RESUMO
Amber codon suppression for the insertion of non-natural amino acids (nnAAs) is limited by competition with release factor 1 (RF1). Here we describe the genome engineering of a RF1 mutant strain that enhances suppression efficiency during cell-free protein synthesis, without significantly impacting cell growth during biomass production. Specifically, an out membrane protease (OmpT) cleavage site was engineered into the switch loop of RF1, which enables its conditional inactivation during cell lysis. This facilitates extract production without additional processing steps, resulting in a scaleable extract production process. The RF1 mutant extract allows nnAA incorporation at previously intractable sites of an IgG1 and at multiple sites in the same polypeptide chain. Conjugation of cytotoxic agents to these nnAAs, yields homogeneous antibody drug conjugates (ADCs) that can be optimized for conjugation site, drug to antibody ratio (DAR) and linker-warheads designed for efficient tumor killing. This platform provides the means to generate therapeutic ADCs inaccessible by other methods that are efficient in their cytotoxin delivery to tumor with reduced dose-limiting toxicities and thus have the potential for better clinical impact.
Assuntos
Aminoácidos/química , Imunoconjugados , Fatores de Terminação de Peptídeos/química , Fatores de Terminação de Peptídeos/genética , Engenharia de Proteínas , Sítios de Ligação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida , Códon de Terminação , Estabilidade de Medicamentos , Humanos , Imunoconjugados/química , Imunoconjugados/isolamento & purificação , Imunoconjugados/metabolismo , Imunoconjugados/farmacologia , Imunoglobulina G/química , Imunoglobulina G/farmacologia , Espectrometria de Massas , Modelos Moleculares , Mutação , Fatores de Terminação de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Trastuzumab/química , Trastuzumab/farmacologiaRESUMO
Antibody drug conjugates (ADCs) have progressed from hypothesis to approved therapeutics in less than 30 years, and the technologies available to modify both the antibodies and the cytotoxic drugs are expanding rapidly. For reasons well reviewed previously, the field is trending strongly toward homogeneous, defined antibody conjugation. In this review we present the antibody and small molecule chemistries that are currently used and being explored to develop specific, homogenous ADCs.
Assuntos
Anticorpos Monoclonais/química , Imunoconjugados/química , Engenharia de Proteínas , Bibliotecas de Moléculas Pequenas/química , Tecnologia Farmacêutica/métodos , Animais , Desenho de Fármacos , HumanosRESUMO
Selection technologies such as ribosome display enable the rapid discovery of novel antibody fragments entirely in vitro. It has been assumed that the open nature of the cell-free reactions used in these technologies limits selections to single-chain protein fragments. We present a simple approach for the selection of multi-chain proteins, such as antibody Fab fragments, using ribosome display. Specifically, we show that a two-chain trastuzumab (Herceptin) Fab domain can be displayed in a format which tethers either the heavy or light chain to the ribosome while retaining functional antigen binding. Then, we constructed synthetic Fab HC and LC libraries and performed test selections against carcinoembryonic antigen (CEA) and vascular endothelial growth factor (VEGF). The Fab selection output was reformatted into full-length immunoglobulin Gs (IgGs) and directly expressed at high levels in an optimized cell-free system for immediate screening, purification and characterization. Several novel IgGs were identified using this cell-free platform that bind to purified CEA, CEA positive cells and VEGF.
Assuntos
Técnicas de Visualização da Superfície Celular/métodos , Sistema Livre de Células , Fragmentos Fab das Imunoglobulinas , Biblioteca de Peptídeos , Anticorpos/genética , Anticorpos Monoclonais Humanizados/genética , Antígeno Carcinoembrionário/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Imunoglobulina G/genética , Trastuzumab , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Antibody-drug conjugates (ADCs) are a targeted chemotherapeutic currently at the cutting edge of oncology medicine. These hybrid molecules consist of a tumor antigen-specific antibody coupled to a chemotherapeutic small molecule. Through targeted delivery of potent cytotoxins, ADCs exhibit improved therapeutic index and enhanced efficacy relative to traditional chemotherapies and monoclonal antibody therapies. The currently FDA-approved ADCs, Kadcyla (Immunogen/Roche) and Adcetris (Seattle Genetics), are produced by conjugation to surface-exposed lysines, or partial disulfide reduction and conjugation to free cysteines, respectively. These stochastic modes of conjugation lead to heterogeneous drug products with varied numbers of drugs conjugated across several possible sites. As a consequence, the field has limited understanding of the relationships between the site and extent of drug loading and ADC attributes such as efficacy, safety, pharmacokinetics, and immunogenicity. A robust platform for rapid production of ADCs with defined and uniform sites of drug conjugation would enable such studies. We have established a cell-free protein expression system for production of antibody drug conjugates through site-specific incorporation of the optimized non-natural amino acid, para-azidomethyl-l-phenylalanine (pAMF). By using our cell-free protein synthesis platform to directly screen a library of aaRS variants, we have discovered a novel variant of the Methanococcus jannaschii tyrosyl tRNA synthetase (TyrRS), with a high activity and specificity toward pAMF. We demonstrate that site-specific incorporation of pAMF facilitates near complete conjugation of a DBCO-PEG-monomethyl auristatin (DBCO-PEG-MMAF) drug to the tumor-specific, Her2-binding IgG Trastuzumab using strain-promoted azide-alkyne cycloaddition (SPAAC) copper-free click chemistry. The resultant ADCs proved highly potent in in vitro cell cytotoxicity assays.
Assuntos
Aminoácidos/química , Imunoconjugados/química , Linhagem Celular , Sistema Livre de Células , Cromatografia Líquida , Ensaios de Triagem em Larga Escala , Humanos , Imunoconjugados/farmacologia , Espectrometria de Massas em TandemRESUMO
We describe protein synthesis, folding and assembly of antibody fragments and full-length aglycosylated antibodies using an Escherichia coli-based open cell-free synthesis (OCFS) system. We use DNA template design and high throughput screening at microliter scale to rapidly optimize production of single-chain Fv (scFv) and Fab antibody fragments that bind to human IL-23 and IL-13α1R, respectively. In addition we demonstrate production of aglycosylated immunoglobulin G (IgG 1) trastuzumab. These antibodies are produced rapidly over several hours in batch mode in standard bioreactors with linear scalable yields of hundreds of milligrams/L over a 1 million-fold change in scales up to pilot scale production. We demonstrate protein expression optimization of translation initiation region (TIR) libraries from gene synthesized linear DNA templates, optimization of the temporal assembly of a Fab from independent heavy chain and light chain plasmids and optimized expression of fully assembled trastuzumab that is equivalent to mammalian expressed material in biophysical and affinity based assays. These results illustrate how the open nature of the cell-free system can be used as a seamless antibody engineering platform from discovery to preclinical development of aglycosylated monoclonal antibodies and antibody fragments as potential therapeutics.
Assuntos
Biossíntese de Proteínas , Anticorpos de Cadeia Única/biossíntese , Transcrição Gênica , Sistema Livre de Células/química , Glicosilação , Humanos , Subunidade alfa1 de Receptor de Interleucina-13/antagonistas & inibidores , Subunidade alfa1 de Receptor de Interleucina-13/genética , Subunidade alfa1 de Receptor de Interleucina-13/imunologia , Interleucina-23/antagonistas & inibidores , Interleucina-23/genética , Interleucina-23/imunologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologiaRESUMO
BACKGROUND: This study compared the angiogenic effects of developmental endothelial locus-1 (DEL-1), vascular endothelial growth factor (VEGF), as well as the negative control, beta-galactosidase (beta-gal), in a porcine model of cardiac ischemia. METHODS: Twenty pigs underwent left circumflex artery occlusions. After 3 weeks, the animals received myocardial injections of adenovirus expressing beta-gal (n=6), DEL-1 (n=7), or VEGF (n=7). At 7 weeks, animals were assessed for both function and coronary flow and compared with baseline measurements. RESULTS: Regional wall motion index and global ejection fraction showed deterioration in function in the beta-gal group and no change in the VEGF and DEL-1 groups between the treatment and harvest time points. Preload recruitable stroke work suggested functional improvement in the VEGF group (35.8 +/- 8.6 vs 56.4 +/- 17.8, p = 0.033). The increase in the DEL-1 group was not statistically significant (27.3 +/- 9.8 vs, 40.2 +/- 19.4, p = 0.067). The beta-gal group exhibited minimal change (30.7 +/- 14.8 vs 35.9 +/- 12.1, p = 0.96). Regional blood flow as assessed by fluorescent microspheres was improved under stress conditions in the VEGF group (1.00 +/- 0.15 vs 1.15 +/-0.22, p = 0.03). CONCLUSIONS: Treatment with VEGF led to a modest improvement in regional blood flow and cardiac function in previously ischemic myocardial tissue.
Assuntos
Proteínas de Transporte/farmacologia , Isquemia Miocárdica/fisiopatologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Adenoviridae/genética , Animais , Angiografia Coronária , Circulação Coronária/efeitos dos fármacos , Feminino , Frequência do Gene , Suínos , Função Ventricular Esquerda/efeitos dos fármacos , Função Ventricular Esquerda/fisiologiaRESUMO
The ECM protein Del-1 is one of several novel ECM proteins that accumulate around angiogenic blood vessels in embryonic and tumor tissue and promote angiogenesis in the absence of exogenous growth factors. Del-1 expressed in mouse or rabbit ischemic hind-limb muscle by gene transfer rapidly promotes new blood vessel formation and restores muscle function. This angiogenic ECM protein initiates angiogenesis by binding to integrin alphavbeta5 on resting endothelium, thereby resulting in expression of the transcription factor Hox D3 and integrin alphavbeta3. Hox D3 converts resting endothelium to angiogenic endothelium by inducing expression of proangiogenic molecules such as integrin alphavbeta3. These findings provide evidence for an angiogenic switch that can be initiated in the absence of exogenous growth factors and indicate that the angiogenic matrix protein Del-1 may be a useful tool for the therapy of ischemic disease.
Assuntos
Proteínas de Transporte/genética , Proteínas de Ligação a DNA , Terapia Genética , Isquemia/terapia , Neovascularização Fisiológica , Animais , Proteínas de Ligação ao Cálcio , Proteínas de Transporte/metabolismo , Moléculas de Adesão Celular , Fatores de Crescimento Endotelial/genética , Membro Posterior/irrigação sanguínea , Proteínas de Homeodomínio/genética , Integrinas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Isquemia/fisiopatologia , Linfocinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Vitronectina/metabolismo , Fatores de Transcrição , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Del1 is a unique alpha v beta 3 integrin ligand that is produced by endothelial cells, and thus provides an autocrine signaling pathway in this cell type. It is expressed transiently in the embryo and mediates cell attachment, migration, and activation of cytoplasmic signaling molecules in focal contacts. Del1 also activates angiogenesis in the chick chorioallantoic membrane assay. Reexpression of this embryonic signaling molecule has now been documented in naturally occurring human tumors, where it is expressed by both tumor cells and angiogenic endothelial cells, suggesting that Del1 is important in mediating angiogenesis under pathophysiological conditions in the adult. To investigate the role of Del1 in tumor growth and angiogenesis, human 143B osteosarcoma cells and murine Lewis lung carcinoma cells were engineered to express Del1 and compared to control transfectants for their ability to produce tumors in nude or syngeneic mice, respectively. Del1 expressing tumors showed a two- to fourfold increase in capillary density and an accelerated rate of growth. Expression of Del1 also correlated with a decrease in apoptosis in tumor cells in vivo. Taken together, these data suggest that Del1 acts as an angiogenic factor in the context of solid tumor formation and that this increase in vascularization accelerates tumor growth through decreased apoptosis.
Assuntos
Proteínas de Transporte/fisiologia , Neoplasias/irrigação sanguínea , Neoplasias/metabolismo , Animais , Apoptose , Western Blotting , Proteínas de Ligação ao Cálcio , Carcinoma Pulmonar de Lewis , Moléculas de Adesão Celular , Divisão Celular , Humanos , Imuno-Histoquímica , Integrina alfaVbeta3/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Microscopia de Fluorescência , Mitose , Modelos Biológicos , Neovascularização Patológica , Fatores de Tempo , Transfecção , Células Tumorais CultivadasRESUMO
Del1 is a matrix protein transiently expressed by embryonic endothelial cells. It was recently demonstrated that vascular endothelial cells adhere and interact with Del1 through alpha(v)beta(3)- integrins, providing an autocrine angiogenic signaling pathway in this cell type. To determine whether Del1 might signal to other cell types in the vessel wall in a paracrine fashion, studies were conducted with vascular smooth muscle cells (VSMC). Del1 promoted adhesion and migration of VSMC in a dose-dependent fashion. These functions were mediated through alpha(v)beta(3)-integrins, as the vitronectin receptor inhibitory peptide containing penacillamine (PCN) arginine-glycine-aspartic acid (PCN-RGD) and an antibody specific for the alpha(v)beta(3)-integrin specifically blocked both adhesion and migration. Adhesion of VSMC to Del1 was associated with organization of actin filaments and formation of focal contacts enriched in vinculin and alpha(v)beta(3). Furthermore, Del1 supported VSMC proliferation at least in part by inhibiting these cells from undergoing apoptosis. These data, in conjunction with evidence that Del1 expression is reactivated in vascular injury, suggest that Del1 may have a paracrine role in vessel wall development and remodeling.