Send Orders for Reprints to [email protected] 6584 Current Pharmaceutical Design, 2014, 20, 6584-6643 Immunotoxins Constructed with Ribosome-Inactivating Proteins and their Enhancers: A Lethal Cocktail with Tumor Specific Efficacy Roger Gilabert-Oriol1, Alexander Weng2, Benedicta von Mallinckrodt1, Matthias F. Melzig3, Hendrik Fuchs1 and Mayank Thakur1* 1Institut für Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie, Charité - Universitätsmedizin Berlin, Campus Virchow- Klinikum, Augustenburger Platz 1, D-13353, Berlin, Germany; 2Wolfson Centre for Gene Therapy of Childhood Disease, University College London - Institute of Child Health, London, 30 Guilford Street, London, WC 1N 1EH, United Kingdom; 3Institute of Pharmacy, Free University Berlin, Koenigin-Luise Str. 2+4, D-14195, Berlin, Germany Abstract: The term ribosome-inactivating protein (RIP) is used to denominate proteins mostly of plant origin, which have N-glycosidase enzymatic activity leading to a complete destruction of the ribosomal function. The discovery of the RIPs was almost a century ago, but their usage has seen transition only in the last four decades. With the advent of antibody therapy, the RIPs have been a subject of exten- sive research especially in targeted tumor therapies, which is the primary focus of this review. In the present work we enumerate 250 RIPs, which have been identified so far. An attempt has been made to identify all the RIPs that have been used for the construction of immunotoxins, which are conjugates or fusion proteins of an antibody or ligand with a toxin. The data from 1960 onwards is reviewed in this paper and an extensive list of more than 450 immunotoxins is reported. The clinical reach of tumor-targeted toxins has been identi- fied and detailed in the work as well. While there is a lot of potential that RIPs embrace for targeted tumor therapies, the success in pre- clinical and clinical evaluations has been limited mainly because of their inability to escape the endo/lysosomal degradation. Various strategies that can increase the efficacy and lower the required dose for targeted toxins have been compiled in this article. It is plausible that with the advancements in platform technologies or improved endosomal escape the usage of tumor targeted RIPs would see the day- light of clinical success. Keywords: Targeted toxins, immunotoxins, ribosome-inactivating proteins, clinical application of toxins, tumor therapy, efficacy enhancer, endosomal escape enhancer. summary of these RIPs with relevant literature reference and the INTRODUCTION botanical description is elaborated in Table 1. The information pro- vided includes the origin of the RIP, its type and the reported usage Ribosome-Inactivating Proteins (RIPs) of this RIP as a targeted toxin. The term ribosome-inactivating protein (RIP) engenders a spe- While type I RIPs generally have lower toxicity, this is not cific class of toxins, mostly of plant origin, which act predomi- nantly on the ribosomal machinery via their N-glycosidase activity predominantly because of their lack of enzymatic activity but con- or polynucleotide adenosine glycosidase activity [1]. Although trastingly due to the missing B-chain making their cellular inter- nalization cumbersome [5]. The missing cell binding domain is a there is varying information about their mechanism of action, their enzymatic activity has drawn the most attention, especially relating blessing in disguise for molecular biologists, and has facilitated to the anti-viral and anti-tumor effects [2]. In general, all RIPs are them to prepare fusion proteins or synthetic analogs of type 1 RIPs together with ligands that are able to facilitate their cellular inter- considered to be N-glycosidases, thus removing adenines from ribo- somal RNA, and depurinating the conserved alpha-sarcin loop of nalization [6]. Moreover, in the recent decade, there has been a the 28S ribosomal RNA (rRNA). This leads to the inhibition of growing evidence that use of endosomal escape enhancers can lead to a significant augmentation of the efficacy of RIPs. This strategy protein synthesis, a vital process for cellular proliferation, and therefore leading to cell death [3]. has also paved a path for an improvement in the therapeutic utility of RIPs as targeted toxins or immunotoxins [5]. The plant RIPs are further classified as type 1, 2 and in rare cases as type 3. Type 1 RIPs are characterized by the presence of Endocytosis, Cytosolic Delivery and Enzymatic Action of RIPs only a toxic domain, whereas type 2 RIPs are the ones consisting of The toxic potential of RIPs is determined by their ability to a toxin domain (A chain) together with a cell binding domain (B reach to the ribosomes, which are located within the cytosol. Thus, chain of lectin type). The B-chain facilitates its binding to the ga- RIPs that are able to overcome cellular barriers end up exhibiting lactose residues on the cellular membrane, thus facilitating the cel- tremendous toxic potential. This overcoming of cellular barriers lular internalization. A further class of RIPs (type 3) has been pro- includes their internalization, which is generally facilitated by their posed but the exact classification and occurrence are ambiguous. B chain. Type 2 RIPs such as ricin from Ricinus communis L., abrin The literature description of type 3 RIP defines it as a protein which from Abrus precatorius L., or volkensin from Adenia volkensii is evolutionarily related to a 60-kDa jasmonate-induced protein Harms. [7] efficiently deliver their N-glycosidase domain (A chain) from barley, with RIP activity [4]. In total, there are nearly 250 into the cytosol of intoxicated cells [8] which is facilitated by their RIPs that are scientifically described and the information pertinent B chains. The B chain serves as galactose/N-acetylgalactosamine to them was retrievable upon an extensive literature search. A binding domain (lectin) and is linked to the A chain via disulfide bonds. *Address correspondence to this author at the Institut für Laboratoriums- After the binding with glycoproteins or glycolipids, which have medizin, Klinische Chemie und Pathobiochemie, Charité - Universitäts- medizin Berlin, Campus Virchow-Klinikum (Forum 4), Augustenburger numerous galactose residues on their surface, ricin is endocytosed Platz 1, D-13353 Berlin, Germany; Tel: +49-30-450-569206; via clathrin-dependent as well as clathrin-independent endocytosis Fax: +49-30-450-569900; E-mail: [email protected] and is thereafter delivered into the early endosomes. From there on 1873-4286/14 $58.00+.00 © 2014 Bentham Science Publishers Immunotoxins Constructed with Ribosome-Inactivating Proteins and their Enhancers Current Pharmaceutical Design, 2014, Vol. 20, No. 42 6585 Table 1. RIPs isolated from different plants, their type and reported absolute molecular masses. Plant RIP Type Ma (kDa) Immunotoxins Ref. Abelmoschus esculentus (L.) Abelesculin 1 30 [94] Moench Abrus precatorius L. Abrin-a 2 63 Yes [95] Abrus precatorius L. Abrin-b 2 67 [95] Abrus precatorius L. Abrin-c 2 63 [95] Abrus precatorius L. Abrin-d 2 67 [95] Abrus precatorius L. Abrin-I 2 64 [96] Abrus precatorius L. Abrin-II 2 63 [96] Abrus precatorius L. Abrin-III 2 63 [96] Abrus precatorius L. APA-I 2 130 [96] Abrus precatorius L. APA-II 2 128 [96] Abrus precatorius L. Abrus agglutinin 2 67 [95] Abrus precatorius L. Abrus agglutinin 2 134 [97] Abrus pulchellus L. Pulchellin 2 61.5 - 63 [98, 99] Adenia digitata Burtt-Davy Modeccin 2 57 [100] Adenia ellenbeckii Harms. Adenia ellenbeckii RIP 1 30 [101] Adenia ellenbeckii Harms. Adenia ellenbeckii RIP 2 60 [101] Adenia fruticosa L. Burtt-Davy Adenia fruticosa RIP 1 30 [101] Adenia goetzii Burtt-Davy Adenia goetzii RIP 1 30 [101] Adenia goetzii Burtt-Davy Adenia goetzii RIP 2 60 [101] Adenia keramanthus Harms. Adenia keramanthus RIP 2 60 - 65 [101] Adenia lanceolata Engl. Adenia lanceolata RIP 2 60 [101] Adenia lanceolata Engl. Lanceolin 2 61.2 [102] Adenia racemosa W.J. de Wilde Adenia racemosa RIP 1 30 [101] Adenia stenodactyla Harms. Adenia stenodactyla RIP 2 60 [101] Adenia stenodactyla Harms. Stenodactylin 2 63.1 [102] Adenia venenata Forssk. Adenia venenata RIP 1 30 [101] Adenia venenata Forssk. Adenia venenata RIP 2 60 [101] Adenia volkensii Harms. Volkensin 2 62 [103, 104] Agrostemma githago L. Agrostin-2 1 30.6 [105] Agrostemma githago L. Agrostin-5 1 29.5 [105] Agrostemma githago L. Agrostin-6 1 29.6 [105] Amaranthin (Amaranthus caudatus aggluti- Amaranthus caudatus L. 1 33 - 36 [106] nin, ACA) Amaranthus tricolor antiviral protein-27 Amaranthus tricolor L. 1 27 [107] (AAP-27) Amaranthus viridis L. Amaranthin 1 30 [108] Aralia elata (Miq.) Seem Aralin (Aralia elata lectin) 2 61.3 [109, 110] 6586 Current Pharmaceutical Design, 2014, Vol. 20, No. 42 Gilabert-Oriol et al. (Table 1) Contd…. Plant RIP Type Ma (kDa) Immunotoxins Ref. Asparagus officinalis L. Asparagus officinalis RIP 1 32.5 [105] Asparagus officinalis L. Asparin 1 1 30.5 [111] Asparagus officinalis L. Asparin 2 1 29.8 [111] Basella rubra Roxb. Basella rubra RIP 2a 1 30.6 [112] Basella rubra Roxb. Basella rubra RIP 2b 1 31.2 [112] Basella rubraRoxb. Basella rubra RIP 3 1 31.2 [112] Benincasa hispida (Thunb.) Cogn. Alpha-benincasin Small RIP 11 [113] Benincasa hispida (Thunb.) Cogn. Beta-benincasin Small RIP 10.6 [113] Benincasa hispida (Thunb.) Cogn. Hispin 1 21 [114] Beta vulgaris L. Betavulgin 1 28 [115] Beta vulgaris L. Beetin 27 1 27 [116, 117] Beta vulgaris L. Beetin 29 1 29 [116, 117] Bougainvillea spectabilis Willd. Bouganin (Bougainvillea spectabilis RIP) 1 26.2 Yes [112, 118] Bougainvillea xbuttiana Willd. Bougainvillea xbuttiana antiviral protein 1 35.5 [119] Bryonia dioica Jacq. Bryodin-L 1 28.8 [111] Bryonia dioica Jacq. Bryodin-1 (BD-1) 1 30 Yes [120] Bryonia dioica Jacq. Bryodin-2 (BD-2) 1 27 Yes [121] Camellia sinensis (L.) Kuntze Camellia sinensis RIP (CS-RIP) 2 63.6 [122] Celosia cristata antiviral protein 25 (CCP- Celosia cristata L. 1 25 [123] 25) Celosia cristata antiviral protein 27 (CCP- Celosia cristata L. 1 27 [124] 27) Charybdis maritima L. Charybdin 1 29 [125] Chenopodium album L. Chenopodium album antiviral RIP (CAP30) 1 30 [126, 127] Cinnamomum camphora (L.) J. Camphorin 1 23 [128] Presl. Cinnamomum camphora (L.) J. Cinnamomin 2 61 [128] Presl. Cinnamomum porrectum L. Porrectin 2 64.5 [129] Citrullus colocynthis Schrad. Colocin 1 1 26.3 Yes [111] Citrullus colocynthis Schrad. Colocin 2 1 26.3 [111] Clerodendrum inerme (L.) Gaertn CIP-29 1 29 [130, 131] Clerodendrum inerme (L.) Gaertn CIP-34 1 34 [130, 131] Croton tiglium L. Crotin I 1 ND [132] Croton tiglium L. Crotin II 1 30.2 [132] Cucumis figarei Naud. Cucumis figarei RIP (CF-RIP) 1 31.8 [133] Cucumis melo L. Melonin 1 23.5 [134, 135] Cucurbita foetidissima Kunth. Foetidissimin 2 63 [136] Immunotoxins Constructed with Ribosome-Inactivating Proteins and their Enhancers Current Pharmaceutical Design, 2014, Vol. 20, No. 42 6587 (Table 1) Contd…. Plant RIP Type Ma (kDa) Immunotoxins Ref. Cucurbita foetidissima Kunth. Foetidissimin II 2 61 [137] Cucurbita maxima L. Cucurmoschin Small RIP 8 [138] Cucurbita moschata Duchesne ex Alpha-moschin Small RIP 12 [139] Poir. Cucurbita moschata Duchesne ex Beta-moschin Small RIP 12 [139] Poir. Cucurbita moschata Duchesne ex Moschatin 1 29 Yes [140] Poir. Cucurbita moschata Duchesne ex Cucurmosin (CUS) 1 27 [141, 142] Poir. Cucurbita moschata Duchesne ex Cucurmosin 2 1 27.2 [143] Poir. Cucurbita moschata Duchesne ex Cucurbita moschata RIP 1 30.7 [144] Poir. Cucurbita pepo L. Pepocin 1 26 [145] Cucurbita texana (Scheele) A. Gray Texanin 1 29.7 [137] Dianthus barbatus L. Dianthin-29 1 29 [146] Dianthus caryophyllus L. Dianthin-30 1 29.5 Yes [147, 148] Dianthus caryophyllus L. Dianthin-32 1 31.7 Yes [147, 148] Dianthus sinensis L. Dianthus sinensis RIP (DsRIP) 1 33.3 [149] Eranthis hyemalis Salisb. Eranthis hyemalis lectin (EHL) 2 62 [150, 151] Gelonium multiflorum A. Juss. Gelonin (GAP31) 1 31 Yes [152, 153] Gynostemma pentaphyllum (Thunb.) Gynostemmin 1 27 [144, 154] Makino Gypsohila elegans Bieb. Gypsophilin 1 28 [155] Barley translation inhibitor (barley toxin I, Hordeum vulgare L. 1 31 Yes [156] BRIP) Hordeum vulgare L. Barley toxin II 1 30 Yes [157] Hordeum vulgare L. Barley toxin III 1 30 [157] Hordeum vulgare L. JIP60 (60 kDa jasmonate-induced protein) 3 60 [158] Hura crepitans L. Hura crepitans RIP 1 28 [105] Iris hollandica L. Iris agglutinin b (IRAb) 2 65 [159] Iris hollandica L. Iris agglutinin r (IRAr) 2 65 [159] Iris hollandica L. Iris RIP A1 (IRIP A1) 1 30.9 [160] Iris hollandica L. Iris RIP A2 (IRIP A2) 1 31 [160] Iris hollandica L. Iris RIP A3 (IRIP A3) 1 30.9 [160] Jatropha curcas L. Curcin 1 28.2 Yes [161, 162] Jatropha curcas L. Jc-SCRIP 1 38.9 [163] Lagenaria siceraria Molina. Lagenin 1 20 [164] Luffa acutangula Roxb. Luffaculin-1 1 28 [165] 6588 Current Pharmaceutical Design, 2014, Vol. 20, No. 42 Gilabert-Oriol et al. (Table 1) Contd…. Plant RIP Type Ma (kDa) Immunotoxins Ref. Luffa acutangula Roxb. Luffaculin-2 1 28 [165] Luffa acutangula Roxb. Luffangulin Small RIP 6.5 [166] Luffa aegyptiaca Mill. Luffin-c 1 ND [167] Luffa aegyptiaca Mill. Luffa ribosomal inhibitory protein (LRIP) 1 30 Yes [168] Luffa cylindrica Mill. Luffacylin Small RIP 7.8 [169] Luffa cylindrica Mill. Luffin-A (alpha-luffin) 1 27 Yes [170, 171] Luffa cylindrica Mill. Luffin-B (beta-luffin) 1 28 Yes [170] Luffa cylindrica Mill. Luffin-P1 Small RIP 5.2 Yes [172] Luffa cylindrica Mill. Luffin-S Small RIP 10 [173] Lychnis chalcedonica L. Lychnin 1 26.1 [111, 174] Malania oleifera Chun & S.K. Lee Malanin 2 61.9 [175] Manihot palmate Mill. Mapalmin 1 32.3 [111] Manihot utilissima Mill. Manutin 1 30.7 [176] Marah oreganus (Torr. ex S. Wats.) MOR-I (Marah oreganus RIP-I) 1 28 [177] Howell Marah oreganus (Torr. ex S. Wats.) MOR-II (Marah oreganus RIP-II) 1 27.6 [177] Howell Mesembryanthemum crystallinum L. RIP1 1 32.7 [178] Mirabilis expansa Standl. ME1 1 27 [179] Mirabilis expansa Standl. ME2 1 27.5 [179] Mirabilis jalapa L. Mirabilis antiviral protein (MAP) 1 27.8 [180] Mirabilis jalapa L. MAP-2 1 30.4 [180] Mirabilis jalapa L. MAP-3 1 29.7 [180] Mirabilis jalapa L. MAP-4 1 29.3 [180] Momordica balsamina L. Momordica balsamina RIP-1 (MbRIP-1) 1 30 [181] Momordica balsamina L. Momordin II 1 32 [182] Momordica balsamina L. Balsamin 1 28 [183] Momordin (Momordica charantia inhibitor, Momordica charantia L. 1 23 Yes [184] momordin-a) Momordica charantia L. Alpha-momorcharin (alpha-MMc) 1 29 [185, 186] Momordica charantia L. Beta-momorcharin (beta-MMc) 1 28 [187, 188] Momordica charantia L. Gamma-momorcharin Small RIP 11.5 [189] Momordica charantia L. Delta-momorcharin 1 30 [190] Momordica charantia L. Epsilon-momorcharin 1 24 [190] Momordica charantia L. Momordica charantia lectin (MCL) 2 130 [122] Momordica charantia L. Charantin Small RIP 9.7 [191] Momordin I (Momordica charanthia inhibi- Momordica charantia L. 1 31 Yes [147, 192] tor) Immunotoxins Constructed with Ribosome-Inactivating Proteins and their Enhancers Current Pharmaceutical Design, 2014, Vol. 20, No. 42 6589 (Table 1) Contd…. Plant RIP Type Ma (kDa) Immunotoxins Ref. Momordica cochinchinensis Spreng Momorcochin-S 1 30 Yes [193] Momordica cochinchinensis Spreng Momorcochin 1 32 Yes [194] Momordica cochinchinensis Spreng Cochinin B 1 28 [195, 196] Momordica grosvenorii Swingle Momorgrosvin 1 27.7 [197] Muscari armeniacum Baker. Musarmin-1 (MU-1) 1 28.7 [198] Muscari armeniacum Baker. Musarmin-2 (MU-2) 1 30 [198] Muscari armeniacum Baker. Musarmin-3 (MU-3) 1 27.6 [198] Nicotiana tabacum L. Tobacco RIP (TRIP) 1 26 [199] Nicotiana tabacum L. CIP31 1 31 [200] Oryza sativa L. Oryza sativa RIP 1 33 [201] Oryza sativa L. Oryza sativa cultivar Kazemi RIP 1 29 [202] Panax ginseng L. Panaxagin RIP-like 52 [203] Panax quinquefolium L. Quinqueginsin RIP-like 53 [204] Petrocoptis glaucifolia (Lag.) Boiss. Petroglaucin-1 1 26.7 [205] Petrocoptis glaucifolia (Lag.) Boiss. Petroglaucin-2 1 27.5 [206] Petrocoptis grandiflora Rothm. Petrograndin 1 28.6 [205] Phoradendron californicum Nutt. Phoradendron californicum lectin (PCL) 2 69 [207] PAP (pokeweed antiviral protein, Phytolacca Phytolacca americana L. 1 29 Yes [208, 209] antiviral protein) Phytolacca americana L. PAP II (pokeweed antiviral protein II) 1 30 Yes [209] Phytolacca americana L. PAP III (pokeweed antiviral protein III) 1 30 [210, 211] Phytolacca americana L. PAP-S 1 30 Yes [212] Phytolacca americana L. PAP-C 1 29 [213] Phytolacca americana L. PAP-R 1 29.8 [111] Phytolacca americana L. PAP-H 1 29.5 [214] Phytolacca dioica L. PD-S1 (Phytolacca dioica RIP 1) 1 30 [215] Phytolacca dioica L. PD-S2 (Phytolacca dioica RIP 2) 1 29.6 Yes [215, 216] Phytolacca dioica L. PD-S3 (Phytolacca dioica RIP 3) 1 30 [215] Phytolacca dioica L. PD-L1 1 32.7 [217, 218] Phytolacca dioica L. PD-L2 1 31.5 [217, 218] Phytolacca dioica L. PD-L3 1 30.4 [217, 218] Phytolacca dioica L. PD-L4 1 29.2 [217, 218] Phytolacca dioica L. Dioicin 1 1 30 [219, 220] Phytolacca dioica L. Dioicin 2 1 29.9 [219, 220] Phytolacca dodecandra L’Herrit Dodecandrin 1 29 [221] Heterotepalin-4 (Mexican pokeweed RIP-4, Phytolacca heterotepala H. Walter Phytolacca heterotepala anti-viral protein 1 29.3 [222] PAP) 6590 Current Pharmaceutical Design, 2014, Vol. 20, No. 42 Gilabert-Oriol et al. (Table 1) Contd…. Plant RIP Type Ma (kDa) Immunotoxins Ref. Heterotepalin-5b (Mexican pokeweed RIP- Phytolacca heterotepala H. Walter 1 30.5 [222] 5b) Phytolacca insularis antiviral protein (PIP, Phytolacca insularis Nakai 1 35 [223] insularin) Phytolacca insularis antiviral protein 2 Phytolacca insularis Nakai 1 35.7 [224] (PIP2) Pisum sativum L. Alpha-pisavin 1 20.5 [225] Pisum sativum L. Beta-pisavin 1 18.7 [225] Pisum sativum L. Sativin 1 38 [226] Polygonatum multiflorum RIP monomer Polygonatum multiflorum Kunth. 2 60 [227] (PMRIPm) Polygonatum multiflorum RIP tetramer Polygonatum multiflorum Kunth. 2 240 [227] (PMRIPt) Ricinus communis L. Ricin 2 62 Yes [228] Ricinus communis L. Ricin 1 2 64 [229] Ricinus communis L. Ricin 2 2 67 [229] Ricinus communis L. Ricin 3 2 66 [229] Ricinus communis L. Ricin D 2 60 [230] Ricinus communis L. Ricin E 2 60 [231] Ricinus communis L. Ricinus agglutinin (RCA120) 2 120 [97] Ricinus communis L. Ricinus agglutinin 1 (RCA1) 2 134 [229] Ricinus communis L. Ricinus agglutinin 2 (RCA2) 2 140 [229] Ricinus sanguineus Hort. ex Groen- Ricin R2 2 63.1 [232] land Ricinus sanguineus Hort. ex Groen- Ricin R11 2 57.8 [232] land Ricinus sanguineus Hort. ex Groen- Ricin R12 2 62.2 [232] land Ricinus sanguineus Hort. ex Groen- Ricinus sanguineus agglutinin 2 120 [233] land Sambucus ebulus L. Ebulin r 2 56 [234] Sambucus ebulus L. Ebulin I (ebulin 1) 2 56 Yes [235] Sambucus ebulus L. Alpha-ebulitin 1 32 [236] Sambucus ebulus L. Beta-ebulitin 1 29 [236] Sambucus ebulus L. Gamma-ebulitin 1 29 [236] Sambucus nigra L. Basic nigrin b 2 63.5 [237] Sambucus nigra L. Nigrin b 2 58 Yes [238] Sambucus nigra L. Nigritin f1 1 24.1 [239] Sambucus nigra L. Nigritin f2 1 23.6 [239] Sambucus nigra L. Sambucus nigra agglutinin I (SNAI) 2 140 [240] Immunotoxins Constructed with Ribosome-Inactivating Proteins and their Enhancers Current Pharmaceutical Design, 2014, Vol. 20, No. 42 6591 (Table 1) Contd…. Plant RIP Type Ma (kDa) Immunotoxins Ref. Sambucus nigra L. SNLRP 2 60 - 62 [241] Sambucus racemosa L. Basic racemosin b 2 58 [242] Sambucus sieboldiana L. Sieboldin-b 2 59.4 [243] Saponaria ocymoides L. Ocymoidine 1 30.2 Yes [244] Saponaria officinalis L. Saporin-6 1 29.5 Yes [105, 245] Saponaria officinalis L. Saporin-9 1 29.5 [105] Saponaria officinalis L. Saporin-L1 1 31.6 Yes [246] Saponaria officinalis L. Saporin-L2 1 31.6 [246] Saponaria officinalis L. Saporin-R1 1 30.2 [246] Saponaria officinalis L. Saporin-R2 1 30.9 [246] Saponaria officinalis L. Saporin-R3 1 30.9 [246] Saponaria officinalis L. Saporin-S5 1 30.9 [246] Saponaria officinalis L. Saporin-S6 1 31.6 Yes [246] Saponaria officinalis L. Saporin-S8 1 29.5 [246] Saponaria officinalis L. Saporin-S9 1 29.5 [246] Secale cereale L. Secale cereale RIP 1 31 [247] Sechium edule (Jacq.) Sw. Sechiumin 1 27 [248] Spinacia oleracea L. Spinacia oleracea RIP1 (SoRIP1, BP31) 1 31 [249] Spinacia oleracea L. Spinacia oleracea RIP2 (SoRIP2) 1 29 [249] Stellaria aquatica Scop. Stellarin 1 ND [250] Stellaria media (L.) Vill. RIP Q3 1 28.2 [251] Trichosanthes anguina L. Trichoanguin 1 35 [252] Trichosanthes cucumerina seed lectin Trichosanthes cucumerina Wall. RIP-like 69 [253] (TCSL) Trichosanthes cucumeroides Maxim. Beta-trichosanthin 1 28 [254] Trichosanthes dioica Roxb. Trichosanthes dioica seed lectin (TDSL) RIP-like 55 [255] Trichosanthes kirilowii Maxim. Alpha-kirilowin 1 28.8 [256] Trichosanthes kirilowii Maxim. Beta-kirilowin 1 27.5 [257] Trichosanthes kirilowii Maxim. Trichosanthin (TCS) 1 25 - 26 Yes [258] Trichosanthes kirilowii Maxim. TAP-29 (Trichosanthes anti-HIV protein 29 1 29 [259] kDa) Trichosanthes kirilowii Maxim. Trichobitacin 1 27.2 [260, 261] Trichosanthes kirilowii Maxim. S-Trichokirin Small RIP 8 [262] Trichosanthes kirilowii Maxim. Trichokirin-S1 Small RIP 11.4 [263] Trichosanthes kirilowii Maxim. Alpha-trichosanthin 1 31.7 [264] Trichosanthes kirilowii Maxim. Karasurin-A 1 27.1 [265, 266] Trichosanthes kirilowii Maxim. Karasurin-B 1 27.2 [267] Trichosanthes kirilowii Maxim. Karasurin-C 1 27.4 [267] 6592 Current Pharmaceutical Design, 2014, Vol. 20, No. 42 Gilabert-Oriol et al. (Table 1) Contd…. Plant RIP Type Ma (kDa) Immunotoxins Ref. Trichosanthes kirilowii Maxim. Trichosanthrip Small RIP 11 [268] Trichosanthes kirilowii Maxim. Trichomislin 1 27.2 [269] Trichosanthes kirilowii Maxim. Trichokirin 1 27 Yes [270] Trichosanthes lepiniate Maxim. Trichomaglin 1 24.7 [271] Trichosanthes sp. Bac Kan 8-98 Trichobakin 1 27 [272] Triticum aestivum L. Tritin 1 30 [273] Vaccaria pyramidata Medik. Pyramidatine 1 28 Yes [244] Viscum album L. Viscumin (mistletoe lectin I) 2 60 Yes [274] Viscum articulatum Burm. F. Articulatin-D 2 66 [275] Ximenia americana L. Riproximin 2 63 [276] Zea mays L. Maize seed RIP (b-32, corn RIP) 1 32.4 [277] Zea mais L. Maize proRIP 3 34 [278] it is transported to the Golgi-apparatus by retrograde transport and only one internalized molecule is sufficient to kill one cell. From an finally reaches the endoplasmic reticulum (ER). Within the ER the evolutionary point of view, it has been suggested that the B chain of disulfide bonds are cleaved by thioredoxin reductases and disulfide ricin was generated by a lateral gene transfer from a bacteria. isomerases [9, 10]. The enzymatically active A chain is released Contrasting to type 2 RIPs, type 1 RIPs are less toxic [21] and and partially unfolded during this process [11]. To facilitate its consist of only the A chain (N-glycosidase), which lacks any spe- entry into the cytosol, the A chain exploits a mechanism, which is cific cell binding properties. The low cytotoxicity of type 1 RIPs is known as ER-associated degradation (ERAD). ERAD is a natural generally attributed to an inefficient endocytosis. However, based mechanism for maintaining the homeostasis of the ER [12]. Pro- on some other reports [22] and our own experiments (Fig. 1), it is teins that are misfolded and thus non-functional are designated for admissible that type 1 RIPs are effectively internalized. The major proteasome degradation within the cytosol. The transport of the problem restricting their efficacy is the inefficient endosomal re- partially unfolded A chain is mediated by the translocon Sec61p lease. [13] and the ER degradation-enhancing (cid:2)-mannosidase-like protein 1 [14]. One of the most important factors for the cytosolic delivery is the recognition of the A chain as a substrate for the ERAD sys- tem. This is achieved by disguising the A chain as a misfolded pro- tein. After reaching the cytosol the partially unfolded A chain is fully refolded to regain the conformational integrity as an enzy- matically active form. This is facilitated by the chaperons Hsc70 and Hsp90 [15]. Genetic interaction maps indicate the involvement of a number of different factors responsible for modulating the ricin trafficking [16]. The cytosolic delivery of the A chain marks the end of a highly efficient molecular strategy that ricin adopts in or- der to direct the catalytic domain to the ribosomes. As mentioned before, a common feature of all the RIPs is their ability to depurinate the rRNA by releasing an adenine residue at their (cid:2)-sarcin/ricin loop. This results in an irreversible inhibition of protein synthesis facilitated by the prevention of eukaryotic elonga- tion factor binding [17]. According to the protein data bank (PDB), RIPs belong to a group of rRNA N-glycosidases (EC 3.2.2.22) that hydrolyze the N-glycosidic bonds at the position 4324 on the 28S Fig. (1). Three-dimensional depiction (z-stacks) of the endosomal network rRNA. The bond between the N9 of adenine and the C1 of ribose is of ECV-304 cells loaded with Alexasaporin. ECV-304 cells were challenged hydrolyzed by a concerted action of an arginine at position 180 for 3 h with 1 (cid:3)M Alexa-Fluor 488 labeled saporin (type I RIP from (R180) and a glutaminic acid at position 177 (E177). E177 is stabi- Saponaria officinalis L.). Cells were co-incubated with pH rodo™ Red lized at a cationic oxocarbenium ribose transition state and R180 is Dextran, a marker for endo/lysosomes and analyzed by confocal live cell responsible for activating water. This facilitates the nucleophilic imaging. Depicted is the endo/lysosomal network of one living ECV-304 attack on the C1 of the oxocarbenium intermediate resulting in the cell. Green: Alexasaporin in celular vesicles, red: pHrodo™ Red Dextran in release of adenine [18]. Mutants lacking E177 and R180 are also endosomes/lysosomes, yellow: co-localization of Alexasaporin and pHrodo™ devoid of the N-glycosidase activity [19]. Recent studies suggest Red Dextran in endosomes/lysosomes. The figure illustrates the fact that that the action of RIPs on ribosomes depends on the ribosomal saporin is internalized and trapped in to the endosomal vesicles, thereafter it stalk, which is a network of different proteins that recruit transla- is degraded by the endo/lysosomal degradation. tional factors to the ribosomes [20]. After gaining access to their substrate, RIPs act as toxic agents. It is further hypothesized that Immunotoxins Constructed with Ribosome-Inactivating Proteins and their Enhancers Current Pharmaceutical Design, 2014, Vol. 20, No. 42 6593 The exact mechanism of the internalization of type 1 RIPs is toxins and have demonstrated high specificity in in vitro and pre- not deciphered so far. Previous studies indicate towards a receptor- clinical evaluations. The ligand, apart from providing selectivity, mediated endocytosis of type I RIPs by low density lipoprotein also helps in cellular internalization of the toxin. There are a num- (LDL) receptors [23-26]. Contrastingly, some other results confirm ber of aspects associated with the internalization and trafficking of a receptor independent endocytosis [22]. However, the exertion of toxins. When the toxins are transformed into targeted toxins, there toxic effects appears to be independent of the internalization are numerous critical elements deciding their fate in vitro and in mechanism. The toxicity determining factor is the ability of type 1 vivo; these events are discussed in detail hereafter. RIPs to cross the endo/lysosomal membrane. Since type 1 RIPs do not contain any transduction domains facilitating the endo/lyso- Antigen Selection and Efficiency of Internalization somal escape into the cytosol, they are less cytotoxic. Upon endo- The analysis of the expression pattern of tumor-associated sur- cytosis, type 1 RIPs are delivered into the cellular compartments face antigens and the knowledge about their ability to promote or that are positive for lysobisphosphatidic acid (LBPA) (a specific modulate the tumor growth are critical for the identification of eukaryotic phospholipid marker for late endosomes) and the novel targets for targeted anti-tumor therapies. For the development lysosomal-associated membrane proteins LAMP1 and LAMP2 [22, of monoclonal antibodies (mAbs) or targeted toxins, it is essential 27]. Type I RIPs are thereafter degraded within the lysosomes [5]. to determine, whether a particular surface antigen undergoes an accelerated internalization or not (Fig. 2). There is a variety of can- Immunotoxins and Targeted Toxins cer-associated antigens that are being targeted by mAbs [32, 33]. Immunotoxins as per definition are conjugates of cell binding For mAbs that mediate their efficacy in part by interaction with antibodies and the complete type 1/2 RIP or the A chain of a type 2 natural killer cells (NK) (antibody dependent cellular cytotoxicity, RIP [6]. In all the reported cases, the complete type 2 RIP has a ADCC), it is important to select antigens, which do not undergo very high cytotoxic effect when conjugated to the antibody. None- rapid down-regulation after binding. This is a feature contrasting theless, there is an increased side effect due to the off-target binding the modality of targeted toxins, where it is desirable to select anti- of the B chain. To circumvent this, a lot of alternative strategies gens that show enhanced endocytosis after ligand binding [34]. This including but not limited to the use of high concentrations of free facilitates a rapid delivery of the toxin into the cancer cells. galactose or lactose as competitive binders have been tested. An- The receptor that is being addressed by the targeted toxin other alternative in overcoming this problem has been the use of should be over-expressed on the tumor cell surface compared to the steric hindrance [28]. Coupling of an antibody or its fragment to the normal tissue. A considerable number of receptors [35] have been isolated A chain via disulfide linkage appears to be the most effec- addressed to date, amongst them are the cytokine receptors [36], tive strategy. RIPs lack thiol groups for a disulfide linkage and it is tumor necrosis factor receptor, growth factor receptors [37, 38] and necessary to synthetically introduce it. Alternatively, other linkages cluster of differentiation CD22 [39], CD25 [40] and CD30 [41]. such as maleimide linkage have also been attempted but are not Contrasting to the numerous advantages listed above, a drawback of successful, mainly due to the inability of cellular enzymes to reduc- antibody-based targeted toxins is their limited ability to induce the tively cleave the bonds [29]. effector functionalities of the naked antibodies. It is in fact a pre- Another important term for the fusion proteins comprising of dominant basis for the concept of targeted toxins, wherein it is en- toxins is targeted toxin. It is a term which coherently finds usage in visaged to outweigh the biological functionalities of the monoclonal the literature to define a generic name for immunotoxins. In gen- antibodies by conjugating them to bacterial toxins such as Pseudo- eral, targeted toxins comprise of tumor specific ligands coupled to monas exotoxin from Pseudomonas aeruginosa [42] or plant toxins polypeptide toxins. They constitute a class of cancer therapeutics such as saporin from Saponaria officinalis L. that leads to the death of cancer cells. They mainly act by the inac- tivation of cytosolic protein synthesis and induction of programmed Release of Targeted Toxins into the Cytosol and their Lysoso- cell death [3]. Immunotoxins are per se, restricted to an antibody or mal Degradation antibody fragment as the targeting moiety whereas, targeted toxins Once internalized, the targeted toxin is delivered into early form a larger domain including the use of antibodies, small anti- endosomes. Early endosomes are part of the endosomal transport body fragments, growth factors, cytokines or small peptides as tar- system, which is an intracellular vesicular and tubular compartment geting moieties. Thus, immunotoxins form a smaller subset of tar- surrounded by cytosol. Within early endosomes, endocytosed geted toxins as a classification in general. ligands (targeted toxins) are either designated for recycling [43, 44] These targeted toxins can either be prepared by chemical con- or they are further transported into late endosomes, and finally jugation as described above, or they can be produced recombinantly lysosomes for degradation. Since targeted toxins exert their anti- as a fusion protein that is expressed in cells [6]. Within the past two tumoral efficacy only in the cytosol, it is a vital prerequisite for decades, significant progress has been made towards proper identi- their efficacy that they are able to escape from the endosomal net- fication of the appropriate cellular target for toxins with target work into the cytosol. Targeted toxins fused to truncated variants of specificity. Moreover, tremendous progress made in the field of bacterial toxins such as diphtheria toxin (DT) from Corynebacte- genetic engineering and a better understanding of receptor physiol- rium diphtheriae utilize the native T-domain of DT to escape from ogy coupled with the single molecule tracking modality have led to early endosomes into the cytosol [42, 45, 46] while other targeted an exponential growth in the scientific output as far as targeted toxins employ a KDEL-like motive of their toxin moieties, which in toxins are concerned. This is further evidenced by an increased turn facilitate their retrograde delivery into the ER and thereafter number of clinical trials which are being conducted on targeted their transport to the cytosol [47]. However, plant-derived toxins toxins, with many of them in Phase 3 [30, 31]. such as saporin and gelonin or the A chain of the type 2 RIP ricin does not comprise of such translocation domains. It can be therefore Plant RIPs constitute a major portion of the therapies with tar- anticipated that the cytosolic delivery of type 1 RIP-based targeted geted toxins, and although there is additional literature available on toxins is attenuated, compared to appropriate bacterial counterparts. bacterial and human toxins, plant RIPs generate a lot of scientific However, comparative studies in this regard have not been under- interest. As listed in Table 2, there are more than 450 targeted tox- taken so far. ins described, which comprise of plant RIPs as a toxic moiety. Amongst various RIPs the leading toxin components are ricin A Several strategies such as photochemical internalization [48], chain from Ricinus communis L., saporin from Saponaria offici- pore formation by listeriolysin O from Listeria monocytogenes [37], nalis L. and gelonin from Gelonium multiflorum A. Juss. A lot of cell penetration by protein transduction domains [49], the use of different targeting ligands have been successfully coupled to these lysosomotropic agents like chloroquine [50] or the use of triterpe-