RESUMO
The use of immunomodulatory agents for the treatment of cancer is gaining a growing biopharmaceutical interest. Antibody-cytokine fusion proteins, namely immunocytokines, represent a promising solution for the regulation of the immune system at the site of disease. The three-dimensional arrangement of these molecules can profoundly influence their biological activity and pharmacokinetic properties. Structural techniques might provide important insight in the 3D arrangement of immunocytokines. Here, we performed structure investigations on clinical grade fusion proteins L19-IL2, IL12-L19L19 and L19L19-IL2 to elucidate their quaternary organization. Crystallographic characterization of the common L19 antibody fragment at a resolution of 2.0-Å was combined with low-resolution studies of the full-length chimeric molecules using small-angle synchrotron X-ray scattering (SAXS) and negative stain electron microscopy. Characterization of the full-length quaternary structures of the immunocytokines in solution by SAXS consistently supported the diabody structure in the L19-IL2 immunocytokine and allowed generation of low-resolution models of the chimeric proteins L19L19-IL2 and IL12-L19L19. Comparison with 3D reconstructions obtained from negative-stain electron microscopy revealed marked flexibility associated to the linker regions connecting the cytokine and the antibody components of the chimeric proteins. Collectively, our results indicate that low-resolution molecular structure characterizations provide useful complementary insights for the quality control of immunocytokines, constituting a powerful tool to guide the design and the subsequent optimization steps towards clinical enhancement of these chimeric protein reagents.
Assuntos
Citocinas/química , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Estrutura Molecular , Proteínas Recombinantes de Fusão/química , Espalhamento a Baixo Ângulo , Difração de Raios X/métodosRESUMO
We describe the cloning and characterization of a novel fusion protein (termed L19-mIL12), consisting of murine interleukin-12 in single-chain format, sequentially fused to the L19 antibody in tandem diabody format. The fusion protein bound avidly to the cognate antigen (the alternatively spliced EDB domain of fibronectin), retained the activity of the parental cytokine and was able to selectively localize to murine tumors in vivo, as shown by quantitative biodistribution analysis. L19-mIL12 exhibited a potent antitumor activity in immunocompetent mice bearing CT26 carcinomas and WEHI-164 sarcomas, which could be boosted by combination with checkpoint blockade, leading to durable cancer eradication. L19-mIL12 also inhibited tumor growth in mice with Lewis lung carcinoma (LLC), but in this case, cancer cures could not be obtained, both in monotherapy and in combination. A microscopic analysis and a depletion experiment of tumor-infiltrating leukocytes illustrated the contribution of NK cells and CD8+ T cells for the anticancer activity observed in both tumor models. Upon L19-mIL12 treatment, the density of regulatory T cells (Tregs) was strongly increased in LLC, but not in CT26 tumors. A FACS analysis also revealed that the majority of CD8+ T cells in CT26 tumors were specific to the retroviral AH1 antigen.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Antineoplásicos Imunológicos/farmacologia , Linfócitos T CD8-Positivos/imunologia , Neoplasias do Colo/imunologia , Sinergismo Farmacológico , Interleucina-12/administração & dosagem , Células Matadoras Naturais/imunologia , Animais , Anticorpos Monoclonais/imunologia , Apoptose , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/patologia , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Carcinoma Pulmonar de Lewis/imunologia , Carcinoma Pulmonar de Lewis/patologia , Proliferação de Células , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Feminino , Fibronectinas/imunologia , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/patologia , Ativação Linfocitária , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Sarcoma/tratamento farmacológico , Sarcoma/imunologia , Sarcoma/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
There is a considerable interest for the discovery and characterization of tumor-associated antigens, which may facilitate antibody-based pharmacodelivery strategies. Thrombospondin-1 and thrombospondin-2 are homologous secreted proteins, which have previously been reported to be overexpressed during remodeling typical for wound healing and tumor progression and to possibly play a functional role in cell proliferation, migration and apoptosis. To our knowledge, a complete immunohistochemical characterization of thrombospondins levels in normal rodent tissues has not been reported so far. Using antibody phage technology, we have generated and characterized monoclonal antibodies specific to murine thrombospondin-1 and thrombospondin-2, two antigens which share 62% aminoacid identity. An immunofluorescence analysis revealed that both antigens are virtually undetectable in normal mouse tissues, except for a weak staining of heart tissue by antibodies specific to thrombospondin-1. The analysis also showed that thrombospondin-1 was strongly expressed in 5/7 human tumors xenografted in nude mice, while it was only barely detectable in 3/8 murine tumors grafted in immunocompetent mice. By contrast, a high-affinity antibody to thrombospondin-2 revealed a much lower level of expression of this antigen in cancer specimens. Our analysis resolves ambiguities related to conflicting reports on thrombosponding expression in health and disease. Based on our findings, thrombospondin-1 (and not thrombospondin-2) may be considered as a target for antibody-based pharmacodelivery strategies, in consideration of its low expression in normal tissues and its upregulation in cancer.
Assuntos
Anticorpos Monoclonais/imunologia , Expressão Gênica/genética , Expressão Gênica/imunologia , Neoplasias/genética , Neoplasias/imunologia , Trombospondina 1/imunologia , Trombospondinas/imunologia , Animais , Antígenos de Neoplasias/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos NusRESUMO
S100A7 (psoriasin) is a calcium-binding protein that is upregulated in many types of cancer and often associated with poor prognosis. Its role in carcinogenesis has been associated with the stimulation of VEGF and EGF activity. The recent research showed that psoriasin directly interacts with αvß6 integrin, a protein related to the invasive phenotype of cancer. Moreover, this interaction promotes the αvß6-dependent invasive activity. The important function of S100A7 in carcinoma development determines a great need for valuable tools enabling its detection, quantification and also activity inhibition. Here, we show the selection of S100A7 specific antibody fragments from the human scFv phage library ETH-2 Gold. We have selected antibody fragments specific for psoriasin, purified them and analyzed by BIAcore affinity measurements. The best clone was subjected to affinity maturation procedure yielding molecule with a subnanomolar affinity towards human S100A7 protein. Selected clone was expressed in a bivalent format and applied for immunostaining analysis, which confirmed the ability of the antigen recognition in physiological conditions. We therefore propose that obtained antibody, that is the first phage display-derived human antibody against psoriasin, can serve as a useful psoriasin binding platform in research, diagnostics and therapy of cancer.
Assuntos
Anticorpos Antineoplásicos/isolamento & purificação , Afinidade de Anticorpos , Biomarcadores Tumorais/imunologia , Neoplasias/imunologia , Proteínas S100/imunologia , Anticorpos de Cadeia Única/isolamento & purificação , Anticorpos Antineoplásicos/química , Anticorpos Antineoplásicos/imunologia , Humanos , Biblioteca de Peptídeos , Proteína A7 Ligante de Cálcio S100 , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/imunologiaRESUMO
Transient transfection allows for fast production of recombinant proteins. However, the current bottlenecks in transient transfection are low titers and low specific productivity compared to stable cell lines. Here, we report an improved transient transfection protocol that yields titers exceeding 1 g/l in HEK293E cells. This was achieved by combining a new highly efficient polyethyleneimine (PEI)-based transfection protocol, optimized gene expression vectors, use of cell cycle regulators p18 and p21, acidic Fibroblast Growth Factor, exposure of cells to valproic acid and consequently the maintenance of cells at high cell densities (4 million cells/ml). This protocol was reproducibly scaled-up to a working volume of 2 l, thus delivering >1 g of purified protein just 2 weeks after transfection. This is the fastest approach to gram quantities of protein ever reported from cultivated mammalian cells and could initiate, upon further scale-up, a paradigm shift in industrial production of such proteins for any application in biotechnology.
Assuntos
Formação de Anticorpos/genética , Vetores Genéticos , Proteínas Recombinantes/biossíntese , Transfecção/métodos , Anticorpos/genética , Linhagem Celular , Meios de Cultura Livres de Soro , Humanos , Proteínas Recombinantes/genética , TransgenesRESUMO
Large-scale transient gene expression (TGE) in mammalian cells is a rapid method to generate recombinant proteins, but the volumetric productivity for secreted proteins is still more than an order of magnitude lower than the yields typically achieved with recombinant cell lines. Here transient recombinant protein production in Chinese hamster ovary cells transfected with linear 25 kDa polyethylenimine was significantly enhanced by incubation of the cells at temperatures ranging from 29 to 33 degrees C after DNA delivery. With this approach, transient recombinant antibody yields of 60-80 mg/L were achieved within 6 days of transfection. The increase in TGE correlated with the accumulation of cells in the G1 phase of the cell cycle, increased cell size, higher cell viability, higher steady-state levels of transgene mRNA, reduced consumption of nutrients, and decreased accumulation of waste products. The enhancement of TGE was not vector-dependent, but the presence of the woodchuck hepatitis virus post-transcriptional regulatory element in the 3' untranslated region of the transgene mRNA increased transient recombinant antibody expression more than 3-fold at 31 degrees C as compared to expression at 37 degrees C. The yields achieved by the low-temperature enhancement of TGE in CHO cells makes this technology feasible for the rapid production of gram amounts of secreted recombinant proteins at large scale (up to 100 L).
Assuntos
Temperatura Baixa , Expressão Gênica/fisiologia , Animais , Anticorpos/genética , Células CHO , Ciclo Celular/genética , Ciclo Celular/fisiologia , Tamanho Celular , Cricetinae , Cricetulus , Glicosilação , Proteínas de Fluorescência Verde/genética , Plasmídeos/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , TransfecçãoRESUMO
Purpose: Antibody-drug conjugates and small-molecule-drug conjugates have been proposed as alternatives to conventional anticancer cytotoxic agents, with the potential to deliver bioactive payloads to the site of disease, helping spare normal tissues.Experimental Design: Here, we describe a novel small-molecule-drug conjugate, based on a high-affinity ligand specific to carbonic anhydrase IX. The product featured a peptidic linker, suitable for cleavage in the tumor extracellular environment, and monomethyl auristatin E as cytotoxic payload.Results: A potent anticancer activity was observed in nude mice bearing SKRC-52 renal cell carcinoma xenografts, but no durable complete responses could be observed in this model. However, when the product was administered together with L19-IL2 (a clinical-stage fusion protein capable of delivering IL2 to the tumor neovasculature), all treated mice in the combination group could be rendered tumor free, in a process that favored the influx of natural killer cells into the tumor mass. The combination of L19-IL2 and the new small-molecule-drug conjugate also eradicated cancer in 100% of immunocompetent mice, bearing subcutaneously grafted CT26 colorectal cancer cells, which stably expressed carbonic anhydrase IX.Conclusions: These findings may be of clinical significance, because carbonic anhydrase IX is overexpressed in the majority of clear cell renal cell carcinomas and in approximately 30% of colorectal cancers. The targeted delivery of IL2 helps potentiate the action of targeted cytotoxics, leading to cancer eradication in models that cannot be cured by conventional chemotherapy. Clin Cancer Res; 24(15); 3656-67. ©2018 AACR.
Assuntos
Anidrase Carbônica IX/antagonistas & inibidores , Carcinoma de Células Renais/tratamento farmacológico , Neoplasias Colorretais/tratamento farmacológico , Interleucina-2/antagonistas & inibidores , Animais , Antígenos de Neoplasias/imunologia , Anidrase Carbônica IX/imunologia , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Imunoterapia/métodos , Interleucina-2/imunologia , Ligantes , Camundongos , Terapia de Alvo Molecular , Peptídeos/imunologia , Peptídeos/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A novel biopharmaceutical, consisting of the F8 mAb (specific to a splice isoform of fibronectin) simultaneously fused to both TNF and IL2, was found to react with the majority of solid tumors and hematologic malignancies in mouse and man, but not with healthy adult tissues. The product selectively localized to neoplastic lesions in vivo, as evidenced by quantitative biodistribution studies using radioiodinated protein preparations. When the potency of the cytokine payloads was matched by a single-point mutation, the resulting fusion protein (IL2-F8-TNFmut) eradicated soft-tissue sarcomas in immunocompetent mice, which did not respond to individual antibody-cytokine fusion proteins or by standard doxorubicin treatment. Durable complete responses were also observed in mice bearing CT26, C1498, and F9 tumors. The simultaneous delivery of multiple proinflammatory payloads to the cancer site conferred protective immunity against subsequent tumor challenges. A fully human homolog of IL2-F8-TNFmut, which retained selectivity similar to its murine counterpart when tested on human material, may open new clinical applications for the immunotherapy of cancer. Mol Cancer Ther; 16(11); 2442-51. ©2017 AACR.
Assuntos
Interleucina-2/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Sarcoma/terapia , Fator de Necrose Tumoral alfa/uso terapêutico , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Linhagem Celular Tumoral , Fibronectinas/genética , Fibronectinas/imunologia , Fibronectinas/uso terapêutico , Humanos , Imunoterapia , Interleucina-2/genética , Interleucina-2/imunologia , Camundongos , Mutação Puntual , Proteínas Recombinantes de Fusão/genética , Sarcoma/genética , Sarcoma/patologia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/uso terapêutico , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
The development of antibody-drug conjugates (ADC), a promising class of anticancer agents, has traditionally relied on the use of antibodies capable of selective internalization in tumor cells. We have recently shown that also noninternalizing antibodies, coupled to cytotoxic drugs by means of disulfide linkers that can be cleaved in the tumor extracellular environment, can display a potent therapeutic activity. Here, we have compared the tumor-targeting properties, drug release rates, and therapeutic performance of two ADCs, based on the maytansinoid DM1 thiol drug and on the F8 antibody, directed against the alternatively spliced Extra Domain A (EDA) domain of fibronectin. The antibody was used in IgG or in small immune protein (SIP) format. In both cases, DM1 was coupled to unpaired cysteine residues, resulting in a drug-antibody ratio of 2. In biodistribution studies, SIP(F8)-SS-DM1 accumulated in the tumor and cleared from circulation more rapidly than IgG(F8)-SS-DM1. However, the ADC based on the IgG format exhibited a higher tumor uptake at later time points (e.g., 33%IA/g against 8%IA/g at 24 hours after intravenous administration). In mouse plasma, surprisingly, the ADC products in IgG format were substantially more stable compared with the SIP format (half-lives >48 hours and <3 hours at 37°C, respectively), revealing a novel mechanism for the control of disulfide-based drug release rates. Therapy experiments in immunocompetent mice bearing murine F9 tumors revealed that SIP(F8)-SS-DM1 was more efficacious than IgG(F8)-SS-DM1 when the two products were compared either in an equimolar basis or at equal milligram doses.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos/farmacologia , Imunoconjugados/farmacologia , Maitansina/farmacologia , Neoplasias Experimentais/tratamento farmacológico , Animais , Anticorpos/metabolismo , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais Humanizados , Peso Corporal/efeitos dos fármacos , Células CHO , Linhagem Celular Tumoral , Cromatografia Líquida , Cricetinae , Cricetulus , Liberação Controlada de Fármacos , Eletroforese em Gel de Poliacrilamida , Feminino , Imunoconjugados/metabolismo , Imunoconjugados/farmacocinética , Espectrometria de Massas/métodos , Maitansina/metabolismo , Maitansina/farmacocinética , Camundongos , Microscopia de Fluorescência , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Distribuição Tecidual , Resultado do Tratamento , Carga Tumoral/efeitos dos fármacosRESUMO
The scFv-Fc format allows for rapid characterization of candidate scFvs isolated from phage display libraries before conversion into a full-length IgG. This format offers several advantages over the phage display-derived scFv, including bivalent binding, longer half-life, and Fc-mediated effector functions. Here, a detailed method is presented, which describes the cloning, expression, and purification of an scFv-Fc fragment, starting from scFv fragments obtained from a phage display library. This method facilitates the rapid screening of candidate antibodies, prior to a more time-consuming conversion into a full IgG format. Alternatively, the scFv-Fc format may be used in the clinic for therapeutic applications.
Assuntos
Fragmentos Fc das Imunoglobulinas/metabolismo , Anticorpos de Cadeia Única/metabolismo , Fragmentos Fc das Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Biblioteca de Peptídeos , Anticorpos de Cadeia Única/genéticaRESUMO
Several synthetic antibody phage display libraries have been created and used for the isolation of human monoclonal antibodies. The performance of antibody libraries, which is usually measured in terms of their ability to yield high-affinity binding specificities against target proteins of interest, depends both on technical aspects (such as library size and quality of cloning) and on design features (which influence the percentage of functional clones in the library and their ability to be used for practical applications). Here, we describe the design, construction and characterization of a combinatorial phage display library, comprising over 40 billion human antibody clones in single-chain fragment variable (scFv) format. The library was designed with the aim to obtain highly stable antibody clones, which can be affinity-purified on protein A supports, even when used in scFv format. The library was found to be highly functional, as >90% of randomly selected clones expressed the corresponding antibody. When selected against more than 15 antigens from various sources, the library always yielded specific and potent binders, at a higher frequency compared to previous antibody libraries. To demonstrate library performance in practical biomedical research projects, we isolated the human antibody G5, which reacts both against human and murine forms of the alternatively spliced BCD segment of tenascin-C, an extracellular matrix component frequently over-expressed in cancer and in chronic inflammation. The new library represents a useful source of binding specificities, both for academic research and for the development of antibody-based therapeutics.
Assuntos
Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Biblioteca de Peptídeos , Animais , Anticorpos Monoclonais/química , Especificidade de Anticorpos , Clonagem Molecular , Humanos , Camundongos , Modelos Moleculares , Estrutura Terciária de Proteína , Tenascina/química , Tenascina/imunologiaRESUMO
Bispecific and bifunctional antibodies are attracting considerable interest as innovative anti-cancer therapeutics, but their ability to selectively localize at the tumor site has rarely been studied by quantitative biodistribution studies in immunocompetent animal models or in patients. Here, we describe the production of a novel bifunctional antibody, consisting of the F8 antibody (specific to the alternatively spliced EDA domain of fibronectin) fused to the extracellular portion of CD86 (co-stimulatory molecule B7.2). However, the fusion molecule was unable to target tumors in vivo. These data suggest that bispecific antibodies do not always localize on tumors and should therefore be characterized by imaging or biodistribution studies.
Assuntos
Anticorpos Biespecíficos/biossíntese , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacocinética , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Humanizados , Linhagem Celular Tumoral , Fibronectinas/genética , Fibronectinas/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Distribuição TecidualRESUMO
PURPOSE: Interleukin-12 (IL12) is a potent proinflammatory cytokine with antitumor activity. Its heterodimeric nature makes it compatible with a large variety of different immunocytokine formats. Here we report the design, production, and characterization of a novel immunocytokine, based on the fusion of the F8 antibody (specific to the alternatively spliced EDA domain of fibronectin, a marker of tumor neovasculature) with IL12 (termed IL12-F8-F8). EXPERIMENTAL DESIGN: We developed a novel immunocytokine based on the sequential fusion of interleukin-12 as a single polypeptide with two F8 antibodies in single-chain Fv (scFv) format. The fusion protein was characterized in vitro, and its targeting performance was assessed in vivo. The immunocytokine antitumor activity was studied as monotherapy as well as in combination therapies in three different murine tumor models. Moreover, depletion experiments and tumor analysis revealed a dominant role of natural killer cells for the mechanism of action. RESULTS: IL12-F8-F8 can be produced in mammalian cells, yielding a product of good pharmaceutical quality, capable of selective localization on the tumor neovasculature in vivo, as judged by quantitative biodistribution analysis with radioiodinated protein preparations. The protein potently inhibited tumor growth in three different immunocompetent syngeneic models of cancer. The treatment was generally well tolerated. Moreover, the IL12-F8-F8 fusion protein could be produced both with murine IL12 (mIL12) and with human IL12 (hIL12). CONCLUSIONS: The potent antitumor activity of mIL12-F8-F8, studied alone or in combination with paclitaxel in different tumor models, paves the way to the clinical development of the fully human immunocytokine.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Interleucina-12/imunologia , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Proteínas Recombinantes de Fusão/imunologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Humanizados , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Modelos Animais de Doenças , Feminino , Humanos , Interferon gama/sangue , Interferon gama/imunologia , Interleucina-12/genética , Interleucina-12/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Neoplasias/irrigação sanguínea , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/imunologia , Paclitaxel/administração & dosagem , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/farmacocinética , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/metabolismo , Distribuição Tecidual , Resultado do Tratamento , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/imunologiaRESUMO
Human monoclonal antibodies (mAbs) can routinely be isolated from phage display libraries against virtually any protein available in sufficient purity and quantity, but library design can influence epitope coverage on the target antigen. Here we describe the construction of a novel synthetic human antibody phage display library that incorporates hydrophilic or charged residues at position 52 of the CDR2 loop of the variable heavy chain domain, instead of the serine residue found in the corresponding germline gene. The novel library was used to isolate human mAbs to various antigens, including the alternatively-spliced EDA domain of fibronectin, a marker of tumor angiogenesis. In particular, the mAb 2H7 was proven to bind to a novel epitope on EDA, which does not overlap with the one recognized by the clinical-stage F8 antibody. F8 and 2H7 were used for the construction of chelating recombinant antibodies (CRAbs), whose tumor-targeting properties were assessed in vivo in biodistribution studies in mice bearing F9 teratocarcinoma, revealing a preferential accumulation at the tumor site.
Assuntos
Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Fibronectinas/imunologia , Biblioteca de Peptídeos , Sequência de Aminoácidos , Animais , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacocinética , Anticorpos Monoclonais/farmacocinética , Especificidade de Anticorpos/imunologia , Sequência de Bases , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Feminino , Humanos , Camundongos , Camundongos da Linhagem 129 , Dados de Sequência Molecular , Transplante de Neoplasias , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/farmacocinética , Distribuição TecidualRESUMO
We generated and characterized novel antibody-cytokine fusion proteins ("immunocytokines") based on murine interleukin-7 (IL7), an immunomodulatory protein which has previously shown anti-cancer activity in preclinical models and whose human counterpart is currently being investigated in clinical trials. The sequential fusion of the clinical-stage antibody fragment scFv(F8), specific to a tumor-associated splice isoform of fibronectin, yielded an immunocytokine (termed "F8-mIL7") of insufficient pharmaceutical quality and in vivo tumor targeting performance, with a striking dose dependence on tumor targeting selectivity. By contrast, a novel immunocytokine design (termed "F8-mIL7-F8"), in which two scFv moieties were fused at the N- and C-terminus of murine IL7, yielded a protein of excellent pharmaceutical quality and with improved tumor-targeting performance [tumor: blood ratio=16:1, 24h after injection]. Both F8-mIL7 and F8-mIL7-F8 could induce tumor growth retardation in immunocompetent mice, but were not able to eradicate F9 tumors. The combination of F8-mIL7-F8 with paclitaxel led to improved therapeutic results, which were significantly better compared to those obtained with saline treatment. The study indicates how the engineering of novel immunocytokine formats may help generate fusion proteins of acceptable pharmaceutical quality, for those immunomodulatory proteins which do not lend themselves to a direct fusion with antibody fragments.
Assuntos
Interleucina-7/uso terapêutico , Neoplasias/tratamento farmacológico , Proteínas Recombinantes de Fusão/uso terapêutico , Animais , Clonagem Molecular , Humanos , Interleucina-7/farmacocinética , Radioisótopos do Iodo , Camundongos , Paclitaxel/farmacologia , Proteínas Recombinantes de Fusão/farmacocinética , Anticorpos de Cadeia Única/imunologia , Distribuição Tecidual/efeitos dos fármacosRESUMO
Valproic acid (VPA) is a small molecule that inhibits histone deacetylase activity. Here we report that VPA increases recombinant mRNA and protein levels in transiently transfected CHO DG44 cells. In the presence of VPA, transient recombinant antibody yields of up to 40 mg/L were achieved in simple batch cultures. The steady-state levels of the IgG light and heavy chain mRNAs were nearly 10 times higher than in the untreated control transfection even though the level of transfected plasmid DNA was the same in the presence or absence of VPA. The combination of VPA treatment and incubation of the transfected cells in mildly hypothermic conditions resulted in recombinant antibody yields of over 90 mg/L by 6 days post-transfection in batch cultures. The results demonstrated that the treatment of transfected CHO DG44 cells with VPA is a cost-effective strategy for enhancing transient gene expression by increasing the transgene mRNA levels.
Assuntos
RNA Mensageiro/metabolismo , Proteínas Recombinantes/biossíntese , Ácido Valproico/farmacologia , Animais , Células CHO , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Cricetulus , DNA/análise , DNA/genética , Humanos , Cadeias Leves de Imunoglobulina/biossíntese , Cadeias Leves de Imunoglobulina/genética , Cadeias gama de Imunoglobulina/biossíntese , Cadeias gama de Imunoglobulina/genética , Reação em Cadeia da Polimerase , Biossíntese de Proteínas , RNA Mensageiro/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura , TransfecçãoRESUMO
Cell expansion, gene transfer and protein production were all executed with a single serum-free, animal protein-free commercial medium designed for suspension-adapted Chinese hamster ovary cells (CHO DG44). This is a most important process to consider for clinical production of recombinant proteins. The transfection with polyethylenimine (PEI) was shown here to be scalable using both stirred-tank bioreactors of 3- and 150-l and novel agitated cultivation vessels (50 ml ventilated centrifuge tubes and 1-l square-shaped glass bottles) that lack any instrumentation. The transient transfections spanned a range of working volumes from 2 ml to 80 l. The maximum transient recombinant antibody yield was 22 mg/l, the highest ever reported for a multiliter transfection in CHO. The transiently expressed protein had the same extent of glycosylation as the same antibody produced from a stably transfected recombinant CHO cell line.