RESUMO
Cross-linking of the Fcγ receptors expressed on the surface of hematopoietic cells by IgG immune complexes triggers the activation of key immune effector mechanisms, including antibody-dependent cell mediated cytotoxicity (ADCC). A conserved N-glycan positioned at the N-terminal region of the IgG CH 2 domain is critical in maintaining the quaternary structure of the molecule for Fcγ receptor engagement. The removal of a single core fucose residue from the N-glycan results in a considerable increase in affinity for FcγRIIIa leading to an enhanced receptor-mediated immunoeffector function. The enhanced potency of the molecule translates into a number of distinct advantages in the development of IgG antibodies for cancer therapy. In an effort to significantly increase the potency of an anti-CD20, IgG1 molecule, we selectively targeted the de novo GDP-fucose biosynthesis pathway of the host CHO cell line to generate >80% afucosylated IgG1 resulting in enhanced FcγRIIIa binding (13-fold) and in vitro ADCC cell-based activity (11-fold). In addition, this effective glycoengineering strategy also allowed for the utilization of the alternate GDP-fucose salvage pathway to provide a fast and efficient mechanism to manipulate the N-glycan fucosylation level to modulate IgG immune effector function.
Assuntos
Cricetulus/metabolismo , Imunoglobulina G/biossíntese , Engenharia de Proteínas , Rituximab/biossíntese , Animais , Cricetulus/genética , Glicosilação , Imunoglobulina G/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Rituximab/genéticaRESUMO
The role of biotherapeutic proteins in the prevention and treatment of diseases such as cancers, infectious diseases, and autoimmune disorders continues to grow. The biological activity or "potency" of a biotherapeutic reflects its mechanism of action and thus its efficacy. The potency of these complex biomolecules cannot be quantitatively correlated to chemical and physical properties and thus must be determined by comparison to a reference standard, typically using a cell-based bioassay. This lack of an absolute method for determining potency, along with test method variability and potential for bias make assignment and monitoring of reference standard potency a major challenge during pharmaceutical development and manufacturing. The reference standard links the potency of dosages administered to the patient with those of original clinical studies. Therefore, the assignment of potency to biotherapeutic reference standards is vital for assuring the quality of medicines for patients. In this work, we propose a comprehensive roadmap for assigning potency to reference standards that is compliant with the two-tier system of standards as recommended in regulatory guidance. The roadmap includes statistical approaches for study design and acceptance criteria that are risk-based and phase-appropriate. It also provides mitigation approaches for potential assay bias.
Assuntos
Bioensaio , Projetos de Pesquisa , Humanos , Proteínas , Padrões de ReferênciaRESUMO
Polyamides are a class of heterocyclic small molecules with the potential of controlling gene expression by binding to the minor groove of DNA in a sequence-specific manner. To evaluate the feasibility of this class of compounds as antiviral therapeutics, molecules were designed to essential sequence elements occurring numerous times in the HPV genome. This sequence element is bound by a virus-encoded transcription and replication factor E2, which binds to a 12 bp recognition site as a homodimeric protein. Here, we take advantage of polyamide:DNA and E2:DNA co-crystal structural information and advances in polyamide synthetic chemistry to design tandem hairpin polyamides that are capable of displacing the major groove-binding E2 homodimer from its DNA binding site. The binding of tandem hairpin polyamides and the E2 DNA binding protein to the DNA site is mutually exclusive even though the two ligands occupy opposite faces of the DNA double helix. We show with circular permutation studies that the tandem hairpin polyamide prevents the intrinsic bending of the E2 DNA site important for binding of the protein. Taken together, these results illustrate the feasibility of inhibiting the binding of homodimeric, major groove-binding transcription factors by altering the local DNA geometry using minor groove-binding tandem hairpin polyamides.
Assuntos
DNA Viral/metabolismo , Proteínas de Ligação a DNA , Nylons/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Sítios de Ligação/genética , DNA Viral/química , Modelos Biológicos , Conformação de Ácido Nucleico , Nylons/química , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Proteínas Oncogênicas Virais/química , Papillomaviridae/genética , Papillomaviridae/metabolismo , Ligação ProteicaRESUMO
Humanized monoclonal antibodies (mAbs) are the fastest growing class of biological therapeutics that are being developed for various medical indications, and more than 30 mAbs are already approved and in the market place. Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important biological function attributed to the mechanism of action of several therapeutic antibodies, particularly oncology targeting mAbs. The ADCC assay is a complicated and highly variable assay. Thus, the use of an ADCC assay as a lot release test or a stability test for clinical trial batches of mAbs has been a substantial challenge to install in quality control laboratories. We describe here the development and validation of an alternate approach, an ADCC-reporter gene assay that is based on the key attributes of the PBMC-based ADCC assay. We tested the biological relevance of this assay using an anti-CD20 based model and demonstrated that this ADCC-reporter assay correlated well with standard ADCC assays when induced with the drugable human isotypes [IgG1, IgG2, IgG4, IgG4S > P (S228P) and IgG4PAA (S228P, F234A, L235A)] and with IgG1 isotype variants with varying amounts of fucosylation. This data demonstrates that the ADCC-reporter gene assay has performance characteristics (accuracy, precision and robustness) to be used not only as a potency assay for lot release and stability testing for antibody therapeutics, but also as a key assay for the characterization and process development of therapeutic molecules.
Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Citotoxicidade Celular Dependente de Anticorpos/genética , Testes Imunológicos de Citotoxicidade , Genes Reporter , Antígenos CD20/imunologia , Estudos de Viabilidade , Humanos , Leucócitos Mononucleares/imunologia , Variações Dependentes do Observador , Controle de Qualidade , Reprodutibilidade dos TestesRESUMO
Brahma (BRM) and Brahma-related gene 1 (BRG1) are the ATP-dependent catalytic subunits of the SWI/SNF family of chromatin-remodeling complexes. These complexes are involved in essential processes such as cell cycle, growth, differentiation, and cancer. Using imaging approaches in a cell line that harbors tandem repeats of stably integrated copies of the steroid responsive MMTV-LTR (mouse mammary tumor virus-long terminal repeat), we show that BRG1 and BRM are recruited to the MMTV promoter in a hormone-dependent manner. The recruitment of BRG1 and BRM resulted in chromatin remodeling and decondensation of the MMTV repeat as demonstrated by an increase in the restriction enzyme accessibility and in the size of DNA fluorescence in situ hybridization (FISH) signals. This chromatin remodeling event was concomitant with an increased occupancy of RNA polymerase II and transcriptional activation at the MMTV promoter. The expression of ATPase-deficient forms of BRG1 (BRG1-K-R) or BRM (BRM-K-R) inhibited the remodeling of local and higher order MMTV chromatin structure and resulted in the attenuation of transcription. In vivo photobleaching experiments provided direct evidence that BRG1, BRG1-K-R, and BRM chromatin-remodeling complexes have distinct kinetic properties on the MMTV array, and they dynamically associate with and dissociate from MMTV chromatin in a manner dependent on hormone and a functional ATPase domain. Our data provide a kinetic and mechanistic basis for the BRG1 and BRM chromatin-remodeling complexes in regulating gene expression at a steroid hormone inducible promoter.
Assuntos
Cromatina/química , DNA Helicases/metabolismo , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Receptores de Glucocorticoides/metabolismo , Fatores de Transcrição/metabolismo , Trifosfato de Adenosina/química , Animais , Linhagem Celular Tumoral , Cromatina/metabolismo , Hidrólise , Hibridização in Situ Fluorescente , Cinética , Vírus do Tumor Mamário do Camundongo/metabolismo , Camundongos , Modelos Biológicos , RNA Polimerase II/químicaRESUMO
Although histone deacetylases (HDACs) are generally viewed as corepressors, we show that HDAC1 serves as a coactivator for the glucocorticoid receptor (GR). Furthermore, a subfraction of cellular HDAC1 is acetylated after association with the GR, and this acetylation event correlates with a decrease in promoter activity. HDAC1 in repressed chromatin is highly acetylated, while the deacetylase found on transcriptionally active chromatin manifests a low level of acetylation. Acetylation of purified HDAC1 inactivates its deacetylase activity, and mutation of the critical acetylation sites abrogates HDAC1 function in vivo. We propose that hormone activation of the receptor leads to progressive acetylation of HDAC1 in vivo, which in turn inhibits the deacetylase activity of the enzyme and prevents a deacetylation event that is required for promoter activation. These findings indicate that HDAC1 is required for the induction of some genes by the GR, and this activator function is dynamically modulated by acetylation.