Soluble Cytoplasmic Expression and Purification of Immunotoxin HER2(scFv)-PE24B as a Maltose Binding Protein Fusion.

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.

Triton X-114 and Amine-Based Wash Strategy Reduces Lipopolysaccharides to FDA Limit and Achieves Purer, More Potent Recombinant Immunotoxin.

Many proteins are still routinely expressed prokaryotically in Escherichia coli, some because they are toxic to eukaryotes. Immunotoxins, which are fusion proteins of a targeting moiety and a truncated Pseudomonas exotoxin A, kill target cells by arresting protein synthesis. Thus, immunotoxins must be expressed in E. coli. Proteins expressed in E. coli are contaminated by endotoxin (also called lipopolysaccharides (LPS)). LPS binds to toll-like receptors, inducing up to life-threatening systemic inflammation in mammals. Therefore, accepted LPS limits for therapeutics as well as for substances used in immunological studies in animals are very low. Here, we report the use of Triton X-114 and polyamine-based wash strategies, which only in combination achieved LPS-contamination well below FDA limits. Resulting LPS-reduced immunotoxins were purer and up to 2.4-fold more active in vitro. Increased activity was associated with a 2.4-fold increase in affinity on cell surface expressed target antigen. The combination method maintained enzymatic function, protein stability, and in vivo efficacy and was effective for Fab as well as dsFv formats. With some modifications, the principle of this novel combination may be applied to any chromatography-based purification process.

Preparation of a novel EGFR specific immunotoxin and its efficacy of anti-colorectal cancer in vitro and in vivo.

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.

An efficient scheme for purification of a novel recombinant immunotoxin, rCCK8PE38, for anti-tumour experiments.

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.

Construction, expression, and activity of a novel immunotoxin comprising a humanized antiepidermal growth factor receptor scFv and modified Pseudomonas aeruginosa exotoxin A.

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.

Immunotoxicity of skin acid secretion produced by the sea slug Berthellina citrina in mice spleen: Histological and Immunohistochemical study.

Acid secretion containing sulfuric and hydrochloric acids is a fascinating defensive phenomenon within many groups of marine organisms. This study aimed to investigate the mice spleen histology and immunotoxicity using skin acid secretion (SAS) of the sea slug Berthellina citrina after oral administration. The spleen showed atrophy in the white pulp, decrease in the splenocytes density, megakaryocytes cytoplasmic degeneration as well as inflammatory cells infiltrations. The white and red pulp splenocytes number decreased time-dependently in the treated spleens. Additionally, the size of the megakaryocytes increased as compared with the control. The administration with SAS increased the number of the IgA(+) cells aggregation in the splenic red pulp. Furthermore, after 7days of the administration, large number of dispersed IgA(+) cells were distributed in splenic parenchyma. The IgA(+) cells numbers increased time-dependently as compared with those in the control. The aggregation sizes and number of the F4/80(+) cell in the splenic red pulp were increased. Furthermore the F4/80(+) cells numbers increased time-dependently as compared with those in the control. The UEAI(+) cells were found as free cells but not in aggregations in the control splenic red pulp. Contradictory to the number of IgA(+) cells and F4/80(+) cells the number of the UEAI(+) cells decreased time-dependently after administration with SAS. Hematologically, abnormal numbers of WBCs different cells were observed after administration with SAS. This study provides new insight about the toxicity of a marine extract may be used in natural products industry or medical applications.

Saponins from Saponaria officinalis L. Augment the Efficacy of a Rituximab-Immunotoxin.

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 50 %) 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.

Development and investigation of recombinant immunotoxin protein 4D5scFv-mCherry-PE(40).

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.

Preparation of an engineered safer immunotoxin against colon carcinoma based on the ribotoxin hirsutellin A.

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.

Development of a recombinant hCG-specific single chain immunotoxin cytotoxic to hCG expressing cancer cells.

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.

Development and scale-up of a commercial fed batch refolding process for an anti-CD22 two chain immunotoxin.

We describe the development and scale-up of a novel two chain immunotoxin refolding process. This work provides a case study comparing a clinical manufacturing process and the commercial process developed to replace it. While the clinical process produced high quality material, it suffered from low yield and high yield variability. A systematic approach to process development and understanding led to a number of improvements that were implemented in the commercial process. These include a shorter inclusion body recovery process, limiting the formation of an undesired deamidated species and the implementation of fed batch dilution refolding for increased refold titers. The use of a combination of urea, arginine and DTT for capture column cleaning restored the binding capacity of the capture step column and resulted in consistent capture step yields compared to the clinical process. Scalability is shown with data from 250 L and 950 L scale refolding processes. Compared to the clinical process it replaces, the commercial process demonstrated a greater than fivefold improvement in volumetric productivity at the 950 L refolding scale.

Purification and characterization of a novel type i ribosome inactivating protein, pachyerosin, from Pachyrhizus erosus seeds, and preparation of its immunotoxin against human hepatoma cells.

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.

Polyionic and cysteine-containing fusion peptides as versatile protein tags.

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.

Abrin immunotoxin: targeted cytotoxicity and intracellular trafficking pathway.

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.

Recombinant expression and purification of a tumor-targeted toxin in Bacillus anthracis.

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.

Production of unique immunotoxin cancer therapeutics in algal chloroplasts.

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.

Development of VEGFR2-specific Nanobody Pseudomonas exotoxin A conjugated to provide efficient inhibition of tumor cell growth.

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.

Process scale separation of an anti-CD22 immunotoxin charge variant.

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.

Human kappa light chain targeted Pseudomonas exotoxin A--identifying human antibodies and Fab fragments with favorable characteristics for antibody-drug conjugate development.

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.

[Genetically encoded photoimmunosensitizer].

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.