ISSN 10681620, Russian Journal of Bioorganic Chemistry, 2012, Vol. 38, No. 6, pp. 565–577. © Pleiades Publishing, Ltd., 2012. Original Russian Text © S.I. Romaniuk, D.V. Kolybo, S.V. Komisarenko, 2012, published in Bioorganicheskaya Khimiya, 2012, Vol. 38, No. 6, pp. 639–652.

Recombinant Derivatives: Perspectives of Application S. I. Romaniuk1, D. V. Kolybo, and S. V. Komisarenko Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv Received October 12, 2011; in final form, November 25, 2011

Abstract—Diphtheria toxin (DT) is a unique bacterial which consists of three domains with various biological functions. Using genetic engineering for the creation of various recombinant constructions of DT with definite features, it is possible to create unique tools for cellular biology and with efficient and selective action on certain populations of cells. The review highlights the structural and functional aspects of the DT molecule, its fragments and domains, as well as the major areas of application of its recombinant derivatives. In particular, the perspectives for practical use of recombinant DT derivatives are discussed for creating immunobiological preparations, cytotoxins, blockers of the heparinbinding epidermal growth fac torlike growth factor (HBEGF), protein constructions for direct delivery of substances into the , and also the possibility to use DT recombinant derivatives for therapy and prevention of a number of diseases.

Keywords: diphtheria toxin, fragments and domains of diphtheria toxin, recombinant , heparinbinding epidermal growth factorlike growth factor (HBEGF), vaccines, cytotoxins DOI: 10.1134/S106816201206012X

1 INTRODUCTION medicine, since they did not satisfy the thermostability requirements and the process to obtain them was not Diphtheria toxin (DT) is a globular protein that is standardized completely. classified to the family of the ABtype bacterial exo toxins [1]. It is the major factor of pathogenicity of the The development of genetic engendering tech Сorynebacterium diphtheriae, diphtheria causative niques made it possible to obtain recombinant diph agent [2]; nevertheless, the toxin is encoded in the theria with mutations in various enzymes that of a corynephage, which infects this bacte are currently more often used in both fundamental rium [3]. DT is the most toxic toxin among protein research and in medical practice (Fig. 1). The fast pace toxins in relation to sensitive cells in vitro (one mole of development in this area, and progress in the field of cule of this toxin is sufficient to kill a cell [4]), molecular biological techniques, make it urgent to although DT yields to the botulism and tetanus neuro estimate the prospects for the use of DT recombinant toxins in its influence on the organism [5]. The high derivatives in various areas of human activity. toxicity of DT opened perspectives on the use of its derivatives in medical practice and attracted the atten 1. Structure of the DT Molecule. Application tion of researchers to the study of the structure and of Recombinant Derivatives of the Toxin mechanism of action of this toxin. for Studying the Mechanism of Its Action When studying the antigenic features of DT, The DT is encoded by the nucleotide sequence of mutant forms of the toxin were found that exhibit no the tox (1683 bp) which is absolutely identical in cytotoxicity and were called CRM (from English— corynephages β [7], γ [8], and ω [9], which evidences crossreacting material) due to their ability of cross the high conservatism of this gene and the importance reactivity in a precipitation reaction with immune sera of all of the elements of the toxin molecule to fulfill its to DT [6]. Nevertheless, the chemically conjugated cytotoxic function. CRMbased chimeric proteins were not widely used in The DT polypeptide has a molecular weight of Abbreviations: DT is diphtheria toxin, CRM is crossreacting 58358 Da and consists of 560 residues, 25 material (mutant diphtheria is similar in its antigenic fea of which comprise the leader [10]. Two disul tures to the diphtheria toxin), HBEGF is heparinbinding epi fide bonds are formed between cysteines at the posi dermal growth factorlike growth factor, IL is interleukin, and scFv is a singlechain fragment variable. tions 186–201 and 461–471 [11]. Figure 2 shows a 1 Corresponding author: phone: +38(044)2343354; fax: scheme of the DT molecular structure, which consists +38(044)2796365; [email protected]. of two fragments: the fragment A (from English–

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Studying the functions of cell receptors Studying the mechanisms Studying the role of DT action of cells in biological processes

Fundamental

Areas for use for the recombinant DT analogues

Vaccines Direct delivery (against of drugs, Applied diphtheria and vaccines, other etc into cells infections)

HBEGF blockers Diagnostic (antitumor and test systems and antidiphtheria) immune therapeutic Cytotoxins for cancer, preparations virus, autoimmune, and other disease treatment

Fig. 1. The areas of recombinant DT derivatives application.

Active) (21164 Da) which presents the catalytic C of the eukaryotic eEF2 domain, and the fragment B (from English–Binding) that results in protein synthesis arrest in the cell [14] (37194 Da) which consists of a transport Tdomain and cell death via apoptosis [15]. and a binding Rdomain [11]. Each of the Using mutant diphtheria toxoids, the hydrophobic three domains of DT performs a specific biological Cterminal fragment (482–535) of the Rdomain of function for cytotoxic action. The Cterminal DT was established to be responsible for interaction Rdomain (386–535) binds with a receptor on the sur with the receptor of the target cell [12] (Fig. 3). The face of the cell [12], the Tdomain (205–378) provides spacious structure of the Rdomain is formed by two the translocation of the Cdomain into the cytosol βsheets which lie close to one another: the fourfold [13], and the Nterminal Cdomain (1–193) catalyzes βsheet is formed by the RB2, RB3, RB5, and RB8 the reaction of NAD+dependent ADPribosylation folds and the fivefold βsheet by the RB4, RB6, RB7, RB9, and RB10 folds [11]. The formation of hydrogen bonds between the RB6 fold and both βsheets results in the formation of the βbarrel with a jellyrolllike A B topology which is found for many proteins which С R interact with carbohydrate residues. The 461–471 dis ulfide bond holds a loop of nine amino acid residues by СRT which the phosphatebinding Psite (456–458–460– 472–474) with unknown function is located [11]. The S SSSLys516 and Phe530 residues, and also possibly the T Tyr514, Val523, Asn524, and Lys526, play an essential role in receptor recognition and are located in the exposed region of the Rdomain [16]. The DT is activated via splitting the peptide bond Fig. 2. Scheme of DT domain structure. C is the catalytic domain, T is the transmembrane domain, R is the recep between the fragments A and B in the region 186–201 torbinding domain, A is fragment A, B is fragment B, and under the action of proteinases, e.g., furin [17] in the –S–S– is bonds. presence of disulfide bond reducers. The uptake of

RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 38 No. 6 2012 RECOMBINANT DIPHTHERIA TOXIN DERIVATIVES 567

Glu148 Tyr54

Active center His21

Tyr65 Thr23

Fragment Hairpin loop Т8/9 Lys516 482–535

Asp352 Glu349

Fig. 3. Structural elements of the DT molecule that are especially essential for catalytic, transport, and receptorbinding functions of the toxin. activated toxin complex with a cellular receptor by the fusion of early [24], and possibly to with lower pH values (4.5–5.5) provides cause a delay in their maturation, which may be essen conformational rearrangements in the Tdomain [18], tial for the translocation process. which is a specialized pHdependent chaperone that Data on the participation of some cytosol proteins provides Cdomain translocation through the endo in the translocation of Cdomain have recently been some membrane into the cytosol [19]. obtained, e.g., vesicular transport proteins COPI, The Tdomain consists of nine helices organized which interact with Lys residues of the TH1 helix [25], into three layers. The first layer forms two long hydro actin [26], Hsp 90 chaperone, and thioredoxin reduc phobic Cterminal TH8 and TH9 helices; the second tase [27]. Nevertheless, a definite concept of the trans layer, three hydrophobic TH5–TH7 helices; and the location mechanism has not been formulated thus far, third, four TH1–TH4 helices with hydrophilic fea which makes it necessary and promising to further tures [11]. The construction of diphtheria toxoids study this process, including the application of recom which are able to bind with the receptor and exhibit binant DT derivatives. catalytic activity, but which would be unable to trans The Cdomain which carries out the NAD+ locate, e.g., the CRM503 [20], was useful for detecting dependent ADPribosylation of the eEF2 elongation the regions of the toxin molecule essential for translo factor consists of two βsheet subdomains, whose folds cation. Protonation of the Glu349 and Asp352 resi are oriented almost perpendicularly and form the core dues at low pH values (Fig. 3) leads to the TH8/9 hair of the domain. The first subdomain consists of the pin loop insertion into the membrane [21]. At a deep CB2, CB4, and CB8 folds which are surrounded by insertion, the TH5–TH7 helices are also inserted into the CH2, CH3, CH6, and CH7 helices. The second the membrane, and the TH5 helix, along with the TH8 subdomain consists of the CB1, CB3, CB5, CB6, and and TH9 helices, participates in pore formation. The CB7 folds which are surrounded by the CH1, CH4, TH6 and TH7 helices may form a “cork”, which par and CH5 helices. Four helices CL1–CL4 connect two tially blocks the transmembrane pore [22]. Protona subdomains providing the flexibility of the Cdomain, tion of the His257 residue is also likely to play an which is important for the transmembrane transfer. essential role in the translocation process, since a The active center of the Cdomain is a cleft formed His257Arg substitution results in a disturbance in the from the CB2, CB3, and CB7 folds and CH3 helix, structure of the Tdomain and interferes with its inser which is enclosed by the CL2 loop and RB6 fold of the tion into the membrane even at neutral pH [23]. The Rdomain [11] that explains the need to cleave the feature of the Tdomain to be inserted into the mem fragment B for toxin activation (Fig. 3). The amino brane is directly associated with its ability to stimulate acid residues of the active center were identified using

RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 38 No. 6 2012 568 ROMANIUK et al. toxoids with mutations in the Cdomain, e.g., joined to the Nterminal of the fragment A of the tox CRM228 [28] and CRM197 [29]. oid and follow the fragment A during transport [39]. It By means of interacting with His21, Thr23, Tyr54, was shown that using these constructions, polypep Tyr65, and Glu148 amino acid residues [30] (Fig. 3), tides with a molecular weight of up to 20 kDa may be the active center of the Cdomain binds NAD+ in a translocated through the cytoplasmic membrane; unique conformation at which the carboxamide group moreover, the double fragment A with a molecular of NAD+ occurs in the transposition, although the weight of 42 kDa may also be translocated through the cisposition is more stable for it [31]. The Tyr27 and membrane [40]. Trp50 amino acid residues are also important for By means of DTbased transport constructions, NAD+ binding [32]. During the interaction of NAD+ proteins which influence the intracellular processes with Pro38, and also possibly with Tyr54 and Trp153, may be delivered into . For example, using a hydrogen bonds are disturbed which stabilize the 39– recombinant protein obtained by joining the BclxL 47 loop in the active center of DT. The disturbance in apoptosis inhibitor to the Nterminal of the Rdomain this loop structure and the interaction with of DT, apoptosis of cells may be inhibited [41]. Similar 2'hydroxyl groups of and negatively charged fused proteins may be applied for disease therapy, phosphate groups of NAD+ is an essential factor for which are associated with disturbances in the expres eEF2 recognition [30]. sion of apoptosis inhibitors and activators. It is theo The ADPribosyl residue is joined to the eEF2 retically possible that this approach will be successful molecule through αglycoside bond with the first for studying the action of other intracellular regulator nitrogen atom of the imidazole ring of a unique amino proteins, including transcriptional factors. acid residue, diphthamide (2[3carboxyamido3 An area of using for transport, diphtheria toxoid (trimethylammonio)propyl]histidine) [33], which is based recombinant constructions, i.e., the administra formed as a result of a multistage sitespecific post tion of into cell cytosol with the aim of translational modification of the His715 [34]. The immune response induction restricted by the MHC I ADPribosylation reaction is carried out via the class antigens, and activation of cytotoxic CD8+ T mechanism of monomolecular nucleophilic substitu lymphocytes which are able to recognize these pep tion SN1: nicotynamide group removal leads to the for tides, are of interest [39]. Thus, experiments with the mation of an intermediate—carbocation, which is CD11cDTR transgenic mice, whose dendritic cells then attacked by nucleophilic diphthamide [35]. It is carried the humantype HBEGF receptors, showed a supposed that during the reaction, DT copies the nat significant stimulation of proliferation of CD8+ T ural conformation of the 80S eukaryotic ribosome for cells specific to ovalbumin after the administration of interaction with eEF2 [36]. The addition of ADP diphtheria toxoid fused with ovalbumin [42]. ribosyl to eEF2 diphthamide results in protein synthe sis arrest owing to the disturbance of peptidyl tRNA Similar recombinant transporters may be created translocation from the Asite to the Psite [37] and based on, not only diphtheria toxoids, but also some also attenuates the interaction of eEF2 with the sar other bacterial toxins, e.g., anthraxtoxin [43]. cin/ domain of 28S ribosome RNA [38]. Hence, clear links are observed between single 3. The Use of Recombinant DT Derivatives for Studying structural elements in the DT molecule and their cer the Expression of Cellular Receptors tain functions needed for the realization of toxic action. The development of molecular and cellular Fused proteins that consist of the fragment A of DT biological techniques provides almost unlimited possi and a specific ligand may be used to study the expres bilities to use the structural components of the DT sion of cell receptors. The cells which carry receptors molecule for construction of its derivatives with defi to this ligand turn out to be sensitive to the toxic action nite features. of this fused protein. This fact makes it possible to use similar recombinant constructions, e.g., to verify the successful expression of receptors by transgenic cells 2. The Use of Recombinant DT Derivatives [44]. for Constructing the Systems of Direct Protein and Peptide Delivery into the Cell The Rdomain itself is a selective ligand for the HBEGF (heparinbinding epidermal growth factor Despite the unclear view of the DT translocation like epidermal growth factor) transmembrane form mechanism, its Tdomain attracts the attention of [45]. We suggested using a fused recombinant protein more researchers as a promising transporter of thera which consists of green fluorescent protein (EGFP) peutic preparations and vaccines into cells. Diphtheria and the fragment B of DT as a fluorescent probe for toxoids with no catalytic activity may be used for direct studying HBEGF expression in cells [46]. These transport, in which the Rdomain is substituted into a probes may be applied in medical practice, since the ligand that provides binding with receptors of certain change in the expression of the HBEGF receptor on types of cells. As a rule, the passenger proteins are cells is a significant diagnostic sign of the development

RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 38 No. 6 2012 RECOMBINANT DIPHTHERIA TOXIN DERIVATIVES 569 of certain pathological processes, particularly, malig 5. The Use of DT Recombinant Derivatives nant transformation of cells. for Studying the Role of Various Populations of Cells in Biological Processes With the development genetic engineering tech 4. Recombinant Derivatives of DT as Blockers niques, the possibility appeared to construct recombi of HBEGF and Antidotes nant DT derivatives for selective killing of cells of a certain type in the organism with the aim of studying HBEGF, the transmembrane form of which is a their functions and role in any biological processes. receptor for DT, carries out a number of physiological Recombinant DT derivatives are used more often for functions in the organism: it stimulates cell migration this aim using two experimental models considered and proliferation, participates in many normal physi below. ological processes, such as blastocyst implantation The first experimental model was based on the use during pregnancy [47], wound healing [48], and neu of transgenic mice which carry the transmembrane ron restoration after hypoxia and ischemia [49]. In form of the human HBEGF, i.e., a receptor to DT, on addition, HBEGF is likely to participate in such the surface of certain types of cells that allowed selec pathological processes as tumor growth [50], hyper tive killing of these cells by administration of the natu plasia of smooth muscle cells, and atherosclerosis ral DT or diphtheria toxoids with attenuated toxic fea [51]. Therefore, there is possibility that HBEGF tures. The application of transgenic mice with the antagonists, including the DT mutant forms may be receptor to DT under the CD11b gene promoter applied for therapeutic purposes. allowed selective killing of cells of the monocyte– macrophage lineage and study of their role in arthro It is known that a nontoxic DT analogue with the sclerosis pathogenesis and the formation of athero Gly52Glu point mutation in the catalytic domain— sclerotic plaques [57]. CRM197—downregulates the mitogenic activity of The second experimental model was based on the the mature and transmembrane HBEGF [52], likely use of transgenic mice, in which the gene that encodes through blockage of its interaction with the HER2 and the fragment A of DT is placed under the control of a HER4 receptors. It has recently been shown that specific promoter of a unique gene expressed in these CRM197 or small interfering RNAs to the HBEGF cells. For example, the integration of this gene under gene may block the peritoneal dissemination of some the control of granzyme A gene promoter allows the types of tumors at various stages, which opens up per killing of cytotoxic Tcells [58]. This approach was spectives for the creation of a novel class of pharma used to study the role of Langerhans cells in the devel ceuticals to cure HBEGF dependent tumors [53]. opment of contact hypersensitivity [59]. Nevertheless, The fragment B of DT also exerts cytotoxic properties it should be noted that the construction of transgenic in terms of malignant cells [54], and similar features mice is quite a laborconsuming process, and the gene are likely to be revealed in its Rdomain. In addition, encoding the fragment A of the toxin may express non a potential possibility exists of using DTbased recom specifically in the cells of other types, as well due to binant blockers of HBEGF as antidotes for diphthe incidental integration that results in unforeseen kill ria treatment. For example, recombinant Rdomain of ing. Hence, research is currently carried out aimed at perfection of this approach [60]. the toxin (378–535) may be used to block DT binding with the HBEGF receptor in target cells [55]. Attempts were also made to use the EGFlike (106– 6. DT Based Cytotoxins for Treatment of Cancer, 149) domain of HBEGF as diphtheria antidote which Autoimmune, Virus, and Other Diseases is characterized by higher affinity to DT compared The major area of application of DT fragments and with the fullsize receptor and is incapable of nonspe recombinant derivatives is the construction of cytok cific binding with heparan sulphate proteoglycans pre ines which consist of two components: mutant diph sented in large quantity on the surface of cells. The theria toxoid with toxicity, but which is unable to EGFlike domain of the mutant HBEGF, with lost interact with the receptor of the native DT, and a com mitogenic activity due to Ile117Ala and Leu148Ala ponent which provides specific binding with a definite amino acid substitutions, was used in order to exclude type of cell. Antibodies to the surface antigens of cells its own biological activity from the recombinant prod or ligands which specifically bind with cell receptors uct [56]. may be used as this component. Various diphtheria toxoids are used for the construction of cytotoxins, A disadvantage of the abovementioned recombi most often DAB388, DAB389 (from English—DAB is nant proteins that impedes wide practical application diphtheria toxin A and B fragments) and CRM107. All is the possibility of adverse affects emerging due to these toxoids are unable to interact with the DT recep unforeseen interactions of these constructions with tor, i.e., DAB388 has no Rdomain [61], DAB389 has other proteins on the surface of and inside the cells. only 50 Cterminal amino acid residues of this domain

RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 38 No. 6 2012 570 ROMANIUK et al. and is characterized by enhanced resistance to pro cytotoxins are employed for Tcell killing during teinases [62], and the CRM107 carries only two point organ transplantation in addition to the introduction mutations in the Rdomain–Leu390Phe and of the donor bone marrow. Moreover, antibodies to the Ser525Phe [63]. Sometimes the use of the natural DT CD20 [83] marker of Bcells and to specific tumor with high toxicity does not allow sufficient selectivity antigens [84], antibodies to the αchain of IL2 recep when killing certain, especially small, populations of tor (CD25) which is expressed on proliferating human cells. Attenuated (15–30 times less toxic) toxoids may Tlymphocytes [85], and also the antibodies to the be used in these cases, e.g., CRM176 which is less gp120 and gp41 glycoproteins of the human immune toxic due to the Gly128Asp amino acid substitution deficiency virus (HIV) and to the surface antigens of near the active center of the Cdomain [64]. HIVinfected cells [86] are also promising compo nents of immunocytotoxins. Also, cytotoxins com Currently, cytotoxins are increasingly being used prising diphtheria toxoid and two domains of the which were obtained through the substitution of the human CD4 receptor interacting with the gp120 HIV Rdomain of DT on a sequence of a certain interleu antigen expressed on the surface of infected cells were kin (IL). A cytotoxin containing the IL2 sequence is designed [87]. the most efficient (DAB389IL2, Denileukin Diftitox, ONTAK) that is able to kill activated Tlymphocytes One of the approaches to protect cytotoxins from and monocytes which express IL2 receptors [65], and antibodies synthesized in the organism in response to produces positive results in transplant grafting, as well their administration is the encapsulation of cytotoxins as in the treatment of Tlymphomas [66], atheroscle into fusogenic liposomes [88]. However, these lipo rosis [67], schistosomiasis [68], acquired immune somes are able to fuse with any cells and may only be deficiency syndrome (AIDS), and some autoimmune used locally, e.g., for solid tumor treatment. Another diseases [69]. It is interesting that the use of solution to this problem is to maximally decrease the DAB389IL2 may enhance the effect of antitumor immunogenicity of cytotoxins. For this purpose, vaccines based on dendritic cells transfected with recombinant scFvantibodies (from English—single RNA of malignant cells [70]. Cytotoxins with the IL3 chain fragment variable) are often used instead of the and IL7 sequences are effective for leukemia treat entire antibody molecules, which are able to bind with ment [71, 72]. Cytotoxins with the IL4 sequence are surface antigens of the cell [85]. In addition, the con used for killing Bcells that produce class E immuno struction of mutant toxoids, which lack the most immu globulins, which play a key role in the development of nogenic epitopes, is one of the promising means of the allergic reaction [73], as well as for cure of decreasing immunogenicity of DTbased cytotoxins. Kaposi’s sarcoma, which often accompanied patients In addition to protein constructions, DNA with AIDS [74]. Cytotoxins with the IL13 sequence sequences that encode toxic DT fragments (DNA are used to cure brain tumors [75]. cytotoxins) are also used for killing certain types of Mutant variants of interleukins with enhanced cells and transported into cells in liposomes, nanopar affinity to a receptor, e.g., DT388IL3[Lys116Trp] are ticles, capsids of various viruses or using special poly often chosen for construction of cytotoxins [61]. In mers [89]. The simplest variant of a DNA cytotoxin is addition to interleukins, the GMCSF granulocyte the DNA sequence of DT fragment A that is integrated and macrophage colonystimulating factor [76], vessel under the promoter of a unique gene for this type of endothelium growth factor [77], transferrin [78], and cells, or under the control of a heatshock protein gene αmelanocyte stimulating hormone [79], are regarded promoter that permits regulating the expression of as promising components of antitumor cytotoxins. cytotoxins by temperature [90]. These DNA cytotox Cytotoxins obtained through the substitution of diph ins are developed for killing certain populations of theria toxoid Rdomain on the epidermal growth fac cells (e.g., mature CD8+ Tcells [58] or Bcells [91]) tor (EGF) may be used to prevent excessive growth of prior transplantation of organs and tissues as well as for smooth muscle cells and the formation of atheroscle cancer treatment. rotic plaques [80], as well as for selective killing of Another variety of DNA cytotoxins is the DNA tumors, which express an increased quantity of recep constructions which do not cause cell death after tors to this growth factor. transformation, but provoke stable expression of cyto Immunocytotoxins comprise a separate class of toxins in them which specifically kill cells of other cytokines, which include immune components— types. For example, in order to cure acute myeloid antibodies, or their recombinant analogues capable of leukemia, DNA cytotoxin was created which induces highly specific binding with an antigen on the surface the synthesis of DAB389IL4 in Tlymphocytes, of certain types of cells, and toxoid delivery into a cell which is toxic in relation to transformed Bcells [92]. through the receptordependent , for The third variety of DNA cytotoxins is the DNA example, among antibodies, monoclonal antibodies to protectors, i.e., plasmids which contain a nucleotide Tlymphocyte antigen, i.e., the CD3 complex [81], sequence of DT fragment A under the control of the and to chains of the Tcell receptor [82], turned out to gene promoter of a certain virus, e.g., HIV [93]. The be efficient components of immunocytotoxins. These cell transformed with this plasmid dies quickly in the

RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 38 No. 6 2012 RECOMBINANT DIPHTHERIA TOXIN DERIVATIVES 571 case when virus enters the cell. Since DT is toxic, not sesses sufficient protectiveness but often causes aller only for human and animal cells but also for cells, gic reactions especially with repeated administrations. DNA protectors were obtained against plant viruses. For One of the approaches to decrease the allergenicity example, the gene of the fragment A of DT was inte and immunogenicity of therapeutic antibodies is to grated into tobacco cells using agrobacteria [94]. decrease their molecular mass or to humanize the It should be noted that DNA constructions created antibodies, i.e., to transfer animal antibody fragments for gene therapy should penetrate the cell highly effec which determine their antigen complementarity into tively, exhibit minimal nonspecific toxicity, and do not the structure of human antibodies using genetic engi express in the healthy cells. neering techniques. The possibilities currently being intensively studied use human monoclonal antibodies, humanized animal antibodies, recombinant scFv 7. The Use of DT Recombinant Derivatives in Test antibodies with a 25–35 kDa molecular mass [102], System and Antidiphtheria Immunotherapeutic and also camel nanoantibodies, which consists of one Preparation Construction variable domain of the immunoglobulin heavy chain with a molecular mass of 15 kDa [103], as toxin neu Nontoxic diphtheria anatoxin is usually used as the tralizing agents. Low molecular mass scFvantibodies, antigen in immuno diagnostic tools (as well as in anti both mono and multivalent, are more widely used in diphtheria vaccines) obtained through treatment of experimental and clinical medicine for Parkinson’s the toxin with formaldehyde [95]. Diphtheria ana disease [104], Alzheimer’s disease [105], AIDS [106] toxin is similar to DT by its antigenic structure. Nev for detecting the antigens of encephalitis virus [107], ertheless, owing to the partial modification of its sur toxin B of difficile [108], toxin of Androcto face groups with formaldehyde, the anatoxin loses nus australis scorpio [109] and other antigens. We used some epitopes of the native DT [96]. nontoxic recombinant DT fragments for the immuni The recombinant analogues of DT differ from the zation of animals with the aim of creating an immuno native DT in terms of antigenic properties, but quite globulin gene library, as well as the selection of specific insignificantly compared with diphtheria anatoxin. In phages from this library, and to obtain antitoxic scFv addition, any diagnostically significant recombinant antibodies [110]. These recombinant antibodies may DT fragments and genetically fused proteins on their be used in test systems for DT detection and possibly basis may be obtained using the approaches of genetic as toxin neutralizing agents. engineering. Thus, we obtained recombinant DT frag ments A and B [97] as well as a fused protein consisting of the fragment B of DT and green fluorescent EGFP 8. Recombinant DT Derivative Based Vaccines against protein [46]. The investigations we performed showed Diphtheria and Other Infectious Diseases that antibodies to the fragment A often dominate the Recombinant derivatives of DT which lack toxic antibodies to the fragment B in patients with diphthe features or the capability of penetrating into the cell ria compared with infection carriers and healthy vac may be used in antidiphtheria vaccines of the new age, cinated patients [98], and recombinant A and B frag and also as carriers in vaccines against other infectious ments of DT may be used in differential diagnostics of diseases. One of the prevailing toxoids in antidiphthe diphtheria [99]. ria vaccines is the CRM197, which differs from DT by Another application of recombinant DT fragments only one amino acid substitution (Gly52Glu) in the is to obtain antibodies specific to certain regions of the catalytic domain [29]. Despite the fact that CRM197 DT molecule which may be used in test systems for relinquishes a little to anatoxin by immunogenicity DT detection using the sandwich immunoenzyme due to the influence of the amino acid substitution on assay (IEA), as well as being a component of immuno the conformation of the fragment B [111, 112], it is therapeutic preparations for treatmenting patients widely used in vaccines and offered for intranasal anti with diphtheria. The antibodies to the Cterminal diphtheria revaccination of the adult population [113]. fragment of the Rdomain of fragment B is known to The vaccine also comprises nontoxic diphtheria tox have the strongest protective features which interfere oid CRM228 [28] and CRM9 [114] and CRM45 [29] with DT binding with receptors of target cells [100]. In toxoids which are unable to bind with the receptor. addition, antibodies to the 188–201 loop, which con In order to enhance the immunogenicity of DT nects the fragments A and B are also able to neutralize mutant forms, various approaches are used: positive DT cytotoxicity, probably via preventing the pro charge increase with modification by ethylene amine teolytic activation of the toxin [101]. Antibodies to the [115], crosslink joining of toxoid molecules or engi active center of the fragment A block toxin action, but neered fusion of several copies of its fragment [116] as are not able to prevent DT binding with specific recep well as the expression of diphtheria toxoids or their tors [100]. fragments in cells of attenuated [117], Antidiphtheria antitoxic horse serum, which is streptococcus [118], salmonella [119], mycobacteria used for diphtheria treatment, contains highly affine [120], or influenza virus capsids [121] that, in addition F(ab')2fragments of horse antibodies to DT and pos to the enhancement of diphtheria toxoid immunoge

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(а) (b) C C

R R

T T

Tepitopes of DT Bepitopes of DT

(c) 271–290 321–350 351–370 411–450 T

50 100 150 200 250 300 350 400 450 500 141–157 188–201 224–237 194–378 386–453 454–481 513–526 30–40 B

C Domain ТDomain RDomain

Fig. 4. The antigenic structure of the diphtheria toxin (DT) (by the data [122–127]). The spatial location of (a) Tepitopes and (b) Bepitopes on the DT molecule (Tepitopes are indicated in dark gray, Bepitopes in black); (c) is the linear arrangement of Tepitopes (above) and Bepitopes (below) in the amino acid sequence of DT. C is the catalytic domain, T is the transmembrane domain, and R is the receptorbinding domain. nicity, induce immune responses against other infec Tepitopes of DT are the regions of the B fragment, tious agents. In order to expose the fragment of mutant which also directly participate in fragment A translo diphtheria toxoid on the surface of bacterial cells or cation through endosome membrane into the cytosol. virus capsids, a fragment of mutant diphtheria toxoid ADPribosylating protein bacterial toxoids and is genetically fused with a surface protein antigen of an their fragments [129] are often used as adjuvants for attenuated strain of the causative agent. The adminis newage vaccines, e.g., the cholerae toxin [130] or its tration of this live vaccine produces an immune fragment B [131], fragment C of the tetanus toxin response, which is enhanced with time due to long [116], partially nontoxic LTR72, LTK63, and term persistence of or viruses in the organism LTR192G mutants of thermolabile Escherichia coli [118]. For exposing on virus capsids or bacterial cells, [132]. The mechanism of the adjuvant both fullsized toxoids [121] and their various frag action of bacterial toxins is not fully clear; however, ments (fragment A [116], fragment B [121], and the stimulation of dendritic cell maturation by chol Rdomain of fragment B [117]) are chosen. erae toxin and thermolabile E. coli enterotoxin is The most immunogenic fragments of the polypep known to be mediated by the cAMP system and tide DT chain, which may be used for vaccine cre depends on the presence of catalytic activity in these ation, are presented in Fig. 4 [122–127]. The majority toxins [133]. of B and Tepitopes of DT compose the fragment B of The majority of current investigations in the area of DT, which explains its strong immunogenic features vaccine development are directed to the construction compared to the A fragment of DT [128]. As a rule, B of complex schemes of immunization with complex epitopes of DT are included in the fragments exposed (multivalent) vaccines which would defend against on the surface of the toxin molecule. The main many infections [134]. In the complex vaccines, diph

RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 38 No. 6 2012 RECOMBINANT DIPHTHERIA TOXIN DERIVATIVES 573 theria toxoids (CRM197, CRM9, CRM228, isms that is able to favor the enhancement of their vir CRMHis21Gly et al.) and their fragments are used, as ulence [156]. Current recombinant vaccines must be a rule, as carriers for weak immunogenic antigens (oli nontoxic, highly efficient, but produce no adverse gosaccharides, peptides, et al.) of various causative effects typical of the traditional vaccines, and not sup agents, due to their marked immunogenic features press the immune response to vaccines against other [135]. For example, they are used for diseases. enhancement against malaria [136], pertussis [137], poliomyelitis [138] causative agents, meningococcus [139], pneumococcus [140], streptococcus [141], shi CONCLUSIONS gella [142], type B Haemophilus influenzae bacteria The DT has been known to humans for more than [143], Moraxella catarrhalis bacteria [144], cholerae a hundred years and it is one of the most studied bac vibrios [145], and pathogenic fungi [146]. Some mul terial protein toxins. Since the fragments and separate tivalent conjugated vaccines based on the CRM197 domains of this molecule are highly specialized, DT diphtheria toxoid are already used in developed coun turned out to be a convenient object for the creation of tries for mass immunization of the population, e.g., various constructions with definite features using pro the Quattvaxem vaccine (Novartis Pharma, Switzer tein engineering. land) against diphtheria, pertussis, tetanus, and type B H. influenzae infection [147] as well as vaccines against Genetically modified toxoids and their fragments meningococcal [148] and pneumococcal infections are currently regarded as more promising components [149]. for vaccines than the chemically modified analogues of the natural toxins, since they are cheaper, quite Another approach in the development of vaccines immunogenic, and cause fewer complications in mul is the engineered substitution of one of the surface tiple vaccinations [157]. epitopes of DT fragment B on an epitope of another protein, e.g., poliomyelitis virus antigen [138] or main Despite broad perspectives of practical application external membrane protein of the meningitis causative of recombinant DT derivatives and its fragments, and agent [150]. Some synthetic vaccines have no carrier also the corresponding nucleotide sequences, a num and consist of peptide epitopes of proteins of various ber of serious problems exists which slow down the causative agents conjugated with one another: DT, the development of this area. First, in some cases the M protein of streptococcus, surface antigen of hepati industrial production of recombinant proteins turns tis B virus, parasitic plasmodium proteins [151]. These out to be complicated owing to the low level of their vaccines may be quite immunogenic, and they often expression and abnormal folding. Second, the meth do not relinquish their effectiveness to the vaccines ods for administering the genetically engineered con with carriers [152]. structions in the organism and their targeted delivery into the target cells remain quite inefficient. Third, the One of the important areas of the current vaccino protein constructions injected into the organism are logy and biotechnology is the development of methods affected by the action of proteolytic enzymes and to obtain cheap recombinant vaccines in bacteria and cause the synthesis of neutralizing antibodies, and . In 2007, recombinant analogues of the DTP DNA sequences may be aborted by the cells and be (diphtheria, pertussis, tetanus) vaccine were obtained eliminated from the organism. Fourth, the possibilities in Escherichia coli and tomatoes. The first experimen of the emergence of adverse effects, which are deter tal analogue of the DTP vaccine was a genetically mined by unforeseen interactions with other function fused protein which contained the immune protective ally active molecules on the surface of, and inside, the subunit S1 of , the fullsized nontoxic cells and incidental incorporation into healthy cells, diphtheria toxoid, and fragment C of the tetanus toxin have been little studied. In addition, the use of cyto [153]. The other analogue of the DTP vaccine was toxic DTbased constructions for virus infection cures obtained by transformation of tomatoes with a syn often appear ineffective due to the selection of infected thetic gene that encodes the six most immunogenic cells which do not express virus antigens on their sur epitopes of the pertussis, diphtheria, and tetanus tox face. Fifth, as a rule, the regulation of the level and ins, and also two adjuvant polypeptides using agrobac duration of DNA construction expression in the cells teria [154]. An alternative variant of antidiphtheria is impossible and the consequences of the interaction vaccine may be DNA vaccines that are able to induce of these constructions with the human genome or the synthesis of the diphtheria toxoid, or diphtheria other organisms entering the environmental ecosys toxoidbased protein constructions, in transformed tem are unpredictable. cells of the organism and to stimulate both humoral Nevertheless, DTbased recombinant proteins are and cell immune response [155]. easily modified allowing us to change the degree of The need to construct novel vaccines is determined their toxicity, remove the sequences which have cross not only by the low efficiency of the current vaccines, reactivity with nonspecific receptors, eliminate but also by the fact that vaccines may be the factor of adverse effects, decrease immunogenicity and allerge the evolutionary selection of pathogenic microorgan nicity, enhance resistance to proteinases and the dura

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