RESUMO
Hu11B6 is a monoclonal antibody that internalizes in cells expressing androgen receptor (AR)-regulated prostate-specific enzyme human kallikrein-related peptidase 2 (hK2; KLK2). In multiple rodent models, Actinium-225-labeled hu11B6-IgG1 ([225Ac]hu11B6-IgG1) has shown promising treatment efficacy. In the present study, we investigated options to enhance and optimize [225Ac]hu11B6 treatment. First, we evaluated the possibility of exploiting IgG3, the IgG subclass with superior activation of complement and ability to mediate FC-γ-receptor binding, for immunotherapeutically enhanced hK2 targeted α-radioimmunotherapy. Second, we compared the therapeutic efficacy of a single high activity vs. fractionated activity. Finally, we used RNA sequencing to analyze the genomic signatures of prostate cancer that progressed after targeted α-therapy. [225Ac]hu11B6-IgG3 was a functionally enhanced alternative to [225Ac]hu11B6-IgG1 but offered no improvement of therapeutic efficacy. Progression-free survival was slightly increased with a single high activity compared to fractionated activity. Tumor-free animals succumbing after treatment revealed no evidence of treatment-associated toxicity. In addition to up-regulation of canonical aggressive prostate cancer genes, such as MMP7, ETV1, NTS, and SCHLAP1, we also noted a significant decrease in both KLK3 (prostate-specific antigen ) and FOLH1 (prostate-specific membrane antigen) but not in AR and KLK2, demonstrating efficacy of sequential [225Ac]hu11B6 in a mouse model.
Assuntos
Actínio/uso terapêutico , Imunoconjugados/uso terapêutico , Antígeno Prostático Específico/imunologia , Neoplasias da Próstata/terapia , Calicreínas Teciduais/metabolismo , Partículas alfa , Animais , Biomarcadores Tumorais , Humanos , Masculino , Camundongos , Camundongos Nus , Neoplasias Experimentais/terapiaRESUMO
Escherichia coli TolA is a cytoplasmic membrane protein required for outer membrane integrity and the translocation of F-specific filamentous (Ff) bacteriophage DNA. Both phage infection and membrane integrity depend on several TolA interactions, e.g. those of the TolA C-terminal domain (TolAIII). Membrane integrity involves interaction with two host proteins and phage translocation requires direct interaction with the N-terminal domain (N1) of Ff phage protein g3p. Although cocrystallization of TolAIII and N1g3p has identified several contact points, it is still uncertain which residues are selectively involved in the different TolA functions. Thus, four different limited substitution libraries of TolA were created, targeting contacts at positions 415-420. These libraries were introduced into the tolA strain K17DE3tolA/F(+) and several variants, containing complementing, multiple amino-acid substitutions, were identified. However, most randomized variants did not complement the tolA strain K17DE3tolA/F(+). The TolA variants that restored sensitivity to phage infection displayed a considerable sequence variation, while the few variants that restored tolerance to detergent were from the same library. A comparison of the generated residue variation and natural variation, suggests that structural dependence overrides contact residue dependence. Thus, library screening can be efficient in identifying TolA variants with different functionally associated characteristics.
Assuntos
Substituição de Aminoácidos , Membrana Celular/fisiologia , Colífagos/fisiologia , Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Receptores Virais/metabolismo , Sequência de Aminoácidos , Colífagos/metabolismo , Escherichia coli/genética , Escherichia coli/virologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Fator F/metabolismo , Biblioteca Gênica , Variação Genética , Modelos Moleculares , Dados de Sequência MolecularRESUMO
Antibody evolution in vivo proceeds mainly by stepwise improvements, accomplished by single base pair substitutions. Lately, receptor revision, i.e. exchange of large parts of the V gene for another sequence, has been suggested to provide a complementary route for affinity maturation. By employing a receptor revision like evolution process in vitro using combinatorial libraries and phage display selection, we demonstrate here that maturation of a clone may preferentially proceed through exchange of a large gene segment rather than via minor sequence changes. These modifications of a CD40-specific human antibody fragment outline how receptor revision like events may provide an advantage to a particular clonotype put under selective pressure.
Assuntos
Anticorpos/genética , Receptores de Antígenos de Linfócitos B/genética , Sequência de Aminoácidos , Anticorpos/química , Especificidade de Anticorpos , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Evolução Molecular , Humanos , Dados de Sequência Molecular , Biblioteca de PeptídeosRESUMO
To identify Escherichia coli genes potentially regulated by filamentous phage infection, we used oligonucleotide microarrays. Genome-wide comparison of phage M13-infected and uninfected E. coli, 2 and 20 min after infection, was performed. The analysis revealed altered transcription levels of 12 E. coli genes in response to phage infection, and the observed regulation of phage genes correlated with the known in vivo pattern of M13 mRNA species. Ten of the 12 host genes affected could be grouped into 3 different categories based on cellular function, suggesting a coordinated response. The significantly upregulated genes encode proteins involved in reactions of the energy-generating phosphotransferase system and transcription processing, which could be related to phage transcription. No genes belonging to any known E. coli stress response pathways were scored as upregulated. Furthermore, phage infection led to significant downregulation of transcripts of the bacterial genes gadA, gadB, hdeA, gadE, slp, and crl. These downregulated genes are normally part of the host stress response mechanisms that protect the bacterium during conditions of acid stress and stationary phase transition. The phage-infected cells demonstrated impaired function of the oxidative and the glutamate-dependent acid resistance systems. Thus, global transcriptional analysis and functional analysis revealed previously unknown host responses to filamentous phage infection.
Assuntos
Bacteriófago M13/fisiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Escherichia coli/virologia , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Transcrição GênicaRESUMO
The early events in filamentous bacteriophage infection of gram-negative bacteria are mediated by the gene 3 protein (g3p) of the virus. This protein has a sophisticated domain organization consisting of two N-terminal domains and one C-terminal domain, separated by flexible linkers. The molecular interactions between these domains and the known bacterial coreceptor protein (TolA) were studied using a biosensor technique, and we report here on interactions of the viral coat protein with TolA, as well as on interactions between the TolA molecules. We detected an interaction between the pilus binding second domain (N2) of protein 3 and the bacterial TolA. This novel interaction was found to depend on the periplasmatic domain of TolA (TolAII). Furthermore, extensive interaction was detected between TolA molecules, demonstrating that bacterial TolA has the ability to interact functionally with itself during phage infection. The kinetics of g3p binding to TolA is also different from that of bacteriocins, since both N-terminal domains of g3p were found to interact with TolA. The multiple roles for each of the separate g3p and TolA domains imply a delicate interaction network during the phage infection process and a model for the infection mechanism is hypothesized.
Assuntos
Capsídeo/metabolismo , Colífagos/patogenicidade , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/virologia , Proteínas Virais de Fusão/metabolismo , Proteínas do Capsídeo , Colífagos/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Escherichia coli/química , Fímbrias Bacterianas/química , Fímbrias Bacterianas/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Periplasma/metabolismo , Ligação Proteica , Ressonância de Plasmônio de Superfície , Proteínas Virais de Fusão/químicaRESUMO
CD40 plays a central regulatory role in the immune system and antibodies able to modulate CD40 signalling may consequently have a potential in immunotherapy, in particular for treatment of lymphomas and autoimmune disease like multiple sclerosis. As a first step to achieve this goal, we describe the selection and characterization of a novel set of fully human anti-CD40 antibody fragments (scFv) from a phage display library (n-CoDeR). In order to determine their biological potential, these antibody fragments have been analysed for their ability to promote B-cell activation, rescue from apoptosis and to block the CD40-CD40 ligand (CD40L) interaction. The selected cohort of human scFv could be subcategorized, each expressing a distinct functional signature. Thus scFv were generated that induced B-cell proliferation, rescued B cells from apoptosis and blocked the CD40-CD40L interaction to different extents. In particular, one of the scFv clones (F33) had the ability to abrogate completely this interaction. The epitope recognition patterns as well as individual rate constants were also determined and the affinity was shown to vary from low to high nanomolar range. In conclusion, this panel of human anti-CD40 scFv fragments displays a number of distinct properties, which may constitute a valuable source when evaluating candidates for in vivo trials.