RESUMO
Human epidermal growth factor receptor 2 (HER-2) is overexpressed in many malignant tumors. The anti-HER2 antibody trastuzumab has been approved for treating HER2-positive early and metastatic breast cancers. Pseudomonas exotoxin A (PE), a bacterial toxin of Pseudomonas aeruginosa, consists of an A-domain with enzymatic activity and a B-domain with cell binding activity. Recombinant immunotoxins comprising the HER2(scFv) single-chain Fv from trastuzumab and the PE24B catalytic fragment of PE display promising cytotoxic effects, but immunotoxins are typically insoluble when expressed in the cytoplasm of Escherichia coli, and thus they require solubilization and refolding. Herein, a recombinant immunotoxin gene was fused with maltose binding protein (MBP) and overexpressed in a soluble form in E. coli. Removal of the MBP yielded stable HER2(scFv)-PE24B at 91% purity; 0.25 mg of pure HER2(scFv)-PE24B was obtained from a 500 mL flask culture. Purified HER2(scFv)-PE24B was tested against four breast cancer cell lines differing in their surface HER2 level. The immunotoxin showed stronger cytotoxicity than HER2(scFv) or PE24B alone. The IC50 values for HER2(scFv)-PE24B were 28.1 ± 2.5 pM (n = 9) and 19 ± 1.4 pM (n = 9) for high HER2-positive cell lines SKBR3 and BT-474, respectively, but its cytotoxicity was lower against MDA-MB-231 and MCF7. Thus, fusion with MBP can facilitate the soluble expression and purification of scFv immunotoxins.
Assuntos
ADP Ribose Transferases , Antineoplásicos Imunológicos/farmacologia , Toxinas Bacterianas , Exotoxinas , Imunotoxinas/farmacologia , Proteínas Ligantes de Maltose , Receptor ErbB-2/antagonistas & inibidores , Proteínas Recombinantes de Fusão/farmacologia , Anticorpos de Cadeia Única , Fatores de Virulência , ADP Ribose Transferases/genética , Toxinas Bacterianas/genética , Linhagem Celular Tumoral , Sobrevivência Celular , Relação Dose-Resposta a Droga , Escherichia coli/genética , Escherichia coli/metabolismo , Exotoxinas/genética , Expressão Gênica , Engenharia Genética , Vetores Genéticos/genética , Humanos , Imunotoxinas/genética , Imunotoxinas/isolamento & purificação , Proteínas Ligantes de Maltose/genética , Espectrometria de Massas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Anticorpos de Cadeia Única/genética , Fatores de Virulência/genética , Exotoxina A de Pseudomonas aeruginosaRESUMO
OBJECTIVES: Epithelial growth factor receptor (EGFR), as a malignancy marker, is overly expressed in multiple solid tumors including colorectal neoplasms, one of the most prevalent malignancies worldwide. The main objective of this study is to enhance the efficacy of anti-tumor therapy targeting EGFR by constructing a novel EGFR-specific immunotoxin (C-CUS245C) based on Cetuximab and recombinant Cucurmosin (CUS245C). METHODS: E. coli BL21 (DE3) PlysS (E. coli) was used to express CUS245C with a cysteine residue inserting to the C-terminus of Cucurmosin. Then immobilized metal ion affinity chromatography (IMAC) was used to purify CUS245C. The chemical conjugation method was used for the preparation of C-CUS245C. Then dialysis and IMAC were used to purify C-CUS245C. Western blot as well as SDS-PAGE was carried out to characterize the formation of C-CUS245C. At last the anti-colorectal cancer activity of C-CUS245C was investigated in vitro and in vivo. RESULTS: CUS245C with high purity could be obtained from the prokaryotic system. C-CUS245C was successfully constructed and highly purified. The cytotoxicity assays in vitro showed a significant proliferation inhibition of C-CUS245C on EGFR-positive cells for 120 h with IC50 values less than 0.1 pM. Besides, the anti-tumor efficacy of C-CUS245C was remarkably more potent than that of Cetuximab, CUS245C, and C + CUS245C (P < 0.001). Whereas the cytotoxicity of C-CUS245C could hardly be detected on EGFR-null cell line. Our results also showed that C-CUS245C had efficacy of anti-colorectal cancer in mouse xenograft model, indicating the therapeutic potential of C-CUS245C for the targeted therapy of colorectal neoplasms. CONCLUSIONS: C-CUS245C exhibits potent and EGFR-specific cytotoxicity. Insertional mutagenesis technique is worthy to be adopted in the preparation of immunotoxin. Immunotoxin can be highly purified through dialysis followed by IMAC.
Assuntos
Cetuximab/uso terapêutico , Neoplasias Colorretais/terapia , Imunotoxinas/uso terapêutico , Terapia de Alvo Molecular/métodos , Proteínas de Plantas/uso terapêutico , Animais , Antineoplásicos Imunológicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cetuximab/farmacologia , Cromatografia de Afinidade/métodos , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Escherichia coli/metabolismo , Humanos , Imunoconjugados/química , Imunoconjugados/uso terapêutico , Imunotoxinas/química , Imunotoxinas/isolamento & purificação , Imunotoxinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mutagênese Insercional/métodos , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Fatores de Tempo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
rCCK8PE38 is a novel immunotoxin that targets choleystokinin B receptor, which is over-expressed in some tumor tissues. Although we constructed a prokaryotic expression vector to express rCCK8PE38 in our laboratory, thorough purification was necessary to quantitatively assess its anti-tumor effect. In this study, we established a purification protocol to obtain rCCK8PE38 with high purity from E. coli. Three different types of chromatography, hydrophobic chromatography, ion exchange chromatography and size exclusion chromatography, were used in combination. The purification technological parameters of each chromatography type were optimized. The whole process of purification was arranged to minimize the purification steps and achieve purity and bioactivity. Finally, through this optimized scheme, we obtained a recombinant protein with a purity of >95%; then, the protein was stored at -80°C after lyophilization. The purified protein was used in a tumor inhibition experiment and was effective in killing tumor cells that over-expressed choleystokinin B receptor. The results of this study may provide some valuable information about protein purification and lay the foundation for further clinical experiments with rCCK8PE38.
Assuntos
Antineoplásicos/isolamento & purificação , Imunotoxinas/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Sulfato de Amônio/química , Antineoplásicos/química , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Precipitação Química , Cromatografia Líquida , Escherichia coli/genética , Humanos , Imunotoxinas/química , Imunotoxinas/genética , Imunotoxinas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Overexpression of epidermal growth factor receptor (EGFR) plays a significant role in the development and metastasis of many solid tumors. Strategies based on anti-EGFR immunotoxins have shown promising results in several studies, but immunogenicity of antibody and toxin moieties is a limitation of this type of therapeutics. In the present study, a novel humanized anti-EGFR immunotoxin (huscFv-PE25) was developed by genetic fusing of a humanized anti-EGFR single-chain variable fragment (huscFv) with a modified Pseudomonas aeruginosa exotoxin A (PE25KDEL). The reactivity and toxicity of this immunotoxin with tumor cells were assessed by dot-blot, enzyme-linked immunosorbent assay, and MTT procedures. Results of enzyme-linked immunosorbent assay and dot-blot assay indicated that the immunotoxin recognizes and efficiently binds to EGFR-overexpressing tumor cells. MTT assay showed a specific growth-inhibitory effect of huscFv-PE25 on EGFR-overexpressing A431 cells, without any inhibitory effect on EGFR-negative cells. In conclusion, the results of this study indicated that huscFv-PE25 can recognize and exert an inhibitory effect on EGFR-overexpressing cancer cells, despite its smaller size and lower immunogenicity. This may provide a basis for the development of novel clinical therapeutic agents against EGFR-overexpressing tumor cells.
Assuntos
ADP Ribose Transferases/farmacologia , Toxinas Bacterianas/farmacologia , Receptores ErbB/imunologia , Exotoxinas/farmacologia , Imunotoxinas/química , Imunotoxinas/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Fatores de Virulência/farmacologia , Células A549 , ADP Ribose Transferases/química , Animais , Toxinas Bacterianas/química , Células CHO , Linhagem Celular Tumoral , Cetuximab/farmacologia , Cricetulus , Ensaio de Imunoadsorção Enzimática , Receptores ErbB/biossíntese , Exotoxinas/química , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/imunologia , Imunotoxinas/imunologia , Imunotoxinas/isolamento & purificação , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/imunologia , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/imunologia , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/imunologia , Fatores de Virulência/química , Exotoxina A de Pseudomonas aeruginosaRESUMO
Triterpenoidal saponins are synthesized in the roots of Saponaria officinalis L. The same plant is also a source for the toxin Saporin, which is a ribosome-inactivating protein. Triterpenoidal saponins are known to increase the cytotoxicity of Saporin by modulating its intracellular trafficking. Here, we investigated if the combinatorial effects elicited by purified saponins and Saporin can be applied to increase the therapeutic efficacy of the immunotoxin Saporin-Rituximab. First, saponins were purified by high-performance liquid chromatography. Thereafter, their intrinsic cytotoxicity was evaluated on Ramos cells with no observed effect up to 5 µg/mL, however, saponins increased the cytotoxicity of Saporin, while no influence was observed on its N-glycosidase activity. Saporin-Rituximab bound to CD20 in Ramos cells and, in the absence of saponins, had a GI50 (concentration inhibiting cell growth to 50â%) of 7 nM. However, in the presence of a nontoxic concentration of saponins, the GI50 of Saporin-Rituximab was 0.01 nM, a nearly 700-fold increase in efficacy. Moreover, two further immunotoxins, namely Saporin-anti-CD22 and Saporin-anti-CD25, were tested in combination with saponins yielding enhancement factors of 170-fold and 25-fold, respectively. All three receptors are present in Ramos cells and the differences in cytotoxicity enhancement may be explained by the differing expression levels of the cellular receptors. The application of purified saponins from S. officinalis L. is therefore a new strategy to potentially improve the cytotoxicity and therapeutic efficacy of Rituximab-immunotoxins for the treatment of B-cell lymphoma.
Assuntos
Imunotoxinas/farmacologia , Linfoma de Células B/patologia , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Rituximab/farmacologia , Saponaria/química , Saponinas/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Sinergismo Farmacológico , Humanos , Imunotoxinas/química , Imunotoxinas/isolamento & purificação , Proteínas Inativadoras de Ribossomos Tipo 1/química , Proteínas Inativadoras de Ribossomos Tipo 1/isolamento & purificação , Saponinas/química , Saponinas/isolamento & purificação , SaporinasRESUMO
Development of agents for theranostics implies combining the targeting module, the effector module, and the detection module within the same complex or recombinant protein. We have constructed, isolated, and characterized the 4D5scFv-mCherry-PE(40) protein, which exhibits fluorescent properties and specifically binds to cancer cells expressing the HER2 receptor and reduces their viability. The ability of the obtained targeted antitumor agent 4D5scFv-mCherry-PE(40) to selectively stain the HER2-positive cells and its highly selective cytotoxicity against these cells make the obtained targeted recombinant protein 4D5scFv-mCherry-PE(40) a promising theranostic agent for the diagnostics and therapy of HER2-positive human tumors.
Assuntos
Imunotoxinas/farmacologia , Proteínas Luminescentes/farmacologia , Anticorpos de Cadeia Única/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Antineoplásicos/toxicidade , Células CHO , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cromatografia de Afinidade , Cricetulus , Escherichia coli , Fluorescência , Vetores Genéticos , Humanos , Imunotoxinas/isolamento & purificação , Imunotoxinas/toxicidade , Proteínas Luminescentes/síntese química , Proteínas Luminescentes/isolamento & purificação , Proteínas Luminescentes/toxicidade , Microscopia de Fluorescência , Receptor ErbB-2/metabolismo , Proteínas Recombinantes/síntese química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/toxicidade , Anticorpos de Cadeia Única/isolamento & purificação , Anticorpos de Cadeia Única/toxicidadeRESUMO
Immunotoxins are chimeric proteins composed of an antibody domain that specifically directs the action of the toxic domain, resulting in the death of the targeted cells. Over recent years, immunotoxins have been widely studied and the number of different constructions has increased exponentially. Protein engineering has allowed the design of optimized versions of immunotoxins with an improved tumor binding affinity, stability or cytotoxic efficacy, although sometimes this has compromised the safety of the patient in terms of undesirable adverse secondary reactions. A triple mutant at three Trp residues (HtA3ΔW) of the ribotoxin hirsutellin A retains its specific ribonucleolytic activity, although cell internalization capacity is lacking. This toxin variant has been fused to the single chain variable fragment A33 (scFvA33). This immunoconjugate (IMTXA33HtA3ΔW) was produced in the methylotrophic yeast Pichia pastoris and purified using nickel-nitrilotriacetic acid affinity chromatography. Both target and toxic domains were characterized. The immunotoxin showed an exquisite specific binding against GPA33-positive culture cells, which results in the death of the targeted cells because of specific ribonucleolytic activity against ribosomes of the engineered hirsutellin A variant. IMTXA33HtA3ΔW represents a promising structure in the search for an improved immunotoxin without compromising the safety of patients.
Assuntos
Proteínas Fúngicas/genética , Imunotoxinas/genética , Substituição de Aminoácidos , Antibacterianos/biossíntese , Antibacterianos/isolamento & purificação , Neoplasias do Colo/tratamento farmacológico , Ensaios de Seleção de Medicamentos Antitumorais , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/isolamento & purificação , Células HT29 , Humanos , Imunotoxinas/isolamento & purificação , Imunotoxinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Ligação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
A large number of cancers express human chorionic gonadotropin (hCG) or its subunits ectopically. Patients harboring such cancers have poor prognosis and adverse survival. PiPP is a monoclonal antibody of high affinity and specificity for hCGß/hCG. Work was carried out to develop a PiPP based recombinant immunotoxin for the immunotherapy of hCG expressing cancers. Recombinant PiPP antibody was constructed in scFv format in which gene encoding the VH and VL domains were joined through a linker. This scFv gene was fused to the gene expressing Pseudomonas exotoxin (PE38), and cloned in a Escherichia coli based expression vector under the control of strong bacteriophage T7 promoter. Immunotoxin conjugating scFv(PiPP) and PE38, was expressed in E. coli as recombinant protein. Recombinant PiPP immunotoxin was purified from the bacterial cell lysate and tested for binding and killing of hCGß expressing lymphoma, T-lymphoblastic leukemia and lung carcinoma cells in vitro. Immunotoxin showed nearly 90% killing on the cells. This is the first ever report on recombinant immunotoxin for binding and cytotoxicity to hCG expressing cancer cells, and thus can be a potential candidate for the immunotherapy of hCG expressing cells.
Assuntos
Gonadotropina Coriônica/imunologia , Imunoterapia/métodos , Imunotoxinas/toxicidade , Proteínas Recombinantes/toxicidade , Anticorpos de Cadeia Única/imunologia , Ligação Competitiva/efeitos dos fármacos , Western Blotting , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Escherichia coli/metabolismo , Humanos , Imunotoxinas/isolamento & purificação , Plasmídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Pachyrhizus erosus seeds have a high protein content and are used in China due to their cytotoxic effect. Here we report the biological and pharmacological activity of the protein extracts from P. erosus seeds. A novel ribosome-inactivating protein, pachyerosin, from P. erosus seeds was successively purified to homogeneity using ammonium sulfate precipitation, DEAE-sepharose FF, and Sephacryl S-200. Pachyerosin showed to be a type I ribosome-inactivating protein with a molecular mass of 29 kDa and an isoelectric point of 9.19. It strongly inhibited protein synthesis of rabbit reticulocyte lysate with an IC50 of 0.37 ng/mL and showed N-glycosidase activity on rat liver ribosomes with an EC50 of 85.9 pM. The N-terminal 27 amino acids of pachyerosin revealed a 60.71% sequence identity with abrin A from the seeds of Abrus precatorius. With the aim of targeting the delivery of pachyerosin, immunotoxin was prepared by conjugating pachyerosin with anti-human AFP monoclonal antibodies SM0736. The immunotoxin pachyerosin-SM0736 efficiently inhibited the growth of the human hepatoma cell line HuH-7 with an IC50 of 0.050 ± 0.004 nM, 2360 times lower than that of pachyerosin and 430 times lower than that of the immunotoxin against human gastric cancer cell line SGC7901. These results imply that pachyerosin may be used as a new promising anticancer agent.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Pachyrhizus/química , Extratos Vegetais/farmacologia , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/farmacologia , Antineoplásicos Fitogênicos/isolamento & purificação , Humanos , Imunotoxinas/isolamento & purificação , Imunotoxinas/farmacologia , Dados de Sequência Molecular , Peso Molecular , Extratos Vegetais/isolamento & purificação , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Plantas Medicinais , Biossíntese de Proteínas/efeitos dos fármacos , Coelhos , Proteínas Inativadoras de Ribossomos Tipo 1/isolamento & purificação , Sementes/química , Alinhamento de SequênciaRESUMO
In response to advances in proteomics research and the use of proteins in medical and biotechnological applications, recombinant protein production and the design of specific protein characteristics and functions has become a widely used technology. In this context, protein fusion tags have been developed as indispensable tools for protein expression, purification, and the design of functionalized surfaces or artificially bifunctional proteins. Here we summarize how positively or negatively charged polyionic fusion peptides with or without an additional cysteine can be used as protein tags for protein expression and purification, for matrix-assisted refolding of aggregated protein, and for coupling of proteins either to technologically relevant matrices or to other proteins. In this context we used cysteine-containing polyionic fusion peptides for the design of immunotoxins. In general, polyionic fusion tags can be considered as a multifunctional module in protein technology.
Assuntos
Cisteína/genética , Peptídeos/genética , Proteínas Recombinantes de Fusão/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular/métodos , Cisteína/química , Cisteína/isolamento & purificação , Cisteína/metabolismo , Humanos , Imunotoxinas/química , Imunotoxinas/genética , Imunotoxinas/isolamento & purificação , Imunotoxinas/metabolismo , Íons/química , Íons/isolamento & purificação , Íons/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/isolamento & purificação , Peptídeos/metabolismo , Renaturação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismoRESUMO
BACKGROUND: Immunotherapy is fast emerging as one of the leading modes of treatment of cancer, in combination with chemotherapy and radiation. Use of immunotoxins, proteins bearing a cell-surface receptor-specific antibody conjugated to a toxin, enhances the efficacy of cancer treatment. The toxin Abrin, isolated from the Abrus precatorius plant, is a type II ribosome inactivating protein, has a catalytic efficiency higher than any other toxin belonging to this class of proteins but has not been exploited much for use in targeted therapy. METHODS: Protein synthesis assay using (3)[H] L-leucine incorporation; construction and purification of immunotoxin; study of cell death using flow cytometry; confocal scanning microscopy and sub-cellular fractionation with immunoblot analysis of localization of proteins. RESULTS: We used the recombinant A chain of abrin to conjugate to antibodies raised against the human gonadotropin releasing hormone receptor. The conjugate inhibited protein synthesis and also induced cell death specifically in cells expressing the receptor. The conjugate exhibited differences in the kinetics of inhibition of protein synthesis, in comparison to abrin, and this was attributed to differences in internalization and trafficking of the conjugate within the cells. Moreover, observations of sequestration of the A chain into the nucleus of cells treated with abrin but not in cells treated with the conjugate reveal a novel pathway for the movement of the conjugate in the cells. CONCLUSIONS: This is one of the first reports on nuclear localization of abrin, a type II RIP. The immunotoxin mAb F1G4-rABRa-A, generated in our laboratory, inhibits protein synthesis specifically on cells expressing the gonadotropin releasing hormone receptor and the pathway of internalization of the protein is distinct from that seen for abrin.
Assuntos
Abrina/farmacologia , Abrus/química , Imunotoxinas/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Abrina/isolamento & purificação , Anticorpos Monoclonais/farmacologia , Transporte Biológico , Linhagem Celular Tumoral , Escherichia coli/metabolismo , Humanos , Imunoconjugados/farmacologia , Imunotoxinas/isolamento & purificação , Terapia de Alvo Molecular , Extratos Vegetais/farmacologia , Receptores LHRH/metabolismo , Proteínas Recombinantes/farmacologiaRESUMO
Angiogenesis targeting is an attractive approach for cancer treatment. Vascular endothelial growth factor receptor 2 (VEGFR2) is such an important target that is overexpressed in tumor vasculature compared to the endothelium cells of resting blood vessels and blocking of its signaling inhibits neovascularization and tumor metastasis. Immunotoxins represent a promising group of targeted therapeutics to combat tumors. They consist of an antibody linked to a toxin and are designed to kill specifically the tumor cells. In this study, we fused a VEGFR2-specific Nanobody, the antigen-binding single-domain fragment derived from functional Heavy-chain antibody of Camelidae, to the truncated form of Pseudomonas exotoxin A and evaluated its ability to bind the VEGFR2 molecule on the cell surface. We demonstrate that this immunotoxin inhibits the proliferation of VEGFR2-expressing cells in vitro. This finding is considered to be a significant achievement in tumor therapy and it forms a basis for further studies in animal models.
Assuntos
ADP Ribose Transferases/uso terapêutico , Toxinas Bacterianas/uso terapêutico , Exotoxinas/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Anticorpos de Domínio Único/uso terapêutico , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/imunologia , Fatores de Virulência/uso terapêutico , ADP Ribose Transferases/farmacologia , Toxinas Bacterianas/farmacologia , Proliferação de Células/efeitos dos fármacos , Cromatografia em Gel , Ensaio de Imunoadsorção Enzimática , Exotoxinas/farmacologia , Células HEK293 , Humanos , Imunotoxinas/isolamento & purificação , Imunotoxinas/farmacologia , Imunotoxinas/uso terapêutico , Proteínas Recombinantes/isolamento & purificação , Fatores de Virulência/farmacologia , Exotoxina A de Pseudomonas aeruginosaRESUMO
The idea of targeted therapy, whereby drug or protein molecules are delivered to specific cells, is a compelling approach to treating disease. Immunotoxins are one such targeted therapeutic, consisting of an antibody domain for binding target cells and molecules of a toxin that inhibits the proliferation of the targeted cell. One major hurdle preventing these therapies from reaching the market has been the lack of a suitable production platform that allows the cost-effective production of these highly complex molecules. The chloroplast of the green alga Chlamydomonas reinhardtii has been shown to contain the machinery necessary to fold and assemble complex eukaryotic proteins. However, the translational apparatus of chloroplasts resembles that of a prokaryote, allowing them to accumulate eukaryotic toxins that otherwise would kill a eukaryotic host. Here we show expression and accumulation of monomeric and dimeric immunotoxin proteins in algal chloroplasts. These fusion proteins contain an antibody domain targeting CD22, a B-cell surface epitope, and the enzymatic domain of exotoxin A from Pseudomonas aeruginosa. We demonstrated that algal-produced immunotoxins accumulate as soluble and enzymatically active proteins that bind target B cells and efficiently kill them in vitro. We also show that treatment with either the mono- or dimeric immunotoxins significantly prolongs the survival of mice with implanted human B-cell tumors.
Assuntos
Chlamydomonas reinhardtii/metabolismo , Cloroplastos/metabolismo , Imunotoxinas/isolamento & purificação , Imunotoxinas/farmacologia , Linfoma/tratamento farmacológico , Engenharia de Proteínas/métodos , Animais , Western Blotting , Cromatografia em Gel , Proteínas de Ligação a DNA/genética , Eletroforese em Gel de Poliacrilamida , Feminino , Citometria de Fluxo , Vetores Genéticos/genética , Humanos , Camundongos , Camundongos Knockout , Organismos Geneticamente Modificados , Proteínas Recombinantes/metabolismo , Transformação Genética , Transplante HeterólogoRESUMO
Many recombinant therapeutic proteins are purified from Escherichia coli. While expression in E. coli is easily achieved, some disadvantages such as protein aggregation, formation of inclusion bodies, and contamination of purified proteins with the lipopolysaccharides arise. Lipopolysaccharides have to be removed to prevent inflammatory responses in patients. Use of the Gram-positive Bacillus anthracis as an expression host offers a solution to circumvent these problems. Using the multiple protease-deficient strain BH460, we expressed a fusion of the N-terminal 254 amino acids of anthrax lethal factor (LFn), the N-terminal 389 amino acids of diphtheria toxin (DT389) and human transforming growth factor alpha (TGFα). The resulting fusion protein was constitutively expressed and successfully secreted by B. anthracis into the culture supernatant. Purification was achieved by anion exchange chromatography and proteolytic cleavage removed LFn from the desired fusion protein (DT389 fused to TGFα). The fusion protein showed the intended specific cytotoxicity to epidermal growth factor receptor-expressing human head and neck cancer cells. Final analyses showed low levels of lipopolysaccharides, originating most likely from contamination during the purification process. Thus, the fusion to LFn for protein secretion and expression in B. anthracis BH460 provides an elegant tool to obtain high levels of lipopolysaccharide-free recombinant protein.
Assuntos
Antígenos de Bactérias/isolamento & purificação , Bacillus anthracis/metabolismo , Toxinas Bacterianas/isolamento & purificação , Toxina Diftérica/isolamento & purificação , Imunotoxinas/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , Fator de Crescimento Transformador alfa/isolamento & purificação , Antígenos de Bactérias/biossíntese , Antígenos de Bactérias/farmacologia , Bacillus anthracis/genética , Toxinas Bacterianas/biossíntese , Toxinas Bacterianas/farmacologia , Linhagem Celular Tumoral , Cromatografia por Troca Iônica , Toxina Diftérica/biossíntese , Toxina Diftérica/farmacologia , Humanos , Imunotoxinas/farmacologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/farmacologia , Fator de Crescimento Transformador alfa/biossíntese , Fator de Crescimento Transformador alfa/farmacologiaRESUMO
We describe the analytical characterization and process scale separation of a deamidated variant of an immunotoxin. The different charge variants of the immunotoxin were separated using analytical ion-exchange HPLC. These charge variants were analyzed by peptide mapping and LC-MS/MS to identify the site of modification, which was determined to reside in the toxin portion of the molecule. Using a cell-based bioassay it was also determined that deamidation led to reduced biological activity, requiring it be controlled during manufacturing. This was accomplished using process scale anion-exchange chromatography. The process was capable of reducing the deamidated form to a level low enough for the resulting product to maintain acceptable biological activity. Keys to the successful control of this impurity at process scale were a good understanding of structure-function relationship and the availability of an analytical HPLC assay to provide a surrogate for the cell-based bioassay.
Assuntos
Toxinas Bacterianas/química , Toxinas Bacterianas/isolamento & purificação , Cromatografia por Troca Iônica/métodos , Exotoxinas/química , Exotoxinas/isolamento & purificação , Imunotoxinas/química , Imunotoxinas/isolamento & purificação , Mapeamento de Peptídeos/métodos , ADP Ribose Transferases/química , Amidas/química , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Espectrometria de Massas em Tandem , Fatores de Virulência/química , Exotoxina A de Pseudomonas aeruginosaRESUMO
Antibody-drug conjugates (ADC) represent promising agents for targeted cancer therapy. To allow rational selection of human antibodies with favorable characteristics for ADC development a screening tool was designed obviating the need of preparing individual covalently linked conjugates. Therefore, α-kappa-ETA' was designed as a fusion protein consisting of a human kappa light chain binding antibody fragment and a truncated version of Pseudomonas exotoxin A. α-kappa-ETA' specifically bound to human kappa light chains of human or human-mouse chimeric antibodies and Fab fragments. Antibody-redirected α-kappa-ETA' specifically inhibited proliferation of antigen-expressing cell lines at low toxin and antibody concentrations. Selected antibodies that efficiently delivered α-kappa-ETA' in the novel assay system were used to generate scFv-based covalently linked immunotoxins. These molecules efficiently triggered apoptosis of target cells, indicating that antibodies identified in our assay system can be converted to functional immunoconjugates. Finally, a panel of human epidermal growth factor receptor (EGFR) antibodies was screened--demonstrating favorable characteristics with antibody 2F8. These data suggest that antibodies with potential for Pseudomonas exotoxin A-based ADC development can be identified using the novel α-kappa-ETA' conjugate.
Assuntos
ADP Ribose Transferases/imunologia , Toxinas Bacterianas/imunologia , Exotoxinas/imunologia , Cadeias kappa de Imunoglobulina/isolamento & purificação , Imunotoxinas/isolamento & purificação , Fatores de Virulência/imunologia , ADP Ribose Transferases/uso terapêutico , Animais , Toxinas Bacterianas/uso terapêutico , Linhagem Celular , Citotoxicidade Imunológica , Ensaio de Imunoadsorção Enzimática , Receptores ErbB/imunologia , Exotoxinas/uso terapêutico , Citometria de Fluxo , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Cadeias kappa de Imunoglobulina/química , Cadeias kappa de Imunoglobulina/uso terapêutico , Imunotoxinas/química , Imunotoxinas/uso terapêutico , Camundongos , Modelos Moleculares , Neoplasias/imunologia , Neoplasias/terapia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/uso terapêutico , Fatores de Virulência/uso terapêutico , Exotoxina A de Pseudomonas aeruginosaRESUMO
Photosensitizer-antibody conjugates are successfully used for targeted elimination of cancer cells bearing specific membrane markers. This method is known as photoimmunotherapy. However, chemical conjugation of photosensitizer and antibody poses a number of complications such as low reproducibility, aggregation and unconjugated photosensitizer impurities. Here we report a fully genetically encoded photoimmunosensitizer, consisting of an anti-HER2/neu miniantibody 4D5scFv and a phototoxic fluorescent protein KillerRed. Both domains in this photoimmunosensitizer retained their functional qualities - high affinity for HER2/neu antigen and phototoxicity respectively. 4D5scFv-KillerRed fusion protein showed high specificity for HER2/neu-over-expressing cells and effectively lowered their viability upon illumination.
Assuntos
Proteínas de Fluorescência Verde/genética , Imunotoxinas/genética , Proteínas Recombinantes/genética , Anticorpos de Cadeia Única/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Imunotoxinas/isolamento & purificação , Imunotoxinas/farmacologia , Luz , Fármacos Fotossensibilizantes/isolamento & purificação , Fármacos Fotossensibilizantes/farmacologia , Receptor ErbB-2/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologiaRESUMO
VB4-845 is a recombinant immunotoxin comprised of an anti-epithelial cell adhesion molecule (EpCAM) scFv fused to a truncated form of the bacterial toxin, Pseudomonas exotoxin A. VB4-845, purified from TB fed-batch fermentation, showed clinical efficacy when administered locally to treat non-muscle invasive bladder cancer (NMIBC) and squamous cell carcinomas of the head and neck (SCCHN). Here, we describe the implementation of an Escherichia coli high cell density (HCD) cultivation and purification process for VB4-845. HCD cultivation was a prerequisite for achieving higher yields necessary for Phase III clinical trials and commercialization. Using this process, the VB4-845 titer in the supernatant was increased by 30-fold over the original TB fed-batch cultivation. To obtain clinical grade material, a process involving a five-step column purification procedure was implemented and led to an overall recovery of â¼ 40%. VB4-845 purity of >97% was achieved after the first three columns following the removal of low-molecular weight product-related impurities and aggregates. Endotoxins were effectively separated from VB4-845 on the Q-columns and by washing the Ni-column with a detergent buffer while host cell proteins were removed using ceramic hydroxyapatite. Comparability studies demonstrated that the purified product from the Phase III process was identical to the Phase II reference standard produced using TB fed-batch fermentation.
Assuntos
Biotecnologia/métodos , Escherichia coli/química , Imunotoxinas/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , Reatores Biológicos , Biotecnologia/normas , Western Blotting , Contagem de Células , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentação , Imunotoxinas/genética , Imunotoxinas/metabolismo , Ligação Proteica , Estabilidade Proteica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Estados Unidos , United States Food and Drug Administration/normasRESUMO
The use of pore-forming toxins from sea anemones (actinoporins) in the construction of immunotoxins (ITs) against tumour cells is an alternative for cancer therapy. However, the main disadvantage of actinoporin-based ITs obtained so far has been the poor cellular specificity associated with the toxin's ability to bind and exert its activity in almost any cell membrane. Our final goal is the construction of tumour proteinase-activated ITs using a cysteine mutant at the membrane binding region of sticholysin-I (StI), a cytolysin isolated from the sea anemone Stichodactyla helianthus. The mutant and the ligand moiety would be linked by proteinase-sensitive peptides through the StI cysteine residue blocking the toxin binding region and hence the IT non-specific killing activity. To accomplish this objective the first step was to obtain the mutant StI W111C, and to evaluate the impact of mutating tryptophan 111 by cysteine on the toxin pore-forming capacity. After proteolysis of the cleavage sequence, a short peptide would remain attached to the toxin. The next step was to evaluate whether this mutant is able to form pores even with a residual peptide linked to cysteine 111. In this work we demonstrated that (i) StI W111C shows pore-forming capacity in a nanomolar range, although it is 8-fold less active than the wild-type recombinant StI, corroborating the previously reported importance of residue 111 for the binding of StI to membranes, and (ii) the mutant is able to form pores even with a residual seven-residue peptide linked to cysteine 111. In addition, it was demonstrated that binding of a large molecule to cysteine 111 renders an inactive toxin that is no longer able to bind to the membrane. These results validate the mutant StI W111C for its use in the construction of tumour proteinase-activated ITs.
Assuntos
Imunotoxinas/química , Proteínas Citotóxicas Formadoras de Poros/química , Animais , Membrana Celular/química , Membrana Celular/metabolismo , Cromatografia em Gel , Cromatografia por Troca Iônica , Dimerização , Imunotoxinas/genética , Imunotoxinas/isolamento & purificação , Imunotoxinas/metabolismo , Modelos Moleculares , Mutação , Compostos Orgânicos/química , Compostos Orgânicos/isolamento & purificação , Compostos Orgânicos/metabolismo , Perforina , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/isolamento & purificação , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Ligação Proteica , Reprodutibilidade dos Testes , Anêmonas-do-MarRESUMO
Ricin is one of the most potent and deadly plant toxins from the seeds of Ricinus communis. In view of its high toxicity, ricin is being used as an immunotoxin in cancer therapy. Ricin also has several isoforms with differential glycosylation depending on the seed variety. Our study shows three isoforms designated 1, 2 and 3, which differed in their surface charge, resulting in a different behavior on cation exchange chromatography, two dimensional (pI 5.5-8.7) and native PAGE. The molecular masses of isoform-1, 2 and 3 were measured as 63.55 kDa, 64.03 kDa and 62.8 kDa, respectively, by MALDI-TOF/MS. In vitro studies with monkey kidney (Vero) cells showed a time dependent increase in cytotoxicity of the isoforms evaluated by extracellular lactate dehydrogenase activity and mitochondrial dehydrogenase assay. These isoforms also induce oxidative stress and DNA damage. Among the isoforms, isoform-3 was quick to generate reactive oxygen species (ROS), (in 90 min) and exhibited maximum cytotoxicity. Morphological changes, catalase activity and DNA fragmentation were significantly higher with isoform-3 treatment compared to others. The glycosylation studies by MALDI-TOF/MS showed that isoform-3 is highly glycosylated with high sugar levels containing more of hybrid/complex type glycopeptides with mannose as hexose units. These experimental evidences clearly suggest that isoform-3 is superior in its early ROS generation, potency to induce oxidative stress and cytotoxicity, that could be due to it's higher glycosylation levels which make isoform-3 as an ideal candidate for immunotoxin studies.