RESUMO
Sortase enzymes play an important role in Gram-positive bacteria. They are responsible for the covalent attachment of proteins to the surface of the bacteria and perform this task via a highly sequence-specific transpeptidation reaction. Since these immobilized proteins are often involved in pathogenicity of Gram-positive bacteria, characterization of this type of enzyme is also of medical relevance. Different classes of sortases (A-F) have been found, which recognize characteristic recognition sequences present in substrate proteins. Up to date, sortase A from Staphylococcus aureus, a housekeeping class A sortase, is the most thoroughly studied representative of the sortase family of enzymes. Here we report the in-depth characterization of the class F sortase from Propionibacterium acnes, a class of sortases that has not been investigated before. As Sortase F is the only transpeptidase found in the P. acnes genome, it is the housekeeping sortase of this organism. Sortase F from P. acnes shows a behavior similar to sortases from class A in terms of pH dependence, recognition sequence and catalytic activity; furthermore, its activity is independent of bivalent ions, which contrasts to sortase A from S. aureus We demonstrate that sortase F is useful for protein engineering applications, by producing a site-specifically conjugated homogenous antibody-drug conjugate with a potency similar to that of a conjugate prepared with sortase A. Thus, the detailed characterization presented here will not only enable the development of anti-virulence agents targeting P. acnes but also provides a powerful alternative to sortase A for protein engineering applications.
Assuntos
Aminoaciltransferases , Proteínas de Bactérias , Cisteína Endopeptidases , Genoma Bacteriano , Propionibacterium acnes , Aminoaciltransferases/química , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Propionibacterium acnes/enzimologia , Propionibacterium acnes/genética , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genéticaRESUMO
A vaccine protecting against all influenza strains is a long-sought goal, particularly for emerging pandemics. As previously shown, vaccines based on the highly conserved extracellular domain of M2 (M2e) may protect against all influenza A strains. Here, we demonstrate that M2e-specific monoclonal antibodies (mAbs) protect mice from a lethal influenza infection. To be protective, antibodies had to be able to bind to Fc receptors and fix complement. Furthermore, mAbs of IgG2c isotype were protective in mice, while antibodies of identical specificity, but of the IgG1 isotype, failed to prevent disease. These findings readily translated into vaccine design. A vaccine targeting M2 in the absence of a toll-like receptor (TLR) 7 ligand primarily induced IgG1, whilst the same vaccine linked to a TLR7 ligand yielded high levels of IgG2c antibodies. Although both vaccines protected mice from a lethal challenge, mice treated with the vaccine containing a TLR7 ligand showed significantly lower morbidity. In accordance with these findings, vaccination of TLR7(-/-) mice with a vaccine containing a TLR7 ligand did not result in protection from a lethal challenge. Hence, the innate immune system is required to direct isotype switching toward the more protective IgG2a/c antibodies.
Assuntos
Anticorpos Antivirais/imunologia , Imunoglobulina G/imunologia , Vírus da Influenza A/imunologia , Vacinas contra Influenza/imunologia , Glicoproteínas de Membrana/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Transdução de Sinais/imunologia , Receptor 7 Toll-Like/imunologia , Animais , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Monoclonais Murinos/farmacologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/genética , Humanos , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/genética , Switching de Imunoglobulina/efeitos dos fármacos , Switching de Imunoglobulina/genética , Imunoglobulina G/sangue , Imunoglobulina G/genética , Vírus da Influenza A/genética , Vírus da Influenza A/metabolismo , Vacinas contra Influenza/farmacologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Infecções por Orthomyxoviridae/sangue , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/imunologia , Pandemias , Transdução de Sinais/genética , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/metabolismo , VacinaçãoRESUMO
Suppression by natural CD4(+)CD25(+) regulatory T cells (Tregs) is one mechanism by which tolerance is maintained. However, the way in which Tregs mediate suppression is not well understood. Here, we show that secreted phospholipase A2 (sPLA2)-IID is selectively produced by Tregs. sPLA2-IID is a potent mediator of Treg function, because it strongly suppressed proliferation of CD4(+) and CD8(+) T cells in vitro and in vivo in a manner independent of its catalytic activity. Furthermore, sPLA2-IID promoted the differentiation of Tregs, presumably via attenuating signaling through the PI3K/Akt/mammalian target of rapamycin pathway. Importantly, administration of a sPLA2-IID-Fc fusion protein inhibited disease development in murine models of colitis and multiple sclerosis, suggesting that sPLA2-IID's immunosuppressive function might be exploited therapeutically.
Assuntos
Diferenciação Celular/imunologia , Proliferação de Células/efeitos dos fármacos , Colite/prevenção & controle , Fosfolipases A2 do Grupo II/farmacologia , Esclerose Múltipla/prevenção & controle , Transdução de Sinais/imunologia , Linfócitos T Reguladores/metabolismo , Transferência Adotiva , Animais , Diferenciação Celular/efeitos dos fármacos , Colite/imunologia , Primers do DNA/genética , Citometria de Fluxo , Fosfolipases A2 do Grupo II/metabolismo , Camundongos , Esclerose Múltipla/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Due to their low immunogenicity in patients, humanized or fully human mAbs are becoming increasingly important for the treatment of a growing number of diseases, including cancer, infections, and immune disorders. Here, we describe a technology allowing for the rapid isolation of fully human mAbs. In contrast to previously described methods, B cells specific for an antigen of interest are directly isolated from peripheral blood mononuclear cells (PBMC) of human donors. Recombinant, antigen-specific single-chain Fv (scFv) libraries are generated from this pool of B cells and screened by mammalian cell surface display by using a Sindbis virus expression system. This method allows isolating antigen-specific antibodies by a single round of FACS. The variable regions (VRs) of the heavy chains (HCs) and light chains (LCs) are isolated from positive clones and recombinant fully human antibodies produced as whole IgG or Fab fragments. In this manner, several hypermutated high-affinity antibodies binding the Qbeta virus like particle (VLP), a model viral antigen, as well as antibodies specific for nicotine were isolated. All antibodies showed high expression levels in cell culture. The human nicotine-specific mAbs were validated preclinically in a mouse model. Thus, the technology presented here allows for rapid isolation of high-affinity, fully human antibodies with therapeutic potential from human volunteers.
Assuntos
Allolevivirus/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Mamíferos , Biblioteca de Peptídeos , Animais , Anticorpos Monoclonais/genética , Anticorpos Antivirais/genética , Encéfalo/metabolismo , Proteínas do Capsídeo/imunologia , Citometria de Fluxo , Humanos , Imunização Passiva , Leucócitos Mononucleares/imunologia , Camundongos , Nicotina/imunologia , Sindbis virus/genética , Sindbis virus/metabolismoRESUMO
BACKGROUND: Allergen-specific desensitization (SIT) is the most effective therapy for allergies. Although allergen-specific antibodies have an important role in the process, mechanisms of IgG-mediated inhibition of allergic reactions are not well defined. OBJECTIVE: We investigated mechanisms by which SIT-induced allergen-specific IgGs inhibit allergic reactions. METHODS: We generated mAbs that recognize 3 nonoverlapping epitopes of the major cat allergen Fel d 1. Each of the mAbs was produced as an IgE and different IgG isotype. RESULTS: IgEs against 2 nonoverlapping epitopes on Fel d 1 are necessary and sufficient to sensitize mast cells for maximal FcepsilonRI signaling and degranulation on exposure to monomeric Fel d 1. IgE antibodies of a third specificity did not further increase mast cell degranulation, indicating that formation of large FcepsilonRI clusters are not required to induce maximal activation of mast cells. A single IgG that was specific for an epitope different from those recognized by the IgEs was a potent inhibitor of Fel d 1-mediated mast cell activation in vitro and in vivo. This inhibition required Fcgamma receptor-IIB. In human beings, IgGs of a single specificity were able to block degranulation of basophils from individuals with cat allergy. The inhibitory potential of these antibodies increased when larger allergen-IgG complexes were formed. CONCLUSIONS: These data reconcile conflicting theories in the literature and might explain the reason IgE levels do not necessarily decrease during therapy, despite clinical efficacy. These findings have important implications for vaccine design.
Assuntos
Alérgenos/imunologia , Anticorpos Monoclonais/farmacologia , Dessensibilização Imunológica , Glicoproteínas/imunologia , Hipersensibilidade/terapia , Imunoglobulina G/farmacologia , Alérgenos/farmacologia , Animais , Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Glicoproteínas/antagonistas & inibidores , Glicoproteínas/farmacologia , Humanos , Hipersensibilidade/genética , Hipersensibilidade/imunologia , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Mastócitos/imunologia , Mastócitos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Receptores de IgE/genética , Receptores de IgE/imunologia , Receptores de IgG/genética , Receptores de IgG/imunologia , Vacinas/genética , Vacinas/imunologiaRESUMO
Antibody-drug conjugates directed against tumor-specific targets have allowed targeted delivery of highly potent chemotherapy to malignant cells while sparing normal cells. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncofetal protein with limited expression on normal adult tissues and is overexpressed on the surface of malignant cells in mantle cell lymphoma, acute lymphocytic leukemia with t(1;19)(q23;p13) translocation, and chronic lymphocytic leukemia. This differential expression makes ROR1 an attractive target for antibody-drug conjugate therapy, especially in malignancies such as mantle cell lymphoma and acute lymphocytic leukemia, in which systemic chemotherapy remains the gold standard. Several preclinical and phase 1 clinical studies have established the safety and effectiveness of anti-ROR1 monoclonal antibody-based therapies. Herein we describe a humanized, first-in-class anti-ROR1 antibody-drug conjugate, huXBR1-402-G5-PNU, which links a novel anti-ROR1 antibody (huXBR1-402) to a highly potent anthracycline derivative (PNU). We found that huXBR1-402-G5-PNU is cytotoxic to proliferating ROR1+ malignant cells in vitro and suppressed leukemia proliferation and extended survival in multiple models of mice engrafted with human ROR1+ leukemia. Lastly, we show that the B-cell lymphoma 2 (BCL2)-dependent cytotoxicity of huXBR1-402-G5-PNU can be leveraged by combined treatment strategies with the BCL2 inhibitor venetoclax. Together, our data present compelling preclinical evidence for the efficacy of huXBR1-402-G5-PNU in treating ROR1+ hematologic malignancies.
Assuntos
Neoplasias Hematológicas , Imunoconjugados , Leucemia Linfocítica Crônica de Células B , Linfoma de Célula do Manto , Animais , Anticorpos Monoclonais , Linfoma de Célula do Manto/tratamento farmacológico , Linfoma de Célula do Manto/genética , CamundongosRESUMO
We report the first method of enzyme protection enabling the production of partially shielded enzymes capable of processing substrates as large as proteins. We show that partially shielded sortase retains its transpeptidase activity and can perform bioconjugation reactions on antibodies. Moreover, a partially shielded trypsin is shown to outperform its soluble counterpart in terms of proteolytic kinetics. Remarkably, partial enzyme shielding results in a drastic increase in temporal stability of the enzyme.
Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Aminoaciltransferases/química , Proteínas de Bactérias/química , Cisteína Endopeptidases/química , Cinética , Tamanho da Partícula , Proteólise , Staphylococcus aureus/enzimologia , Especificidade por Substrato , Propriedades de SuperfícieRESUMO
T cell activation by APCs is positively and negatively regulated by members of the B7 family. We have identified a previously unknown function for B7 family-related protein V-set and Ig domain-containing 4 (VSIG4). In vitro experiments using VSIG4-Ig fusion molecules showed that VSIG4 is a strong negative regulator of murine and human T cell proliferation and IL-2 production. Administration to mice of soluble VSIG4-Ig fusion molecules reduced the induction of T cell responses in vivo and inhibited the production of Th cell-dependent IgG responses. Unlike that of B7 family members, surface expression of VSIG4 was restricted to resting tissue macrophages and absent upon activation by LPS or in autoimmune inflammatory foci. The specific expression of VSIG4 on resting macrophages in tissue suggests that this inhibitory ligand may be important for the maintenance of T cell unresponsiveness in healthy tissues.
Assuntos
Imunoglobulinas/fisiologia , Ativação Linfocitária/imunologia , Receptores de Complemento/fisiologia , Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Doenças Autoimunes/induzido quimicamente , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Antígeno B7-1/farmacologia , Antígeno B7-H1 , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Feminino , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/imunologia , Humanos , Imunoglobulinas/genética , Imunoglobulinas/farmacologia , Interferon gama/metabolismo , Interleucina-2/metabolismo , Interleucina-2/farmacologia , Lipopolissacarídeos/farmacologia , Fígado/citologia , Fígado/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Glicoproteínas de Membrana/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Miocardite/induzido quimicamente , Miocardite/imunologia , Miocardite/metabolismo , Peptídeos/farmacologia , Proteína 2 Ligante de Morte Celular Programada 1 , Receptores de Complemento/genética , Receptores de Complemento/metabolismo , Homologia de Sequência de Aminoácidos , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Tioglicolatos/farmacologiaRESUMO
Influenza virus infection is a prevalent disease in humans. Antibodies against hemagglutinin have been shown to prevent infection and hence hemagglutinin is the major constituent of current vaccines. Antibodies directed against the highly conserved extracellular domain of M2 have also been shown to mediate protection against Influenza A infection in various animal models. Active vaccination is generally considered the best approach to combat viral diseases. However, passive immunization is an attractive alternative, particularly in acutely exposed or immune compromized individuals, young children and the elderly. We recently described a novel method for the rapid isolation of natural human antibodies by mammalian cell display. Here we used this approach to isolate human monoclonal antibodies directed against the highly conserved extracellular domain of the Influenza A M2 protein. The identified antibodies bound M2 peptide with high affinities, recognized native cell-surface expressed M2 and protected mice from a lethal influenza virus challenge. Moreover, therapeutic treatment up to 2 days after infection was effective, suggesting that M2-specific monoclonals have a great potential as immunotherapeutic agents against Influenza infection.
Assuntos
Anticorpos Monoclonais , Vírus da Influenza A Subtipo H1N1/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Infecções por Orthomyxoviridae , Proteínas da Matriz Viral/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Especificidade de Anticorpos , Linhagem Celular , Cricetinae , Modelos Animais de Doenças , Feminino , Humanos , Imunização Passiva , Virus da Influenza A Subtipo H5N1/patogenicidade , Vacinas contra Influenza , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/virologia , Resultado do Tratamento , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genéticaRESUMO
Plasma cells are the main producers of antibody and key effector cells of the immune system. Despite their importance, analytics of plasma cells still suffers from the limited availability of specific markers. Currently, plasma cell identification relies on the expression of a single marker, CD138/syndecan-1. However, syndecan-1 is widely expressed on various cell types outside the hematopoietic compartment, and furthermore, not expressed on all subsets of plasma cells. To discover novel surface markers, a differential screening followed by signal sequence trap cloning was developed, leading to the identification of mouse Ly-6K (mLy-6K). Expression profiling confirmed that mLy-6K is expressed by plasma cells but not B cells or tissues not containing plasma cells. Expression at the surface of plasma cells isolated from spleen, lymph node, bone marrow, and lamina propria of the small intestine was demonstrated at the protein level using a polyclonal rabbit antibody. This novel plasma cell marker shows promise to help broaden our understanding of plasma cell differentiation and function.
Assuntos
Antígenos Ly/metabolismo , Plasmócitos/metabolismo , Sequência de Aminoácidos , Animais , Antígenos Ly/química , Antígenos Ly/genética , Biomarcadores/química , Biomarcadores/metabolismo , Northern Blotting , Linhagem Celular , Membrana Celular/metabolismo , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Plasmócitos/citologia , Alinhamento de SequênciaRESUMO
Antibody-drug conjugates (ADCs) are highly potent targeted anticancer therapies. They rely on the linking of a selectively targeting antibody moiety with potent cytotoxic payloads to effect antitumoral activity. In recent years, one focus in the ADC field was to create novel methods for site-specifically conjugating payloads to antibodies. The method presented here is based on the S. aureus sortase A-mediated transpeptidation reaction. This method requires antibodies to be engineered in such a way that they possess the sortase recognition pentapeptide motif LPETG on the C-terminus of the immunoglobulin heavy and/or light chains. In addition, the toxin must contain an oligoglycine motif in order to make it a suitable substrate for sortase A. Here we describe a detailed method to conjugate a pentaglycine-modified toxin to the C-termini of LPETG-tagged antibody heavy and light chains using sortase-mediated antibody conjugation (SMAC-Technology™). Highly homogenous, site-specifically conjugated ADCs with controlled drug to antibody ratio and improved overall properties can be obtained with this method.
Assuntos
Aminoaciltransferases/química , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacologia , Proteínas de Bactérias/química , Cisteína Endopeptidases/química , Imunoconjugados/química , Imunoconjugados/farmacologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Composição de Medicamentos , Humanos , Imunoconjugados/imunologia , Imunoconjugados/isolamento & purificação , Estrutura MolecularRESUMO
Increasing evidence suggests that antibody-drug conjugates (ADCs) can enhance anti-tumor immunity and improve clinical outcome. Here, we elucidate the therapeutic efficacy and immune-mediated mechanisms of a novel HER2-targeting ADC bearing a potent anthracycline derivate as payload (T-PNU) in a human HER2-expressing syngeneic breast cancer model resistant to trastuzumab and ado-trastuzumab emtansine. Mechanistically, the anthracycline component of the novel ADC induced immunogenic cell death leading to exposure and secretion of danger-associated molecular signals. RNA sequencing derived immunogenomic signatures and TCRß clonotype analysis of tumor-infiltrating lymphocytes revealed a prominent role of the adaptive immune system in the regulation of T-PNU mediated anti-cancer activity. Depletion of CD8 T cells severely reduced T-PNU efficacy, thus confirming the role of cytotoxic T cells as drivers of the T-PNU mediated anti-tumor immune response. Furthermore, T-PNU therapy promoted immunological memory formation in tumor-bearing animals protecting those from tumor rechallenge. Finally, the combination of T-PNU and checkpoint inhibition, such as α-PD1, significantly enhanced tumor eradication following the treatment. In summary, a novel PNU-armed, HER2-targeting ADC elicited long-lasting immune protection in a murine orthotopic breast cancer model resistant to other HER2-directed therapies. Our findings delineate the therapeutic potential of this novel ADC payload and support its clinical development for breast cancer patients and potentially other HER2 expressing malignancies.
Assuntos
Antraciclinas/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Imunoconjugados/uso terapêutico , Neoplasias Mamárias Experimentais/tratamento farmacológico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor ErbB-2/antagonistas & inibidores , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Feminino , Humanos , Memória Imunológica/efeitos dos fármacos , Neoplasias Mamárias Experimentais/imunologia , Camundongos Endogâmicos BALB C , Receptor ErbB-2/genética , Trastuzumab/uso terapêuticoRESUMO
Receptor tyrosine kinase-like orphan receptor 2 (ROR2) has been identified as a highly relevant tumor-associated antigen in a variety of cancer indications of high unmet medical need, including renal cell carcinoma and osteosarcoma, making it an attractive target for targeted cancer therapy. Here, we describe the de novo discovery of fully human ROR2-specific antibodies and potent antibody drug conjugates (ADCs) derived thereof by combining antibody discovery from immune libraries of human immunoglobulin transgenic animals using the Transpo-mAb mammalian cell-based IgG display platform with functional screening for internalizing antibodies using a secondary ADC assay. The discovery strategy entailed immunization of transgenic mice with the cancer antigen ROR2, harboring transgenic IgH and IgL chain gene loci with limited number of fully human V, D, and J gene segments. This was followed by recovering antibody repertoires from the immunized animals, expressing and screening them as full-length human IgG libraries by transposon-mediated display in progenitor B lymphocytes ("Transpo-mAb Display") for ROR2 binding. Individual cellular "Transpo-mAb" clones isolated by single cell sorting and capable of expressing membrane-bound as well as secreted human IgG were directly screened during antibody discovery, not only for high affinity binding to human ROR2, but also functionally as ADCs using a cytotoxicity assay with a secondary anti-human IgG-toxin-conjugate. Using this strategy, we identified and validated 12 fully human, monoclonal anti-human ROR2 antibodies with nanomolar affinities that are highly potent as ADCs and could be promising candidates for the therapy of human cancer. The screening for functional and internalizing antibodies during the early phase of antibody discovery demonstrates the utility of the mammalian cell-based Transpo-mAb Display platform to select for functional binders and as a powerful tool to improve the efficiency for the development of therapeutically relevant ADCs.
Assuntos
Anticorpos Monoclonais Humanizados/isolamento & purificação , Anticorpos Monoclonais/isolamento & purificação , Imunoconjugados/isolamento & purificação , Neoplasias/terapia , Células Precursoras de Linfócitos B/fisiologia , Animais , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Humanos , Imunização , Imunoconjugados/farmacologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Imunotoxinas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias/imunologia , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/imunologia , Análise de Célula Única , Éxons VDJ/genéticaRESUMO
The availability of rapid and robust methods for controlling gene function is of prime importance not only for assigning functions to newly discovered genes, but also for therapeutic intervention. Traditionally, gene function has been probed by often-laborious methods that either increase the level of a gene product or decrease it. Advances now make it possible to rapidly produce zinc-finger proteins capable of recognizing virtually any 18 bp stretch of DNA--a sequence long enough to specify a unique address in any genome. The attachment of functional domains also allows the design of tailor-made transcription factors for specific genes. Recent studies demonstrate that artificial transcription factors are capable of controlling the expression of endogenous genes in their native chromosomal context with a high degree of specificity in both animals and plants. Dominant regulatory control of expression of any endogenous gene can be achieved rapidly and can be also placed under chemical control. A wide range of potential applications is now within reach.
Assuntos
Engenharia de Proteínas/métodos , Fatores de Transcrição , Dedos de Zinco , Animais , Sítios de Ligação , DNA/metabolismo , Regulação da Expressão Gênica , Humanos , Regiões Promotoras Genéticas , Ligação Proteica , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Transcrição GênicaRESUMO
Antibody-drug conjugates (ADC) are highly potent and specific antitumor drugs, combining the specific targeting of mAbs with the potency of small-molecule toxic payloads. ADCs generated by conventional chemical conjugation yield heterogeneous mixtures with variable pharmacokinetics, stability, safety, and efficacy profiles. To address these issues, numerous site-specific conjugation technologies are currently being developed allowing the manufacturing of homogeneous ADCs with predetermined drug-to-antibody ratios. Here, we used sortase-mediated antibody conjugation (SMAC) technology to generate homogeneous ADCs based on a derivative of the highly potent anthracycline toxin PNU-159682 and a noncleavable peptide linker, using the anti-HER2 antibody trastuzumab (part of Kadcyla) and the anti-CD30 antibody cAC10 (part of Adcetris). Characterization of the resulting ADCs in vitro and in vivo showed that they were highly stable and exhibited potencies exceeding those of ADCs based on conventional tubulin-targeting payloads, such as Kadcyla and Adcetris. The data presented here suggest that such novel and highly potent ADC formats may help to increase the number of targets available to ADC approaches, by reducing the threshold levels of target expression required. Mol Cancer Ther; 16(5); 879-92. ©2017 AACR.
Assuntos
Antraciclinas/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Imunoconjugados/administração & dosagem , Neoplasias/tratamento farmacológico , Ado-Trastuzumab Emtansina , Aminoaciltransferases/química , Animais , Antraciclinas/química , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Proteínas de Bactérias/química , Brentuximab Vedotin , Linhagem Celular Tumoral , Cisteína Endopeptidases/química , Doxorrubicina/administração & dosagem , Doxorrubicina/análogos & derivados , Doxorrubicina/química , Humanos , Imunoconjugados/química , Imunoconjugados/imunologia , Antígeno Ki-1/química , Antígeno Ki-1/imunologia , Maitansina/análogos & derivados , Maitansina/química , Maitansina/imunologia , Camundongos , Neoplasias/imunologia , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/imunologia , Trastuzumab/administração & dosagem , Trastuzumab/química , Trastuzumab/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The antigen-specific targeting of autoreactive B cells via their unique B cell receptors (BCRs) is a novel and promising alternative to the systemic suppression of humoral immunity. We generated and characterized cytolytic fusion proteins based on an existing immunotoxin comprising tetanus toxoid fragment C (TTC) as the targeting component and the modified Pseudomonas aeruginosa exotoxin A (ETA') as the cytotoxic component. The immunotoxin was reconfigured to replace ETA' with either the granzyme B mutant R201K or MAPTau as human effector domains. The novel cytolytic fusion proteins were characterized with a recombinant human lymphocytic cell line developed using Transpo-mAb™ technology. Genes encoding a chimeric TTC-reactive immunoglobulin G were successfully integrated into the genome of the precursor B cell line REH so that the cells could present TTC-reactive BCRs on their surface. These cells were used to investigate the specific cytotoxicity of GrB(R201K)-TTC and TTC-MAPTau, revealing that the serpin proteinase inhibitor 9-resistant granzyme B R201K mutant induced apoptosis specifically in the lymphocytic cell line. Our data confirm that antigen-based fusion proteins containing granzyme B (R201K) are suitable candidates for the depletion of autoreactive B cells.
Assuntos
Linfócitos B/citologia , Engenharia Celular/métodos , Granzimas/genética , Toxoide Tetânico/imunologia , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Granzimas/metabolismo , Humanos , Imunotoxinas/metabolismo , Receptores de Antígenos de Linfócitos B/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Cadeia Única/metabolismo , Toxoide Tetânico/metabolismoRESUMO
Owing to their high affinities and specificities, rabbit monoclonal antibodies (mAbs) have demonstrated value and potential primarily as basic research and diagnostic reagents, but, in some cases, also as therapeutics. To accelerate access to rabbit mAbs bypassing immunization, we generated a large naïve rabbit antibody repertoire represented by a phage display library encompassing >10 billion independent antibodies in chimeric rabbit/human Fab format and validated it by next-generation sequencing. Panels of rabbit mAbs selected from this library against two emerging cancer targets, ROR1 and ROR2, revealed high diversity, affinity, and specificity. Moreover, ROR1- and ROR2-targeting rabbit mAbs demonstrated therapeutic utility as components of chimeric antigen receptor-engineered T cells, further corroborating the value of the naïve rabbit antibody library as a rich and virtually unlimited source of rabbit mAbs.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Fatores Imunológicos/isolamento & purificação , Animais , Anticorpos Monoclonais/uso terapêutico , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Proliferação de Células , Citocinas/metabolismo , Testes Imunológicos de Citotoxicidade , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fatores Imunológicos/uso terapêutico , Imunoterapia/métodos , Biblioteca de Peptídeos , Coelhos , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/uso terapêutico , Linfócitos T/imunologiaRESUMO
Misfolding of the cellular prion protein (PrPC) into the scrapie prion protein (PrPSc) results in progressive, fatal, transmissible neurodegenerative conditions termed prion diseases. Experimental and epidemiological evidence point toward a protracted, clinically silent phase in prion diseases, yet there is no diagnostic test capable of identifying asymptomatic individuals incubating prions. In an effort to identify early biomarkers of prion diseases, we have compared global transcriptional profiles in brains from pre-symptomatic prion-infected mice and controls. We identified Cst7, which encodes cystatin F, as the most strongly upregulated transcript in this model. Early and robust upregulation of Cst7 mRNA levels and of its cognate protein was validated in additional mouse models of prion disease. Surprisingly, we found no significant increase in cystatin F levels in both cerebrospinal fluid or brain parenchyma of patients with Creutzfeldt-Jakob disease compared to Alzheimer's disease or non-demented controls. Our results validate cystatin F as a useful biomarker of early pathogenesis in experimental models of prion disease, and point to unexpected species-specific differences in the transcriptional responses to prion infections.
Assuntos
Cistatinas/metabolismo , Doenças Priônicas/metabolismo , Animais , Biomarcadores , Encéfalo/metabolismo , Encéfalo/patologia , Cistatinas/líquido cefalorraquidiano , Cistatinas/genética , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Doenças Priônicas/líquido cefalorraquidiano , Doenças Priônicas/genética , Doenças Priônicas/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
In vitro antibody display and screening technologies geared toward the discovery and engineering of clinically applicable antibodies have evolved from screening artificial antibody formats, powered by microbial display technologies, to screening of natural, full-IgG molecules expressed in mammalian cells to readily yield lead antibodies with favorable properties in production and clinical applications. Here, we report the development and characterization of a novel, next-generation mammalian cell-based antibody display and screening platform called Transpo-mAb Display, offering straightforward and efficient generation of cellular libraries by using non-viral transposition technology to obtain stable antibody expression. Because Transpo-mAb Display uses DNA-transposable vectors with substantial cargo capacity, genomic antibody heavy chain expression constructs can be utilized that undergo the natural switch from membrane bound to secreted antibody expression in B cells by way of alternative splicing of Ig-heavy chain transcripts from the same genomic expression cassette. We demonstrate that stably transposed cells co-express transmembrane and secreted antibodies at levels comparable to those provided by dedicated constructs for secreted and membrane-associated IgGs. This unique feature expedites the screening and antibody characterization process by obviating the need for intermediate sequencing and re-cloning of individual antibody clones into separate expression vectors for functional screening purposes. In a series of proof-of-concept experiments, we demonstrate the seamless integration of antibody discovery with functional screening for various antibody properties, including binding affinity and suitability for preparation of antibody-drug conjugates.
Assuntos
Anticorpos Monoclonais/análise , Técnicas de Visualização da Superfície Celular/métodos , Ensaios de Triagem em Larga Escala/métodos , Imunoglobulina G/análise , Animais , Elementos de DNA Transponíveis , Avaliação Pré-Clínica de Medicamentos/métodos , Vetores Genéticos , HumanosRESUMO
Intracellular expression of single-chain antibodies (scFvs) represents a promising approach for selective interference with cellular proto-oncogenes such as the epidermal growth factor receptor (EGFR). Previously, we have shown that intrabodies targeted to the lumen of the endoplasmic reticulum prevent the transit of EGFR or the related ErbB2 molecule to the cell surface, thereby inactivating their transforming potential. While intramolecular disulfide bridges important for antibody stability are correctly formed during expression in the secretory pathway, scFvs expressed in the reducing environment of the cytosol are often inactive. To overcome this problem and to generate antibody fragments that interact with the intracellular domain of human EGFR in the cytoplasm, here we have chosen a two-step approach combining classical selection of scFvs by phage display with subsequent expression in yeast. After enrichment of EGFR-specific antibody fragments from a combinatorial library by biopanning, a yeast two-hybrid screen was performed using the intracellular domain of EGFR as bait. Screening of 1.5 x 10(5) preselected scFv plasmids under highly stringent conditions yielded 223 colonies that represented at least five independent scFv clones functional in the intracellular milieu of eukaryotic cells. Interaction of selected antibody fragments with the intracellular domain of EGFR was confirmed in GST pull-down and coimmunoprecipitation experiments. Upon cytoplasmic expression in human tumor cells, scFvs colocalized with EGFR at the plasma membrane demonstrating their functionality in vivo.