In polarized epithelial cells, another pathway emerges from the sorting endosome and leads to membrane transport across the cell by transcytosis. The same GM1 species with cis -unsaturated or short-chain fatty acids that efficiently enter the recycling endosome also sort into this pathway Saslowsky et al.
By analogy with the bacterial toxins and viruses that bind glycosphingolipids for trafficking into host cells Chinnapen et al. Our first attempt to test this idea showed that these glycosphingolipid species were capable of sorting a therapeutic cargo into the transcytotic pathway. But release into solution to effect transport across epithelial barriers in vitro, or absorption into the systemic circulation in vivo was not detectable te Welscher et al.
To solve this problem, we conducted additional structure-function studies for the glycosphingolipids in intracellular sorting and discovered modifications of the ceramide and oligosaccharide domains that enable the lipids to act as molecular carriers for mucosal absorption of therapeutic peptides, achieving levels of bioavailability comparable to that of intraperitoneal injection. To test if GM1 glycosphingolipids can be harnessed for biologic drug delivery, we first developed a non-degradable all D-isomer reporter peptide for structure-function studies on the ceramide domain.
The reporter peptide was designed to contain two functional groups, a biotin for high-affinity streptavidin-enrichment, and an alkyne reactive group for chemical ligation to fluorophore molecules and quantitative detection. A C-terminal reactive aminooxy was used for coupling the reporter peptide to the oligosaccharide domain of the different GM1 species Figure 1A and Figure 1—figure supplement 1A te Welscher et al.
This was assessed by confocal microscopy for endosome sorting and transcytosis, using fluorescent cholera toxin B-subunit to label the GM1-peptide fusion molecules Figure 1—figure supplement 1C. These results are consistent with our previous studies Chinnapen et al. A Representative structure of GM1 sphingolipids fused to an all-D amino acid reporter peptide. The reporter peptide contains a lysine-linked biotin red circle used for affinity purification and an N-terminal Alexa Fluor green circle. B GM1-peptide fusions or unfused reporter peptide were added apically to MDCK-II cells grown on filter supports and imaged by live cell confocal microscopy.
Transcytosis of the CGM1-reporter peptide fusion with either a C or C sphingosine is evident by basolateral membrane fluorescence. The CGM1 reporter molecule is delivered to intracellular puncta, presumably lysosomes. Scale bars 10 um C Transport of the indicated GM1-reporter peptide fusions across T84 cell monolayers with the indicated fatty acid chain length and degree of saturation.
D Transepithelial transport of the CGM1 peptide fusion across T84 monolayers is dose-dependent and far exceeds transport of the unconjugated reporter peptide.
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E T84 monolayers were simultaneously treated with unfused peptide and unfused CGM1. Mixing experiments confirm that fusion of the reporter peptide to the glycosphingolipid carrier is required for amplified transcellular transport. All glycosphingolipid-peptide fusion molecules subsequently prepared were coupled to Alexa Fluor AF , purified by HPLC, and structures confirmed by mass spectrometry Figure 1—figure supplement 1A and Material and methods.
The GM1 species containing long saturated fatty acids CGM1 were localized to intracellular puncta consistent with sorting to the lysosome Figure 1B , bottom panels , and the GM1 species containing short fatty acids were sorted into the recycling and transcytotic pathways as evidenced by localization to apical and basolateral plasma membranes and small intracellular vesicles Figure 1B middle panels.
This interpretation was confirmed using lysotracker to mark the lysosome and the transferrin receptor to mark the recycling endosome Figure 1—figure supplement 1D. The peptide alone did not bind or enter cell monolayers Figure 1B , top panels. Because we use GM1 originally purified from bovine brain to synthesize the different GM1 species, the end products comprise two isoforms of the long chain base: one containing a sphingosine chain of C and the other of C For the GM1 species containing C fatty acids, the two sphingosine-isoforms were purified and found to track identically in transcytosis Figure 1B , middle two panels.
Thus, it is the structure of the fatty acid that dominates in the sorting reactions Chinnapen et al. To test structure-function of the ceramide fatty acid chain, we developed a quantitative assay for transcytosis Figure 1—figure supplement 2A—B and Material and methods. The assay is sensitive to picomolar concentrations and linear over a large 6-log dynamic range Figure 1—figure supplement 2B. Different GM1-peptide fusions 0. When tested on human intestinal T84 cell monolayers, we found approximately a fold increase in transepithelial transport PAPP for the GM1 ceramide species containing C, C, C, fatty acids or lyso-GM1 as compared to controls.
Introduction of an unsaturated cis -double bond to the short chain ceramide fatty acids C and C had no effect on transcytosis in comparison to the saturated species C and C Figure 1C. This result is in contrast to the dramatic effect the cis -double bond induces in trafficking of the long fatty-acid chain GM1 glycosphingolipids Chinnapen et al. Transepithelial transport was dose-dependent for the CGM1-peptide fusion grey bars and greatly exceeded transport of the unconjugated reporter peptide white bars over a wide range of concentrations Figure 1D.
Mixing experiments using unconjugated GM1 and reporter peptide as individual molecules confirmed that transcellular transport of the peptide cargo was dependent on fusion to the GM1 glycosphingolipid Figure 1E. Neither the unfused reporter peptide nor the GM1-peptide fusion had any detectable confounding effects on cell viability as determined by measurement of metabolic activity MTT assay , or monolayer integrity and tight junction function assessed as trans-epithelial resistance TEER or dextran flux Figure 1—figure supplement 2E—G.
Several approaches were used to confirm that the mechanism of cargo transport across epithelial cell monolayers was by transcytosis and not by paracellular leak. Such low temperature effectively stops all forms of membrane dynamics including transcytosis, but has minimal effects on paracellular solute diffusion. The same results were obtained when transcytosis was measured by live cell confocal microscopy. In the presence of disrupted tight junctions i. In a third approach, we blocked endocytosis at physiologic temperature using the dynamin inhibitor Dyngo-4A. For the C and C GM1-peptide fusion molecules, transport into the basolateral reservoir was strongly inhibited by Dyngo-4A treatment, consistent with active transcellular transport by transcytosis Figure 2C.
In contrast, Dyngo-4A had no detectable effect on transport of the reporter peptide alone, as expected for diffusion of solutes by paracellular leak. Similar results were obtained using a genetic approach. The exocyst complex is necessary for efficient receptor-mediated transcytosis of immunoglobulins Oztan et al. In contrast, transport of the unfused reporter peptide was not affected by exocyst KD. Thus, fusion of a peptide cargo to certain GM1 species enables active transport of the peptide across the epithelial barrier by transcytosis.
To explain how the very short chain fatty acids amplified transport across epithelial cell monolayers, we first measured the rate of transcytosis by pulse chase. Transcytosis was measured as fluorescence at basolateral membranes. By this method, we found no detectable difference among the two GM1 species in the rate of transcytosis Figure 3—figure supplement 1A. In both cases, basolateral membranes were fluorescent after a 10 min chase. At longer chase times, however, we observed a dramatic difference between the C and Cpeptide fusion molecules Figure 3A.
Monolayers loaded with the CGM1 peptide fusion stained brightly at both the apical bottom left panel and basolateral membranes bottom right panel. In stark contrast, monolayers loaded with the CGM1 peptide fusion showed no fluorescence middle panels. We interpreted this result as indicating a higher rate of release from cell membranes to the basolateral solution and emptying the cell of the peptide-GM1 fusion over time. To test this idea, we quantified the rate of release from cell membranes for the fluorescent GM1-peptide fusion molecules Material and Methods.
Results show a faster and more complete diffusion from membrane to solution for the CGM1 fusion molecule Figure 3B and C ; red curve compared to the longer chain CGM1-peptide blue curve. Faster and more complete release into solution was also observed for the CGM1-peptide fusion molecule Figure 3B and C ; purple curve. Thus, the greater efficiency for transepithelial transport by the short chain GM1 species is largely explained by their greater efficiency of diffusion from membrane to solution after transcytosis.
Linkers, resins, and general procedures for solid-phase peptide synthesis.
Glycosphingolipids contain another major functional domain in addition to the ceramide, the extracellular oligosaccharide head group. In all cases, however, the oligosaccharide head group acts to trap sphingolipids in the outer leaflet of cell membranes, thus rendering the lipids dependent on membrane trafficking for their distribution across the cell.
To test if the effects of ceramide structure on transcytosis and membrane-release were specific to GM1 glycosphingolipids, or could be generalized to other glycosphingolipid species, we fused our reporter peptide to a GM3 ganglioside synthesized to contain ceramide domains with either C or C fatty acids. The oligosaccharide domain of GM3 differs from GM1 by the absence of two sugars and thus lacks the terminal galactose and GalNAc that functions strongly as a lectin-binding site in GM1.
When tested for transcytosis, we unexpectedly found that the GM3-Cpeptide fusion molecule crosses epithelial monolayers far more efficiently that the closely related GM1-Cpeptide; and as efficiently as the GM1-C and C species Figure 3D. Similarly, transepithelial transport for the GM3-Cpeptide was approximately 2-fold greater than that observed for the GM1-Cpeptide when compared directly Figure 3—figure supplement 1B. Transport was strongly inhibited by pretreatment with the dynamin inhibitor Dyngo-4A, implicating active transcellular trafficking by transcytosis.
In membrane-release studies, we found a higher rate of release to solution for the CGM3-peptide fusion green curve when compared to the GM1 fusion molecule Figure 3B and C. Thus, the GM1 glycosphingolipid species appear to be retained in the membrane more tightly than the GM3 species containing the same ceramide domains.
Because GM3 lacks a free terminal galactose, we hypothesized the GM1 lipids, which contain the terminal galactose, might be further tethered to the membrane by a form of lectin-binding at the cell surface. To test this idea, we studied the rate of membrane release for the CGM1 species in the presence or absence of mM lactose Glc-Gal disaccharide as a competitive ligand Figure 3E.
These studies show enhanced release from the membrane in the presence of excess free lactose, but not excess mannitol, implicating interaction with a galactose-specific lectin membrane tether Figure 3E. Here we find that lactose at high mM concentrations competed both the GM1 and GM3 species off the membrane not shown but at lower doses 5 mM lactose released only the GM1-fusion Figure 3—figure supplement 1C—D. Thus, the oligosaccharide domain of the glycosphingolipids can also affect the efficiency of transport across epithelial barriers, we propose by interacting with lectin-like molecules at the cell surface.
To test for glycosphingolipid-mediated transport across the intestine in vivo, the unfused reporter peptide or the CGM1-peptide fusion molecule were intragastrically gavaged to mice at equal doses 0. We also measured uptake into the liver, where at 1 hr after gastric gavage we find the glycosphingolipid-peptide fusion molecule, but not the unfused peptide Figure 4C.
Thus, fusion to the glycosphingolipids facilitated absorption of the peptide cargo across the intestine and into the two tissues we sampled, blood and liver. The reporter peptide on its own was not detectably absorbed. A In vivo studies showing absorption across intestinal epithelial barriers into blood after gastric administration of the CGM1-peptide fusion, vehicle alone, or unfused reporter peptide five independent experiments. C 1 hr after gastric gavage the CGM1 peptide fusion is absorbed to the liver whereas the unfused reporter peptide is not detected four independent experiments.
D Uptake into nasal epithelium.
Images by two-photon microscopy comparing transport of the unfused reporter peptide left panels and CGM1 peptide fusion green labeling, right panels Scale bars 20 um upper panels, 10 um lower panels. E Biochemical analysis of blood 30 min after nasal administration shows systemic absorption of C and CGM1 peptide fusions two independent experiments. To test if these results can be generalized, we applied the C and CGM1-peptide fusions to the nasal epithelium, another tight epithelial mucosal surface. In this case, the CGM1-peptide green labeling; Figure 4D could be visualized by two-photon microscopy within the epithelial barrier in all regions of the nasal epithelium Figure 4D , including in areas of pseudostratified top right panels and simple columnar epithelial tissues bottom right panels.
Uptake of the unfused peptide, applied at the same dose, was very rarely detected left panels. Absorption to the systemic circulation for the GM1-peptide fusion molecules was confirmed biochemically by measuring content in the blood 15 min after nasal administration Figure 4E. Here, we find approximately a fold increase in blood levels for the GM1-peptide fusion molecules, compared to peptide alone which is close to background. Unexpectedly, in the nasal epithelium, we find evidence for efficient absorption of the CGM1-peptide fusion molecules, similar to our results with the CGM3-peptide species in the intestine.
The result suggests that different tissues may interact in different ways with the oligosaccharide domains of glycosphingolipids. In this case, the nasal epithelium may not bind the GM1 oligosaccharide, thus allowing for more efficient release from cell membranes into solution after transcytosis and systemic absorption. Glucagon-like peptide-1 GLP-1 and related peptides have become important drugs in the management of type two diabetes mellitus, by both promoting weight reduction and sensitizing glucose-stimulated insulin release van Bloemendaal et al.
A major factor limiting the clinical utility in many individuals is the fact that all currently available preparations must be delivered by subcutaneous injection. To test if the properties of glycosphingolipid trafficking could be applied to enable oral absorption of GLP-1, we coupled a long-half-life version of GLP-1 Figure 5A with C-terminal peptide linker termed here GLP-1 for simplicity to the CGM1 ceramide species as described te Welscher et al. The fusion of CGM1 to GLP-1 caused some loss of function, but the molecule remained highly potent as an incretin hormone, closely comparable to that of the controls.
E GLP-1 in blood 15 min after gastric gavage quantified for each species using the luciferase bioassay fmols of compound per uls blood for three independent experiments. F Systemic absorption of an all D-isomer of GLP-1 used to directly measure the cargo in blood three independent experiments. The GLP-1 peptide cargo is 40 residues, approximately 4-fold greater in size compared to the reporter peptide. We first studied transport across intestinal T84 cell monolayers in vitro to test if GM1 glycosphingolipids could transport such a larger cargo.
In these studies, GLP-1 transport was quantified by luciferase bioassay as previously described te Welscher et al. Here, we find an even greater effect of the glycosphingolipids on transepithelial GLP-1 transport 20—fold above controls. This is explained by a much lower rate of paracellular leak for the larger sized residue GLP-1 peptide.
Such size-exclusion from tight junctions is a well-known determinant of paracellular solute diffusion across intact epithelial barriers. The effect on glucose tolerance by gastrically administered CGM1-GLP-1 was similar to the effect achieved by the intraperitoneal injection of GLP-1 peptide alone, implicating an equally high level of bioavailability for the gastrically-delivered GM1-fusion molecule Figure 5D.
First, we measured GLP-1 activity in blood samples by streptavidin capture and quantitative luciferase bioassay Figure 5E. In a second approach, we synthesized an all D-amino acid non-degradable isomer of GLP-1 coupled to AF to allow for direct quantitative measurement of the residue isomer in the blood using the same streptavidin capture assay as described for our reporter peptide Figure 5A.
These experiments were performed in two different laboratories, using two different animal facilities with the same results. In all assays Figure 5C—5F , we find the efficiency of intestinal absorption enabled by fusion to CGM1 was again almost as efficient as for IP injection of the peptide alone, implicating a high level of bioavailability for the GM1-fusion molecules applied by gastric gavage. Notably, however, the CGM1-GLP-1 fusion molecule had no effect on glucose tolerance Figure 5D and was not detectably absorbed after gastric gavage Figure 5—figure supplement 1B , even though this molecule was readily transported across epithelial monolayers in vitro Figure 1C.
This may be explained by lower affinity of the C and lyso- ceramide domains for incorporation into cell membranes, as inferred from membrane loading and release assays Figure 3B and Figure 1—figure supplement 2D. The difference in biology transcytosis in vitro versus absorption in vivo becomes apparent only in vivo where the conditions for epithelial uptake and transport are not optimized as they are in vitro. Thus, although it seemed at first glance that further shortening of the fatty acid beyond C should amplify transepithelial transport and thus clinical utility, this was not the case and the result informs further development of the technology.
In summary, we find that fusion of therapeutic peptides to GM1 and GM3 glycosphingolipids with short fatty acids enables their active transport across tight epithelial barriers by transcytosis. In the case of the incretin hormone GLP-1, fusion to the lipid carriers allows for gastric oral absorption with high bioavailability and the expected effects on blood glucose, highlighting the potential use of this technology in clinical applications.
Our findings delineate a novel synthetic method for enabling absorption of therapeutic peptides across mucosal surfaces in vivo. The approach is based on the natural biology of lipid sorting for the glycosphingolipids, which depends primarily on the structure of the ceramide domain to allow for trafficking in the transcytotic pathway, and thus active transport across mucosal surfaces without barrier disruption. For applications requiring systemic drug delivery, non-native glycosphingolipid carriers with ceramide domains containing short-chain fatty acids are required to allow for efficient release from cell membranes into the circulation after transcytosis.
The apparent high level of intestinal bioavailability enabled by the glycosphingolipid carriers is unprecedented. The mechanism s for transcellular trafficking co-opted by the cis -unsaturated or short chain fatty acid glycosphingolipids are not fully understood. It is possible the unsaturated and short-chain ceramide domains engage sorting mechanisms that dictate their trafficking to the recycling endosome and elsewhere, but we suggest it also possible that their trafficking might be stochastic after escape from the lysosomal pathway, essentially tracking along with bulk membrane flow.
In other words, the robust sorting event may occur only for the long chain saturated glycosphingolipids, directing them to the lysosome. Another key structural feature enabling this technology must be the oligosaccharide head group. This domain traps the ceramide lipid in the outer membrane leaflet, preventing flip-flop between leaflets and thus rendering the molecule dependent on membrane dynamics for movement throughout the cell — an essential feature for a trafficking vehicle.
As shown by our studies using GM3, the extracellular oligosaccharide can in some cell types also affect the efficiency of transepithelial transport. One way, we suggest, may be by binding to adjacent membrane lectins, thus enhancing the tethering of the lipid to the membrane surface.
In the case of transport across mucosal barriers, we envision several applications for the glycosphingolipids of relevance to clinical medicine. The direct delivery of intrabodies as proteins could become therapeutically interesting to specifically target signaling cascade components and to mediate instantaneous biological effects.
The use of intrabodies as therapeutic molecules brings solutions to overcome challenges currently faced by the pharmaceutical industry with small molecules and antibodies targeting extracellular substrates.
An antibody has the advantage, over classical small molecule drugs, to be highly specific for a single protein. They can be designed to discriminate between splice protein variants and even post-translational subpopulation eg, a phosphorylated protein. With the exponential growth of the therapeutic monoclonal antibody market, the technologies to design and produce antibodies enable antibody manufacturing scale up and are clinically approved.
Clinical trials show that antibodies, which can be recombinantly produced and humanized, are generally well-tolerated and have high substrate specificity that significantly reduce the risk of deleterious side-effects compared to many others types of therapeutic products. The immediate but transient blocking effect of the targeted protein by intrabodies also provides an advantage over more indirect and less-specific RNA interference technologies or permanent modification provided by CRISPR nucleases. Usually, proteins, including antibodies, do not have the innate capacity to penetrate cells by themselves in sufficient therapeutic concentration.
Unfortunately, current viral and non-viral delivery methods inactive virus, electroporation, chemical agents have mainly been developed to introduce foreign genetic material but are poorly efficient to directly deliver proteins in cells, leaving few options for the delivery of purified antibody. The feasibility of targeting intracellular proteins using intrabodies has been proven using either intracellular overexpression of antibody fragments through a gene-based approach or microinjection.
Few corporate and academic groups also reported that some cationic lipid agents could be used to translocate antibodies, but the toxicity associated with their use prohibits their transfer in clinical trials for both ex vivo and in vivo studies. Cell-Penetrating Peptides as Intrabody Carriers Using short protein domains as intracellular carriers is a strategy discovered in that was rationally considered 30 years ago after the identification of the first cell-penetrating peptide CPP , isolated from the HIV-transactivator TAT.
However, CPPs mediate intracellular uptake through endocytic processes that lead to the sequestration and the enzymatic degradation of proteins into vesicular compartments, named endosomes, that substantially reduces the likelihood to get functional effect. The Endosomal Escape Strategy Indeed, the endosomal escape of delivered proteins is a challenging and limiting step that requires endosome destabilizing strategies.
Indeed, using both CPPs and ELDs could be an approach resulting in a peptide with both protein uptake and endosomal escape properties Figure 1. Recently, the fusion of CPPs with ELDs was validated as a promising peptide-based delivery strategy with easily adaptable protocols for macromolecule delivery that provided exciting results into mammalian cells. Toward a New Generation of Intrabody Carriers The research division of Feldan aims at developing and patenting a new generation of peptides named Feldan Shuttles that provides the efficient, safe, and fast intracellular delivery of proteins, like antibodies, in several mammalian cells, including human stem cells, lymphocytes, myeloma, and primary cells.
Exempt from chemical modifications, the Feldan Shuttle is degraded after transient active use, a characteristic that considerably decreases regulatory burdens for human applications. Feldan Shuttle technology opens new avenues allowing the modification of hard-to-transfect cells with high therapeutic potential. These easily soluble designed peptides can deliver diverse antibodies in the cytoplasm of adherent and suspension cells using a simple co-incubation protocol.
The Feldan Shuttles offer a high level of adjustment and accuracy to reach functional effects in cells. Indeed, this technology is in continuous improvement using both sequence analysis and computational learning approaches. Thereby, the Feldan Shuttle is a promising and effective peptide-based alternative to safely bring antibodies and other proteins in the cytoplasm of cells. Our main objective is to use the Feldan Shuttle platform to develop a high throughput technology that will bring a new strategy to target intracellular epitopes and to modulate signalling pathways, enabling the use of intrabodies as a drug to prevent, treat, or cure diseases.
Initial results using the Feldan Shuttle demonstrates that intrabodies can be internalized while keeping their ability to bind a specific target to mediate cellular activity. The experiments presented in this review show that the Feldan Shuttle efficiently delivered the following functional antibodies: an anti-tubulin antibody Figure 3A , an anti-NUP98 antibody that label the perinuclear protein nucleoporin Figure 3B , and most importantly, two anti-Active Caspase 3 monoclonal and polyclonal antibodies that bind and inactivate the pro-apoptotic Caspase 3 protein in human monocyte THP1 and human immortalized CD4 T lymphocyte Jurkat Figure 4.
In presence of the cytotoxic inducer actinomycin D, the Feldan Shuttle-mediated delivery of each anti-Active Caspase 3 monoclonal mAb and polyclonal pAb antibodies in THP1 and Jurkat cells, respectively, resulted in cell protective effect via the reduction of the basal level of apoptosis compared to the non-specific IgG antibody control condition. The simple and adaptable co-incubation protocol used here present many advantages to preserve cells from toxicity. Indeed, the Feldan Shuttle and each antibody were co-incubated with adherent or suspension cells only for 1 to 10 minutes, illustrating the fast delivery action mode of the technology.
These results confirmed that the Feldan Shuttle technology efficiently and safely delivers functional intrabodies in mammalian cells. Because the vast majority of therapeutic antibodies still aim at extracellular targets, the implementation of the Feldan Shuttle technology to deliver intrabodies in mammalian cells provides huge interests in the antibody market.
The development of a Feldan Shuttle platform for the screening of functional intrabodies should result in the identification of intracellular protein targets with therapeutic interest. In the development process, multiple antibodies need to be generated against one target and subsequently screened for their specificity.
The access to high-quality intrabodies will also allow Feldan Therapeutics to transiently modulate the cell machinery to get functional changes. Moreover, the Feldan Shuttle technology could deliver engineered intrabodies to transiently modulate signaling pathways at different cell cycle stages during cell proliferation and differentiation.
This may be achieved with the delivery of specific intrabodies that could trap protein targets into specific intracellular compartments. The use of the Feldan Shuttle Technology for intrabody delivery can be directly used for ex vivo manipulation of mammalian cells to support the development of the screening platform. Its low cost of manufacturing, ease of use, and innocuity also provide this tractable method toward an industrial platform for the development of topical therapeutic products and local injections of intrabodies to treat problematics like skin inflammation in psoriasis, pain in osteoarthritis, and neovascular age-related macular degeneration.
Injected proteins diffused in tissues and provided functional activity, indicating the potential of this technology for in vivo applications. However, given the more complex environment, the systemic injection of the Feldan Shuttle product is still limited and needs improvements with peptide protection strategies like the use of peptide-coating agents and non-permanent peptide-cargo linking strategies. In this regard, Feldan Therapeutics continuously develops partnerships to seize exciting opportunities and expertise to combine the Feldan Shuttle technology with other approaches and to raise it toward intrabody-based clinical applications.
Therapeutic antibodies: successes, limitations and hopes for the future. Br J Pharmacol.
Serum Stability of Peptides | Springer Nature Experiments
May ; 2 Engineering antibody therapeutics. Curr Opin Struct Biol. Jun ; The therapeutic monoclonal antibody market. Reichert JM. Antibodies to watch in Miersch S, Sidhu SS.