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1.
Anal Chem ; 92(13): 8827-8835, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32453570

RESUMO

Most of the current FDA and EMA approved therapeutic monoclonal antibodies (mAbs) are based on humanized or human IgG1, 2, or 4 subclasses and engineered variants. On the structural side, these subclasses are characterized by specific interchain disulfide bridge connections. Different analytical techniques have been reported to assess intact IgGs subclasses, with recently special interest in native ion mobility (IM) and collision induced unfolding (CIU) mass spectrometry (MS). However, these two techniques exhibit significant limitations to differentiate mAb subclasses at the intact level. In the present work, we aimed at developing a unique IM-MS-based approach for the characterization of mAb subclasses at the middle level. Upon IdeS-digestion, the unfolding patterns of the F(ab')2 and Fc domains were simultaneously analyzed in a single run to provide deeper structural insights of the mAb scaffold. The unfolding patterns associated with the F(ab')2 domains are completely different in terms of unfolding energies and number of transitions. Thereby, F(ab')2 regions are the diagnostic domain to provide specific unfolding signatures to differentiate IgG subclasses and provide more confident subclass categorization than CIU on intact mAbs. In addition, the potential of middle-level CIU was evaluated through the characterization of eculizumab, a hybrid therapeutic IgG2/4 mAb. The unfolding signatures of both domains were allowed to corroborate, within a single run, the hybrid nature of eculizumab as well as specific subclass domain assignments to the F(ab')2 and Fc regions. Altogether, our results confirm the suitability of middle-level CIU of F(ab')2 domains for subclass categorization of canonical and more complex new generation engineered antibodies and related products.


Assuntos
Anticorpos Monoclonais/análise , Imunoglobulina G/análise , Espectrometria de Massas/métodos , Adalimumab/análise , Adalimumab/química , Adalimumab/classificação , Anticorpos Monoclonais/química , Anticorpos Monoclonais/classificação , Anticorpos Monoclonais Humanizados/análise , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/classificação , Cromatografia Líquida de Alta Pressão , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Imunoglobulina G/classificação , Espectrometria de Mobilidade Iônica , Desdobramento de Proteína
2.
Mol Cancer Ther ; 19(1): 168-177, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31594825

RESUMO

The insulin-like growth factor type 1 receptor (IGF-1R) is important in tumorigenesis, and its overexpression occurs in numerous tumor tissues. To date, therapeutic approaches based on mAbs and tyrosine kinase inhibitors targeting IGF-1R have only shown clinical benefit in specific patient populations. We report a unique IGF-1R-targeted antibody-drug conjugate (ADC), W0101, designed to deliver a highly potent cytotoxic auristatin derivative selectively to IGF-1R overexpressing tumor cells. The mAb (hz208F2-4) used to prepare the ADC was selected for its specific binding properties to IGF-1R compared with the insulin receptor, and for its internalization properties. Conjugation of a novel auristatin derivative drug linker to hz208F2-4 did not alter its binding and internalization properties. W0101 induced receptor-dependent cell cytotoxicity in vitro when applied to various cell lines overexpressing IGF-1R, but it did not affect normal cells. Efficacy studies were conducted in several mouse models expressing different levels of IGF-1R to determine the sensitivity of the tumors to W0101. W0101 induced potent tumor regression in certain mouse models. Interestingly, the potency of W0101 correlated with the expression level of IGF-1R evaluated by IHC. In an MCF-7 breast cancer model with high-level IGF-1R expression, a single injection of W0101 3 mg/kg led to strong inhibition of tumor growth. W0101 provides a potential new therapeutic option for patients overexpressing IGF-1R. A first-in-human trial of W0101 is currently ongoing to address clinical safety.


Assuntos
Imunoconjugados/uso terapêutico , Neoplasias/tratamento farmacológico , Receptor IGF Tipo 1/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Imunoconjugados/farmacologia , Camundongos , Camundongos Nus , Neoplasias/patologia
3.
Front Pharmacol ; 7: 27, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26924984

RESUMO

Based on the recent development of NanoLuc luciferase (Nluc), a small (19 kDa), highly stable, ATP independent, bioluminescent protein, an extremely robust and ultra high sensitivity screening system has been developed whereby primary hits of therapeutic antibodies and antibody fragments could be characterized and quantified without purification. This system is very versatile allowing cellular and solid phase ELISA but also homogeneous BRET based screening assays, relative affinity determinations with competition ELISA and direct Western blotting. The new Nluc protein fusion represents a "swiss army knife solution" for today and future high throughput antibody drug screenings.

4.
Int J Cancer ; 132(6): 1463-74, 2013 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-22886345

RESUMO

To identify new potential targets in oncology, functional approaches were developed using tumor cells as immunogens to select monoclonal antibodies targeting membrane receptors involved in cell proliferation. For that purpose cancer cells were injected into mice and resulting hybridomas were screened for their ability to inhibit cell proliferation in vitro. Based on this functional approach coupled to proteomic analysis, a monoclonal antibody specifically recognizing the human junctional adhesion molecule-A (JAM-A) was defined. Interestingly, compared to both normal and tumor tissues, we observed that JAM-A was mainly overexpressed on breast, lung and kidney tumor tissues. In vivo experiments demonstrated that injections of anti-JAM-A antibody resulted in a significant tumor growth inhibition of xenograft human tumors. Treatment with monoclonal antibody induced a decrease of the Ki67 expression and downregulated JAM-A levels. All together, our results show for the first time that JAM-A can interfere with tumor proliferation and suggest that JAM-A is a potential novel target in oncology. The results also demonstrate that a functional approach coupled to a robust proteomic analysis can be successful to identify new antibody target molecules that lead to promising new antibody-based therapies against cancers.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Moléculas de Adesão Celular/fisiologia , Neoplasias/tratamento farmacológico , Receptores de Superfície Celular/fisiologia , Animais , Moléculas de Adesão Celular/análise , Moléculas de Adesão Celular/antagonistas & inibidores , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Antígeno Ki-67/análise , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/patologia , Receptores de Superfície Celular/análise , Receptores de Superfície Celular/antagonistas & inibidores
5.
Anal Chem ; 81(15): 6364-73, 2009 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-19588976

RESUMO

Monoclonal antibodies (mAbs) have taken on an increasing importance for the treatment of various diseases including cancers, immunological disorders, and other pathologies. These large biomolecules display specific structural features, which affect their efficiency and need, therefore, to be extensively characterized using sensitive and orthogonal analytical techniques. Among them, mass spectrometry (MS) has become the method of choice to study mAb amino acid sequences as well as their post-translational modifications. In the present work, recent noncovalent MS-technologies including automated chip-based nanoelectrospray MS and traveling wave ion mobility MS were used for the first time to characterize immune complexes involving both murine and humanized mAb 6F4 directed against human JAM-A, a newly identified antigenic protein (Ag) overexpressed in tumor cells. MS-based structural insights evidenced that heterogeneous disulfide bridge pairings of recombinant JAM-A alter neither its native structure nor mAbs 6F4 recognition properties. Investigations focused on mAb:Ag complexes revealed that, similarly to murine mAb, humanized mAb 6F4 binds selectively up to four antigen molecules with a similar affinity, confirming in this way the reliability of the humanization process. Noncovalent MS appears as an additional supporting technique for therapeutic mAbs lead characterization and development.


Assuntos
Anticorpos Monoclonais/química , Complexo Antígeno-Anticorpo/química , Dissulfetos/química , Imunoglobulinas/química , Preparações Farmacêuticas/química , Proteínas Recombinantes/química , Espectrometria de Massas por Ionização por Electrospray , Animais , Anticorpos Monoclonais/isolamento & purificação , Moléculas de Adesão Celular/imunologia , Humanos , Imunoglobulinas/imunologia , Camundongos , Fragmentos de Peptídeos/química , Ligação Proteica , Receptores de Superfície Celular
6.
Blood ; 104(6): 1778-83, 2004 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-15166032

RESUMO

Although human CD56(+)CD3(-) natural killer (NK) cells participate in immune responses against microorganisms, their capacity to directly recognize and be activated by pathogens remains unclear. These cells encode members of the Toll-like receptor (TLR) family, involved in innate cell activation on recognition of pathogen-associated molecular patterns (PAMPs). We therefore evaluated whether the 2 bacterial protein PAMPs, the outer membrane protein A from Klebsiella pneumoniae (KpOmpA) and flagellin, which signal through TLR2 and TLR5, respectively, may directly stimulate human NK cells. These proteins induce interferon-gamma (IFN-gamma) production by NK cells and synergize with interleukin-2 (IL-2) and proinflammatory cytokines in PAMP-induced activation. Similar results were obtained using CD56(+)CD3(+) (NKR-expressing) T cells. NK cells from TLR2(-/-) mice fail to respond to KpOmpA, demonstrating TLR involvement in this effect. Defensins are antimicrobial peptides expressed mainly by epithelial cells and neutrophils that disrupt the bacterial membrane, leading to pathogen death. We show that NK cells and NKR-expressing T cells constitutively express alpha-defensins and that KpOmpA and flagellin rapidly induce their release. These data demonstrate for the first time that highly purified NK cells directly recognize and respond to pathogen components through TLRs and evidence defensins as a novel and direct cytotoxic pathway involved in NK cell-mediated protection against microorganisms.


Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Flagelina/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , alfa-Defensinas/biossíntese , Animais , Células Cultivadas , Citocinas/metabolismo , Humanos , Células Matadoras Naturais/citologia , Ativação Linfocitária , Camundongos , Receptor 2 Toll-Like , Receptor 5 Toll-Like , Receptores Toll-Like , alfa-Defensinas/genética , alfa-Defensinas/metabolismo
7.
Vaccine ; 21(25-26): 3765-74, 2003 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-12922110

RESUMO

Outer membrane proteins (OMP) are expressed in Gram-negative bacterial cell wall. OmpA from Klebsiella pneumoniae (KpOmpA) has been shown to bind and to activate selectively antigen presenting cells (APCs), eliciting protective CTL responses. In this study, we investigated whether OmpX, another member of the OMP family and structurally related to OmpA, exhibits the same immune properties. Using recombinant OmpX from Escherichia coli (EcOmpX), we report that EcOmpX binds to and is internalized by human APCs. However, EcOmpX does not activate APCs. EcOmpX acts as an efficient carrier protein as it induces a potent and Th1/Th2 mixed anti-TNP humoral response. However, adjuvant is required to generate a protective anti-tumoral immune response in mice injected with a tumor model antigen coupled to EcOmpX. Collectively, these data show that EcOmpX is recognized by innate cells but does not activate them, suggesting that EcOmpX does not provide a signal danger to APCs. In conclusion, this study provides information on the molecular mechanisms involved in the recognition and activation of innate cells by bacterial outer membrane proteins.


Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Escherichia coli/imunologia , Escherichia coli/imunologia , Hidrolases , Animais , Anticorpos Antineoplásicos/biossíntese , Formação de Anticorpos/imunologia , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/biossíntese , Biotina , DNA Complementar/biossíntese , DNA Complementar/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/biossíntese , Citometria de Fluxo , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Haptenos/imunologia , Humanos , Imunidade Celular/imunologia , Ativação de Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Monócitos/imunologia , Transplante de Neoplasias , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Toxoide Tetânico/imunologia
8.
J Immunol Methods ; 272(1-2): 199-210, 2003 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-12505724

RESUMO

Mutations in the Escherichia coli (E. coli) and Salmonella lpxM gene have been shown to result in strains which grow normally and which produce a non-myristoylated lipopolysaccharide (nmLPS) with strongly reduced endotoxicity. Using homologous recombination, we inactivated the lpxM gene in BL21 (DE3), a strain widely used for the production of recombinant proteins. This led to a derivative unaffected in its capacity to support the production of recombinant proteins. This new strain expresses non-myristoylated LPS that induces markedly less activation and maturation of monocyte-derived dendritic cells (DC), as assessed by nuclear translocation of nuclear factor kappa B (NF-kappaB), production of TNF-alpha and IL-8 or expression of CD86. Activation of the main signal transducing receptor for extracellular LPS, Toll like receptor (TLR) 4 in conjunction with the soluble accessory protein MD-2 was also markedly decreased. The modified BL21 strain represents a new application of lpxM inactivation for the expression of proteins to be tested on dendritic cells or other LPS sensitive cells/receptor complexes. It is likely to be useful for the identification of new proteins activating the innate immune response and to reducing the risk linked with low level of endotoxin contamination in therapeutic recombinant proteins.


Assuntos
Células Dendríticas/citologia , Células Dendríticas/imunologia , Proteínas de Drosophila , Lipídeo A/genética , Lipídeo A/imunologia , Antígenos CD/metabolismo , Antígenos de Superfície/metabolismo , Antígeno B7-2 , Sequência de Bases , Diferenciação Celular , DNA Bacteriano/genética , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Escherichia coli/genética , Escherichia coli/imunologia , Genes Bacterianos , Humanos , Técnicas In Vitro , Interleucina-8/biossíntese , Lipídeo A/farmacologia , Antígeno 96 de Linfócito , Glicoproteínas de Membrana/metabolismo , Mutagênese Insercional , Mutação , NF-kappa B/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Receptor 4 Toll-Like , Receptores Toll-Like , Fator de Necrose Tumoral alfa/biossíntese
9.
Eur J Biochem ; 269(1): 233-9, 2002 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11784317

RESUMO

The beta 1,3-glycosyltransferase enzymes identified to date share several conserved regions and conserved cysteine residues, all being located in the putative catalytic domain. To investigate the importance of these motifs and cysteines for the enzymatic activity, 14 mutants of the murine beta 1,3-galactosyltransferase-I gene were constructed and expressed in Sf9 insect cells. Seven mutations abolished the galactosyltransferase activity. Kinetic analysis of the other seven active mutants revealed that three of them showed a threefold to 21-fold higher apparent K(m) with regard to the donor substrate UDP-galactose relative to the wild-type enzyme, while two mutants had a sixfold to 7.5-fold increase of the apparent K(m) value for the acceptor substrate N-acetylglucosamine-beta-p-nitrophenol. Taken together, our results indicate that the conserved residues W101 and W162 are involved in the binding of the UDP-galactose donor, the residue W315 in the binding of the N-acetylglucosamine-beta-p-nitrophenol acceptor, and the domain including E264 appears to participate in the binding of both substrates.


Assuntos
Acetilglucosamina/metabolismo , Galactosiltransferases/química , Uridina Difosfato Galactose/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência de Bases , Sequência Conservada , Galactosiltransferases/fisiologia , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Spodoptera
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