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1.
Semin Immunopathol ; 38(1): 57-74, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26597100

RESUMO

Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are two severe autoimmune bullous diseases of the mucosae and/or skin associated with autoantibodies directed against desmoglein (Dsg) 3 and/or Dsg1. These two desmosomal cadherins, typifying stratified epithelia, are components of cell adhesion complexes called desmosomes and represent extra-desmosomal adhesion receptors. We herein review the advances in our understanding of the immune response underlying pemphigus, including human leucocyte antigen (HLA) class II-associated genetic susceptibility, characteristics of pathogenic anti-Dsg antibodies, antigenic mapping studies as well as findings about Dsg-specific B and T cells. The pathogenicity of anti-Dsg autoantibodies has been convincingly demonstrated. Disease activity and clinical phenotype correlate with anti-Dsg antibody titers and profile while passive transfer of anti-Dsg IgG from pemphigus patients' results in pemphigus-like lesions in neonatal and adult mice. Finally, adoptive transfer of splenocytes from Dsg3-knockout mice immunized with murine Dsg3 into immunodeficient mice phenotypically recapitulates PV. Although the exact pathogenic mechanisms leading to blister formation have not been fully elucidated, intracellular signaling following antibody binding has been found to be necessary for inducing cell-cell dissociation, at least for PV. These new insights not only highlight the key role of Dsgs in maintenance of tissue homeostasis but are expected to progressively change pemphigus management, paving the way for novel targeted immunologic and pharmacologic therapies.


Assuntos
Pênfigo/diagnóstico , Pênfigo/etiologia , Animais , Anticorpos Monoclonais/imunologia , Autoanticorpos/genética , Autoanticorpos/imunologia , Autoantígenos/imunologia , Desmogleínas/imunologia , Progressão da Doença , Epitopos/imunologia , Predisposição Genética para Doença , Humanos , Soros Imunes/imunologia , Idiótipos de Imunoglobulinas/genética , Idiótipos de Imunoglobulinas/imunologia , Mutação , Especificidade de Órgãos/imunologia , Pênfigo/epidemiologia , Pênfigo/terapia , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Pesquisa Translacional Biomédica
2.
Cancer Immunol Immunother ; 64(8): 1021-32, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25982371

RESUMO

We report on the safety and immunogenicity of idiotypic DNA vaccination in a phase I, non-randomised, open-label study in patients with multiple myeloma. The study used DNA fusion gene vaccines encoding patient-specific single chain variable fragment, or idiotype (Id), linked to fragment C (FrC) of tetanus toxin. Patients in complete or partial response following high-dose chemotherapy and autologous stem cell transplant were vaccinated intramuscularly with 1 mg DNA on six occasions, beginning at least 6 months post-transplant; follow-up was to week 52. Fourteen patients were enrolled on study and completed vaccinations. Idiotypic DNA vaccines were well tolerated with vaccine-related adverse events limited to low-grade constitutional symptoms. FrC- and Id-specific T-cell responses were detected by ex vivo ELISPOT in 9/14 and 3/14 patients, respectively. A boost of pre-existing anti-FrC antibody (Ab) was detected by ELISA in 8/14 patients, whilst anti-Id Ab was generated in 1/13 patients. Overall, four patients (29 %) made an immune response to FrC and Id, with six patients (43 %) responding to FrC alone. Over the 52-week study period, serum paraprotein was undetectable, decreased or remained stable for ten patients (71 %), whilst ongoing CR/PR was maintained for 11 patients (79 %). The median time to progression was 38.0 months for 13/14 patients. Overall survival was 64 % after a median follow-up of 85.6 months.


Assuntos
Vacinas Anticâncer/uso terapêutico , Mieloma Múltiplo/terapia , Linfócitos T/imunologia , Vacinas de DNA/uso terapêutico , Adulto , Idoso , Feminino , Seguimentos , Humanos , Imunidade Humoral , Idiótipos de Imunoglobulinas/genética , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/mortalidade , Estadiamento de Neoplasias , Fragmentos de Peptídeos/genética , Proteínas Recombinantes de Fusão/genética , Análise de Sobrevida , Toxina Tetânica/genética
3.
J Immunol ; 193(6): 2691-8, 2014 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-25127856

RESUMO

Systemic lupus erythematosus (SLE) is marked by a Th cell-dependent B cell hyperresponsiveness, with frequent germinal center reactions and hypergammaglobulinemia. The specificity of Th cells in lupus remains unclear, but B cell Ids have been suggested. A hallmark is the presence of anti-dsDNA, mutated IgG autoantibodies with a preponderance of arginines in CDR3 of the Ig variable H chain (IgVH). B cells can present V region-derived Id peptides on their MHC class II molecules to Id-specific Th cells. We show that Id-specific Th cells support the proliferation of anti-dsDNA Id(+) B cells in mice suffering from systemic autoimmune disease with SLE-like features. Mice developed marked clonal expansions of B cells; half of the IgVH sequences were clonally related. Anti-dsDNA B cells made up 40% of B cells in end-stage disease. The B cells expressed mutated IgVH with multiple arginines in CDR3. Hence, Id-driven T cell-B cell collaboration supported the production of classical anti-dsDNA Abs, recapitulating the characteristics of such Abs in SLE. The results support the concept that Id-specific Th cells may trigger the development of SLE and suggest that manipulation of the Id-specific T cell repertoire could play a role in treatment.


Assuntos
Anticorpos Antinucleares/imunologia , Seleção Clonal Mediada por Antígeno/imunologia , DNA/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Anticorpos Antinucleares/biossíntese , Arginina/química , Linfócitos B/imunologia , Sequência de Bases , Hipergamaglobulinemia/imunologia , Imunoglobulina G/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Idiótipos de Imunoglobulinas/genética , Idiótipos de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Análise de Sequência de DNA
4.
Methods Mol Biol ; 1139: 289-303, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24619688

RESUMO

Available therapies for lymphoplasmacytic lymphoma (LPL) provide no survival advantage if started before signs or symptoms of end-organ damage develop; hence, current recommendations are to follow a program of observation while patients are in the asymptomatic phase of disease. We hypothesize that using idiotypic determinants of a B-cell lymphoma's surface immunoglobulin as a tumor-specific marker, we can develop patient-specific chemokine-idiotype fusion DNA vaccines that induce an immune response against LPL. By activating the host immune system against the tumor antigen, we postulate that disease control of asymptomatic phase lymphoplasmacytic lymphoma can be maintained. These chemokine-idiotype fusion DNA vaccines provide protection in a lymphoma mouse model and have recently entered clinical trials. Herein, we describe procedures for the generation of therapeutic vaccines, particularly "second-generation" recombinant vaccines. Specifically, in the Methods section we describe how to identify lymphoma-associated immunoglobulin V (IgV) genes from patient biopsy and how to assemble these genes as single-chain variable gene fragment (scFv) in-frame with MIP-3α to generate novel DNA fusion vaccines.


Assuntos
Vacinas Anticâncer/genética , Idiótipos de Imunoglobulinas/genética , Linfoma/imunologia , Linfoma/patologia , Medicina de Precisão/métodos , Anticorpos de Cadeia Única/genética , Vacinas de DNA/genética , Vacinas de DNA/imunologia , Sequência de Aminoácidos , Vacinas Anticâncer/imunologia , Células Clonais/metabolismo , Clonagem Molecular , DNA Complementar/genética , Humanos , Idiótipos de Imunoglobulinas/química , Idiótipos de Imunoglobulinas/imunologia , Dados de Sequência Molecular , Plasmídeos/genética , Análise de Sequência de DNA , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/imunologia
5.
Methods Mol Biol ; 1139: 367-87, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24619694

RESUMO

Non-Hodgkin's lymphoma (NHL) is the most common hematological malignancy both in Europe and in the United States. Follicular lymphoma (FL), a tumor comprised of mature B cells, represents one fourth of all NHL and, despite good response rates to standard treatments, tends to frequently relapse to such an extent that it is still considered incurable. Among several alternative therapeutic options actively being pursued, immunotherapy by idiotypic vaccination is in the forefront of clinical experimental medicine. The idiotype vaccine consists of the tumor-specific immunoglobulin conjugated with keyhole limpet hemocyanin (KLH) and administered together with an adjuvant. Over the last 20 years, researchers have proven that this vaccine can induce specific immune responses. Too, those patients with such responses experience a disease-free survival longer than normally achievable, although these latter results require further confirmation in large clinical trials. Traditionally, idiotype vaccines have been produced through hybridoma technology. In this chapter this technology is described.


Assuntos
Vacinas Anticâncer/biossíntese , Hibridomas/metabolismo , Idiótipos de Imunoglobulinas/biossíntese , Animais , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/metabolismo , Fusão Celular , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Criopreservação , Meios de Cultivo Condicionados , Eletroforese , Hemocianinas/metabolismo , Humanos , Hibridomas/citologia , Idiótipos de Imunoglobulinas/genética , Idiótipos de Imunoglobulinas/imunologia , Idiótipos de Imunoglobulinas/metabolismo , Linfonodos/patologia , Camundongos , Análise de Sequência
6.
Exp Oncol ; 35(1): 8-14, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23528309

RESUMO

AIM: Idiotype, the unique part of immunoglobulin molecule expressed on the surface of B-cells, represents a specific antigen for vaccination against lymphoma. We have developed a rapid method for immunoglobulin variable fragments cloning, assembling and expression of recombinant idiotype protein in Escherichia coli. METHODS: PCR with specially designed panel of primers was used for direct amplification of variable regions of tumor immunoglobulin. Overlapping extension PCR, restriction and ligation was applied for assembling and cloning of vaccine construction. Idiotype protein was purified by metal-chelate chromatography. RESULTS: Methods of idiotype cloning from lymphoma cells and production of recombinant protein were developed and optimized. Several samples of idiotypic proteins originating from B-cell lines and lymphoma patients were produced. CONCLUSION: The proposed method of vaccine production is relatively cheap, not very laborious and requires as long as 6-7 week to perform. The expressed protein was soluble, did not accumulate in inclusion bodies and harvested at sufficient for vaccination quantity and concentration.


Assuntos
Anticorpos Antineoplásicos/genética , Vacinas Anticâncer , Idiótipos de Imunoglobulinas/genética , Linfoma de Células B/imunologia , Anticorpos de Cadeia Única/genética , Anticorpos Antineoplásicos/imunologia , Linfócitos B/imunologia , Sequência de Bases , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Clonagem Molecular , Escherichia coli/genética , Amplificação de Genes , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Idiótipos de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/imunologia , Imunoglobulina M/genética , Imunoglobulina M/imunologia , Cadeias kappa de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/imunologia , Anticorpos de Cadeia Única/imunologia
7.
Int Immunol ; 25(6): 345-52, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23382353

RESUMO

A methodology based on the representation of each amino acid of a protein sequence by the electron-ion interaction potential and subsequent analysis by signal processing was used to determine the characteristic or common frequency (in Hz) that reflects the biological activity shared among phosphocholine (PC)-binding antibodies. The common frequency for the variable portion of the heavy chain (VH) of the PC-specific antibodies is found to be at f = 0.37 Hz. The VH sequences of the PC-binding antibodies exhibit three subsites for the PC moiety where hypervariable region 2 (CDR2) plays a role in the interaction with the phosphate group. Mutations in this VH region have an impact on the ability of mutant variants to bind PC and its carrier molecule, as well as on the characteristic frequency shift toward f = 0.12 Hz for mutants failing to bind both hapten and carrier. The VH sequence of mutants that retain the ability to bind PC still shows f = 0.37 Hz, suggesting that this frequency determines PC binding. However, this statement was not confirmed as mutation in another PC subsite impairs PC binding but retains both the phosphate-group recognition and the frequency at f = 0.37 Hz. Herein, this finding is discussed to promote the idea that the VH sequence of the PC-binding antibodies encodes the subsite for phosphate-group binding as a dominant functional activity and that only CDR2 of the T15-idiotype antibodies together with FR3 region form an autonomous self-association function represented by the T15VH50-73 peptide with f = 0.37±0.05 Hz. Thus, these data confirmed that T15VH50-73 peptide might be used in superantibody technology.


Assuntos
Anticorpos/imunologia , Idiótipos de Imunoglobulinas/química , Idiótipos de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/imunologia , Fosforilcolina/imunologia , Animais , Sítios de Ligação/imunologia , Idiótipos de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Camundongos
8.
Int Arch Allergy Immunol ; 161(2): 122-6, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23343692

RESUMO

BACKGROUND: The mechanisms driving the development of immunoglobulin E (IgE) antibody repertoires are a matter of debate. Alternatives to the classical view on antibody development, involving somatic mutation and antigen-driven selection of high-affinity variants in germinal centers, have been proposed. METHODS: We have re-analyzed the pattern of mutations in previously isolated and characterized human clonally unrelated IgE-encoding transcripts using the validated focused binomial methodology to find evidence in such genes of antigen-specific selection. RESULTS: As expected there is a selection against replacement mutations in IgE framework regions. In contrast, in all examined cases but one (assessing IgE repertoires of parasite-infected individuals) there was no evidence in favor of either positive or negative selection in complementarity determining regions. Importantly, however, the validated method also failed to detect selection for replacement mutations in two, non-IgE, hypermutated antibody populations targeting tetanus toxoid and vaccinia virus, respectively. CONCLUSIONS: Current methodology is unable to define with certainty, using commonly assessed IgE repertoire sizes, whether antigen selection is or is not a major driving force in the establishment of human IgE. New approaches are needed to address this matter.


Assuntos
Imunoglobulina E/genética , Imunoglobulina E/imunologia , Mucosa Nasal/imunologia , Rinite Alérgica Perene/imunologia , Códon , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Epitopos/genética , Epitopos/imunologia , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Idiótipos de Imunoglobulinas/genética , Idiótipos de Imunoglobulinas/imunologia , Leucócitos Mononucleares/imunologia , Modelos Estatísticos , Mutação , Rinite Alérgica , Rinite Alérgica Perene/genética
9.
J Neurosci ; 32(41): 14402-14, 2012 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-23055510

RESUMO

Bipolar, amacrine, and retinal ganglion cells elaborate arbors and form synapses within the inner plexiform layer (IPL) of the vertebrate retina. Specific subsets of these neuronal types synapse in one or a few of the ≥10 sublaminae of the IPL. Four closely related Ig superfamily transmembrane adhesion molecules--Sidekick1 (Sdk1), Sdk2, Dscam, and DscamL--are expressed by non-overlapping subsets of chick retinal neurons and promote their lamina-specific arborization (Yamagata and Sanes, 2008). Here, we asked whether contactins (Cntns), six homologs of Sdks and Dscams, are expressed by and play roles in other subsets. In situ hybridization showed that cntn1-5 were differentially expressed by subsets of amacrine cells. Immunohistochemistry showed that each Cntn protein was concentrated in a subset of IPL sublaminae. To assess roles of Cntns in retinal development, we focused on Cntn2. Depletion of Cntn2 by RNA interference markedly reduced the ability of Cntn2-positive cells to restrict their arbors to appropriate sublaminae. Conversely, ectopic expression of cntn2 redirected neurites of transduced neurons to the Cntn2-positive sublaminae. Thus, both loss- and gain-of-function strategies implicate Cntn2 in lamina-specific neurite targeting. Studies in heterologous cells showed that Cntn2 mediates homophilic adhesion, but does not bind detectably to Sdks, Dscams, or other Cntns. Overexpression analysis showed that Cntns1 and 3 can also redirect neurites to appropriate sublaminae. We propose that Cntns, Sdks, and Dscams comprise an Ig superfamily code that uses homophilic interactions to promote lamina-specific targeting of retinal dendrites in IPL.


Assuntos
Contactina 2/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Idiótipos de Imunoglobulinas/genética , Retina/embriologia , Retina/metabolismo , Animais , Membrana Basal/metabolismo , Galinhas , Contactina 2/biossíntese , Contactina 2/genética , Contactinas/biossíntese , Contactinas/fisiologia , Feminino , Células HEK293 , Humanos , Idiótipos de Imunoglobulinas/biossíntese , Células K562 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout
10.
J Autoimmun ; 39(4): 466-70, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22871259

RESUMO

Long-lived secreted autoantibody responses in systemic autoimmunity are generally regarded to be polyclonal and to express a diverse B-cell repertoire. Here, we have used a proteomic approach based on de novo sequencing to determine the clonality and V region structures of human autoantibodies directed against a prototypic systemic autoantigen, Ro52 (TRIM21). Remarkably, anti-Ro52 autoantibodies from patients with primary Sjögren's syndrome, systemic lupus erythematosus, systemic sclerosis or polymyositis were restricted to two IgG1 kappa clonotypes that migrated as a single species on isoelectric focusing; shared a common light chain paired with one of two closely-related heavy chains; and were public in unrelated patients. Targeted mass spectrometry using these uniquely mutated V region peptides as surrogates detected anti-Ro52 autoantibodies in human sera with high sensitivity and specificity compared with traditional ELISA. Mass spectrometry-based detection of specific autoantibody motifs provides a powerful new tool for analysis of humoral autoimmunity.


Assuntos
Autoanticorpos/imunologia , Imunoglobulina G/imunologia , Idiótipos de Imunoglobulinas/imunologia , Cadeias kappa de Imunoglobulina/imunologia , Proteoma/imunologia , Ribonucleoproteínas/imunologia , Síndrome de Sjogren/imunologia , Adulto , Idoso , Autoanticorpos/sangue , Autoanticorpos/genética , Autoimunidade/genética , Estudos de Casos e Controles , Feminino , Expressão Gênica/imunologia , Humanos , Imunidade Humoral/genética , Imunoglobulina G/sangue , Imunoglobulina G/genética , Idiótipos de Imunoglobulinas/sangue , Idiótipos de Imunoglobulinas/genética , Cadeias kappa de Imunoglobulina/sangue , Cadeias kappa de Imunoglobulina/genética , Focalização Isoelétrica , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Peptídeos/imunologia , Proteoma/genética , Ribonucleoproteínas/genética , Sensibilidade e Especificidade , Síndrome de Sjogren/sangue , Síndrome de Sjogren/diagnóstico , Síndrome de Sjogren/genética
11.
Protein Expr Purif ; 75(1): 15-20, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20851769

RESUMO

The unique immunoglobulin idiotype expressed on the surface of B lymphoma cells can be used as an effective antigen in tumor-specific vaccines when fused to immunostimulatory proteins and cytokines. A DNA vaccine encoding for an idiotype antibody single chain Fv (scFv) fragment fused to the Tetanus Toxin Fragment C (TTFrC) has been shown to induce protective anti-tumor responses. Protein-based strategies may be more desirable since they provide greater control over dosage, duration of exposure, and in vivo distribution of the vaccine. However, production of fusion protein vaccines containing complex disulfide bonded idiotype antibodies and antibody-derived fragments is challenging. We use an Escherichia coli-based cell-free protein synthesis platform as well as high-level expression of E. coli inclusion bodies followed by refolding for the rapid generation of an antibody fragment - TTFrC fusion protein vaccine. Vaccine proteins produced using both methods were shown to elicit anti-tumor humoral responses as well as protect from tumor challenge in an established B cell lymphoma mouse model. The development of technologies for the rapid production of effective patient-specific tumor idiotype-based fusion protein vaccines provides opportunities for clinical application.


Assuntos
Vacinas Anticâncer/genética , Escherichia coli/genética , Idiótipos de Imunoglobulinas/genética , Linfoma de Células B/prevenção & controle , Fragmentos de Peptídeos/genética , Toxina Tetânica/genética , Vacinas de DNA/genética , Animais , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/isolamento & purificação , Vacinas Anticâncer/uso terapêutico , Feminino , Humanos , Imunização , Idiótipos de Imunoglobulinas/imunologia , Idiótipos de Imunoglobulinas/isolamento & purificação , Idiótipos de Imunoglobulinas/uso terapêutico , Linfoma de Células B/imunologia , Camundongos , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/uso terapêutico , Dobramento de Proteína , Toxina Tetânica/imunologia , Toxina Tetânica/isolamento & purificação , Toxina Tetânica/uso terapêutico , Vacinas de DNA/imunologia , Vacinas de DNA/isolamento & purificação , Vacinas de DNA/uso terapêutico
12.
Blood ; 116(10): 1734-6, 2010 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-20522710

RESUMO

Active immunization with the idiotype of follicular lymphoma induces tumor-specific immunity. T cells induced in vivo by idiotype vaccination recognize human leukocyte antigen (HLA)--restricted hypervariable but not conserved idiotype peptides. We hypothesized that idiotype-directed T-cell immunity occurs naturally and performed a reverse immunology analysis of idiotype HLA binding in 39 follicular lymphoma patients. For every idiotype, the sum of HLA-A or -B binding scores of the 20 highest-scoring peptides was calculated for all 39 HLA types through the BIMAS algorithm. The idiotype sum score of every patient's lymphoma was compared on the respective patient's HLA type to the mean of the sum scores of the remaining 38 idiotypes. Autologous idiotypes had lower immunogenicity than allogeneic idiotypes. Differential immunogenicity resided predominantly in all 3 complementarity-determining regions rather than in framework peptides. Idiotype immunogenicity was not changed by somatic hypermutation. These findings indicate T cell-mediated immunosurveillance of follicular lymphoma directed specifically against individual idiotype epitopes.


Assuntos
Antígenos HLA/imunologia , Idiótipos de Imunoglobulinas/imunologia , Linfoma Folicular/imunologia , Linfócitos T/imunologia , Adulto , Idoso , Algoritmos , Alelos , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Feminino , Antígenos HLA/genética , Antígenos HLA/metabolismo , Antígenos HLA-A/genética , Antígenos HLA-A/imunologia , Antígenos HLA-A/metabolismo , Antígenos HLA-B/genética , Antígenos HLA-B/imunologia , Antígenos HLA-B/metabolismo , Humanos , Idiótipos de Imunoglobulinas/genética , Idiótipos de Imunoglobulinas/metabolismo , Linfoma Folicular/genética , Linfoma Folicular/metabolismo , Masculino , Pessoa de Meia-Idade , Monitorização Imunológica/estatística & dados numéricos , Ligação Proteica , Linfócitos T/citologia , Linfócitos T/metabolismo
13.
Ann Oncol ; 21(12): 2420-2427, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20494963

RESUMO

BACKGROUND: Animal and clinical studies with plant-produced single-chain variable fragment lymphoma vaccines have demonstrated specific immunogenicity and safety. However, the expression levels of such fragments were highly variable and required complex engineering of the linkers. Moreover, the downstream processing could not be built around standard methods like protein A affinity capture. DESIGN: We report a novel vaccine manufacturing process, magnifection, devoid of the above-mentioned shortcomings and allowing consistent and efficient expression in plants of whole immunoglobulins (Igs). RESULTS: Full idiotype (Id)-containing IgG molecules of 20 lymphoma patients and 2 mouse lymphoma models were expressed at levels between 0.5 and 4.8 g/kg of leaf biomass. Protein A affinity capture purification yielded antigens of pharmaceutical purity. Several patient Igs produced in plants showed specific cross-reactivity with sera derived from the same patients immunized with hybridoma-produced Id vaccine. Mice vaccinated with plant- or hybridoma-produced Igs showed comparable protection levels in tumor challenge studies. CONCLUSIONS: This manufacturing process is reliable and robust, the manufacturing time from biopsy to vaccine is <12 weeks and the expression and purification of antigens require only 2 weeks. The process is also broadly applicable for manufacturing monoclonal antibodies in plants, providing 50- to 1000-fold higher yields than alternative plant expression methods.


Assuntos
Vacinas Anticâncer/biossíntese , Idiótipos de Imunoglobulinas/metabolismo , Linfoma não Hodgkin/imunologia , Linfoma não Hodgkin/terapia , Planticorpos/metabolismo , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/imunologia , Agrobacterium tumefaciens/metabolismo , Animais , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/isolamento & purificação , Clonagem Molecular , Eficiência , Regulação da Expressão Gênica de Plantas , Humanos , Idiótipos de Imunoglobulinas/genética , Idiótipos de Imunoglobulinas/imunologia , Individualidade , Camundongos , Camundongos Endogâmicos C3H , Planticorpos/genética , Planticorpos/isolamento & purificação , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/imunologia , Plantas Geneticamente Modificadas/metabolismo , Fatores de Tempo , Vacinas Sintéticas/biossíntese , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificação
14.
J Immunother ; 33(2): 178-84, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20145546

RESUMO

We evaluated the efficacy and safety of patient-specific immunotherapy with mitumprotimut-T idiotype keyhole limpet hemocyanin and granulocyte-monocyte colony-stimulating factor (GM-CSF) following rituximab in patients with follicular B-cell lymphoma. Patients with previously untreated or relapsed/refractory CD20+ follicular lymphoma received 4 weekly infusions of rituximab and those with a complete response (CR), partial response (PR), or stable disease received mitumprotimut-T and GM-CSF injections subcutaneously. Courses were given monthly for 6 doses, every 2 months for 6 doses, and then every 3 months until disease progression. Computed tomography scans were obtained every 3 to 6 months and reviewed centrally. The primary endpoint was event-free survival (EFS). Among 103 patients treated with rituximab, 92 (54 relapsed/refractory and 38 previously untreated) received mitumprotimut-T/GM-CSF; median age was 53 years, 91% had stage III to IV disease, and 59% had failed earlier therapy. The premitumprotimut-T objective response rate was 47% (2 CRs, 41 PRs). During the mitumprotimut-T treatment phase, 16 patients converted to CR resulting in an overall objective response rate of 60% (18 CRs, 37 PRs). Median EFS was 15.2, 20.8, and 13.5 months for all, treatment-naive, and relapsed/refractory disease patients, respectively. Anti-Id cellular immune responses were detected in 13 of 18 (72%) patients and humoral immune responses in 17 of 83 (20%) patients. Adverse events were usually mild-to-moderate. The most common adverse event was injection site reactions. Mitumprotimut-T/GM-CSF-induced anti-Id cellular immune responses in most patients. The occurrence of late CRs and favorable EFS suggested a clinical benefit of active immunotherapy and led to a placebo-controlled phase 3 trial.


Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Imunoterapia , Linfoma de Células B/terapia , Linfoma Folicular/terapia , Proteínas Recombinantes de Fusão/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Anti-Idiotípicos/sangue , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais Murinos , Antígenos CD20/biossíntese , Antígenos CD20/imunologia , Antígenos de Neoplasias/imunologia , Intervalo Livre de Doença , Feminino , Hemocianinas/genética , Hemocianinas/imunologia , Hemocianinas/metabolismo , Humanos , Imunidade Humoral/efeitos dos fármacos , Idiótipos de Imunoglobulinas/genética , Idiótipos de Imunoglobulinas/imunologia , Idiótipos de Imunoglobulinas/metabolismo , Injeções Subcutâneas , Linfoma de Células B/imunologia , Linfoma de Células B/patologia , Linfoma de Células B/fisiopatologia , Linfoma Folicular/imunologia , Linfoma Folicular/patologia , Linfoma Folicular/fisiopatologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Proteínas Recombinantes , Rituximab
15.
Eur J Immunol ; 40(2): 366-77, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19946883

RESUMO

Rapidly induced, specific Ab generated in extrafollicular foci are important components of early immune protection to influenza virus. The signal(s) that prompt B cells to participate in extrafollicular rather than germinal center responses are incompletely understood. To study the regulation of early B-cell differentiation events following influenza infection, we exploited earlier findings of a strong contribution of C12 idiotype-expressing B cells to the primary HA-specific response against influenza A/PR/8/34. Using an idiotype-specific mAb to C12 and labeled HA, in conjunction with multicolor flow cytometry, we followed the fate of C12Id-expressing influenza HA-specific B cells in WT BALB/c mice, requiring neither genetic manipulation nor adoptive cell transfer. Our studies demonstrate that HA-specific C12Id(+) B cells are phenotypically indistinguishable from follicular B cells. While they induced both extrafollicular and germinal center responses, extrafollicular responses were strongly predominant. Provision of increased HA-specific T-cell help increased the magnitude of the extrafollicular response, but did not shift the C12Id(+) response toward germinal center formation. Collectively the data are consistent with the hypothesis that B-cell fate determination following activation is a stochastic process in which infection-induced innate signals might drive the preferential expansion of the early extrafollicular response.


Assuntos
Anticorpos Antivirais/imunologia , Linfócitos B/imunologia , Vírus da Influenza A/imunologia , Infecções por Orthomyxoviridae/imunologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/genética , Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Idiótipos de Imunoglobulinas/genética , Idiótipos de Imunoglobulinas/imunologia , Isotipos de Imunoglobulinas/genética , Isotipos de Imunoglobulinas/imunologia , Linfonodos/imunologia , Linfonodos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Infecções por Orthomyxoviridae/virologia
16.
Crit Rev Immunol ; 29(5): 399-418, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-20001888

RESUMO

Therapeutic vaccines have been developed to induce immune responses capable of eradicating lymphoma and myeloma tumors. Most of these vaccines target the immunoglobulin idiotype (Id) as a tumor-specific antigen. Phase I/II clinical trials of Id vaccination in lymphoma demonstrated that some lymphoma patients could mount immune responses that were correlated with a favorable clinical outcome. These encouraging results initiated phase III trials of Id vaccination in lymphoma, the results of which have been recently released. Disappointingly, only one of three phase III studies achieved the primary end point of progression-free survival. Detailed analysis of these results is awaited to help identify factors that determine clinical efficacy of Id vaccination as reflected in the three trials. Unlike lymphoma, studies of Id vaccination in multiple myeloma have yielded far less evidence of clinical benefit. Although Id vaccines induce immune responses in myeloma patients, their efficacy is insufficient to provide a clear clinical benefit. Strategies to improve lymphoma and myeloma vaccines are currently tested with an emphasis on optimization of antigen delivery and presentation and modulation of the immune system toward enhancement of T-cell function. Despite many hurdles yet to overcome, it is hoped that newly developed strategies that augment both immune and clinical responses will allow effective vaccination resulting in tumor eradication.


Assuntos
Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Idiótipos de Imunoglobulinas/imunologia , Linfoma de Células B/imunologia , Linfoma de Células B/terapia , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/terapia , Animais , Ensaios Clínicos como Assunto , Humanos , Idiótipos de Imunoglobulinas/genética , Linfoma de Células B/diagnóstico , Linfoma de Células B/genética , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/genética , Resultado do Tratamento
17.
Biochem Biophys Res Commun ; 390(3): 971-6, 2009 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-19852937

RESUMO

Antibody fragments (scFvs) fused to luciferase reporter proteins have been used as highly sensitive optical imaging probes. Gaussia princeps luciferase (GLuc) is an attractive choice for a reporter protein because it is small and bright and does not require ATP to stimulate bioluminescence-producing reactions. Both GLuc and scFv proteins contain multiple disulfide bonds, and consequently the production of active and properly folded GLuc-scFv fusions is challenging. We therefore produced both proteins individually in active form, followed by covalent coupling to produce the intended conjugate. We used an Escherichia coli-based cell-free protein synthesis (CFPS) platform to produce GLuc and scFv proteins containing non-natural amino acids (nnAAs) for subsequent conjugation by azide-alkyne click chemistry. GLuc mutants with exposed alkyne reactive groups were produced by global replacement of methionine residues in CFPS. Antibody fragment scFvs contained a single exposed azide group using a scheme for site-specific incorporation of tyrosine analogs. Incorporation of tyrosine analogs at specific sites in proteins was performed using an engineered orthogonal tRNA-tRNA synthetase pair from an archaebacterium. The unique azide and alkyne side chains in GLuc and the antibody fragment scFv facilitated conjugation by click chemistry. GLuc-scFv conjugates were shown to differentiate between cells expressing a surface target of the scFv and cells that did not carry this marker.


Assuntos
Anticorpos Antineoplásicos/biossíntese , Copépodes/enzimologia , Região Variável de Imunoglobulina/biossíntese , Luciferases/biossíntese , Linfoma de Células B/diagnóstico , Engenharia de Proteínas , Sequência de Aminoácidos , Animais , Anticorpos Antineoplásicos/genética , Anticorpos Antineoplásicos/imunologia , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Idiótipos de Imunoglobulinas/biossíntese , Idiótipos de Imunoglobulinas/genética , Idiótipos de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Luciferases/genética , Luciferases/imunologia , Camundongos , Dados de Sequência Molecular , Biossíntese de Proteínas , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia
18.
Ann N Y Acad Sci ; 1173: 152-60, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19758144

RESUMO

Autoimmune type-II cryoglobulinemia (II-MC) is sustained by hepatitis C virus (HCV) infection and B-cell (oligo)clones. This is the reason why the disease may be considered an "indolent B-cell lymphoma (NHL)." B clones show a restricted use of immunoglobulin variable genes (BCR), in particular in the use of the variable kappa (VK)3-20/15 light chain, and show a homology between their BCR functional regions and those of autoimmune rheumatoid factors. We underlined the BCR unique repertoire with frequent rheumatoid factor activity also observed in other autoimmune disorders associated with NHL. The immunoglobulin idiotype is a clonal B-cell marker and an ideal target for immunotherapy. Five monoclonal antibodies were produced in our laboratory toward the VK3-20 of a subject with HCV infection and a II-MC-associated NHL. Epitope determination was performed using the epitope excision approach. Monoclonal antibody reactivity was tested in vitro in ELISA, Western blot, and cytofluorimetry. Data confirmed that a panel of antibodies, reactive against shared idiotypes, can be produced from patients with HCV-associated B-cell lymphoproliferative diseases, thus obviating the need to produce an anti-idiotype antibody for each patient.


Assuntos
Crioglobulinemia/imunologia , Idiótipos de Imunoglobulinas/imunologia , Linfoma de Células B/imunologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Linfócitos B/imunologia , Linfócitos B/patologia , Linfócitos B/virologia , Proliferação de Células , Ensaio de Imunoadsorção Enzimática , Epitopos/genética , Epitopos/imunologia , Citometria de Fluxo , Hepacivirus/imunologia , Hepatite C/imunologia , Hepatite C/virologia , Humanos , Imunização , Immunoblotting , Fragmentos de Imunoglobulinas/genética , Idiótipos de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/genética , Camundongos , Camundongos Endogâmicos BALB C
19.
Immunol Cell Biol ; 87(6): 457-63, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19333248

RESUMO

Antibodies against different chronic viruses, including hepatitis C virus (HCV), express a public cross-reactive idiotype (Id) designated as 1F7. The prominence of this Id may reflect selective engagement of B1 B cells by chronic pathogens. We investigated this by comparing 1F7 Id expression on CD5(+) and CD5(-) B cells, total IgG, total IgM and anti-HCV core antibodies in different HCV exposure settings. By flow cytometry, we observed a selective increase in 1F7 Id(+)CD5(+) B cells in chronic HCV infection. 1F7 Id levels in different immunoglobulin compartments were measured by enzyme-linked immunosorbent assay. 1F7 Id expression was prominent in anti-HCV core antibodies of approximately 90% of 141 HCV-exposed individuals tested. In the Canadian and Armenian study groups, participants who spontaneously cleared HCV infection had lower median 1F7 Id levels on total plasma IgG and anti-HCV core antibodies. Armenian spontaneous clearers, who were younger and more recently infected than their Canadian counterparts, also had had lower median 1F7 Id levels on total plasma IgM. Engagement by HCV of B-cell receptors within, or overlapping with the CD5(+) B1 B-cell repertoire is reflected in the production of 1F7 Id(+) anti-HCV antibodies and expansion of 1F7 Id(+)CD5(+) B cells. Higher 1F7 Id expression levels are associated with chronic infection.


Assuntos
Linfócitos B/metabolismo , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/metabolismo , Hepatite C Crônica/imunologia , Idiótipos de Imunoglobulinas/metabolismo , Adulto , Armênia , Linfócitos B/imunologia , Linfócitos B/patologia , Antígenos CD5/biossíntese , Canadá , Separação Celular , Reações Cruzadas , Feminino , Citometria de Fluxo , Hepacivirus/patogenicidade , Anticorpos Anti-Hepatite C/genética , Anticorpos Anti-Hepatite C/imunologia , Antígenos da Hepatite C/imunologia , Hepatite C Crônica/sangue , Hepatite C Crônica/patologia , Hepatite C Crônica/fisiopatologia , Humanos , Imunoglobulina G/sangue , Idiótipos de Imunoglobulinas/genética , Idiótipos de Imunoglobulinas/imunologia , Imunoglobulina M/sangue , Masculino , Pessoa de Meia-Idade , Proteínas do Core Viral/imunologia
20.
J Huazhong Univ Sci Technolog Med Sci ; 28(5): 495-8, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18846324

RESUMO

The purpose of this study was to construct expression vectors of idiotype (Id) SmIg in patients with B-chronic lymphocytic leukemia and to express them in E.coli to obtain recombinant Id, and to investigate the effect of the protein on the proliferation and secretion of IL-2 and IFN-gamma of stimulated peripheral blood mononuclear cells (PBMC) in vitro. Light chain gene and Fd fragment of heavy chain gene were inserted into fd-tet-DOG2 vector to construct fd-tet-DOG2-Fab. Fab gene was further cloned into expression vector pHEN2 to construct the soluble expression vector pHEN2-Fab. After induction by IPTG, Fab protein was purified by Ni-NTA-chromatography. MTT was used to determine the effects of purified protein on the proliferation of stimulated PBMC in vitro and the concentrations of IL-2 and IFN-gamma in the culture supernatants were detected by ELISA. The results showed that recombinant pHEN2-Fab expression vector was constructed successfully. Fab protein was expressed in positive clone after induced by IPTG and two specific bands at 24-25 kD position were observed by SDS-PAGE electrophoresis. Proliferation of PBMC could be induced by purified Fab and the concentrations of IL-2 and IFN-gamma in culture supernatants were increased significantly after induction. It was suggested that the expression vector of SmIg Fab fragment was constructed successfully, and expressed and secreted from E. coli. The Fab protein could induce proliferation of PBMC and promote secretion of IL-2 and IFN-gamma.


Assuntos
Vetores Genéticos/genética , Fragmentos Fab das Imunoglobulinas/biossíntese , Idiótipos de Imunoglobulinas/biossíntese , Região Variável de Imunoglobulina/genética , Leucemia Linfocítica Crônica de Células B/imunologia , Proliferação de Células , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Idiótipos de Imunoglobulinas/genética , Região Variável de Imunoglobulina/biossíntese , Interferon gama/metabolismo , Interleucina-2/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucócitos Mononucleares/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética
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