drug delivery strategy utilizing conjugation via reversible disulfide linkages: role and site of...

Advanced Drug Delivery Reviews 55 (2003) 199–215www.elsevier.com/ locate/addr

D rug delivery strategy utilizing conjugation via reversibledisulfide linkages: role and site of cellular reducing activities

a,c b a ,*Go Saito , Joel A. Swanson , Kyung-Dall LeeaDepartment of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109-1065,USA

bDepartment of Microbiology and Immunology, University of Michigan, Ann Arbor, MI 48109,USAcPharmacokinetics and Drug Delivery Research Laboratories, Sankyo Co., Ltd, Tokyo, Japan

Received 25 April 2002; accepted 13 July 2002

Abstract

The first disulfide linkage-employing drug conjugate that exploits the reversible nature of this unique covalent bond wasrecently approved for human use. Increasing numbers of drug formulations that incorporate disulfide bonds have beenreported, particularly in the next generation macromolecular pharmaceuticals. These are designed to exploit differences inthe reduction potential at different locations within and upon cells. The recent characterization of a novel redox enzyme inendosomes and lysosomes adds more excitement to this approach. This review focuses on understanding where and how thedisulfide bond in the bioconjugate is reduced upon contact with biological milieu, which affects delivery design and theinterpretation of the delivery strategies. 2002 Elsevier Science B.V. All rights reserved.

Keywords: Disulfide bond; Reduction; Bioconjugate; Macromolecular delivery;g-Interferon-inducible lysosomal thiol reductase (GILT);Protein disulfide isomerase (PDI); Glutathione (GSH)

Contents

1 . Introduction ............................................................................................................................................................................ 2001 .1. Bioconjugation strategies for drug delivery and choice of linkage ........................................................................................ 2001 .2. Disulfide bond-based bioconjugation ................................................................................................................................. 200

2 . Cellular redox enzymes and redox agents .................................................................................................................................. 2012 .1. Reducing cytosolic space and oxidizing ER space .............................................................................................................. 2012 .2. Disulfide reduction at cell surface and early endosomes ...................................................................................................... 2022 .3. Disulfide reduction in endosomes and lysosomes................................................................................................................ 2032 .4. Characterization of redox enzyme in the endocytic pathway: GILT...................................................................................... 204

3 . Creating sulfhydryls via chemical and molecular approaches ...................................................................................................... 2053 .1. Generating sulfhydryls through chemical linkers ................................................................................................................ 2053 .2. Site-directed conjugation using endogenous sulfhydryls or mutagenetically inserted cysteines ............................................... 206

4 . Disulfide linkage-employing drug delivery systems.................................................................................................................... 206

*Corresponding author. Tel.:11-734-647-4941; fax:1 1-734-615-6162.E-mail address: [email protected](K.-D. Lee).

0169-409X/02/$ – see front matter 2002 Elsevier Science B.V. All rights reserved.PI I : S0169-409X( 02 )00179-5

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200 G. Saito et al. / Advanced Drug Delivery Reviews 55 (2003) 199–215

4 .1. Macromolecular delivery systems via endocytosis .............................................................................................................. 2074 .1.1. Attachment of cellular targeting moiety through sulfhydryls ...................................................................................... 2074 .1.2. Conjugation of membrane-disrupting agents for enhanced cytosolic delivery .............................................................. 2074 .1.3. Bestowing stability and instability upon drug delivery systems via disulfides.............................................................. 208

4 .2. Direct cytosolic delivery of macromolecules across the plasma membrane ........................................................................... 2094 .3. Controlled disulfide cleavage in the systemic circulation ..................................................................................................... 209

5 . In vivo applications ................................................................................................................................................................. 2095 .1. Antibody-S-S-toxin .......................................................................................................................................................... 2095 .2. Antibody-S-S-drug ........................................................................................................................................................... 210

6 . Conclusion.............................................................................................................................................................................. 211References .................................................................................................................................................................................. 211

1 . Introduction aspects of this subject, we will briefly discuss theexisting examples of disulfide linkage-employing

1 .1. Bioconjugation strategies for drug delivery drug delivery with the mechanism in mind.and choice of linkage

1 .2. Disulfide bond-based bioconjugationAttachment of cellular and subcellular targeting

moiety, delivery-enhancing molecules, or functional A disulfide bond (–S–S–) is a covalent linkageentities to drugs or their delivery systems has which arises as a result of the oxidation of twobecome an essential and important approach in the sulfhydryl (SH) groups of cysteines or other SH-field of modern drug delivery. Its need is more acute containing material. In bacterial and eukaryotic cells,for the strategies required for macromolecular deliv- they are often found in secretory proteins andery, as unique problems arise as the molecular exoplasmic domains of membrane proteins, whichweight of the drugs increases, including changes in face a harsh extracellular environment. In eukaryoticcytotoxicity, pharmacokinetics, dynamics and metab- cells, cysteines are correctly bridged in the endo-olism. For bioconjugates, the nature of the linker plasmic reticulum (ER) via the disulfide bond, whichbetween the pharmacologic agent and the delivery- functions primarily to fortify the protein tertiaryaugmenting moiety dictates the degree of successful structure. Two distinct characteristics that render thisdelivery and its outcome. In this review, we have bond attractive in designing drug delivery systemsattempted to focus only on covalent linkages, rather are its reversibility and its relative stability inthan the less stable yet sometimes advantageous plasma. Covalently bonded disulfides can be formednon-covalent linkages such as high affinity ligand– spontaneously by autoxidation of sulfhydryls, pri-receptor interactions and electrostatic complexation. marily via oxidation upon exposure to air, which can

Among various covalent linkages, the primary reversibly be cleaved in the presence of reducingfocus of this review is the readily reversible yet agents such as dithiothreitol (DTT) andb-mercap-relatively stable linkage of disulfide bonds. As there toethanol. The presence of a high redox potentialexist several reviews on this subject as well as difference between the oxidizing extracellular spacecomparisons among various covalent linkages uti- and the reducing intracellular space makes thelized for drug delivery (for reviews on bioconjugate disulfide bond intriguing as a potential delivery tool.strategies see Refs. [1,2]), the attention here is Thus, the covalent linkage is dependent on the localedirected to the mechanism of disulfide bond reduc- of the construct relative to the cellular compartments;tion after the drug delivery system contacts the a controlled cleavage and release of reduced com-biological milieu. The key subjects of this review are ponents can occur upon cell entry. Indeed, a numberthe questions of where and how the disulfide bond in of bacterial toxins, such as diphtheria and cholerathe bioconjugate is reduced, which impacts the toxins and plant toxins such as ricin, consist of twodesign strategy as well as interpretation of the protein subunits linked via a disulfide bond. Theyexperimental results. After reviewing the mechanistic take advantage of the reversible breakage of the

G. Saito et al. / Advanced Drug Delivery Reviews 55 (2003) 199–215 201

disulfide bond during the process of translocation membrane is very low, and instead they are generallyacross cellular membranes into the cytosol of host endocytosed, carried to the lysosomes, and degraded.cells [3]. Studies on toxin conjugates using disulfide The lumen of endosomes and lysosomes are insidebonds were published as early as the late 1970s the cell, but still topologically extracellular. These[4–6], and ever since the disulfide-based bioconjuga- spaces are connected to the lumenal spaces of the ERtion approach has been a popular conjugation method and the Golgi apparatus, but separated from theapplied in a variety of cellular drug delivery systems. cytosolic space by membrane. The redox potential ofSuccessful applications of thiol-based conjugation the lumen of the endocytic compartments is muchhave obtained targeted delivery and enhanced cyto- less well characterized than the reducing cytosolicsolic delivery, improved pharmacokinetics and in- space and the oxidizing ER [8], or even the mitoch-creased stability. Moreover, such conjugates are ondrion [9,10], which will not be discussed in thisbeing used more frequently in polypeptide or review. We will start by reviewing how known redoxprotein-based systems and in plasmid gene and potential differences inside the cell, between cytosololigonucleotide formulations, some of which are versus ER, are maintained.discussed later in this review. These macromolecular In general, disulfide bond reduction, oxidation andagents are membrane-impermeant, due to their large isomerization are mediated by small redox moleculesmolecular size or polyanionic nature, and are typical- alone, or with the help of redox proteins. Redoxly endocytosed by cells. However, despite the sub- proteins typically require the co-presence of smallstantial biological evidence for reductive cleavage of redox molecules or other enzymes to regenerate anddisulfide bonds occurring in the endocytosed sub- retain their activity. Both factors seem to play majorstrates, the subcellular locations of these putative roles in maintaining a high free SH level in thereduction sites and the mechanisms by which endo- cytosolic space [11]. The thioredoxin family en-cytosed macromolecules are reduced remain poorly zymes regulate these processes [12]. Members of thisunderstood [7]. This review focuses primarily on family include ubiquitous cytosolic enzymes such asrecent discoveries regarding the disulfide reduction thioredoxin and glutaredoxin [13], all of which sharein the endocytic pathway, and on the related disulfide a common active site motif –Cys–Gly–His /Pro–conjugation-based delivery strategies for macromole- Cys–, two cysteines flanking two amino acids [12].cules. In particular, we concentrate on the redox Although these enzymes can catalyze reactions inenzymes and small redox buffers that reduce disul- both redox directions, the process in the cytosol isfides at cell surfaces, in various endocytic compart- predominantly reducing. This is due to the abundantments, and in the cytosol. presence of reduced glutathione (GSH) and thiore-

doxin reductase, which regenerate and maintain thecysteine catalytic active sites in the reduced state[12,14]. Glutathione (L-g-glutamyl-L-

2 . Cellular redox enzymes and redox agents cysteinylglycine) is the most abundant non-proteinthiol-source in mammalian cells, reaching millimolar

2 .1. Reducing cytosolic space and oxidizing ER concentrations [15], and thus is another importantspace determining factor in controlling cytosolic redox

potential. The redox state of glutathione in theFrom the perspective of cellular drug delivery, cytosol is, in turn, biased to the reduced state by the

access to the cytosolic space of eukaryotic cells is ratio of GSH to GSSG, which is greater than 100 inrestricted primarily to hydrophobic small drugs, most cells. This ratio is maintained catalyticallywhich have relatively high membrane partition co- GSSG→ GSH by glutathione reductase and NADPHefficients and permeability constants, i.e. they diffuse [15,16]. Consequently, the vast excess of reducedpassively across the lipid membrane. Macromolecu- GSH over oxidized GSSG, and the thioredoxinlar drugs have low diffusivity, and the plasma enzymes that are kept in the reduced state by themembrane is the primary barrier to entering the GSH, maintain a high reducing potential in thecytosolic space. Their uptake via permeation through cytosol. As a result, the environment in the cytosol,


and the topologically connected nuclear space, pre- 2 .2. Disulfide reduction at cell surface and earlyvents disulfide bond formation. endosomes

The lumen of the ER is maintained in an oxidizingenvironment. This is the intracytoplasmic compart- In contrast to the cytosol, GSH concentrations inment for synthesizing and processing secretory and the extracellular space are much lower; concentra-membrane proteins. In mammalian cells, disulfide tions in plasma typically being| 10 mM [17]. Thebonds are confined to secretory proteins and to the oxidizing environment in the extracellular spaceexoplasmic domains of membrane proteins that are generally favors the maintenance of disulfide bondsexposed to and processed in the oxidizing milieu of in cell surface proteins, as in the exoplasmic domainsthe ER. Hwang et al. [8] demonstrated that gluta- of membrane receptors, and in secreted proteins likethione was also responsible for the redox potential in insulin and immunoglobulins. Nevertheless, somethe ER. The GSH/GSSG ratio was estimated to be cell surface-associated redox enzymes possess re-between 1 and 3, supporting a redox state| 100 duced thiols [18]. PDI is expressed at plasma mem-times more oxidizing than that in the cytosol. Protein brane in addition to its major site of localization infolding and formation of disulfides are thus favored ER [19]. It contains a KDEL sequence at the Cin this oxidizing environment. Protein disulfide terminus which facilitates its retention in the ERisomerase (PDI), an ER protein of the thioredoxin lumen, causing it to cycle between the cis-Golgi andfamily, plays an important role in these activities. In the ER through its interaction with KDEL receptorsummary, the redox potential of the cytosol and the [20]. Despite its KDEL sequence and ER retentionER are largely controlled by the redox status of mechanism, however, PDI is also found throughoutglutathione, i.e. GSH/GSSG with thioredoxin family the secretory pathway and at the plasma membrane.enzymes acting as catalytic effector of that redox Although these distributions may reflect saturation ofpotential (Fig. 1). KDEL receptors [21], PDI secretion may also be a

regulated event, or perhaps cell type-dependent, asnot all KDEL-sequence containing proteins overflowfrom the ER to the cell surface [22]. Recent reportsindicate that plasma membrane-associated PDI in-deed plays important specific roles at the cell sur-face. Couet [23] has suggested that the thyrotropin(TSH) receptor in human thyroid cells undergoespartial shedding catalyzed by surface PDI; based ontheir observations that shedding can be inhibited byanti-PDI antibodies or by a membrane-impermeantsulfhydryl blocker, and can be increased by inhib-iting endocytosis and recycling. Cell surface PDI hasalso been identified in human B lymphocytes [24],platelets [25], rat hepatocytes [26] and rat pancreaticcells [27].

Involvement of surface-associated PDI in thedisulfide bond reduction of endocytosed material hasbeen demonstrated [28]. In Chinese hamster ovary

Fig. 1. Cellular redox enzymes and redox agents at different (CHO) cells, anti-PDI monoclonal antibodies and alocations within a cell. Reduction of the disulfide bonds in PDI-inhibitor, bacitracin, partially inhibited cleavageendocytosed macromolecular conjugates occurs via surface-associ-of disulfide bonds in the membrane-adsorptive conju-ated redox enzymes such as PDI and endosome/ lysosome redox 125gate, [ I]tyramine-S-S-poly(D-lysine). The role andenzyme, GILT with its putative co-factor cysteine. Redox po-

involvement of plasma membrane PDI in the endo-tentials of the reducing cytosolic space and the oxidizing ER spaceare regulated by the ratio of GSH and GSSG. cytic compartment were also demonstrated for


diphtheria toxin, which consists of two domains, A complicates efficient function at the cell surface. Infact, a number of studies indicate existence ofand B chains, connected via a disulfide bond.additional reduction schemes in the endosome/ lyso-Reduction of the disulfide bond in this toxin is asome compartments. Feener et al. [7] demonstratedprerequisite for release of A chain from the endo-that when CHO cells were incubated with disulfidesome into the cytosol, which is required for the

125linked conjugates, [ I]tyramine-S-S-poly(D-lysine),biological and pharmacological function of the toxinreduction started immediately (which was shownthat leads to cytotoxicity [29]. Although other pro-later to be suppressible by anti-PDI antibody [28])posed mechanisms have been suggested for thisand continued for 6 h. Addition of the membrane-reduction [30,31], cytotoxicity could be inhibitedimpermeant sulfhydryl inhibitor, DTNB, blocked thewith anti-PDI monoclonal antibodies [28] and byinitial phase of disulfide cleavage, but after a| 30-membrane-impermeant sulfhydryl blockers such asmin lag-time, the intracellular phase of reductionDTNB andp-chloromercuriphenylsulfonic acid [32].resumed, indicating that the reduction process con-These studies indicated that disulfide bond reductiontinued in endosomal / lysosomal pathways [7]. Shenof endocytosed macromolecules begins at the cellet al. [40] assessed the significance of surfacesurface, and may continue after macromolecules aredisulfide bond reduction using methotrexate trans-internalized, together with surface enzymes, intoport-defective CHO cells and methotrexate-S-S-poly-early endosomes (Fig. 1).(D-lysine) conjugates. The conjugates were cytotoxicMore recently described NADH-oxidase (NOX) isupon internalization and subsequent reduction, butanother cell surface-associated protein with disul-not when pretreated with a reducing agent,b-mer-fide–thiol interchange activity similar to PDI [33,34].captoethanol. The authors interpreted this to indicateInterestingly, activity of this enzyme in normal cellsthat reduction at cell surface was insignificant.is regulated by hormone or growth factors, but is

Antigen presentation provides further evidence forconstitutively activated in cancerous cells such asdisulfide bond reduction in the endocytic pathwayHeLa and hepatoma cells [35]. Thioredoxin, a small[41,42]. In antigen presenting cells (APCs), endo-ubiquitous protein (| 12 kDa) in the cytosol, wascytosed protein antigens are degraded into smallalso localized to surfaces of human white blood cellpeptides by proteases before binding to MHC class IIlines such as human B and T lymphocytes, mono-molecules. During this process, denaturation andcytes and granulocytes [36]. Other redox-active cellunfolding of the internalized proteins are facilitatedsurface enzymes may exist in addition to theseby the acidic environment of these compartmentsenzymes [22]. The role of these enzymes in disulfide[43]. Disulfide bonds, however, are not susceptible toreduction of endocytosed material has not beenthe lysosomal proteolysis and remain chemicallyinvestigated.stable in the acidic environment; they must becleaved by different process(es) [44,45]. In earlier2 .3. Disulfide reduction in endosomes and

125 131lysosomes studies using [ I]tyrosine-S-S-[ I]a -macroglobu-2125 131lin and [ I]tyrosine-S-S-[ I]transferrin to investi-

The significance of the cell surface enzyme-me- gate reduction after internalization by primary cul-diated reduction of endocytosed material remains tures of mouse peritoneal macrophages, Collins et al.unclear [37]. First, the functional activity of the [42] suggested that lysosomes are the primary site ofthioredoxin family members is optimal at neutral pH disulfide reduction of antigens. The disulfide bonds[38,39]. Thus, upon internalization, the catalytic in the transferrin conjugates, which recycled betweenactivity is most likely reduced rapidly as the pH in cell surface and early endosomes, were not reduced,the endosome/ lysosome compartments drops, favor- whereas those in thea -macroglobulin conjugates2

ing reduction on cell surface and in the earliest were reduced only after they reached the lysosomes,endocytic compartments. Also, optimal catalytic 15–20 min after uptake. Lloyd [46] and Pisoni et al.activity of these enzymes requires regeneration by [47] demonstrated that cysteine is actively trans-other accessory molecules such as glutathione. This ported from the cytosol to the lysosome lumen via a


specific transporter in a normal human fibroblast. expression also colocalized with MHC class II andGainey et al. [48] reported cysteine-specific transport the lysosomal marker Lamp-2 in mouse dendriticin lysosomes of a murine macrophage hybridoma cells [55]. It was suggested that this enzyme is alsoand in endosomes and lysosomes of a B cell involved in disulfide reduction of protein antigens inhybridoma cell line. The existence of these active early endosomes as well, since the precursor GILTtransport systems and accumulation of cysteine was catalytically active [51]. This thiol reductasemolecules in lysosomes and endosomes implied that possesses distinctive characteristics compared tocysteine-dependent disulfide reduction accompanies other known thioredoxin members. Firstly, its cata-endosomal / lysosomal proteolysis, and may help to lytic active site, –Cys–X–X–Cys–, does not havemaintain the catalytic activity of the lysosomal the common motif of –Cys–Gly–His /Pro–Cys–cysteine proteases (e.g. cathepsins B, H, and L). shared by members of the thioredoxin family. Sec-Incidentally and perhaps relevant, the resulting thiol- ondly, the optimal pH for thioredoxin family mem-oxidized cysteine dimer, cystine, was shown to be bers is typically neutral [37–39], and GILT has thepumped out from the lysosome lumen back to the pH activity optimum of 4.0–5.5. For instance, PDI, acytosol via a distinct transporter, where it may again member of the thioredoxin family, contains thebe reduced to cysteine by glutathione [49]. Defects –Cys–Gly–His–Cys– sequence with the pK ofa

in this cystine transport system are associated with cysteine at 7.3 [39], which is significantly lower thancystinosis [49]. the typical pK of | 8.5 observed in most thiols ina

proteins or peptides. This low pK favors PDI to bea

2 .4. Characterization of redox enzyme in the active at physiological pH in the ER, since theendocytic pathway: GILT cysteines require a deprotonation step in the process

of nucleophilic attack in order to form a covalentAlthough cysteine was claimed to be the physio- enzyme-S-S-substrate intermediate, followed by the

logical reducing agent in lysosomes [42,46,48], the adjacent second thiol attack, resulting in the reducedco-presence of redox enzymes with a small reducing substrate [56]. The question of how GILT, despiteagent such as cysteine or glutathione could facilitate having a thioredoxin-like structure, achieves an eventhe process more efficiently [50]. In addition, reduc- lower optimal pH remains to be elucidated [51].tion is inefficient in acidic environments since it Thirdly, in vitro studies have shown that GILTrequires deprotonation of thiols [7]. The presence of requires the co-presence of a reducing agent, such assuch enzymes, therefore, had been inferred for a long DTT or cysteine (but not glutathione) to regeneratetime, yet only recently was the first of its kind and retain its activity. Arunachalam et al. [38]identified and characterized [38]. Gamma-interferon- pointed out that cysteine may be one of its endogen-inducible lysosomal thiol reductase (GILT) is a 30- ous reducing buffers in vivo, since the presence of akDa soluble glycoprotein, which is expressed con- high concentration of cysteine has been postulated institutively in endosomes/ lysosomes of APCs in lysosomes and also in the endosomes of certain cellmultiple species including mice and humans [51,52], types [46–48] (Fig. 1).and is inducible by interferon (IFN)-g in other cell The importance of GILT in reducing disulfides intypes such as fibroblasts, endothelial cells and kera- endocytosed material in vivo is indicated by studiestinocytes [53]. This is notably the first reducing of GILT-knockout mice [55]. In wild-type mice,enzyme identified primarily in the endocytic path- GILT was markedly expressed in lymph nodes,way. GILT is synthesized as a 35-kDa molecular spleen and lung, where APCs play major roles, andweight precursor containing a mannose-6-phosphate only expressed weakly in kidney, liver, and musclesignal sequence (for delivery to lysosomes). Electron [55]. Using a well-characterized egg lysozymemicroscopic studies revealed that the precursor GILT (HEL) as a model antigen, it was shown that thecolocalized with early endosomes, while the mature presentation of two of the four chosen HEL epitopesGILT was only found in MHC class II-containing by spleen-derived APCs (e.g. B cells and macro-compartments (MIICs) [54], indicating that GILT is phages) from GILT knockout mice was abrogated.trafficked through the endocytic pathway [38]. GILT These results, along with the information from the


HEL epitope sequences, showed that the observed cates that disulfide-based drug delivery systemsdisruptions in stimulating their epitope-specific T cell require careful attention to the choice of target cell.clones were due to the lack of disulfide bond The significance of this issue will be further dis-reduction within or near those HEL epitopes in the cussed in the development of the recently FDA

APCs of the GILT knockout mice. approved antibody-based cancer drug, Mylotarg .The existence of GILT explains, at least in part,

the reduction process for endocytosed protein an-tigens in APCs (e.g. macrophages, B cells and 3 . Creating sulfhydryls via chemical anddendritic cells). Of interest is the expression and molecular approacheslocation of similar, related redox proteins in endo-somes/ lysosomes of other cells. A study that may 3 .1. Generating sulfhydryls through chemicaladd some clues in this respect is that of a CHO linkersfibroblast cell line. Merkel et al. [45] prepared twotypes of CHO cell transformants: cell line A, a CHO To conjugate two molecules via a disulfide bond,cell transfected with murine MHC class II gene, thiol groups need to be introduced into both struc-which convert this cell effectively to APC; and cell tures, unless endogenous thiols such as cysteines areline B, a hybrid of cell line A fused with a murine L already present. Different approaches are used tocell fibroblast. The CHO cell line A could elicit a incorporate a thiol or cysteine moiety in a drug. For

1CD4 T cell response only to the antigens lacking oligopeptides and oligonucleotides, it is easy todisulfide bonds, whereas the cell line B efficiently introduce a cysteine or to derivatize with a thiolprocessed even the antigens containing disulfide group during chemical synthesis. It is more difficultbonds, suggesting that some cells have decreased for small drugs, however, since they rarely possess aendosomal / lysosomal reduction capacity which thiol moiety for conjugation, and modification oftencould be complemented genetically. Moreover, when diminishes efficacy. There are, however, some ex-

125cleavage of disulfides in [ I]tyramine-S-S-poly(D- ceptions, such as maytansinoid [57] and CC-1065lysine) conjugates was compared, cell line A showed thiol-derivatives [58] and calicheamicin [59] intro-significantly less cleavage than cell line B. It is not duced in antibody-targeted drug delivery. Thiol(s) inknown which gene from the L cell fibroblast pro- a protein can be prepared in different ways. Whenvided the activity to CHO cells. Cell lineage-depen- free sulfhydryls are absent, they can be chemicallydent schemes of disulfide reduction in the endocytic generated; a popular approach is to modify primarypathway can be further supported indirectly from the amines found in surface exposed lysine residues or infinding that a B cell hybridoma transported cysteine the N-terminal residue with commercially availableboth into endosomes and lysosomes, while in a heterobifunctional linkers. These includeN-suc-macrophage hybridoma the transport activity was cinimidyl-3-(2-pyridyldithio)propionate (SPDP) withlimited to lysosomes [48]. In fibroblasts, endothelial a thiol-reactive pyridyl-disulfide moiety,N-suc-cells and keratinocytes, the induction levels of GILT cinimidyl S-acethylthioacetate (SATA) that can bemay vary depending on the cytokine levels such as deprotected after reaction with hydroxylamine toIFN-g in the surrounding milieu [53]. These observa- give a free thiol, or 2-iminothiolane which cantions imply that cell type-dependent variations in the complement the cationic charge of a primary aminedisulfide reduction mechanism of endocytosed lost upon conjugation [60]. Activated pyridyl disul-macromolecules could derive from a number of fides are convenient for creating a disulfide linkagefactors, including expression levels of redox proteins between two molecules [61]. The reaction with asuch as GILT and surface PDI, location of amino thiol becomes far more efficient with the leavingacid transporters in vesicular compartments, kinetics group, pyridine-2-thione, even in a pH-neutral buf-of vesicular trafficking, concentrations of disulfide- fer, and this can be followed spectrophotometrically.containing drugs at cell surface, position of disulfides In fact, this popular chemical synthetic approach isin the conjugates, and perhaps differences between utilized in most disulfide-based bioconjugate sys-transformed cell lines and primary cells. This indi- tems. Stability of disulfide bonds in vivo can be


problematic especially when the half-life of a deliv- substitution. This has been a powerful approach,ery vehicle in the systemic circulation is long. especially when combined with thiol-specificSterically hindered pyridyl disulfide linkers are also bioconjugate techniques, now that genetically alteredavailable, such as 4-succinimydyl oxy-carbonyl-2- proteins have become easier to design and prepare. Apyridyldithio toluene (SMPT) and possess increased cysteine can be inserted in appropriate positions of aserum stability [62]. If reversibility is inessential in a protein molecule to achieve site specificity anddelivery scheme, non-cleavable thio-ether bonds with optimal molecular orientation for recognition be-increased stability in vivo are more often adopted. tween ligands and receptors. Cochran et al. [66,67]This can be accomplished simply by reacting sulf- recently investigated the importance of molecularhydryls with sulfhydryl-reactive maleimide or distance and orientation in T cell recognition ofhaloacetyl derivatives instead of pyridyl disulfide MHC class II-antigen peptide complex conjugated toderivatives. various maleimide linkers using the MHC molecules

Chemical approaches also have drawbacks. Since that contained a cysteine genetically inserted atmost of the cross-linkers are based on conjugation to different sites. The site-directed bioconjugationprimary amine of lysine, a commonly found amino strategy can be found in numerous drug deliveryacid residue in a protein that exists at multiple sites, systems, particularly in attaching engineered anti-this method typically yields heterogeneity in the bodies (e.g. Fab9, single chain Fv, etc.) to a deliverydegree and position of substitution. vehicle [68–70]. A group at Celltech Therapeutics

demonstrated a site-specific pegylation (derivatiza-3 .2. Site-directed conjugation using endogenous tion with polyethylene glycol, PEG) via a thio-ethersulfhydryls or mutagenetically inserted cysteines bond toE. coli-expressed recombinant Fab9, posses-

sing a free cysteine at the hinge region; this did notAnother approach is to utilize endogenous cys- compromise binding activity and significantly im-

teines, if they exist in the protein structure. Since proved pharmacokinetic profiles [71]. Pegylation to acysteines normally occur at low frequencies in genetically introduced cysteine in staphylokinase, aproteins, site-specific end products can be expected. 136-amino acid profibrinolytic agent, via disulfideFor instance, a group from Bristol-Myers Squibb and thio-ether bonds was also shown to increasedeveloped doxorubicin (Dox)-antibody conjugates by plasma circulation time by decreased hepatic clear-selectively reducing the interchain disulfides, a total ance [72]. Since the approval of PEG-ADA (adeno-of four S–S bonds, in the hinge region and between sine deaminase) in 1990, pegylation chemistry hasthe light and heavy chains. They showed that eight developed based on covalent attachment via lysineequivalent Dox derivatives could be consistently residues, including recently approved pegylated IFN-conjugated to various antibodies using this method, a for chronic hepatitis C viral infections [73,74].while SH groups chemically obtained from modify- This method, however, as already discussed, general-ing primary amines with SPDP resulted in a rather ly results in heterogeneous end products. Thus, theinconsistent outcome [63]. The thio-ether bonded thiol-targeted, site-specific and controlled conjuga-conjugates to the endogenous cysteines, in which tion, which is essential for pharmaceutical manufac-Dox is cleaved from the antibody in the acid-labile ture and consistent therapeutic effect, may becomeacylhydrazone bond, are presently in human clinical the standard conjugation chemistry of the nexttrials [64]. The endogenous single cysteine in a generation pharmaceuticals.sulfhydryl-activated bacterial cytolysin (G. Saito etal. 2002, Gene Therapy, in press) and cysteines inpapain [65] have been demonstrated to be convenient4 . Disulfide linkage-employing drug deliverysites for reversibly conjugating polycation or PEG systemsvia a disulfide bond.

Even if cysteine is not readily available for In addition to the advantage of site-specific conju-conjugation, it can be incorporated into recombi- gation, the reversible nature of the disulfide bond isnantly expressed proteins by site-directed amino acid exploited in a number of ways for drug delivery. As


described in the previous sections, disulfides can be delivery schemes is also achieved with similarreduced by different mechanisms in different en- bioconjugation methods [79,80]. Cleavage of avironments. Depending on the locale of a drug targeting moiety from a delivery vehicle, however, isconjugate at which the reduction and cleavage of typically inessential, and more stable thio-etherdisulfides is anticipated, the delivery strategies are bonds are still more often utilized. For example, fordivided into three groups. Some examples are briefly more recent in vivo studies, Wagner’s group hasdiscussed below. switched the conjugation method from a random

SPDP modification of the targeting ligand to a more4 .1. Macromolecular delivery systems via site-specific approach; attaching polyethylenimineendocytosis (PEI) to the sodium periodate-treated carbohydrate

moiety of transferrin to give a Schiff base, followedThe use of disulfide bonds for delivery of macro- by reduction [81]. In addition to the controlled

molecular drugs via endocytosis has been supported conjugation benefit, the conjugates are more stable inby studies in vitro and in vivo. The first antibody- the systemic circulation [82].targeted chemotherapy drug, discussed in detail inthe in vivo application section, that utilizes disulfide 4 .1.2. Conjugation of membrane-disrupting agentsbonds to release anti-cancer drugs upon cellular for enhanced cytosolic deliveryinternalization, recently received FDA approval [75]. Secondly, bioconjugation schemes are used toAlthough various endocytic compartments have been incorporate membrane-disrupting agents that candemonstrated to possess reducing activities, includ- breach the endocytic membrane barrier. Delivery ofing cell surface, endosomes, lysosomes and the Golgi internalized cargo from the endocytic compartmentsapparatus, for the majority of the delivery schemes into cytosolic space has used membrane-activereviewed below, it is mechanistically unclear which fusogenic peptides [83]. Membrane active peptide-S-redox enzymes or agents are involved in that reduc- S-polycation incorporated in the gene and oligo-tion process. We will start from some of the recent nucleotide delivery systems was reported using theapplications in nucleic acid delivery. peptides derived from viruses such as influenza

hemagglutinin subunit HA-2 [84,85], and other4 .1.1. Attachment of cellular targeting moiety synthetic sequences such as amphipathic peptidesthrough sulfhydryls including GALA and KALA [86]. A more recently

In non-viral gene delivery systems, disulfide-based characterized cationic peptide from bee sting venom,conjugation techniques have aided development of melittin, capable of both membrane-disruption andformulations with high transfection potency. A key nuclear targeting [87], was incorporated into melit-weakness of non-viral vectors is their low transfec- tin-S-S-PEI /DNA complex and dioleoyl-S-S-melittintion efficiency compared to viral vectors. Disulfide- cationic lipid /DNA complex [88]. The endo-based conjugation methods have helped improve somolytic activities of these membrane-active pep-efficacy. First, conjugation of a targeting moiety is tides are largely controlled by their concentrationsroutinely used to enhance receptor-mediated cellular and conformation changes in the acidic environment,uptake. Wagner et al. [76,77] demonstrated increased which trigger membrane insertion and aggregationexpression of reporter genes after incorporating [89]. The importance of the disulfide bond cleavagetransferrin-S-S-polylysine conjugates to condense and release of peptides in these conjugates uponand complex with plasmid DNA. Erbacher et al. [78] cellular internalization, however, is not clear; as theused integrin-binding peptide (RGD)-S-S-PEI conju- disulfide bonded conjugates alone or the conjugatesgates prepared from the cysteine-containing peptide complexed to DNA are hemolytically active at lowand SPDP-modified PEI. Similarly, single chain Fv, pH without the presence of reducing agentspossessing a genetically engineered cysteine at the [84,87,88,90]. The peptides are also incorporatedC-terminus, was conjugated to SPDP-polylysine and through more non-specific conjugation approachesused to enhance receptor-mediated gene delivery such as biotin-streptavidin non-covalent linkage [90],[70]. Attachment of a targeting moiety to other and non-covalent ionic interactions between nega-


tively charged peptides and positively charged DNA/ in mammalian sperm. Sperm protamines are highlypolycation complexes [90,91]. cationic small proteins with over 50% arginine,

We recently developed a unique gene delivery which renders them capable of condensing DNAsystem that utilizes the endosomolytic mechanism of through ionic interaction. In mammals, protaminesLLO, a pore-forming protein from intracellular bac- also possess six to nine conserved cysteine residues,terium, Listeria monocytogenes, to enhance the which further compact and package DNA tightly bycytosolic delivery of plasmid DNA (G. Saito et al. forming inter- and intra-molecular disulfide bridges2002, Gene Therapy, in press). Our delivery system during spermatogenesis [95]. Reduction by cytosolictakes advantage of the reversible nature of disulfide glutathione has been implicated in the decondensa-bonds and is distinct from the fusogenic peptide- tion of sperm DNA after fertilization [96]. Disulfidebased delivery. The functional hemolytic activity of bonds also appear to be important in assembly andLLO, a member of sulfhydryl-activated hemolysins stabilization of viral particles [97]. Similar strategies[92], is regulated by the redox state of its single have been applied to non-viral vectors and shown tocysteine residue within its conserved domain near the be effective in stabilizing aggregation-prone DNA-C-terminus [93,94]. Thus, to complex with DNA, a polycation formulations. Blessing et al. [98] used apyridyl-dithio derivative of protamine was reacted to cysteine-containing cationic detergent to condensethe cysteine to prepare LLO with a disulfide link to DNA, which quickly dimerized via autoxidation intoprotamine, LLO-S-S-protamine. We demonstrated stable 23 nm DNA particles. DNA/polycation com-that the LLO-S-S-protamine conjugate or its DNA plexes were also prepared by covalently cross-link-complex completely lacks pore-forming activity of ing polyamines with disulfide bond-containingLLO, yet regains activity upon reduction. In a chemical linkers such as a bis-imidoester cross-linkerreducing environment, the disulfide bond is cleaved (DTBP) [99–101], and using synthetic oligolysinebetween the cysteine of LLO and the protamine, and peptides incorporated with varying numbers of cys-LLO is released as its hemolytically active form. The teines [102] and thiolated PEG-block-polylysine1:1 conjugation allowed us to estimate that only [103]. Although cross-linking DNA polyplexes40–160 LLO-S-S-protamine molecules in each through disulfides improves stability, diminishesprotamine/DNA complex of| 100 nm were suffi- aggregation, and extends the half-life in the systemiccient to produce significant enhancement in the circulation [100], the gene transfer efficiency de-reporter gene expression, in contrast to the require- creases with increasing numbers of disulfide bondsment of relatively high peptide amounts in fusogenic [100,102]. Oupicky et al. [100] suggested that thepeptide-based delivery. Reduced, active LLO ad- diminished transfection efficiency was due to theministered along with protamine/DNA complex decreased endosomal escape of the cross-linkedcaused rapid toxicity due to plasma membrane complex. Perhaps too much stability via disulfidesdamage, while the same dose of LLO-S-S-protamine leads to inefficient reduction and decondensation ofproduced high gene expression without any cytotox- DNA complexes in the endosomal environment.icity. This indicates that reduction occurs after the Promising results in the in vivo application ofcomplex is internalized, such that activated LLO disulfide bonds to liposomal delivery systems havemolecules are released from the complex and form been recently reported [104]. Pegylated liposomespores in the membranes of endocytic / lysosomal with prolonged circulation lifetimes broadened thecompartments. liposome-based drug delivery applications [105].

However, grafting PEG on the liposome surface4 .1.3. Bestowing stability and instability upon drug decreases the effectiveness of ‘pH-sensitive lipo-delivery systems via disulfides somes’ in the endosome [106]. To solve this prob-

Another application of disulfide bond usage for lem, Kirpotin et al. [107] synthesized a cleavabledrug delivery exploits its relatively stable covalent PEG-S-S-PE (phosphatidylethanolamine) lipid andbond to modulate the stability of formulations. An demonstrated in vitro that the destabilizing propertyexample in nature is in the structure of protamines, of pH-sensitive liposomes, consisting of PE andwhich condense and stabilize the chromosomal DNA cholesteryl hemisuccinate (CHEMS), could be re-


stored in the presence of DTT at pH 5.5 with 4 .3. Controlled disulfide cleavage in the systemiccleavable disulfide-containing lipids. Ishida et al. circulation[104] further extended its in vivo applicability byshowing the improved therapeutic efficacy of PEG- Despite the oxidizing environment, reduction ofS-S-PE over PEG-PE as a stabilizer for delivery of disulfide bonds does occur in the systemic circula-doxorubicin-loaded, anti-CD19-targeted, pH-sensi- tion, due to the presence of low concentrations oftive, PE/CHEMS liposomes. Because the drug was cysteine (| 8 mM in humans) and glutathione (| 2released swiftly from the vehicle, and its clearance mM in humans) [116,117]. This may inactivate thewas high due to the rapid cleavage of disulfides in disulfide bond-based delivery systems with longblood, the authors suggested that more significant blood-circulating times [62,104]. Zalipsky et al.improvements may be obtained using more stabilized [118] have taken advantage of this weakly reducingformulations in blood circulation. environment, and have developed a prodrug ap-

proach to conjugating PEG or lipids via a4 .2. Direct cytosolic delivery of macromolecules dithiobenzyl carbamate linkage [119] to an amine-across the plasma membrane containing small drug [120] or protein [121]. The

disulfide bonds are gradually reduced to release theTo overcome the plasma membrane barrier for drug or protein in circulation. The linker is designed

delivering macromolecules into the cytosolic space such that disulfide bond cleavage triggers decompo-of cells, physical methods such as microinjection, sition, resulting in release of the amino component ofscrape-loading and electroporation have been often the conjugate in its original structure. Trimble et al.used in vitro. In contrast to these conventional [122] investigated the release kinetics of a modeltechniques, there are unique biological approaches to peptide from cysteine mutants of hemoglobin-S-S-delivery across plasma membranes, bypassing the peptide conjugates in the presence of low concen-endosomal pathway, that employ ‘cell-penetrating’ trations of glutathione. They showed that the peptide[108], ‘fusogenic’ [109], or ‘pore-forming’ [110] release rates can be controlled by varying the sites offeatures of viruses and bacteria. Among these ap- attachment on the hemoglobin surface or by insertingproaches, disulfide bond can be and has been used aspartates in the peptide linker region. Charge repul-for conjugating cell-penetrating peptides (CPPs) sion between the nucleophilic glutathiolate anion and[111] such as HIV tat protein-derived peptides and the aspartates was postulated to be responsible forpenetratin. Although the recombinant DNA ap- the retarded release in the latter case. The concept isproaches can effectively produce CPP-cargo fusion attractive, but in vivo studies have yet to be done.proteins in large quantities, their applications arelimited to peptide or protein cargos. Chemical conju-gation via disulfides, on the other hand, enables the 5 . In vivo applicationsdelivery of non-protein macromolecules such asoligonucleotides [112,113], in addition to proteins 5 .1. Antibody-S-S-toxinand peptides [114]. Reduction of the disulfide bondin these conjugates takes place quickly once the While many of the investigations reviewed thusconstructs are delivered to the reducing milieu of the far have been limited to demonstrating their feasibili-cytosol. Hallbrink et al. [115] compared the penetra- ty in a cell culture model, in vivo studies of disulfidetion kinetics of different CPPs using (fluoro- bond-based bioconjugates have centered aroundphore)cargo-S-S-CPP(quencher) probes. CPPs in the those of antibody-S-S-toxin, immunotoxins thatconjugate were labeled with a fluorescent quencher, target diseased cells [123]. These delivery schemes3-nitrotyrosine, and the cargo was labeled with a are based on the efficient internalization of anti-2-amino benzoic acid fluorophore. The disulfide bodies before release of cytoxic components. Al-cleavage in the cytosol relieved the quenched fluo- though the first-generation immunotoxins, murinerescence, which was monitored in real time as monoclonal antibodies chemically coupled toincreased fluorescence intensity. subunits of ribosome-inactivating proteins, per-


formed well in vitro, the in vivo studies were not as human clinical studies have been encouraging. Insuccessful. This was due in part to the instability of fact, the first reversibly linked antibody-targeteddisulfides in the blood circulation [62]. To improve drug, anti-CD33 antibody-S-S-calicheamicin,

the stability, sterically hindered linkers, such as Mylotarg , developed by Celltech Group and Ameri-SMTP inserted with a methyl group adjacent to the can Home Products for treatment of acute myeloiddisulfide, have been employed. These immunotoxins leukemia, won FDA approval recently [75]. To ourwith methyl-hindered linkers have produced signifi- best knowledge, this is the first drug on the marketcantly greater anti-tumor activity compared to those that contains a synthetic disulfide bond in its struc-with unhindered linkers, in both preclinical and ture (the linker also contains cleavable acylhydra-clinical studies of non-Hodgkin’s (B cell) lymphoma zone bond). Immunogen is also licensing to apatients [62,124,125]. More recently, disulfide link- number of biotechnology companies a technologyers with higher hindrance, including a geminal based on a maytansinoid derivative, another potentdimethyl group, two methyl groups at each disulfide anti-cancer drug that can be conjugated to antibodiesend or a phenyl group, have been compared and via a disulfide. The disulfide bonds used in thesetested for their relevance to immunotoxin delivery conjugates are also stabilized with either a mono-[126,127]. The anti-tumor efficacies of these im- methyl or geminal dimethyl group attached to themunotoxins have not yet been evaluated. However, adjacent carbon.in a mouse model,b-phase half-life of the conjugates It is strategically important to know which of theseincreased in correlation with hindrance degree, cleavable linkers perform best in vivo. Unfortuna-which also correlated with disulfide bond stability tely, there are no publications that compare the sameagainst cysteine or glutathione. Importantly, the IC drug conjugates with different linkers. Cleavage of50

for cytotoxicity was comparable among these toxin these linkers releases different drug componentsconjugates; intracellularly unbreakable thio-ether resulting in varying efficacy and metabolism. Thislinked conjugates produced an inactive immuno- makes direct comparisons difficult, as in the case oftoxin, strongly indicating the significance of disulfide methotrexate-antibody conjugates containing a disul-bond cleavage in the endocytic pathway. fide or degradable peptidic linker [131,132]. On the

other hand, Hamann et al. [129,133] have recently5 .2. Antibody-S-S-drug reported important findings on the choice of linker

used in calicheamicin-antibody conjugates. The anti-A number of small anti-cancer drugs have been CD33 antibody-conjugate optimized for targeting

tested both in vitro and in vivo. Early antibody-drug leukemic cells (Mylotarg ) contains two cleavableconjugates contained clinically-used anti-cancer sites in the linker, a disulfide bond and an acyl-drugs such as methotrexate, doxorubicin and hydrazone bond. The latter linkage chemically un-mitomycin C conjugated via either non-cleavable dergoes pH-dependent hydrolysis, which is favored(e.g. amide and succinimide bonds) or cleavable in the acidic pH of endosome/ lysosome compart-linkage (e.g. acid-labile linkers such as hydrazone ments, a strategy also employed in the doxorubicinand cis-aconitic bonds, and lysosomally degradable immunoconjugate [134]. Conjugates were preparedpeptide linkers and disulfide bonds) (for review, see with a non-cleavable amide bond that replaced theRefs. [1,2]). The well-accepted linkers meet the hydrazone bond. In these, hydrolytic release of theessential criterion that they remain stable in the drug via a hydrazone linkage was required to retaincirculation for long periods, yet specifically break the conjugate’s high potency both in vitro, in vivoupon cellular internalization. These are sterically and ex vivo [129]. Interestingly, when the samestabilized disulfide bonds (discussed below), acyl- drug-linker was conjugated to an anti-MUC1 anti-hydrazone bonds [64,128,129] and degradable pep- body, which recognizes mucin epithelial antigentide linkers [130] (used in HPMA polymer drug expressed in many solid tumors, the hydrolyzableconjugates). With the use of humanized antibodies bond conferred no advantage; the disulfide bondand discovery of highly potent cytotoxic drugs, alone was sufficient to provide good results in allrecent results of a so-called ‘magic bullet’ strategy in preclinical models. The authors pointed out that


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