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1.
Cancer Res ; 83(11): 1883-1904, 2023 06 02.
Artículo en Inglés | MEDLINE | ID: mdl-37074042

RESUMEN

The EGFR and TGFß signaling pathways are important mediators of tumorigenesis, and cross-talk between them contributes to cancer progression and drug resistance. Therapies capable of simultaneously targeting EGFR and TGFß could help improve patient outcomes across various cancer types. Here, we developed BCA101, an anti-EGFR IgG1 mAb linked to an extracellular domain of human TGFßRII. The TGFß "trap" fused to the light chain in BCA101 did not sterically interfere with its ability to bind EGFR, inhibit cell proliferation, or mediate antibody-dependent cellular cytotoxicity. Functional neutralization of TGFß by BCA101 was demonstrated by several in vitro assays. BCA101 increased production of proinflammatory cytokines and key markers associated with T-cell and natural killer-cell activation, while suppressing VEGF secretion. In addition, BCA101 inhibited differentiation of naïve CD4+ T cells to inducible regulatory T cells (iTreg) more strongly than the anti-EGFR antibody cetuximab. BCA101 localized to tumor tissues in xenograft mouse models with comparable kinetics to cetuximab, both having better tumor tissue retention over TGFß "trap." TGFß in tumors was neutralized by approximately 90% in animals dosed with 10 mg/kg of BCA101 compared with 54% in animals dosed with equimolar TGFßRII-Fc. In patient-derived xenograft mouse models of head and neck squamous cell carcinoma, BCA101 showed durable response after dose cessation. The combination of BCA101 and anti-PD1 antibody improved tumor inhibition in both B16-hEGFR-expressing syngeneic mouse models and in humanized HuNOG-EXL mice bearing human PC-3 xenografts. Together, these results support the clinical development of BCA101 as a monotherapy and in combination with immune checkpoint therapy. SIGNIFICANCE: The bifunctional mAb fusion design of BCA101 targets it to the tumor microenvironment where it inhibits EGFR and neutralizes TGFß to induce immune activation and to suppress tumor growth.


Asunto(s)
Anticuerpos Monoclonales Humanizados , Carcinoma de Células Escamosas , Neoplasias de Cabeza y Cuello , Neoplasias , Animales , Humanos , Ratones , Anticuerpos Monoclonales Humanizados/uso terapéutico , Carcinoma de Células Escamosas/terapia , Línea Celular Tumoral , Cetuximab/farmacología , Cetuximab/uso terapéutico , Receptores ErbB/metabolismo , Neoplasias de Cabeza y Cuello/terapia , Factor de Crecimiento Transformador beta , Microambiente Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Neoplasias/terapia
3.
Sci Rep ; 11(1): 13233, 2021 06 24.
Artículo en Inglés | MEDLINE | ID: mdl-34168178

RESUMEN

Sequence variants (SV) in protein bio therapeutics can be categorized as unwanted impurities and may raise serious concerns in efficacy and safety of the product. Early detection of specific sequence modifications, that can result in altered physicochemical and or biological properties, is therefore desirable in product manufacturing. Because of their low abundance, and finite resolving power of conventional analytical techniques, they are often overlooked in early drug development. Here, we present a case study where trace amount of a sequence variant is identified in a monoclonal antibody (mAb) based therapeutic protein by LC-MS/MS and the structural and functional features of the SV containing mAb is assessed using appropriate analytical techniques. Further, a very sensitive selected reaction monitoring (SRM) technique is developed to quantify the SV which revealed both prominent and inconspicuous nature of the variant in process chromatography. We present the extensive characterization of a sequence variant in protein biopharmaceutical and first report on control of sequence variants to < 0.05% in final drug product by utilizing SRM based mass spectrometry method during the purification steps.


Asunto(s)
Anticuerpos Monoclonales/genética , Variación Genética/genética , Anticuerpos Monoclonales/aislamiento & purificación , Contaminación de Medicamentos , Cromatografía de Gases y Espectrometría de Masas , Espectrometría de Masas , Mapeo Peptídico , Péptidos/genética
4.
Appl Microbiol Biotechnol ; 105(8): 3061-3074, 2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-33821296

RESUMEN

Manufacturing of insulin and its analogues relied upon in vitro enzymatic cleavages of its precursor forms (single chain precursor, SCP) at both ends of a connecting peptide (C-peptide) that links the respective B-chain and A-chains to corresponding final forms. We have demonstrated a simplified approach of cleaving P. pastoris expressed SCP, distinctly at one site for conversion to insulin glargine. The design of the precursor was made in such a way that there is no C-peptide in the precursor which needs to be removed in the final product. Instead of traditional both side cleavage of the C-peptide and removing the C-peptide (by trypsin), followed by 2nd enzyme reaction (typically carboxipeptidase B), present work established only one side cleavage of the sequence by only trypsin converts the precursor to final insulin glargine product. The novel design of the precursor helped in producing insulin glargine in a single step with an application of single enzyme brought high degree of process efficiencies. Highly purified product was generated through two reversed phase high pressure chromatographic steps. Purified product was compared with the reference product Lantus®, for various physico-chemical and biological properties. Primary, secondary and tertiary structures as well as biological pharmaco-dynamic effects were found comparable. High cell density fermentation that gave a good yield of the SCP, a single step conversion to insulin glargine, enabled by a unique design of SCP and a distinct purification approach, has led to a simplified and economical manufacturing process of this important drug used to treat diabetes. KEY POINTS: • Novel concept for processing single chain precursor of insulin glargine • Simple and economic process for insulin glargine • Physicochemical characterization and animal Pharmacodynamics show similarity to Lantus.


Asunto(s)
Insulina , Pichia , Hipoglucemiantes , Insulina Glargina , Péptidos , Pichia/genética , Saccharomycetales
5.
Protein Expr Purif ; 118: 1-9, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26470649

RESUMEN

Insulin glargine is a slow acting analog of insulin used in diabetes therapy. It is produced by recombinant DNA technology in different hosts namely E. coli and Pichia pastoris. In our previous study, we have described the secretion of fully folded two-chain Insulin glargine into the medium by over-expression of Kex2 protease. The enhanced levels of the Kex2 protease was responsible for the processing of the glargine precursor with in the host. Apart from the two-chain glargine product we observed a small proportion of arginine clipped species. This might be due to the clipping of arginine present at the C-terminus of the B-chain as it is exposed upon Kex2 cleavage. The carboxypeptidase precursor Kex1 is known to be responsible for clipping of C-terminal lysine or arginine of the proteins or peptides. In order to address this issue we created a Kex1 knock out in the host using Cre/loxP mechanism of targeted gene deletion. When two-chain glargine was expressed in the Kex1 knock out host of P. pastoris GS115 the C-terminal clipped species reduced by ∼80%. This modification further improved the process by reducing the levels of product related impurities.


Asunto(s)
Carboxipeptidasas/genética , Proteínas Fúngicas/genética , Insulina Glargina/metabolismo , Pichia/enzimología , Carboxipeptidasas/metabolismo , Proteínas Fúngicas/metabolismo , Silenciador del Gen , Humanos , Pichia/genética , Pichia/metabolismo , Transporte de Proteínas , Proteolisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
6.
Appl Microbiol Biotechnol ; 99(1): 327-36, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25239036

RESUMEN

Glargine is an analog of Insulin currently being produced by recombinant DNA technology using two different hosts namely Escherichia coli and Pichia pastoris. Production from E. coli involves the steps of extraction of inclusion bodies by cell lysis, refolding, proteolytic cleavage and purification. In P. pastoris, a single-chain precursor with appropriate disulfide bonding is secreted to the medium. Downstream processing currently involves use of trypsin which converts the precursor into two-chain final product. The use of trypsin in the process generates additional impurities due to presence of Lys and Arg residues in the Glargine molecule. In this study, we describe an alternate approach involving over-expression of endogenous Kex2 proprotein convertase, taking advantage of dibasic amino acid sequence (Arg-Arg) at the end of B-chain of Glargine. KEX2 gene over-expression in Pichia was accomplished by using promoters of varying strengths to ensure production of greater levels of fully functional two-chain Glargine product, confirmed by HPLC and mass analysis. In conclusion, this new production process involving Kex2 protease over-expression improves the downstream process efficiency, reduces the levels of impurities generated and decreases the use of raw materials.


Asunto(s)
Biotecnología/métodos , Expresión Génica , Insulina de Acción Prolongada/metabolismo , Péptido Hidrolasas/metabolismo , Pichia/metabolismo , Tecnología Farmacéutica/métodos , Cromatografía Líquida de Alta Presión , Insulina Glargina , Insulina de Acción Prolongada/genética , Insulina de Acción Prolongada/aislamiento & purificación , Espectrometría de Masas , Péptido Hidrolasas/genética , Pichia/genética , Regiones Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo
7.
J Microbiol Biotechnol ; 24(3): 337-45, 2014 Mar 28.
Artículo en Inglés | MEDLINE | ID: mdl-24317479

RESUMEN

Pichia pastoris is one of the most widely used expression systems for the secretory expression of recombinant proteins. The secretory expression in P. pastoris usually makes use of the prepro MATα sequence from Saccharomyces cerevisiae, which has a dibasic amino acid cleavage site at the end of the signal sequence. This is efficiently processed by Kex2 protease, resulting in the secretion of high levels of proteins to the medium. However, the proteins that are having the internal accessible dibasic amino acids such as KR and RR in the coding region cannot be expressed using this signal sequence, as the protein will be fragmented. We have identified a new signal sequence of 18 amino acids from a P. pastoris protein that can secrete proteins to the medium efficiently. The PMT1-gene-inactivated P. pastoris strain secretes a ~30 kDa protein into the extracellular medium. We have identified this protein by determining its N-terminal amino acid sequence. The protein secreted has four DDDK concatameric internal repeats. This protein was not secreted in the wild-type P. pastoris under normal culture conditions. We show that the 18-amino-acid signal peptide at the N-terminal of this protein is useful for secretion of heterologous proteins in Pichia.


Asunto(s)
Pichia/genética , Pichia/metabolismo , Señales de Clasificación de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
8.
Protein Expr Purif ; 92(1): 128-33, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-24075932

RESUMEN

Sorbitol is used as a non-repressive carbon source to develop fermentation process for Mut(s) recombinant clones obtained using the AOX1 promoter in Pichia pastoris. Sorbitol dehydrogenase is an enzyme in the carbohydrate metabolism that catalyzes reduction of D-fructose into D-sorbitol in the presence of NADH. The small stretch of 211bps upstream region of sorbitol dehydrogenase coding gene has all the promoter elements like CAAT box, GC box, etc. It is able to promote protein production under repressive as well as non-repressive carbon sources. In this study, the strength of the sorbitol dehydrogenase promoter was evaluated by expression of two heterologous proteins: human serum albumin and erythrina trypsin inhibitor. Sorbitol dehydrogenase promoter allowed constitutive expression of recombinant proteins in all carbon sources that were tested to grow P. pastoris and showed activity similar to GAP promoter. The sorbitol dehydrogenase promoter was active in all the growth phases of the P. pastoris.


Asunto(s)
L-Iditol 2-Deshidrogenasa/genética , Pichia/enzimología , Pichia/genética , Regiones Promotoras Genéticas , Secuencia de Bases , Carbono/metabolismo , Clonación Molecular , Erythrina/genética , Expresión Génica , Humanos , L-Iditol 2-Deshidrogenasa/metabolismo , Datos de Secuencia Molecular , Pichia/metabolismo , Proteínas de Plantas/genética , Proteínas Recombinantes/genética , Albúmina Sérica/genética
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