se demostró además que, en un modelo experimental, los factores reumatoides con las características mencionadas pueden inducir factores heterogéneos, con baja afinidad por IgG y reactividad contra nucleoproteinas no-histonas, muy probablemente a través de circuitos anti-idiotípicos. El mecanismo podría explicar la diversificación serológica que existe en la artritis reumatoide

La red de idiotipos-antiidiotipos: I. su relación con la autoinmunidad / The idiotype-antiidiotype network: I. Its association with autoimmunity

Las inmunoglobulinas presentan determinantes antigénicos en su región variable que estimulan la producción de anticuerpos específicos con lo cual se establece una red de estimulaciones y supresiones sucesivas, que es funcionalmente cerrada y con predominio de las interacciones supresoras. Sobre la base de recientes investigaciones se ha planteado la hipótesis de que la red inmunológica no se establece por generación al azar de los paratopes, sino de modo determinista a través del código de reconocimiento molecular. La red inmunològica tiene carácter regulador, se describen redes estimuladoras de la respuesta inmune y redes supresoras. Especial interés cobra la presencia de idiotipos comunes en los autoanticuerpos presentes en diversas enfermedades autoinmunes para la comprensión de su patogenia