RESUMO
The primary therapeutic goal for the treatment of diabetes is maintenance of a long-term, near-normoglycemic condition and prevention of the onset or progression of the complications associated with the disease. Although several analogs of human insulin have been developed, the currently prescribed long-acting insulin analogs do not provide a stable basal glycemia for more than a few hours. Here, we report the development of Albulin, a long-acting insulin analog obtained by direct gene fusion of a single-chain human insulin to human serum albumin. Albulin showed an elimination t(1/2) of approximately 7 h in normoglycemic mice. In vitro pharmacodynamic profiles for Albulin characterized by receptor binding, inhibition of gluconeogenesis, induction of glucose uptake, and global regulation of gene expression in relevant cell types showed that Albulin produced similar activity profiles compared with that of recombinant human insulin. A single Albulin administration in vivo normalized blood glucose level in diabetic mice in a relatively peakless and sustained (24-h) fashion. A further reduction in glucose levels was achieved by administering a recombinant human insulin a few hours after Albulin injection in mice, indicating the potential for Albulin therapy in combination with available fast-acting insulin derivatives. In summary, Albulin displays characteristics of a potent long-acting insulin analog that can be evaluated for use as a novel insulin therapy for patients with insulin-dependent diabetes.
Assuntos
Insulina/genética , Insulina/farmacocinética , Proteínas Recombinantes de Fusão/farmacocinética , Albumina Sérica/genética , Albumina Sérica/farmacocinética , Células 3T3 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Clonagem Molecular , Escherichia coli , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Sintéticos , Glucose/metabolismo , Humanos , Insulina/farmacologia , Insulina de Ação Prolongada , Insulina Regular Humana , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Receptor de Insulina/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Albumina Sérica/farmacologia , Albumina Sérica HumanaRESUMO
We identified seven putative splice variants of the human IG20 gene. Four variants namely, IG20, MADD, IG20-SV2 and DENN-SV are expressed in human tissues. While DENN-SV is constitutively expressed in all tissues, expression of IG20 appears to be regulated. Interestingly, overexpression of DENN-SV enhanced cell replication and resistance to treatments with TNFalpha, vinblastine, etoposide and gamma-radiation. In contrast, IG20 expression suppressed cell replication and increased susceptibility to the above treatments. Moreover, cells that were resistant and susceptible to TNFalpha-induced apoptosis exclusively expressed endogenous DENN-SV and IG20, respectively. When PA-1 ovarian cancer cells that are devoid of endogenous IG20 variant, but express higher levels of DENN-SV, were transfected with IG20, they showed reduced cell proliferation and increased susceptibility to apoptosis induced by TNFalpha, TRAIL and gamma-radiation. This indicated that overexpression of IG20 can override endogenous DENN-SV function. CrmA reversed the effects of IG20, but not DENN-SV. In contrast, dominant-negative-I-kappa B reversed the effects of DENN-SV, but not IG20, and showed that DENN-SV most likely exerted its effects through NFkappaB activation. Together, our data show that IG20 gene can play a novel and significant role in regulating cell proliferation, survival and death through alternative mRNA splicing.
Assuntos
Processamento Alternativo , Apoptose/efeitos dos fármacos , Variação Genética , Fatores de Troca do Nucleotídeo Guanina/genética , Antineoplásicos/farmacologia , Divisão Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte , Regulação da Expressão Gênica , Células HeLa , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/farmacologia , NF-kappa B/metabolismo , Transfecção , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The recognition that few human diseases are thoroughly addressed by mono-specific, monoclonal antibodies (mAbs) continues to drive the development of antibody therapeutics with additional specificities and enhanced activity. Historically, efforts to engineer additional antigen recognition into molecules have relied predominantly on the reformatting of immunoglobulin domains. In this report we describe a series of fully functional mAbs to which additional specificities have been imparted through the recombinant fusion of relatively short polypeptides sequences. The sequences are selected for binding to a particular target from combinatorial libraries that express linear, disulfide-constrained, or domain-based structures. The potential for fusion of peptides to the N- and C- termini of both the heavy and light chains affords the bivalent expression of up to four different peptides. The resulting molecules, called zybodies, can gain up to four additional specificities, while retaining the original functionality and specificity of the scaffold antibody. We explore the use of two clinically significant oncology antibodies, trastuzumab and cetuximab, as zybody scaffolds and demonstrate functional enhancements in each case. The affect of fusion position on both peptide and scaffold function is explored, and penta-specific zybodies are demonstrated to simultaneously engage five targets (ErbB2, EGFR, IGF-1R, Ang2 and integrin αvß3). Bispecific, trastuzumab-based zybodies targeting ErbB2 and Ang2 are shown to exhibit superior efficacy to trastuzumab in an angiogenesis-dependent xenograft tumor model. A cetuximab-based bispecific zybody that targeting EGFR and ErbB3 simultaneously disrupted multiple intracellular signaling pathways; inhibited tumor cell proliferation; and showed efficacy superior to that of cetuximab in a xenograft tumor model.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Especificidade de Anticorpos , Neoplasias/terapia , Peptídeos/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Sequência de Aminoácidos , Angiopoietina-2/química , Angiopoietina-2/genética , Angiopoietina-2/imunologia , Animais , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/metabolismo , Anticorpos Biespecíficos/uso terapêutico , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Cetuximab , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Dados de Sequência Molecular , Neovascularização Patológica , Peptídeos/genética , Peptídeos/imunologia , Peptídeos/metabolismo , Engenharia de Proteínas/métodos , Receptor ErbB-2/química , Receptor ErbB-2/genética , Receptor ErbB-2/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Transdução de Sinais , Trastuzumab , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Despite the clinical success of anti-tumor necrosis factor (TNF) therapies in the treatment of inflammatory conditions such as rheumatoid arthritis, Crohn disease and psoriasis, full control of the diseases only occurs in a subset of patients and there is a need for new therapeutics with improved efficacy against broader patient populations. One possible approach is to combine biological therapeutics, but both the cost of the therapeutics and the potential for additional toxicities needs to be considered. In addition to the various mediators of immune and inflammatory pathways, angiogenesis is reported to contribute substantially to the overall pathogenesis of inflammatory diseases. The combination of an anti-angiogenic agent with anti-TNF into one molecule could be more efficacious without the risk of severe immunosuppression. To evaluate this approach with our Zybody technology, we generated bispecific antibodies that contain an Ang2 targeting peptide genetically fused to the anti-TNF antibody adalimumab (Humira®). The bispecific molecules retain the binding and functional characteristics of the anti-TNF antibody, but with additional activity that neutralizes Ang2. In a TNF transgenic mouse model of arthritis, the bispecific anti-TNF-Ang2 molecules showed a dose-dependent reduction in both clinical symptoms and histological scores that were significantly better than that achieved by adalimumab alone.
Assuntos
Angiopoietina-2/imunologia , Anticorpos Biespecíficos/uso terapêutico , Anticorpos Monoclonais Humanizados/imunologia , Proteínas Recombinantes de Fusão/uso terapêutico , Fator de Necrose Tumoral alfa/imunologia , Adalimumab , Angiopoietina-2/genética , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais Humanizados/genética , Artrite Reumatoide/imunologia , Artrite Reumatoide/terapia , Linhagem Celular , Modelos Animais de Doenças , Humanos , Inflamação/terapia , Camundongos , Camundongos Transgênicos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Resultado do TratamentoRESUMO
CD80 and CD86 expressed on the surface of antigen-presenting cells interact with cytotoxic T lymphocyte antigen-4 [CTLA-4 (CD152)] expressed on activated T cells and mediate critical T cell inhibitory signals. CD80 and CD86 are type I glycoproteins, and are made up of two extracellular (EC) Ig-like domains-a transmembrane region and a cytoplasmic tail. The N-terminal (V domain) and membrane-proximal (C) domains share homology with the variable region (V) and the constant region (C) of Ig respectively. Co-crystallographic structures of both CD80 and CD86 bound to CTLA-4 indicate that there is no direct interaction of the C domain of either CD80 or CD86 with the CTLA-4. In contrast, previous mutagenesis studies have identified specific amino acids within the C domain of CD80 that are critical for CTLA-4 binding. To further understand the importance of C domains in the functioning of CD80 and CD86, we constructed chimeric human CD80 and CD86 molecules by swapping their respective C domains, and tested their ability to stimulate T cells. A Chinese hamster ovary (CHO) cell line expressing CD86 activated murine T cells. In contrast, CHO cells expressing either CD80 or a chimeric construct of the CD86 V domain and the CD80 C domain showed a significantly reduced activation. Our studies further demonstrated that the decreased activation by cells expressing the CD80 or a chimera containing CD80 C domain is most likely due to enhanced CTLA-4 binding. From these results we conclude that C domains play a critical, albeit indirect, role in determining CTLA-4 binding affinities and co-stimulatory properties.