Immunotoxins Constructed with Ribosome-Inactivating Proteins and their Enhancers: A Lethal Cocktail with Tumor Specific Efficacy

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 extensive 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 identified and detailed in the work as well. While there is a lot of potential that RIPs embrace for targeted tumor therapies, the success in preclinical 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 daylight of clinical success.

INTRODUCTION

Ribosome-Inactivating Proteins (RIPs)

The term ribosome-inactivating protein (RIP) engenders a specific class of toxins, mostly of plant origin, which act predominantly on the ribosomal machinery via their N-glycosidase activity or polynucleotide adenosine glycosidase activity [1]. Although there is varying information about their mechanism of action, their enzymatic activity has drawn the most attention, especially relating to the anti-viral and anti-tumor effects [2]. In general, all RIPs are considered to be N-glycosidases, thus removing adenines from ribosomal RNA, and depurinating the conserved alpha-sarcin loop of the 28S ribosomal RNA (rRNA). This leads to the inhibition of protein synthesis, a vital process for cellular proliferation, and therefore leading to cell death [3].

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 only a toxic domain, whereas type 2 RIPs are the ones consisting of a toxin domain (A chain) together with a cell binding domain (B chain of lectin type). The B-chain facilitates its binding to the galactose residues on the cellular membrane, thus facilitating the cellular internalization. A further class of RIPs (type 3) has been proposed but the exact classification and occurrence are ambiguous. The literature description of type 3 RIP defines it as a protein which is evolutionarily related to a 60-kDa jasmonate-induced protein from barley, with RIP activity [4]. In total, there are nearly 250 RIPs that are scientifically described and the information pertinent to them was retrievable upon an extensive literature search. A summary of these RIPs with relevant literature reference and the botanical description is elaborated in Table 1. The information provided includes the origin of the RIP, its type and the reported usage of this RIP as a targeted toxin.

Table 1.

RIPs isolated from different plants, their type and reported absolute molecular masses.

Plant	RIP	Type	Ma (kDa)	Immunotoxins	Ref.
Abelmoschus esculentus (L.) Moench	Abelesculin	1	30		[94]
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]
Amaranthus caudatus L.	Amaranthin (Amaranthus caudatus agglutinin, ACA)	1	33 - 36		[106]
Amaranthus tricolor L.	Amaranthus tricolor antiviral protein-27 (AAP-27)	1	27		[107]
Amaranthus viridis L.	Amaranthin	1	30		[108]
Aralia elata (Miq.) Seem	Aralin (Aralia elata lectin)	2	61.3		[109, 110]
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 L.	Celosia cristata antiviral protein 25 (CCP-25)	1	25		[123]
Celosia cristata L.	Celosia cristata antiviral protein 27 (CCP-27)	1	27		[124]
Charybdis maritima L.	Charybdin	1	29		[125]
Chenopodium album L.	Chenopodium album antiviral RIP (CAP30)	1	30		[126, 127]
Cinnamomum camphora (L.) J. Presl.	Camphorin	1	23		[128]
Cinnamomum camphora (L.) J. Presl.	Cinnamomin	2	61		[128]
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]
Cucurbita foetidissima Kunth.	Foetidissimin II	2	61		[137]
Cucurbita maxima L.	Cucurmoschin	Small RIP	8		[138]
Cucurbita moschata Duchesne ex Poir.	Alpha-moschin	Small RIP	12		[139]
Cucurbita moschata Duchesne ex Poir.	Beta-moschin	Small RIP	12		[139]
Cucurbita moschata Duchesne ex Poir.	Moschatin	1	29	Yes	[140]
Cucurbita moschata Duchesne ex Poir.	Cucurmosin (CUS)	1	27		[141, 142]
Cucurbita moschata Duchesne ex Poir.	Cucurmosin 2	1	27.2		[143]
Cucurbita moschata Duchesne ex Poir.	Cucurbita moschata RIP	1	30.7		[144]
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.) Makino	Gynostemmin	1	27		[144, 154]
Gypsohila elegans Bieb.	Gypsophilin	1	28		[155]
Hordeum vulgare L.	Barley translation inhibitor (barley toxin I, BRIP)	1	31	Yes	[156]
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]
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.) Howell	MOR-I (Marah oreganus RIP-I)	1	28		[177]
Marah oreganus (Torr. ex S. Wats.) Howell	MOR-II (Marah oreganus RIP-II)	1	27.6		[177]
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]
Momordica charantia L.	Momordin (Momordica charantia inhibitor, momordin-a)	1	23	Yes	[184]
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]
Momordica charantia L.	Momordin I (Momordica charanthia inhibitor)	1	31	Yes	[147, 192]
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]
Phytolacca americana L.	PAP (pokeweed antiviral protein, Phytolacca antiviral protein)	1	29	Yes	[208, 209]
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]
Phytolacca heterotepala H. Walter	Heterotepalin-4 (Mexican pokeweed RIP-4, Phytolacca heterotepala anti-viral protein PAP)	1	29.3		[222]
Phytolacca heterotepala H. Walter	Heterotepalin-5b (Mexican pokeweed RIP-5b)	1	30.5		[222]
Phytolacca insularis Nakai	Phytolacca insularis antiviral protein (PIP, insularin)	1	35		[223]
Phytolacca insularis Nakai	Phytolacca insularis antiviral protein 2 (PIP2)	1	35.7		[224]
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 Kunth.	Polygonatum multiflorum RIP monomer (PMRIPm)	2	60		[227]
Polygonatum multiflorum Kunth.	Polygonatum multiflorum RIP tetramer (PMRIPt)	2	240		[227]
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 Groenland	Ricin R2	2	63.1		[232]
Ricinus sanguineus Hort. ex Groenland	Ricin R11	2	57.8		[232]
Ricinus sanguineus Hort. ex Groenland	Ricin R12	2	62.2		[232]
Ricinus sanguineus Hort. ex Groenland	Ricinus sanguineus agglutinin	2	120		[233]
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]
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 Wall.	Trichosanthes cucumerina seed lectin (TCSL)	RIP-like	69		[253]
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 kDa)	1	29		[259]
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]
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]

While type I RIPs generally have lower toxicity, this is not predominantly because of their lack of enzymatic activity but contrastingly due to the missing B-chain making their cellular internalization cumbersome [5]. The missing cell binding domain is a blessing in disguise for molecular biologists, and has facilitated them to prepare fusion proteins or synthetic analogs of type 1 RIPs together with ligands that are able to facilitate their cellular internalization [6]. Moreover, in the recent decade, there has been a growing evidence that use of endosomal escape enhancers can lead to a significant augmentation of the efficacy of RIPs. This strategy has also paved a path for an improvement in the therapeutic utility of RIPs as targeted toxins or immunotoxins [5].

Endocytosis, Cytosolic Delivery and Enzymatic Action of RIPs

The toxic potential of RIPs is determined by their ability to reach to the ribosomes, which are located within the cytosol. Thus, RIPs that are able to overcome cellular barriers end up exhibiting tremendous toxic potential. This overcoming of cellular barriers includes their internalization, which is generally facilitated by their B chain. Type 2 RIPs such as ricin from Ricinus communis L., abrin from Abrus precatorius L., or volkensin from Adenia volkensii Harms. [7] efficiently deliver their N-glycosidase domain (A chain) into the cytosol of intoxicated cells [8] which is facilitated by their B chains. The B chain serves as galactose/N-acetylgalactosamine binding domain (lectin) and is linked to the A chain via disulfide bonds.

After the binding with glycoproteins or glycolipids, which have numerous galactose residues on their surface, ricin is endocytosed via clathrin-dependent as well as clathrin-independent endocytosis and is thereafter delivered into the early endosomes. From there on it is transported to the Golgi-apparatus by retrograde transport and finally reaches the endoplasmic reticulum (ER). Within the ER the disulfide bonds are cleaved by thioredoxin reductases and disulfide isomerases [9, 10]. The enzymatically active A chain is released and partially unfolded during this process [11]. To facilitate its entry into the cytosol, the A chain exploits a mechanism, which is known as ER-associated degradation (ERAD). ERAD is a natural mechanism for maintaining the homeostasis of the ER [12]. Proteins that are misfolded and thus non-functional are designated for proteasome degradation within the cytosol. The transport of the partially unfolded A chain is mediated by the translocon Sec61p [13] and the ER degradation-enhancing α-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 system. This is achieved by disguising the A chain as a misfolded protein. After reaching the cytosol the partially unfolded A chain is fully refolded to regain the conformational integrity as an enzymatically 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 order 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 α-sarcin/ricin loop. This results in an irreversible inhibition of protein synthesis facilitated by the prevention of eukaryotic elongation 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 rRNA. The bond between the N9 of adenine and the C1 of ribose is hydrolyzed by a concerted action of an arginine at position 180 (R180) and a glutaminic acid at position 177 (E177). E177 is stabilized at a cationic oxocarbenium ribose transition state and R180 is responsible for activating water. This facilitates the nucleophilic attack on the C1 of the oxocarbenium intermediate resulting in the release of adenine [18]. Mutants lacking E177 and R180 are also devoid of the N-glycosidase activity [19]. Recent studies suggest that the action of RIPs on ribosomes depends on the ribosomal stalk, which is a network of different proteins that recruit translational factors to the ribosomes [20]. After gaining access to their substrate, RIPs act as toxic agents. It is further hypothesized that only one internalized molecule is sufficient to kill one cell. From an evolutionary point of view, it has been suggested that the B chain of ricin was generated by a lateral gene transfer from a bacteria.

Contrasting to type 2 RIPs, type 1 RIPs are less toxic [21] and consist of only the A chain (N-glycosidase), which lacks any specific cell binding properties. The low cytotoxicity of type 1 RIPs is generally attributed to an inefficient endocytosis. However, based on some other reports [22] and our own experiments (Fig. 1), it is admissible that type 1 RIPs are effectively internalized. The major problem restricting their efficacy is the inefficient endosomal release.

Fig. (1).

Three-dimensional depiction (z-stacks) of the endosomal network of ECV-304 cells loaded with Alexasaporin. ECV-304 cells were challenged for 3 h with 1 µM Alexa-Fluor 488 labeled saporin (type I RIP from Saponaria officinalis L.). Cells were co-incubated with pH rodo™ Red Dextran, a marker for endo/lysosomes and analyzed by confocal live cell imaging. Depicted is the endo/lysosomal network of one living ECV-304 cell. Green: Alexasaporin in celular vesicles, red: pHrodo™ Red Dextran in endosomes/lysosomes, yellow: co-localization of Alexasaporin and pHrodo™ Red Dextran in endosomes/lysosomes. The figure illustrates the fact that saporin is internalized and trapped in to the endosomal vesicles, thereafter it is degraded by the endo/lysosomal degradation.

The exact mechanism of the internalization of type 1 RIPs is not deciphered so far. Previous studies indicate towards a receptor-mediated endocytosis of type I RIPs by low density lipoprotein (LDL) receptors [23-26]. Contrastingly, some other results confirm a receptor independent endocytosis [22]. However, the exertion of toxic effects appears to be independent of the internalization mechanism. The toxicity determining factor is the ability of type 1 RIPs to cross the endo/lysosomal membrane. Since type 1 RIPs do not contain any transduction domains facilitating the endo/lyso-somal escape into the cytosol, they are less cytotoxic. Upon endocytosis, type 1 RIPs are delivered into the cellular compartments that are positive for lysobisphosphatidic acid (LBPA) (a specific eukaryotic phospholipid marker for late endosomes) and the lysosomal-associated membrane proteins LAMP1 and LAMP2 [22, 27]. Type I RIPs are thereafter degraded within the lysosomes [5].

Immunotoxins and Targeted Toxins

Immunotoxins as per definition are conjugates of cell binding antibodies and the complete type 1/2 RIP or the A chain of a type 2 RIP [6]. In all the reported cases, the complete type 2 RIP has a very high cytotoxic effect when conjugated to the antibody. Nonetheless, there is an increased side effect due to the off-target binding of the B chain. To circumvent this, a lot of alternative strategies including but not limited to the use of high concentrations of free galactose or lactose as competitive binders have been tested. Another alternative in overcoming this problem has been the use of steric hindrance [28]. Coupling of an antibody or its fragment to the isolated A chain via disulfide linkage appears to be the most effective strategy. RIPs lack thiol groups for a disulfide linkage and it is necessary to synthetically introduce it. Alternatively, other linkages such as maleimide linkage have also been attempted but are not successful, mainly due to the inability of cellular enzymes to reductively cleave the bonds [29].

Another important term for the fusion proteins comprising of toxins is targeted toxin. It is a term which coherently finds usage in the literature to define a generic name for immunotoxins. In general, targeted toxins comprise of tumor specific ligands coupled to polypeptide toxins. They constitute a class of cancer therapeutics that leads to the death of cancer cells. They mainly act by the inactivation of cytosolic protein synthesis and induction of programmed cell death [3]. Immunotoxins are per se, restricted to an antibody or antibody fragment as the targeting moiety whereas, targeted toxins form a larger domain including the use of antibodies, small antibody fragments, growth factors, cytokines or small peptides as targeting moieties. Thus, immunotoxins form a smaller subset of targeted toxins as a classification in general.

These targeted toxins can either be prepared by chemical conjugation as described above, or they can be produced recombinantly as a fusion protein that is expressed in cells [6]. Within the past two decades, significant progress has been made towards proper identification of the appropriate cellular target for toxins with target specificity. Moreover, tremendous progress made in the field of genetic engineering and a better understanding of receptor physiology coupled with the single molecule tracking modality have led to an exponential growth in the scientific output as far as targeted toxins are concerned. This is further evidenced by an increased number of clinical trials which are being conducted on targeted toxins, with many of them in Phase 3 [30, 31].

Plant RIPs constitute a major portion of the therapies with targeted toxins, and although there is additional literature available on bacterial and human toxins, plant RIPs generate a lot of scientific interest. As listed in Table 2, there are more than 450 targeted toxins described, which comprise of plant RIPs as a toxic moiety. Amongst various RIPs the leading toxin components are ricin A chain from Ricinus communis L., saporin from Saponaria officinalis L. and gelonin from Gelonium multiflorum A. Juss. A lot of different targeting ligands have been successfully coupled to these toxins and have demonstrated high specificity in in vitro and preclinical evaluations. The ligand, apart from providing selectivity, also helps in cellular internalization of the toxin. There are a number of aspects associated with the internalization and trafficking of toxins. When the toxins are transformed into targeted toxins, there are numerous critical elements deciding their fate in vitro and in vivo; these events are discussed in detail hereafter.

Table 2.

A comprehensive list of all the targeted toxins based on plant RIPs investigated so far.

Toxin	Immunotoxin	Ligand	Target antigen	Tumor/Disease	In vitro	In vivo	Clinical trial status	Ref.
Abrin	Abrin-9.2.27	mAb (9.2.27)	Melanoma-associated antigen (p250)	Melanoma	Yes	Yes		[279, 280]
Abrin	Abrin-NR-ML-05	mAb (NR-ML-05)	Melanoma-associated antigen (p250)	Melanoma	Yes			[281]
Abrin A-chain	Fib 75-abrin A chain	mAb (LICR-LOND Fib 75)	Bladder cancer antigen	EJ bladder cancer	Yes	Yes		[282-284]
Abrin A-chain	C27-Abrin A chain (MAAC)	mAb (C27)	Carcinoembryonic antigen (CEA)	Colorectal cancer	Yes	Yes		[285]
Abrin A-chain	Anti-Thy 1.1-Abrin A-chain	mAb (anti-Thy 1.1) (OX7)	CD90.1 (Thy 1.1)	AKR-A lymphoma	Yes	Yes		[286]
Abrin A-chain	Anti-Hepatoma-associated Antigen-Abrin A-chain	mAb (anti-hepatoma-associated antigen L10 190 kDa glycoprotein)	Hepatoma-associated antigen L10 190 kDa glycoprotein	Hepatocarcinoma	Yes			[287]
Abrin A-chain	ITA	IgG (anti-Trypanosoma cruzi surface antigens)	Trypanosoma cruzi surface antigens	Trypanosoma cruzi	Yes			[288]
Abrin A-chain	F1G4-rABRa-A	mAb (F1G4)	Gonadotropin releasing hormone (GnRH) receptor	Breast cancer, hepatocarcinoma	Yes			[289]
Abrin A-chain	SWA11-SPDB-abrin A	mAb (SWA11)	CD24	SCLC	Yes			[290]
Abrin A-chain	ABRaA-VEGF121	VEGF121	VEGFR-2	Melanoma	Yes	Yes		[291]
Abrin variant	Tfn-abrin variant	Human diferric transferrin (Tfn)	TfR	Glioblastoma multiforme, melanoma	Yes			[292]
Barley toxin I	H65-MM-rBRIP	mAb (H65)	CD5	ALL	Yes			[293]
Barley toxin I	4A2-MM-rBRIP	mAb (4A2)	CD7	ALL	Yes			[293]
Barley toxin I	Anti-melanoma-BRIP	mAb (anti-melanoma)	Melanoma antigen	Melanoma	Yes			[294]
Barley toxin II	5E9C11-Barley toxin II	mAb (HB21) (5E9)	TfR	Colon cancer	Yes			[157]
Bouganin	Anti-CD80/bouganin (M24-bouganin)	mAb (M24)	CD80	Hodgkin's lymphoma, Burkitt's lymphoma	Yes			[295]
Bouganin	Anti-CD86/bouganin	mAb (anti-CD86) (1G10)	CD86	Hodgkin's lymphoma, Burkitt's lymphoma	Yes			[295]
deBouganin	VB6-845	Fab (4D5MOCB)	EpCAM	Solid tumors of epithelial origin	Yes	Yes	Phase I	[296, 297]
Bryodin-1	OX7-bryodin	mAb (anti-Thy 1.1) (OX7)	CD90.1 (Thy 1.1)	AKR-A lymphoma	Yes			[298]
Bryodin-1	BD1-G28-5 sFv	scFv (G28-5)	CD40	B-cell non-Hodgkin’s lymphoma, multiple myeloma	Yes			[299, 300]
Bryodin-1	chiBR96-BD-1	scFv (BR96)	Ley antigen	Breast cancer	Yes			[301]
Bryodin-1	Anti-epithelial antigen-bryodin	mAb (anti-epithelial antigen)	Epithelial antigen	Colon cancer, epidermoid carcinoma	Yes			[302]
Gelonin	Lym-I-gelonin	mAb (Lym-1)	HLA-DR	Burkitt’s lymphoma cells	Yes			[318]
Gelonin	B4G7-gelonin	mAb (B4G7)	EGFR	Lung cancer	Yes	Yes		[319]
Gelonin	80G-gelonin	mAb (80G)	Alpha-fetoprotein	Hepatoma	Yes	Yes		[320]
Gelonin	ZME-gelonin	mAb (ZME-018)	Proteoglycan, p250	Melanoma	Yes	Yes		[321, 322]
Gelonin	Gelonin-9.2.27	mAb (9.2.27)	Melanoma-associated antigen (p250)	Melanoma	Yes	Yes		[280]
Gelonin	AChR-gelonin	AChR (nicotinic acetylcholine receptor)	IgG (anti-AChR)	Experimental autoimmune myasthenia gravis (EAMG)	Yes	Yes		[323]
Gelonin	38.13-gelonin	mAb (38.13)	TH ceramide (Pk antigen)	Burkitt's lymphoma	Yes			[324]
Gelonin	Anti-T11-gelonin	mAb (OKT11)	CD2	T cells	Yes	Yes		[325, 326]
Gelonin	Tf-gelonin	Transferrin	TfR	Malaria (Plasmodium falciparum)	Yes			[327]
Gelonin	AR3-gelonin	mAb (AR3)	CAR-3	Gastric cancer	Yes	Yes		[328]
Gelonin	15A8-gelonin	mAb (15A8)	Breast cancer antigen	Breast cancer, cervical cancer	Yes			[329]
Gelonin	HB5-gelonin	mAb (HB5)	Cd3 receptor	EBV infection	Yes			[330]
Gelonin	Anti-Lyt 2.2-gelonin	mAb (anti-Lyt 2.2) (19/178C1)	Lyt2.2	T-cell lymphoma	Yes			[331]
Gelonin	Anti-Thy 1.2-gelonin	mAb (anti-Thy 1.2) (AT15E)	CD90.2 (Thy 1.2)	T-cell lymphoma	Yes			[331]
Gelonin	Anti-Thy 1-gelonin	mAb (anti-Thy 1) (M549)	CD90 (Thy 1.1 and 1.2)	Leukemia	Yes	Yes		[332]
Gelonin	LG 2/72-gelonin	mAb (LG 2/72)	HLA-DR	Lymphoma	Yes			[331]
Gelonin	Anti-MCMV-gelonin	IgG (anti-MCMV)	MCMV antigen (murine cytomegalovirus antigen)	CMV infection	Yes			[333]
Gelonin	Anti-HCMV-gelonin	IgG (anti-HCMV)	HCMV antigen (human cytomegalovirus antigen)	CMV infection	Yes			[333]
Gelonin	Anti-JL1-gelonin	mAb (anti-JL1)	JL1	Leukemia	Yes			[334]
Gelonin	oLH-gelonin (lutropin-SS-gelonin)	Ovine luteinizing hormone (oLH)	Ovine LH receptor	Leydig cell tumor (testicular cancer)	Yes			[335]
Gelonin	hCG-gelonin	Human chorionic gonadotropin (hCG)	LH receptor	Leydig cell tumor (testicular cancer)	Yes			[335]
Gelonin	Gelonin-gp330	gp330 (renal brush border antigen)	Anti-gp330 Ig	Heymann's nephritis	Yes	Yes		[336]
Gelonin	Anti-PCV-gelonin	IgG (anti-PCV)	Pichinde virus (PCV)	Pichinde virus (PCV)	Yes			[337]
Gelonin	PC4.9A6-gelonin	mAb (PC4.9A6)	Pichinde virus (PCV)	Pichinde virus (PCV)	Yes			[337]
Gelonin	14G2a-gelonin	mAb (14G2a)	Disialoganglioside GD2	Neuroblastoma, melanoma	Yes			[338]
Gelonin	MSN-1-gelonin	mAb (MSN-1)	Endometrial adenocarcinoma antigen	Endometrial adenocarcinoma	Yes	Yes		[339]
Gelonin	F(ab')2-gelonin/UCHT1	F(ab')2 (anti-IgG) / mAb (UCHT1)	CD3	T-cell lymphoma	Yes			[303]
Gelonin	H65-gelonin	mAb (H65)	CD5	T-cell ALL	Yes	Yes		[340]
Gelonin	BACH-250/rGel	mAb (BACH-250)	HER2	Breast cancer	Yes	Yes		[341]
Gelonin	TAB-250/rGel	mAb (TAB-250)	HER2	Breast cancer	Yes	Yes		[341]
Gelonin	VEGF121/rGel	VEGF121	KDR Flk-1 receptor	Tumor neovasculature, melanoma, prostate cancer	Yes	Yes		[342]
Gelonin	HuM195/rGel	mAb (HuM-195)	CD33	AML, CML, myelodysplastic syndrome	Yes	Yes	Phase I	[343-346]
Gelonin	MEL scFv-rGel	scFv (MEL)	gp240	Melanoma, brain cancer, lobular breast cancer	Yes	Yes		[347]
Gelonin	BLyS-gelonin	B lymphocyte stimulator (BLyS)	BR3/BAFF-R, TACI and BCMA	B-NHL subtypes mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), B-cell precursor-acute lymphocytic leukemia (BCP-ALL)	Yes	Yes		[348-350]
Gelonin	C6.5-rGel	scFv (C6.5)	HER2	Breast cancer, gastric cancer, lung cancer, ovarian cancer	Yes	Yes		[351]
Gelonin	e23-L-rGel	scFv (e23)	HER2	Breast cancer, gastric cancer, lung cancer, ovarian cancer	Yes			[352]
Gelonin	ML3-9-rGel	scFv (ML3-9)	HER2	Breast cancer, gastric cancer, lung cancer	Yes	Yes		[351]
Gelonin	MH3-B1-rGel	scFv (MH3-B1)	HER2	Breast cancer, gastric cancer, lung cancer	Yes	Yes		[351]
Gelonin	B1D3-rGel	scFv (B1D3)	HER2	Breast cancer, gastric cancer, lung cancer	Yes	Yes		[351]
Gelonin	3ErGel	scFv (sm3E)	Carcinoembryonic antigen (CEA)	Colorectal cancer	Yes			[353]
Gelonin	FErGel	scFv (shMFE)	Carcinoembryonic antigen (CEA)	Colorectal cancer	Yes			[353]
Gelonin	C7rGel	FN3 fragment (C743)	Carcinoembryonic antigen (CEA)	Colorectal cancer	Yes	Yes		[353, 354]
Gelonin	E4rGel	FN3 fragment (E246)	EGFR	Colorectal cancer	Yes	Yes		[353, 354]
Gelonin	3C/rGel	scFv (3C)	FGFR3	Multiple myeloma, hepatocellular carcinoma, bladder cancer	Yes	Yes		[355, 356]
Gelonin	7D/rGel	scFv (7D)	FGFR3	Multiple myeloma, hepatocellular carcinoma, bladder cancer	Yes	Yes		[355]
Gelonin	H45-rGeloninD274C	mAb (H45)	CD5	ALL	Yes	Yes		[357]
Gelonin	MOC31-gelonin	mAb (MOC31)	Epithelial glycoprotein-2 (EGP-2)	SCLC, colon cancer, breast cancer	Yes			[358]
Luffa ribosomal inhibitory protein (LRIP)	HB21-LRIP	mAb (HB21) (5E9)	TfR	T lymphoblastic leukemia	Yes			[168]
Luffin-A	Luffin A-Ng76	mAb (Ng76)	Melanoma antigen	Melanoma	Yes			[359]
Luffin-B	Luffin B-Ng76	mAb (Ng76)	Melanoma antigen	Melanoma	Yes			[360]
Luffin-B	LKP (Luffin-β-KDEL-uPAcs)	uPAcs (urokinase-type plasminogen activator)	Urokinase receptor	Non-small cell lung cancer (NSCLC)	Yes			[361]
Luffin-P1	hIL-2-Luffin P1	IL-2	CD25 (IL-2 receptor)	Activated lymphocytes	Yes	Yes		[362-364]
Luffin-P1	EBI3-Luffin P1	EBI3 (Epstein-Barr virus (EBV)-induced gene 3)	CD25 (IL-2 receptor)	Immunological diseases, erythroleukemia	Yes			[365]
Mistletoe lectin I A-chain	Anti-CD5/MLIA	mAb (anti-CD5)	CD5	T-lymphocytes	Yes			[366]
Mistletoe lectin I A-chain	Anti-CD25/MLIA (Anti-CD25-MLA)	mAb (anti-CD25)	CD25 (IL-2 receptor)	Activated lymphocytes	Yes			[367]
Mistletoe lectin I A-chain	MoAb-16-MLIA	mAb (16)	Oncofetal antigen	Leukemia	Yes			[368]
Mistletoe lectin I A-chain	BMAC1/MLA	mAb (BMCA1)	CD45	Allograft rejection	Yes			[369]
Mistletoe lectin I A-chain	OX1/MLA	mAb (OX1)	rat CD45	Allograft rejection	Yes			[369]
Momorcochin	Anti-epithelial antigen-momorcochin	mAb (anti-epithelial antigen)	Epithelial antigen	Colon cancer, epidermoid carcinoma	Yes			[302]
Momorcochin	F(ab')2-momorcochin/UCHT1	F(ab')2 (anti-IgG) / mAb (UCHT1)	CD3	T-cell lymphoma	Yes			[303]
Momorcochin-S	Momorcochin-S-A8	mAb (8A)	8A myeloma antigen	Burkitt lymphoma	Yes	Yes		[193]
Momordin	OX7-momordin	mAb (anti-Thy 1.1) (OX7)	CD90.1 (Thy 1.1)	AKR-A lymphoma	Yes			[298]
Momordin	Fib 75-momordin	mAb (LICR-LOND Fib 75)	Bladder cancer antigen	EJ bladder cancer	Yes	Yes		[284, 312]
Bryodin-1	F(ab')2-bryodin/UCHT1	F(ab')2 (anti-IgG) / mAb (UCHT1)	CD3	T-cell lymphoma	Yes			[303]
Bryodin-2	chiBR96-BD-2	scFv (BR96)	Ley antigen	Breast cancer	Yes			[301]
Bryodin-2	HB21-bryodin-II	mAb (HB21) (5E9)	TfR	Breast cancer	Yes			[304]
Colocin 1	Anti-epithelial antigen-colocin 1	mAb (anti-epithelial antigen)	Epithelial antigen	Colon cancer, epidermoid carcinoma	Yes			[302]
Curcin	Curcin-TfRBP9	TfRBP9 [transferrin receptor (TfR) binding peptide]	TfR	Hepatocellular carcinoma	Yes			[305]
Dianthin 30	BerH2-dianthin	mAb (Ber-H2)	CD30	Lymphoblastoid, Hodgkin's lymphoma	Yes			[306, 307]
Dianthin 30	Dianthin-EGF	EGF	EGFR	EGFR overexpressing cells	Yes			[84, 308]
Dianthin 30	Tfn-dianthin	Transferrin	TfR	T-cell leukemia	Yes			[309]
Dianthin 32	F(ab')2-dianthin 32/UCHT1	F(ab')2 (anti-IgG) / mAb (UCHT1)	CD3	T-cell lymphoma	Yes			[303]
Ebulin l	Ebulin l-transferrin	Transferrin	TfR	TfR-over-expressing cancer cells	Yes			[310]
Ebulin l	44G4-ebulin	mAb (44G4)	CD105 (endoglin)	Tumor neovasculature	Yes			[311]
Gelonin	Fib 75-gelonin	mAb (LICR-LOND Fib 75)	Bladder cancer antigen	EJ bladder cancer	Yes	Yes		[284, 312]
Gelonin	Anti-CD86/gelonin (αCD86-gelonin)	mAb (anti-CD86) (1G10)	CD86	Hodgkin's lymphoma, Burkitt's lymphoma	Yes	Yes		[295, 313]
Gelonin	Anti-CD80/gelonin (M24-gelonin)	mAb (M24)	CD80	Hodgkin's lymphoma, Burkitt's lymphoma	Yes			[295]
Gelonin	αCD80-gelonin	mAb (B5B)	CD80	Hodgkin's lymphoma, Burkitt's lymphoma	Yes			[313]
Gelonin	J5/gelonin	mAb (J5)	CD10 (CALLA)	Lymphoma	Yes			[314]
Gelonin	I-2/gelonin	mAb (I-2)	Ia antigen	Lymphoma	Yes			[314]
Gelonin	J30/gelonin	mAb (J30)	gp26 cell surface glycoprotein	Lymphoma	Yes			[314]
Gelonin	BerH2-gelonin	mAb (Ber-H2)	CD30	Hodgkin's lymphoma	Yes			[307]
Gelonin	NDA4-gelonin	mAb (NDA4)	NDA4 antigen	EBV-transformed lymphoblastoid, gibbon MLA leukemia	Yes			[315]
Gelonin	HB21-gelonin (5E9-gelonin)	mAb (HB21) (5E9)	TfR	Colon cancer, Burkitt's lymphoma	Yes	Yes		[157, 316]
Gelonin	OKT9-gelonin	mAb (OKT9)	TfR	Cervical cancer	Yes			[317]
Momordin	OM124-momordin	mAb (anti-CD22) (OM124)	CD22	Burkitt's B-cell lymphoma, Epstein-Barr virus-infected B lymphoblastoid cells	Yes	Yes		[370]
Momordin	8A-Momordin	mAb (8A)	8A myeloma antigen	Multiple myeloma	Yes			[371]
Momordin	Anti-CD5-Momordin	mAb (anti-CD5)	CD5	T-cell leukemia	Yes	Yes		[372]
Momordin	Anti-CD30-Momordin (Ber-H2-Momordin)	mAb (Ber-H2)	CD30	Hodgkin's lymphoma, anaplastic large-cell lymphoma(ALCL)	Yes	Yes		[307, 373, 374]
Momordin	BDI-1-momordin	mAb (BDI-1)	Bladder cancer antigen	Bladder cancer	Yes	Yes	Phase I	[375, 376]
Momordin	Folate-momordin	Folate	Folate receptor	Cervical cancer, ovarian cancer	Yes			[377, 378]
Momordin	Anti-epithelial antigen-momordin	mAb (anti-epithelial antigen)	Epithelial antigen	Colon cancer, epidermoid carcinoma	Yes			[302]
Momordin	F(ab')2-momordin/UCHT1	F(ab')2 (anti-IgG) / mAb (UCHT1)	CD3	T-cell lymphoma	Yes			[303]
Momordin I	48-127/momordin I	mAb (48-127)	gp54	Bladder cancer	Yes			[379]
Moschatin	Moschatin-Ng76	mAb (Ng76)	Melanoma antigen	Melanoma	Yes			[380]
Nigrin b	44G4-nigrin b	mAb (44G4)	CD105 (endoglin)	Tumor neovasculature	Yes			[381]
Nigrin b	MJ7-Ngb	mAb (MJ7/18)	CD105 (endoglin)	Tumor neovasculature, melanoma	Yes	Yes		[382, 383]
Nigrin b	Nigrin b-transferrin	Transferrin	TfR	TfR-over-expressing cancer cells	Yes			[310]
Ocymoidine	Mint-Ocy	mAb (Mint5)	EGFR	Breast cancer	Yes	Yes		[384]
PAP	B43-PAP	mAb (B43)	CD19	Leukemia, B-cell ALL	Yes	Yes	Phase I	[385-388]
PAP	TXU-PAP	mAb (TXU)	CD7	T-NHL, HIV type I	Yes	Yes	Phase I	[389-391]
PAP	Anti-Thy 1.1 (mAb)-PAP	mAb (anti-Thy 1.1) (OX7)	CD90.1 (Thy 1.1)	Leukemia	Yes			[392]
PAP	Anti-Thy 1.1 (F(ab')2)-PAP	F(ab')2 (anti-Thy 1.1) (OX7)	CD90.1 (Thy 1.1)	Leukemia	Yes			[392]
PAP	GnRH-PAP	Gonadotropinreleasing hormone (GnRH)	GnRH receptor	Breast cancer	Yes			[393, 394]
PAP	TP3-PAP	mAb (TP3)	p80	Osteosarcoma	Yes	Yes		[395]
PAP	J3-109-PAP	mAb (J3-109)	CD72	B-cell ALL	Yes			[396]
PAP	74-12-4-PAP	mAb (74-12-4)	porcine CD4	Transplants		Yes		[397]
PAP	Anti-CD4-PAP	mAb (MT151)	CD4	HIV	Yes			[398]
PAP	PAP-9.2.27	mAb (9.2.27)	Melanoma-associated antigen (p250)	Melanoma	Yes	Yes		[280, 399]
PAP	J5/PAP	mAb (J5)	CD10 (CALLA)	Lymphoma	Yes			[314]
PAP9 (High expressed mutated PAP)	PAP9-IL-2	IL-2	CD25 (IL-2 receptor)	T-cell lymphoma	Yes			[400]
PAP II	J5/PAP II	mAb (J5)	CD10 (CALLA)	Lymphoma	Yes			[314]
PAP-S	OM124-PAP-S	mAb (anti-CD22) (OM124)	CD22	Burkitt's B-cell lymphoma, Epstein-Barr virus-infected B lymphoblastoid cells, Hodgkin's lymphoma	Yes	Yes		[307, 370]
PAP-S	Anti-CD30-PAP-S (Ber-H2-PAP-S)	mAb (Ber-H2)	CD30	Hodgkin's lymphoma, anaplastic large-cell lymphoma(ALCL)	Yes	Yes		[373, 401]
PAP-S	48-127/PAP-S	mAb (48-127)	gp54	Bladder cancer	Yes			[379]
PAP-S	Anti-epithelial antigen-PAP-S	mAb (anti-epithelial antigen)	Epithelial antigen	Colon cancer, epidermoid carcinoma	Yes			[302]
PAP-S	F(ab')2-PAP-S/UCHT1	F(ab')2 (anti-IgG) / mAb (UCHT1)	CD3	T-cell lymphoma	Yes			[303]
PAP-S	J5/PAP-S	mAb (J5)	CD10 (CALLA)	Lymphoma	Yes			[314]
PD-S2	Ber-H2-PD-S2	mAb (Ber-H2)	CD30	Hodgkin's lymphoma	Yes			[307]
Pyramidatine	Mint-Pyra	mAb (Mint5)	EGFR	Breast cancer	Yes	Yes		[384]
Ricin	Anti-Ly2.1-ricin	mAb (anti-Ly2.1)	Murine T-cell antigen	T-cell ALL	Yes	Yes		[402]
Ricin	Anti-CD8-ricin	mAb (B9.4.2)	CD8	PBMCs	Yes			[403]
Ricin	Anti-CD4-ricin	mAb (HP2/6)	CD4	PBMCs	Yes			[403]
Ricin	Anti-CD3-ricin	mAb (SPV-T3b)	CD3	PBMCs	Yes			[403]
Ricin	Anti-CD3-ricin	mAb (11D8)	CD3	PBMCs	Yes			[403]
Ricin	UCHT1-ricin	mAb (UCHT1)	CD3ε	GVHD	Yes			[404]
Ricin	35.1-ricin	mAb (35.1)	CD2	GVHD	Yes			[404]
Ricin	T101-ricin	mAb (T101)	CD5	GVHD	Yes	Yes		[404, 405]
Ricin	Ricin-HB55	mAb (BH55)	HLA-DR	B-cell leukemia, lymphoma	Yes			[406]
Ricin	IL2-lectin-deficient RTB-RTA	IL-2	CD25 (IL-2 receptor)	Leukemia	Yes			[407]
Ricin	GMCSF-ricin	GMCSF	GMCSF receptor	AML	Yes			[408]
Ricin	M6-ricin	mAb (M6)	L2C IgM idiotype	B-cell leukemia	Yes	Yes		[409]
Ricin	Anti-GE2-ricin	mAb (anti-GE2)	GE2	Glioma	Yes			[410]
Ricin	AR3-ricin	mAb (AR3)	CAR-3	Gastric cancer, colorectal cancer	Yes			[411]
Ricin	BDI-1-ricin	mAb (BDI-1)	Bladder cancer antigen	Bladder cancer	Yes			[412]
Ricin	Ricin-mAb 35	mAb (35)	AChR (nicotinic acetylcholine receptor)	Strabismus	Yes	Yes		[413, 414]
Ricin	Anti-Lyt 2.2-ricin	mAb (anti-Lyt 2.2) (19/178C1)	Lyt2.2	T-cell lymphoma	Yes			[331]
Ricin	IgE-intact ricin	mAb (IR162)	IgE Fc receptor	Allergies, basophil leukemia	Yes			[415]
Ricin	L6-ricin	mAb (L6)	Lung canger antigen	Lung cancer	Yes	Yes		[416]
Ricin	Ricin-EGF	EGF	EGFR	Epidermoid carcinoma	Yes			[417]
Ricin	Anti-CD6-bR	mAb (anti-CD6)	CD6	CTCL, ALL	Yes	Yes	Phase I	[418, 419]
Ricin	Anti-B4-bR	mAb (anti-B4)	CD19	B-NHL	Yes	Yes	Phase III	[420-425]
Ricin	Anti-My9-bR	mAb (anti-My9)	CD33	AML	Yes	Yes	Phase I	[418, 426, 427]
Ricin	N901-bR	mAb (N901)	CD56 (N-CAM)	SCLC	Yes	Yes	Phase II	[418, 428-431]
Ricin	Anti-CEA-bR	mAb (I-1)	Carcinoembryonic antigen (CEA)	Colorectal cancer	Yes	Yes	Phase I/II	[432]
Ricin	IF7-bR	mAb (IF7)	CD26	T cells	Yes			[433]
Ricin	4B4-bR	mAb (4B4)	CD29	Lymphocytes, endothelium	Yes			[304]
Ricin	MT151-blocked ricin	mAb (MT151)	CD4	ALL	Yes			[434]
Ricin	Anti-CD4.CD26-bRicin	Bispecific mAb (anti-CD4 x CD26)	CD4 + CD26	GVHD	Yes			[433]
Ricin	Anti-CD4-bRicin	Fab' (19thy5D7)	CD4	GVHD	Yes			[433]
Ricin	Anti-CD26-bRicin	Fab' (1F7)	CD26	GVHD	Yes			[433]
Ricin	Anti-CD4.CD29-bRicin	Bispecific mAb (anti-CD4 x CD29)	CD4 + CD29	Tissue allografts	Yes			[435]
Ricin	SEN31-bR	mAb (SEN31)	Cluster-5a antigen	SCLC	Yes	Yes		[436]
Ricin	HB7-blocked ricin	mAb (HB7)	CD38	Multiple myeloma, lymphoma	Yes			[437]
RTA	Anti-Thy 1.1-dgRTA	mAb (anti-Thy 1.1) (OX7)	CD90.1 (Thy 1.1)	AKR-A lymphoma	Yes	Yes		[438]
RTA	Anti-CD7-dgA (DA7)	mAb (3A1e)	CD7	T-NHL, leukemia, GVHD	Yes	Yes	Phase I	[439]
RTA	HD37-dgA (IMTOX-19)	mAb (HD37)	CD19	B-NHL, ALL	Yes	Yes	Phase I	[440, 441]
RTA	RFB4-Fab'-dgA	Fab’ (RFB4)	CD22	B-NHL, leukemia, lymphoma	Yes	Yes	Phase I	[442, 443]
RTA	RFT5-dgA (IMTOX-25)	mAb (RFT5)	CD25	Hodgkin's lymphoma, CTCL, melanoma, GVHD	Yes	Yes	Phase II	[444-448]
RTA	Ki-4.dgA	mAb (Ki-4)	CD30	Hodgkin's lymphoma, NHL	Yes	Yes	Phase I	[447, 449, 450]
RTA	RFB4-dgA (IMTOX-22)	mAb (RFB4)	CD22	B-NHL, CLL, ALL, leukemia, lymphoma, myeloma	Yes	Yes	Phase I	[443, 451, 452]
RTA	Combotox (RFB4-dgA / HD37-dgA)	mAb (RFB4) + mAb (HD37)	CD22, CD19	NHL, ALL	Yes	Yes	Phase I	[453, 454]
RTA	SPV-T3a-dgA + WT1-dgA	mAb (SPV-T3a) + mAb (WT1)	CD3, CD7	GVHD	Yes	Yes	Phase I/II	[455, 456]
RTA	3A1e-dgRTA	scFv (3A1e)	CD7	T-cell leukemia	Yes			[457]
RTA	3AIf-dgRTA	scFv (3A1f)	CD7	T-cell leukemia	Yes			[457]
RTA	UV3-dgRTA	mAb (UV3)	CD54 (ICAM-1)	Myeloma, grnulocytes, monocytes	Yes			[458]
RTA	H22-dgRTA (CD64-RiA)	mAb (H22)	CD64	AML, rheumatoid arthritis, monocytes, macrophages	Yes	Yes		[459-461]
RTA	D5-dgA	mAb (D5)	Cytomegalovirus	Cytomegalovirus (MCMV)	Yes			[462]
RTA	C34-dgA	mAb (C34)	Cytomegalovirus	Cytomegalovirus (MCMV)	Yes			[462]
RTA	HMS-dgA	IgG (HMS)	Cytomegalovirus	Cytomegalovirus (MCMV)	Yes			[462]
RTA	64.1-dgRTA	mAb (64.1)	CD3	Lymphoproliferative disease (LPD)	Yes	Yes		[463, 464]
RTA	HD6-dgA	mAb (HD6)	CD22	Leukemia, lymphoma	Yes			[443]
RTA	HD6-Fab'-dgA	Fab’ (HD6)	CD22	Leukemia, lymphoma	Yes			[443]
RTA	UV22-1-dgA	mAb (UV22-1)	CD22	Leukemia, lymphoma	Yes			[443]
RTA	UV22-1-Fab'-dgA	Fab’ (UV22-1)	CD22	Leukemia, lymphoma	Yes			[443]
RTA	UV22-2-dgA	mAb (UV22-2)	CD22	Leukemia, lymphoma	Yes			[443]
RTA	UV22-2-Fab'-dgA	Fab’ (UV22-2)	CD22	Leukemia, lymphoma	Yes			[443]
RTA	p67.7-dgA	mAb (p67.7)	CD33	AML	Yes			[465]
RTA	120-2A3-dgA	mAb (120-2A3)	TfR	Myeloma, Hodgkin's lymphoma	Yes			[465]
RTA	B-B10-dgA	mAb (B-B10)	CD25 (IL-2 receptor)	Myeloma, Hodgkin's lymphoma	Yes			[465]
RTA	TDR31-1-dgA	mAb (TDR31-1)	MHC class II	Myeloma, Hodgkin's lymphoma	Yes			[465]
RTA	SWA11-dg.RTA	mAb (SWA11)	CD24	SCLC	Yes	Yes		[466, 467]
RTA	M5/114-dgA	mAb (M5/114)	MCH Class II antigens (I-Ad, I-Ed)	Endothelial cells	Yes	Yes		[468]
RTA	11-4.1-dgA	mAb (11-4.1)	MCH Class I antigen (H-2Kk)	Neuroblastoma	Yes	Yes		[468, 469]
RTA	E6-dgA	mAb (E6)	Prostate-specific membrane antigen (PSMA)	Prostate cancer	Yes	Yes		[470]
RTA	14G2a.dgA	mAb (14G2a)	Disialoganglioside GD2	Neuroblastoma	Yes	Yes		[471]
RTA	ch14.18.dgA	mAb (ch14.18)	Disialoganglioside	Neuroblastoma	Yes			[471]
RTA	BW704.dgA	mAb (BW704)	Disialoganglioside	Neuroblastoma	Yes			[471]
RTA	chCE7.dgA	mAb (chCE7)	190 kDa Glycoprotein (gp190)	Neuroblastoma	Yes			[471]
RTA	FVS191cys-dgRTA	scFv (FVS191)	CD19	Leukemia	Yes			[472]
RTA	K4-2C10-dgRA	mAb (K4-2C10)	CD105 (endoglin)	Tumor neovasculature, breast cancer	Yes	Yes		[473]
RTA	SN6j-dgRA	mAb (SN6j)	CD105 (endoglin)	Tumor neovasculature, breast cancer	Yes	Yes		[474]
RTA	SN6k-dgRA	mAb (SN6k)	CD105 (endoglin)	Tumor neovasculature, breast cancer	Yes	Yes		[474]
RTA	D5-dgA	mAb (D5)	MCMV antigen (murine cytomegalovirus antigen)	CMV infection	Yes	Yes		[462, 475]
RTA	C34-dgA	mAb (C34)	MCMV antigen (murine cytomegalovirus antigen)	CMV infection	Yes	Yes		[462, 475]
RTA	FF1-4D5-dgA	mAb (FF1-4D5)	mouse δ H chain of surface IgD (mδsIgD), domain Fd	B-cells	Yes			[476]
RTA	AMS-15.1-dgA	mAb (AMS-15.1)	mouse δ H chain of surface IgD (mδsIgD), domain Fd	B-cells	Yes			[476]
RTA	11-26-dgA	mAb (11-26)	mouse δ H chain of surface IgD (mδsIgD), domain Fd	B-cells	Yes			[476]
RTA	JA12.5-dgA	mAb (JA12.5)	mouse δ H chain of surface IgD (mδsIgD), domain Fd	B-cells	Yes			[476]
RTA	AMS-9.1-dgA	mAb (AMS-9.1)	mouse δ H chain of surface IgD (mδsIgD), domain Fc	B-cells	Yes			[476]
RTA	AMS-28.1-dgA	mAb (AMS-28.1)	mouse δ H chain of surface IgD (mδsIgD), domain Fc	B-cells	Yes			[476]
RTA	Hδa/1-dgA	mAb (Hδa/1)	mouse δ H chain of surface IgD (mδsIgD), domain Fc	B-cells	Yes			[476]
RTA	UCHL1-dgA	mAb (UCHL1)	CD45RO	HIV	Yes			[477-479]
RTA	My7/Fab' GAMIg.dgA	mAb (My7) / Fab' (GAM Ig)	CD13	Myeloid leukemia	Yes			[465]
RTA	1G10/Fab' GAMIg.dgA	mAb (My7) / Fab' (GAM Ig)	CD15	Myeloid leukemia	Yes			[465]
RTA	rCD4-dgA	rCD4 (recombinant CD4)	HIVgp120	HIV	Yes			[480]
RTA	Fib 75-ricin A chain	mAb (LICR-LOND Fib 75)	Bladder cancer antigen	Bladder cancer	Yes	Yes		[282-284]
RTA	ITR	IgG (anti-Trypanosoma cruzi surface antigens)	Trypanosoma cruzi surface antigens	Trypanosoma cruzi	Yes			[288]
RTA	Anti-CD25/RTA	mAb (anti-CD25)	CD25 (IL-2 receptor)	Activated lymphocytes	Yes			[367, 407]
RTA	Anti-CD5/RTA	mAb (anti-CD5)	CD5	T-lymphocytes	Yes			[366]
RTA	BerH2-RTA	mAb (Ber-H2)	CD30	Lymphoblastoid, Hodgkin's lymphoma	Yes			[374, 481]
RTA	H65-RTA (CD5 Plus) (XomaZyme-CD5 Plus)	mAb (H65)	CD5	GVHD, CTCL, CLL, rheumatoid arthritis, systemic lupus erythematosus (SLE), diabetes mellitus	Yes	Yes	Phase II	[482-487]
RTA	454A12-rRA	mAb (454A12)	TfR	Breast cancer, leptomeningeal neoplasia	Yes	Yes	Phase I	[488, 489]
RTA	260F9-rRTA	mAb (260F9)	55 kDa breast cancer antigen (p55)	Breast cancer, ovarian cancer	Yes	Yes	Phase I	[490-492]
RTA	XMMME-001-RTA (XomaZyme-Mel)	mAb (XMMME-001)	Melanoma antigen (Proteoglycan)	Melanoma	Yes	Yes	Phase I/II	[493-498]
RTA	791T/36-RTA (XomaZyme-791)	mAb (791T/36)	72 kDa cancer antigen (72 kDa TAA) (p72)	Colorectal cancer	Yes	Yes	Phase I	[499, 500]
RTA	T101-RTA	mAb (T101)	CD5	CLL	Yes	Yes	Phase I	[501-503]
RTA	T101-RTA	Fab (T101)	CD5	T-cell leukemia	Yes			[504]
RTA	T101-RTA	F(ab')2 (T101)	CD5	T-cell leukemia	Yes			[504]
RTA	MDX-RA (4197X-RA)	mAb (4197X)	Human lens epithelial antigen	Posterior capsule opacification (secondary cataract)		Yes	Phase III	[505-507]
RTA	RTA-EGF	EGF	EGFR	Epidermoid carcinoma, EGFR+ cells	Yes			[84, 417, 508]
RTA	WT82-RTA	mAb (WT82)	CD8	T-cell ALL	Yes			[509]
RTA	2G5-RTA	mAb (2G5)	HLA-DR	Lymphoma, B cells, T cells, dendritic cells	Yes			[510]
RTA	CLL2m-RTA	mAb (CLL2m)	CLL2m antigen	ND, CLL	Yes			[511]
RTA	HAE3-RTA	mAb (HAE3)	Glycophorin-A	Erythromyeloblastoid leukemia	Yes			[512]
RTA	HAE9-RTA	mAb (HAE9)	Erythroid antigen	Erythromyeloblastoid leukemia	Yes			[512]
RTA	BMAC1/RTA	mAb (BMCA1)	CD45	Allograft rejection	Yes			[369]
RTA	OX1/RTA	mAb (OX1)	rat CD45	Allograft rejection	Yes			[369]
RTA	SN7-RTA	mAb (SN7)	SN7 B-cell antigen	B-cell leukemia, B-cell lymphoma	Yes	Yes		[513]
RTA	HB21-RTA	mAb (HB21) (5E9)	TfR	Ovarian cancer, epidermoid carcinoma	Yes			[492]
RTA	R17217-rRTA	mAb (R17217)	Murine TfR	Lymphoma	Yes	Yes		[514]
RTA	YE1/9.9-rRTA	mAb (YE1/9.9)	Murine TfR	Lymphoma	Yes			[514]
RTA	0.5beta-RTA	mAb (0.5beta)	HIV gp120	HIV	Yes			[515]
RTA	Anti-gp120-RTA	mAb (anti-gp120)	HIV gp120	HIV	Yes			[516]
RTA	Anti-gp120-RTA	IgG (anti-gp120)	HIV gp120	HIV	Yes			[517]
RTA	Anti-gp41-RTA	mAb (7B2)	HIV gp120	HIV	Yes	Yes		[516, 518, 519]
RTA	171A-RTA	mAb (171A)	EpCAM	Colorectal cancer	Yes			[520]
RTA	MT151-RTA	mAb (MT151)	CD4	ALL	Yes			[434]
RTA	MRK-RTA	mAb (MRK16)	P-glycoprotein	Kidney cancer	Yes			[521]
RTA	KM231-RTA	mAb (KM231)	Sialyl-Lea-antigen	Gastric cancer, colorectal cancer	Yes	Yes		[522]
RTA	UCHT1 F(ab')2-RTA	F(ab')2 (UCHT1)	CD3ε	GVHD	Yes	Yes		[523]
RTA	WT32-RTA	mAb (WT32)	CD3	T-cell ALL	Yes			[524]
RTA	WT1-RTA	mAb (WT1)	CD7	T-cell ALL, lymphoma	Yes			[524, 525]
RTA	528-rRA	mAb (528)	EGFR	Lung cancer	Yes	Yes		[526]
RTA	Anti-Tac-RTA	mAb (anti-CD25)	CD25 (IL-2 receptor)	T-cell leukemia, activated lymphocytes	Yes			[367, 527]
RTA	Tf-RTA	Transferrin	TfR	T-cell ALL, prostate cancer, malaria (Plasmodium falciparum)	Yes			[327, 528, 529]
RTA	Tf-KFT25-RTA	Transferrin	TfR	T-cell ALL	Yes			[528]
RTA	520C9-RTA	mAb (520C9)	HER2	Breast cancer	Yes			[530]
RTA	741 F8-RTA	mAb (741 F8)	HER2	Breast cancer	Yes			[530]
RTA	454C11-RTA	mAb (454C11)	HER2	Breast cancer	Yes			[530]
RTA	STI-RTA	mAb (STI)	CD5	T-cell ALL	Yes			[531]
RTA	RTA-9.2.27	mAb (9.2.27)	Melanoma-associated antigen (p250)	Melanoma	Yes	Yes		[280]
RTA	BrE-3-RTA	mAb (BrE-3)	Mucin, MUC1	SCLC	Yes			[532]
RTA	C242-RTA (ICI D0490)	mAb (C242)	Mucin	Colorectal cancer	Yes	Yes		[533]
RTA	84.1c-RTA	mAb (84.1c)	mIgE	Allergies	Yes	Yes		[534]
RTA	HRS-3.dgA	mAb (HRS-3)	CD30	Hodgkin's lymphoma, myeloma	Yes			[465, 535]
RTA	HRS-3Fab'.dgA	Fab' (HRS-3)	CD30	Hodgkin's lymphoma	Yes			[535]
RTA	HRS-4.dgA	mAb (HRS-4)	CD30	Hodgkin's lymphoma	Yes			[535]
RTA	HRS-4Fab'.dgA	Fab' (HRS-4)	CD30	Hodgkin's lymphoma	Yes			[535]
RTA	HRS-1.dgA	mAb (HRS-1)	CD30	Hodgkin's lymphoma	Yes			[535]
RTA	90Y-C110-RTA	mAb (C110)	Carcinoembryonic antigen (CEA)	Colon cancer	Yes	Yes		[536]
RTA	C19-RTA	mAb (C19)	Carcinoembryonic antigen (CEA)	Colorectal cancer	Yes			[537]
RTA	M6-RTA	mAb (M6)	L2C IgM idiotype	B-cell leukemia	Yes	Yes		[409]
RTA	38.13-RTA	mAb (38.13)	TH ceramide (Pk antigen)	Burkitt's lymphoma	Yes			[324]
RTA	Fab'-anti-L3T4-A	Fab' (anti-L3T4)	Murine T-cell antigen (limpet hemocyanin-specific T-helper lymphocytes)	Lymphoma	Yes			[538]
RTA	486P-RTA	mAb (486P 3-12-1)	Bladder cancer antigen	Bladder cancer	Yes			[539]
RTA	RFT11-A	mAb (RFT11)	CD2	T-cell ALL	Yes			[540]
RTA	35.1-A	mAb (35.1)	CD2	T-cell ALL	Yes			[464, 540]
RTA	9.6-A	mAb (9.6)	CD2	T-cell ALL	Yes			[464, 540]
RTA	10.2-A	mAb (10.2)	CD5	T cells	Yes			[464]
RTA	452-D9-RTA	mAb (452-D9)	gp74	c-Ha-ras expression tumors, Kirsten sarcoma	Yes	Yes		[541, 542]
RTA	Thyroglobulin-RTA	Thyroglobulin	Ig (anti-thyroglobulin)	Thyroiditis	Yes			[543]
RTA	96.5-RTA	mAb (96.5)	p97	Melanoma	Yes			[544]
RTA	SN5d-RTA	mAb (SN5d)	CD10 (CALLA)	Pre-B-cell ALL	Yes	Yes		[545]
RTA	SN5-RTA	mAb (SN5)	CD10 (CALLA)	Pre-B-cell ALL	Yes	Yes		[545]
RTA	Anti-CALLA-RTA	mAb (anti-CALLA)	CD10 (CALLA)	Burkitt's lymphoma, (pre-B-cell ALL)	Yes			[546]
RTA	Anti-CALLA-RTA	Fab' (anti-CALLA)	CD10 (CALLA)	Burkitt's lymphoma, (pre-B-cell ALL)	Yes			[546]
RTA	Anti-GE2-RTA	mAb (anti-GE2)	GE2	Glioma	Yes			[410]
RTA	D1/12-RTA	mAb (D1/12)	HLA-DR	Glioma	Yes			[410]
RTA	AR3-RTA	mAb (AR3)	CAR-3	Gastric cancer	Yes			[411]
RTA	8C-RTA	mAb (8C)	Ovarian cancer antigen	Ovarian cancer	Yes	Yes		[547]
RTA	M2A-RTA	mAb (M2A)	Ovarian cancer antigen	Ovarian cancer	Yes	Yes		[547]
RTA	Anti-vasopressin-RTA	mAb (anti-vasopressin)	Vasopressin	Pituitary cancer	Yes	Yes		[548]
RTA	Cluster 2 Mab-Fab'-Anti-Mouse/RAT-RTA	mAb (Cluster 2)	Cluster 2 antigen-SCLC	SCLC	Yes			[549]
RTA	SOKT1-RTA	mAb (SOKT1)	T-cell antigen	T cells	Yes			[550]
RTA	MGb2-RTA	mAb (MGb2)	Gastric antigen	Gastric cancer	Yes			[551]
RTA	MG11-RTA	mAb (MG11)	Gastric antigen	Gastric cancer	Yes			[551]
RTA	MoAb-16-RTA	mAb (16)	Oncofetal antigen	Leukemia	Yes	Yes		[368, 552]
RTA	Anti-laryngeal cancer-RTA	mAb (anti-laryngeal cancer)	Laryngeal cancer antigen	Laryngeal cancer	Yes			[553, 554]
RTA	317G5-RTA	mAb (317G5)	42 kDa glycoprotein (p42)	Breast cancer	Yes			[555]
RTA	SEN36-RTA	mAb (SEN36)	CD56 (N-CAM)	SCLC	Yes			[556]
RTA	Anti-mu-RTA	mAb (anti-mu)	Mu chain of IgM	Myeloma	Yes			[557]
RTA	SEN7-bR	mAb (SEN7)	CD56 (N-CAM)	SCLC	Yes			[558]
RTA	Anti-CRF-RTA	mAb (anti-CRF)	CRF (corticotropin-releasing factor)	Immunolesioning (CRF neurons within the paraventricular nucleus of the hypothalamus)		Yes		[559]
RTA	Anti-asialo-GM2-RTA	mAb (anti-asialo-GM2)	Asialo-GM2	Lymphoma	Yes			[560]
RTA	Anti-H-2d-RTA	mAb (anti-H-2d)	H-2d	Lymphoma	Yes			[560]
RTA	V beta 6-specific immunotoxin (VIT6)	mAb (anti-V beta 6-specific)	V beta-associated antigen receptor	Myasthenia gravis	Yes			[561]
RTA	schM21-ricin A	scFv (schM21)	Astrocytoma- and medulloblastoma-associated antigen	Medulloblastoma	Yes			[562]
RTA	ONS-M21-RTA (ORA)	mAb (ONS-M21)	Astrocytoma- and medulloblastoma-associated antigen	Medulloblastoma	Yes			[563]
RTA	Anti-VIP-RTA	mAb (anti-VIP)	Vasoactive intestinal polypeptide (VIP)	Pheochromocytoma, immunolesioning (neurons within the SCN) (suprachiasmatic nucleus of the hypothalamus)	Yes	Yes		[564]
RTA	Anti-Thy 1.2-RTA	IgG (anti-Thy 1.2)	CD90.2 (Thy 1.2)	Leukemia	Yes	Yes		[565]
RTA	IgE-ricin A-chain	mAb (IR162)	IgE Fc receptor	Allergies, basophil leukemia	Yes	Yes		[566, 567]
RTA	OX-40-ricin A	mAb (anti-OX-40)	OX-40	Autoimmune encephalomyelitis (EAE)	Yes	Yes		[568]
RTA	SWA20-RTA	mAb (SWA20)	CD24	SCLC	Yes			[467]
RTA	Anti-T. cruzi-RTA	IgG (anti-Trypanosoma cruzi surface antigens)	Trypanosoma cruzi surface antigens	Trypanosoma cruzi	Yes	Yes		[288]
RTA	UCHT1/F(ab')2-ricin A chain	mAb (UCHT1) / F(ab')2 (anti-IgG)	CD3	T-cell lymphoma	Yes			[303]
RTA	RTA-NIM-R7	mAb (NIM-R7)	p58	Lymphoma	Yes			[569]
Saporin	Anti-Thy 1.1 (F(ab')2)-saporin	F(ab')2 (anti-Thy 1.1) (OX7)	CD90.1 (Thy 1.1)	AKR-A lymphoma	Yes	Yes		[570]
Saporin	Anti-Thy 1.1 (mAb)-saporin	mAb (anti-Thy 1.1) (OX7)	CD90.1 (Thy 1.1)	AKR-A lymphoma	Yes	Yes		[570]
Saporin	192 IgG-saporin (192-IgG-SAP) (IgG-192)	mAb (192)	Rat nerve growth factor receptor (p75NTR)	Immunolesioning (cholinergic basal forebrain neurons), Alzheimer's disease	Yes	Yes		[571-574]
Saporin	OM124-saporin	mAb (OM124)	CD22	Burkitt's B-cell lymphoma, Epstein-Barr virus-infected B lymphoblastoid cells	Yes	Yes		[370]
Saporin	M24-saporin (anti-CD80/saporin)	mAb (M24)	CD80	Hodgkin's lymphoma, Burkitt's lymphoma	Yes			[295]
Saporin	1G10-saporin (anti-CD86/saporin)	mAb (1G10)	CD86	Hodgkin's lymphoma, Burkitt's lymphoma	Yes			[295]
Saporin	M24-saporin / 1G10-saporin	mAb (M24) / mAb (1G10)	CD80 + CD86	Burkitt's lymphoma, Hodgkin's lymphoma	Yes			[295]
Saporin	OKT11-saporin	mAb (OKT11)	CD2	T-CLL	Yes			[575, 576]
Saporin	7A10C9-saporin	mAb (7A10C9)	CD2	T-CLL	Yes			[575]
Saporin	OKT1-saporin	OKT1	CD5	T-lymphocytes, B-CLL	Yes	Yes		[577-579]
Saporin	BsAb (HB2 x anti-saporin)/(OKT10 x anti-saporin)/saporin	Bispecific F(ab')2 (HB2 x anti-saporin)/(OKT10 x anti-saporin)	CD7 + CD38	T-ALL	Yes			[580]
Saporin	BsAb (HB2 x anti-saporin)/saporin	Bispecific F(ab')2 (HB2 x anti-saporin)	CD7	T-ALL	Yes			[581]
Saporin	BsAb (OKT10 x anti-saporin)/saporin	Bispecific F(ab')2 (OKT10 x anti-saporin)	CD38	T-ALL	Yes			[580]
Saporin	HB2-saporin	mAb (HB2)	CD7	Lymphoma, T-ALL	Yes	Yes		[582-584]
Saporin	BU12-saporin	mAb (BU12)	CD19	B-LL, Burkitt's lymphoma	Yes	Yes		[585-587]
Saporin	Rituximab/saporin-S6	mAb (rituximab)	CD20	NHL	Yes			[588]
Saporin	BsAb (4KB128 x anti-saporin)/saporin	Bispecific F(ab')2 (4KB128 x anti-saporin)	CD22	Burkitt's lymphoma	Yes			[589]
Saporin	BsAb (HD37 x anti-saporin)/saporin	Bispecific F(ab')2 (4KB128 x anti-saporin)	CD19	Burkitt's lymphoma	Yes			[589]
Saporin	BsAb (MB-1 x anti-saporin)/saporin	Bispecific F(ab')2 (4KB128 x anti-saporin)	CD37	Burkitt's lymphoma	Yes			[589]
Saporin	BsAb (4KB128 x anti-saporin)/(RFB9 x anti-saporin)/saporin	Bispecific F(ab')2 (4KB128 x anti-saporin)/(RFB9 x anti-saporin)	CD22	Lymphoma, CLL	Yes	Yes	Phase I	[590]
Saporin	BsAb (4KB128 x anti-saporin)/(HD6 x anti-saporin)/saporin	Bispecific F(ab')2 (4KB128 x anti-saporin)/(HD6 x anti-saporin)	CD22	B-cell lymphoma	Yes	Yes	Phase I	[591]
Saporin	IB4/saporin-S6	mAb (IB4)	CD38 (alpha-D-Galactopyranoside residues)	NHL	Yes			[592]
Saporin	Anti-B7-1-saporin	mAb (B7-24)	CD80	Burkitt's lymphoma, Hodgkin's lymphoma	Yes			[593]
Saporin	Anti-CTLA-4 (83)-saporin (83-saporin)	scFv (83)	CD152 (Cytotoxic T-lymphocyte antigen-4, CTLA-4)	Transplantation tolerance, leukemia, EBV-positive B-cell lymphoblastoid	Yes	Yes		[594-596]
Saporin	Anti-CTLA-4 (40)-saporin (40-saporin)	scFv (40)	CD152 (Cytotoxic T-lymphocyte antigen-4, CTLA-4)	Transplantation tolerance, EBV-positive B-cell lymphoblastoid	Yes	Yes		[594, 595]
Saporin	Anti-CTLA-4 (67)-saporin (67-saporin)	scFv (67)	CD152 (Cytotoxic T-lymphocyte antigen-4, CTLA-4)	Transplantation tolerance, leukemia	Yes			[596]
Saporin	ATG-saporin-S6	Antithymocyte globulin (ATG)	Thymocyte	Lymphoma, leukemia	Yes			[597]
Saporin	HD6-saporin	mAb (HD6)	CD22	Lymphoma, B-CLL	Yes			[598]
Saporin	HD39-saporin	mAb (HD39)	CD22	Lymphoma, B-CLL	Yes			[598]
Saporin	HD37-saporin	mAb (HD37)	CD19	B-cell lymphoma	Yes			[598]
Saporin	Saporin-EGF (SE)	EGF	EGFR	Breast cancer, sarcoma, adenocarcinoma, cervical cancer	Yes	Yes		[599-602]
Saporin	SA2E	EGF	EGFR	Breast cancer	Yes	Yes		[599-601]
Saporin	FGF-SAP	FGF	FGFR	Melanoma, teratocarcinoma, neuroblastoma	Yes	Yes		[603]
Saporin	FGF2-SAP	FGF-2	FGFR	Bladder cancer	Yes			[604]
Saporin	bFGF-saporin	bFGF	bFGFR	Prostate cancer	Yes	Yes		[605]
Saporin	ch25A11-Sap	mAb (ch25A11)	CUB domain-containing protein 1 (CDCP1)	Prostate cancer	Yes	Yes		[606]
Saporin	hJ591-saporin	mAb (hj591)	Prostate-specific membrane antigen (PSMA)	Prostate cancer	Yes	Yes		[607]
Saporin	Ep2-saporin	mAb (Ep2)	Proteoglycan, p250	Melanoma	Yes			[608]
Saporin	ML30-saporin	mAb (ML30)	Heat shock protein 65 kDa (HSP65)	Leukemic monocyte lymphoma, pancreatic cancer	Yes	Yes		[609, 610]
Saporin	48-127/saporin-S6	mAb (48-127)	gp54	Bladder cancer	Yes			[379]
Saporin	Anti-ALCAM/CD166 scFv-saporin	scFv (I/F8)	CD166 (activated leukocyte cell adhesion molecule, ALCAM)	SCLC, ovarian cancer	Yes			[611]
Saporin	7E4B11-saporin	mAb (7E4B11)	RPTPβ	Astrocytic tumor, glioblastoma	Yes	Yes		[612]
Saporin	Ber-H2-Saporin	mAb (Ber-H2)	CD30	Hodgkin's lymphoma, anaplastic large-cell lymphoma(ALCL)	Yes	Yes	Phase I	[374, 613-616]
Saporin	Sap-ac-LDL	Acetylated LDL	Rat scavenger receptor	Immunolesioning (microglia)	Yes			[617, 618]
Saporin	Anti-basigin-2-saporin	mAb (anti-basigin-2)	Human basigin-2 (CD147) (EMMPRIN)	Ovarian cancer	Yes			[619]
Saporin	M290-SAP	mAb (M290)	CD103	Organ allograft rejection and GVHD	Yes	Yes		[620]
Saporin	Anti-ChAT IgG-saporin	mAb (anti-ChAT)	Choline acetyltransferase (ChAT)	Parkinson's and schizophrenia		Yes		[621-623]
Saporin	Anti-DAT-saporin	mAb (anti-DAT)	Dopamine transporter (DAT)	Immunolesioning (dopaminergic neurons)		Yes		[624]
Saporin	Anti-DBH-saporin	mAb (anti-DBH)	Dopamine beta-hydroxylase (DBH)	Immunolesioning (noradrenergic neurons)		Yes		[625-627]
Saporin	Anti-SERT-SAP	mAb (anti-SERT)	Serotonin reuptake transporter (SERT)	Immunolesioning (serotonergic neurons)	Yes	Yes		[628]
Saporin	Bombesin-SAP	Bombesin	Gastrin-releasing peptide receptor (GRPR)	Immunolesioning (GRPR+ neurons)	Yes	Yes		[629, 630]
Saporin	CCK-saporin	CCK (cholecystokinin)	Cholecystokinin type 2 receptor (CCK2)	Immunolesioning (CCK+ neurons)		Yes		[631]
Saporin	CRF-SAP	CRF (corticotropin-releasing factor)	CRF receptor	Immunolesioning (CRFR+ cells)	Yes	Yes		[632, 633]
Saporin	CTB-SAP	CTB (cholera toxin B-subunit)	GM1 ganglioside	Immunolesioning (paraplegia)		Yes		[634]
Saporin	Dermorphin-saporin (MOR-SAP)	Dermorphin	Mu opioid receptor (MOR)	Immunolesioning (MOR+ neurons)		Yes		[631]
Saporin	Galanin-saporin (Gal-sap)	Galanin	Galanin-1 receptor (GalR1)	Immunolesioning (GalR1+ neurons)		Yes		[635]
Saporin	GAT1-saporin	IgG (GAT1)	GABA-transporter-1	Immunolesioning (MSDB neurons), Alzheimer's disease		Yes		[636]
Saporin	Lep-SAP	Leptin	Leptin receptor	Immunolesioning (leptin receptor+ neuons)		Yes		[637, 638]
Saporin	Anti-Mac-1-SAP	mAb (anti-Mac-1)	CD11b (Mac-1)	Immunolesioning (Mac-1+ neuons, microglia)	Yes	Yes		[639-642]
Saporin	ME20.4 IgG-saporin	mAb (ME20.4)	Primate p75 low-affinity neurotrophin receptor (p75NTR)	Immunolesioning (p75NTR+ neuons)		Yes		[643, 644]
Saporin	UF008/SAP	IgG (UF008)	Melanopsin	Immunolesioning (intrinsically photosensitive retinal ganglion cells, ipRGCs)	Yes	Yes		[645, 646]
Saporin	NK3-SAP	Neurokinin-3 (NK3)	Neurokinin-3 receptor (NK3R)	Immunolesioning (NK3R+ neuons)		Yes		[647]
Saporin	NPY-SAP	Neuropeptide Y (NPY)	Neuropeptide Y receptor (NPYR)	Immunolesioning (NPYR+ neuons)		Yes		[648, 649]
Saporin	OXY-SAP	Oxytocin	Oxytocin receptors (OXYR)	Immunolesioning (OXYR+ neuons)	Yes	Yes		[650]
Saporin	Substance P-saporin	Substance P	Neurokinin-1 receptor (NK1R) (Substance P receptor)	Immunolesioning (NK1R+ neurons), hyperalgesia		Yes		[651-653]
Saporin	Hypocretin-saporin	Hypocretin (orexin)	Hypocretin-2 receptor	Narcolepsy (parvalbumin and cholinergic neurons)		Yes		[654]
Saporin	TEC-T4-saporin	mAb (TEC-T4)	CD4	T cells	Yes			[655]
Saporin	MB-1 x anti-sap-1/saporin	Bispecific mAb (MB-1 x anti-sap-1)	CD37	Burkitt's lymphoma	Yes			[589]
Saporin	OKT10-saporin	mAb (OKT10)	CD38	T-cell ALL, lymphocytes, macrophages	Yes	Yes		[584]
Saporin	Campath-1-saporin	mAb (Campath-1)	CD52	GVHD, myeloid cells	Yes	Yes		[656]
Saporin	TEC IgM-SAP	mAb (TEC IgM)	Immunoglobulin heavy chain	Burkitt’s lymphoma	Yes			[657]
Saporin	8A-saporin 6	mAb (8A)	8A plasma cell-associated antigens	Multiple myeloma, Burkitt’s lymphoma	Yes			[658]
Saporin	62B1-saporin	mAb (62B1)	62B1 plasma cell-associated antigens	Multiple myeloma, Burkitt’s lymphoma	Yes			[658]
Saporin	3BIT (BU12-saporin / OKT10-saporin + 4KB128-saporin)	mAb (BU12) / (OKT10) / (4KB128)	CD19 + CD22 + CD38	Burkitt’s lymphoma	Yes	Yes		[659]
Saporin	BU12-saporin / OKT10-saporin	mAb (BU12) / (OKT10)	CD19 + CD38	Burkitt’s lymphoma	Yes	Yes		[586]
Saporin	HB2-saporin / OKT10-saporin	mAb (HB2) / (OKT10)	CD7 + CD38	T-cell ALL	Yes	Yes		[584]
Saporin	B3/25-SO6	mAb (B3/25)	TfR	Leukemia	Yes			[660]
Saporin	LAM3/saporin	mAb (LAM3)	M5b leukemia antigen	Acute non-lymphoid leukemia (ANLL)	Yes			[610, 661]
Saporin	Tf-saporin	Transferrin	TfR	Prostate cancer	Yes			[529]
Saporin	uPA-SAP	uPAcs (urokinase-type plasminogen activator)	Urokinase receptor	Lymphoma	Yes			[662]
Saporin	11A8-saporin	mAb (11A8)	bFGFR	Ovarian cancer	Yes	Yes		[663]
Saporin	Anti-CD8-saporin	mAb (anti-CD8)	CD8	T-cell lymphoma	Yes			[655]
Saporin	HBEGF-saporin	HB-EGF	EGFR	Breast cancer, bladder cancer, melanoma, leukemia, colon cancer, renal cancer, ovarian cancer, prostate cancer, non-small cell lung cancer (NSCLC), brain cancer	Yes			[664]
Saporin	HBEGF-L22-saporin	HB-EGF	EGFR	Breast cancer, bladder cancer, melanoma, leukemia, colon cancer, renal cancer, ovarian cancer, prostate cancer, non-small cell lung cancer (NSCLC), brain cancer	Yes	Yes		[664]
Saporin	B-B10-saporin	mAb (B-B10)	CD25 (IL-2 receptor)	GVHD	Yes			[665]
Saporin	W6/800E6-SAP	mAb (W6/800E6)	HER2	Breast cancer	Yes			[666]
Saporin	W6/900H1-SAP	mAb (W6/900H1)	HER2	Breast cancer	Yes			[666]
Saporin	H2-Dd-saporin	H2-Dd MHC class I tetramer	T-cell receptor (TCR)	diabetes mellitus, CD8+ T cells	Yes			[667]
Saporin	2F8-saporin	mAb (2F8)	CD163 (SR-A)	Ovarian cancer	Yes	Yes		[668]
Saporin	Insulin-saporin (saporin insulin complex, SIC)	Insulin	Insulin receptor	Ovarian cancer, hepatocellular carcinoma	Yes			[669]
Saporin	B-B2-saporin	mAb (BB2)	Myeloma antigen	Multiple myeloma	Yes			[670]
Saporin	B-B4-saporin	mAb (BB4)	Myeloma antigen	Multiple myeloma	Yes			[670]
Saporin	Anti-epithelial antigen-saporin 6	mAb (anti-epithelial antigen)	Epithelial antigen	Colon cancer, epidermoid carcinoma	Yes			[302]
Saporin	Anti-SA-1-saporin	mAb (anti-SA-1)	mAb (SA-1) (16/6 idiotype binding to DNA)	Systemic lupus erythematosus (SLE)	Yes	Yes		[671]
Saporin	Anti-Id-saporin	mAb (anti-Id)	mAb (Anti-Id) (anti-lymphoma idiotype)	B-cell leukemia	Yes	Yes		[672]
Saporin	HB6-1 x anti-sap-1/saporin	Bispecific mAb (HB6-1 x anti-sap-1)	κ-chain	Burkitt's lymphoma	Yes			[589]
Saporin	M15-8 x anti-sap-1/saporin	Bispecific mAb (M15-8 x anti-sap-1)	µ-chain	Burkitt's lymphoma	Yes			[589]
Saporin	RFB-9 x anti-sap-1/saporin	Bispecific mAb (RFB-9 x anti-sap-1)	CD19	Burkitt's lymphoma	Yes			[589]
Saporin	WR17 x anti-sap-1/saporin	Bispecific mAb (WR17 x anti-sap-1)	CD37	Burkitt's lymphoma	Yes			[589]
Saporin	LAM7/saporin	mAb (LAM7)	M5b leukemia antigen	Acute non-lymphoid leukemia (ANLL)	Yes			[610, 661]
Saporin	62B8-saporin 6	mAb (62B8)	62B8 myeloma antigen	Multiple myeloma	Yes			[658]
Saporin	F(ab')2-saporin/UCHT1	F(ab')2 (anti-IgG) / mAb (UCHT1)	CD3	T-cell lymphoma	Yes			[303]
Saporin	F(ab')2-saporin/anti-CD2	F(ab')2 (anti-IgG) / mAb (anti-CD2)	CD2	T-cell lymphoma	Yes			[303]
Saporin	F(ab')2-saporin/anti-CD5	F(ab')2 (anti-IgG) / mAb (anti-CD5)	CD5	T-cell lymphoma	Yes			[303]
Saporin	F(ab')2-saporin/C11	F(ab')2 (anti-IgG) / mAb (C11)	CD45	Hodgkin's lymphoma	Yes			[303]
Saporin	F(ab')2-saporin/TEC-T4	F(ab')2 (anti-IgG) / mAb (TEC-T4)	CD4	T-cell lymphoma	Yes			[303]
Saporin	F(ab')2-saporin/HSR-3	F(ab')2 (anti-IgG) / mAb (HSR-3)	CD30	Hodgkin's lymphoma	Yes			[303]
Saporin	F(ab')2-saporin/8A	F(ab')2 (anti-IgG) / mAb (8A)	8A myeloma antigen	Burkitt lymphoma, multiple myeloma	Yes			[303]
Saporin	F(ab')2-saporin/62B1	F(ab')2 (anti-IgG) / mAb (62B1)	62B1 myeloma antigen	Multiple myeloma	Yes			[303]
Saporin	PlGF-2-saporin	Placental growth factor-2 (PlGF-2)	PlGF-2 receptor	Tumor neovascularization	Yes			[673]
Saporin	ATF-saporin	ATF (amino-terminal fragment of human urokinase)	Urokinase receptor	Metastasis	Yes			[674]
Saporin	Cetuximab-saporin	mAb (cetuximab)	EGFR	Colorectal cancer, prostate cancer, epidermoid carcinoma, breast cancer	Yes			[675, 676]
Saporin	Trastuzumab-saporin	mAb (trastuzumab)	HER2	Breast cancer	Yes			[676, 677]
Saporin	2H8/anti-GAM IgG-saporin	mAb (2H8) / IgG (anti-GAM IgG)	Tomoregulin	Prostate cancer	Yes			[678]
Saporin	By114/anti-IgG-saporin	mAb (By114) / IgG (anti-IgG)	Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6)	Pancreatic cancer	Yes	Yes		[679]
Saporin	6-22 IgG/anti-GAH IgG-saporin	mAb (6-22 IgG) / IgG (anti-GAH IgG)	Human aspartyl (asparaginyl) β-hydroxylase (HAAH)	Hepatocellular carcinoma	Yes			[680]
Saporin	Anti-endosialin/anti-IgG-saporin	mAb (anti-endosialin) / IgG (anti-IgG)	Endosialin (CD248, tumor endothelial marker 1, TEM1)	Ewing's sarcoma, neuroblastoma	Yes			[681]
Saporin	Anti-TCblR-saporin	mAb (anti-TCblR)	CD320 (transcobalamin receptor, TCblR)	CML, colon cancer	Yes			[682]
Saporin	AF334-saporin	mAb (AF334)	Tumor endothelial marker 8 (TEM8)	Tumor neovascularization	Yes			[683]
Saporin	MRK16/anti-IgG-saporin	mAb (MRK16) / IgG (anti-IgG)	170 kDa glycoprotein (gp170)	Colon cancer	Yes			[684]
Trichokirin	AT15E-TKR (AT15E-Trichokirin)	mAb (anti-Thy 1.2) (AT15E)	CD90.2 (Thy 1.2)	Leukemia	Yes	Yes		[270]
Trichokirin	F(ab')2-trichokirin/UCHT1	F(ab')2 (anti-IgG) / mAb (UCHT1)	CD3	T-cell lymphoma	Yes			[303]
Trichosanthin	TCS-Hepama-1 (Hepama-1-trichosanthin)	mAb (Hepama-1)	Hepatoma-associated antigen 43 kDa glycoprotein	Hepatoma	Yes	Yes		[685, 686]
Trichosanthin	p75-TCS (anti-p75-anti-mouse IgG-trichosanthin)	mAb (192) / IgG (anti-mouse)	Rat nerve growth factor (NGF) receptor (p75 receptor) (p75NTR)	Immunolesioning (cholinergic basal forebrain neurons)	Yes			[574]
Trichosanthin	CMU15—TCS	mAb (CMU15A)	Lung cancer antigen	Lung cancer	Yes	Yes		[687, 688]
Trichosanthin	TCS-Ng76	mAb (Ng76)	Melanoma antigen	Melanoma	Yes			[689]
Trichosanthin	EGF-TCS	EGF	EGFR	Hepatocellular carcinoma	Yes	Yes		[690, 691]
Trichosanthin	EGF-TCSredlk	EGF	EGFR	Hepatocellular carcinoma	Yes	Yes		[692]

Antigen Selection and Efficiency of Internalization

The analysis of the expression pattern of tumor-associated surface antigens and the knowledge about their ability to promote or modulate the tumor growth are critical for the identification of novel targets for targeted anti-tumor therapies. For the development of monoclonal antibodies (mAbs) or targeted toxins, it is essential to determine, whether a particular surface antigen undergoes an accelerated internalization or not (Fig. 2). There is a variety of cancer-associated antigens that are being targeted by mAbs [32, 33]. For mAbs that mediate their efficacy in part by interaction with natural killer cells (NK) (antibody dependent cellular cytotoxicity, ADCC), it is important to select antigens, which do not undergo rapid down-regulation after binding. This is a feature contrasting the modality of targeted toxins, where it is desirable to select antigens that show enhanced endocytosis after ligand binding [34]. This facilitates a rapid delivery of the toxin into the cancer cells.

Fig. (2).

An overview of the steps required for the recognition and internalization of the targeted toxin in the tumor cells. The critical step of target recognition determines the specificity, thereafter the celular machinery takes over and in the ideal case the toxin escapes from the endosomal membrane thus inactivating the ribosomes via N-glycosidase activity.

The receptor that is being addressed by the targeted toxin should be over-expressed on the tumor cell surface compared to the normal tissue. A considerable number of receptors [35] have been addressed to date, amongst them are the cytokine receptors [36], tumor necrosis factor receptor, growth factor receptors [37, 38] and cluster of differentiation CD22 [39], CD25 [40] and CD30 [41]. Contrasting to the numerous advantages listed above, a drawback of antibody-based targeted toxins is their limited ability to induce the effector functionalities of the naked antibodies. It is in fact a predominant basis for the concept of targeted toxins, wherein it is envisaged to outweigh the biological functionalities of the monoclonal antibodies by conjugating them to bacterial toxins such as Pseudomonas exotoxin from Pseudomonas aeruginosa [42] or plant toxins such as saporin from Saponaria officinalis L.

Release of Targeted Toxins into the Cytosol and their Lysosomal Degradation

Once internalized, the targeted toxin is delivered into early endosomes. Early endosomes are part of the endosomal transport system, which is an intracellular vesicular and tubular compartment surrounded by cytosol. Within early endosomes, endocytosed ligands (targeted toxins) are either designated for recycling [43, 44] or they are further transported into late endosomes, and finally lysosomes for degradation. Since targeted toxins exert their anti-tumoral efficacy only in the cytosol, it is a vital prerequisite for their efficacy that they are able to escape from the endosomal network into the cytosol. Targeted toxins fused to truncated variants of bacterial toxins such as diphtheria toxin (DT) from Corynebacterium diphtheriae utilize the native T-domain of DT to escape from early endosomes into the cytosol [42, 45, 46] while other targeted toxins employ a KDEL-like motive of their toxin moieties, which in turn facilitate their retrograde delivery into the ER and thereafter their transport to the cytosol [47]. However, plant-derived toxins 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 anticipated that the cytosolic delivery of type 1 RIP-based targeted toxins is attenuated, compared to appropriate bacterial counterparts. However, comparative studies in this regard have not been undertaken so far.

Several strategies such as photochemical internalization [48], pore formation by listeriolysin O from Listeria monocytogenes [37], cell penetration by protein transduction domains [49], the use of lysosomotropic agents like chloroquine [50] or the use of triterpenoidal saponins from Saponaria officinalis L. and Gypsophila paniculata L. [51, 52] have been developed to facilitate the escape of targeted toxins from endosomal vesicles (a schematic overview on the obstacles for the cytosolic delivery of targeted toxins is depicted below above). All these methods prevent the lysosomal degradation of targeted toxins by mediating their endosomal escape into the cytosol. This results in a significant augmentation of the anti-tumoral efficacy of the targeted toxin.

Lysosomal degradation is one of the main issues in targeted tumor therapies [53]. It may be compensated by increasing the dosage of the targeted toxins, however, this does promote undesirable side effects. As mentioned above, lysosomal degradation can be outweighed by combination strategies that mediate the endosomal escape of targeted toxins. The generation of modified targeted toxins that are resistant against lysosomal degradation is a further attractive strategy to increase the efficacy of targeted toxins [54].

Advancement in the Use of RIPs as Therapeutic Agent

Initially, targeted toxins were constructed with native ricin and were tested in vitro in the presence of high concentrations of lactose which prevented the non-specific binding of ricin B-chain. Blocking of the oligosaccharide binding sites was used to prevent off-target ricin uptake and provided the possibility of applying the immunotoxins in vivo [55]. The separation of RTA and ricin B-chain by chemical reduction allowed conjugation of the antibody to the catalytic subunit, mainly through cross-linkers containing a disulfide bond. Despite the high yield and good stability of these targeted toxins, one of the main disadvantages for them was a heterogeneous composition [28]. Furthermore, it is well known that the glycosylated residues of RTA also facilitate non-specific uptake by macrophages. Therefore, in order to prevent the non-specific uptake, RTA was submitted to a process of deglycosylation before conjugation to the antibody and formation of the immunotoxin [56].

The advancement of recombinant tools has led to a rather ubiquitous utilization of these techniques for the production of toxins. For generating these targeted toxins, the gene portion encoding the antigen-binding fragments of an antibody (Fab or Fv) is generally coupled to the gene encoding for native catalytic domain. In another case it may be linked to the mutated version of the toxin. Once the construct is available it can be proliferated in any expression system such as bacteria, yeast or algae [57, 58]. The first generated recombinant immunotoxins were mostly formed using the single-chain variable fragments (scFvs), thereafter they were substituted by disulfide-stabilized Fvs (dsFvs). The scFvs have a peptide linker compared to the disulfide bridge in dsFvs which makes the conformation more stable.

Future Perspectives and Opinions on Targeted Toxins

Cancer is an expended burden in an ageing population. In the fight against this complex phenomenon, it would be a misjudgment to believe that one day a single strategy such as the use of targeted toxins will be able to defeat this disease. Thus, different complementary strategies are required to overcome all the hurdles that impede recovery. Surgical intervention, chemotherapy and radiation constitute the traditional troika of cancer therapies that are used as commonly for a wide variety of tumors.

Regrettably, in many cases this combination is not sufficient for a complete remission. Novel chemotherapeutics such as kinase inhibitors [59] and biological molecules such as antibodies [60] essentially improve the treatment of particular cancer entities. While kinase inhibitors are highly potent in suppressing cellular proliferation, antibodies are in particular characterized by their specificity for target cells. Targeted toxins combine the idea of tumor targeting and potent cytotoxicity in a single molecule. Thus, these molecules can be considered as an important addendum to complement the traditional troika. However, it must be stated that a promising therapeutic approach is finally characterized by its clinical success, but only a few targeted toxins have so far been approved by the Food and Drug Administration of the U.S., only one of them contains a protein-based toxin (denileukin diftitox) [61] and none of them is composed of a RIP. Nevertheless, targeted toxins containing RIPs are known from a number of clinical trials and a very large number of preclinical studies, indicating the great expected potential of this class of targeted toxins. Therefore, further research is needed to optimize current developments and to bring RIP-based anti-tumor drugs into the clinical routine. Although Moolten & Cooperband described as early as in 1970 the selective destruction of target cells by diphtheria toxin which was specifically linked to antibodies directed against specific antigens on the surface of tumor cells [62], the main obstacles for protein-based targeted toxins are still unsolved, which includes expensive production, unstable proteins and short biological half-life, immunogenicity and insufficient endosomal escape. Hundreds of research groups in the world are working on these problems, a substantial number of fruitful ideas have been published to date and the techniques to investigate and to manipulate such molecules are incredible compared to 1970 so that we can be confident that we must not wait further 40 years until targeted toxins will have their breakthrough.

Molecular Aspects and Mechanisms in Targeted Toxin Therapy

Although the ultimate goal in a targeted tumor therapy is to kill the tumor cells, the modality of cell death must not be underestimated. Uncontrolled cell killing can result in colliquation, tyromatosis or coagulative necrosis, which may finally end up in causing life threatening or highly degenerative situation for the organism affected. On the contrary, apoptosis is a strictly controlled process for cell death. It can be induced by intracellular and/or extracellular signals resulting in systematic cell degradation with no damage of neighboring cells. While it is a commonly occurring process in numerous cells on a daily basis, it may be impaired in tumor cells [63].

A similar process for cellular degradation is autophagy which involves the activity of lysosomal machinery which digests different cellular organelles. It is a process dependent on internal or external cellular environment and may lead to either cell death or the promotion of cellular survival [64]. Apoptosis and autophagy are stimulated or suppressed by similar pathways. The way a cell responds to these pathways determines its survival or death (this has been extensively reviewed in [65]). In general, the toxin's primary target such as the ribosomal RNA for RIPs is not directly involved in necrosis, apoptosis or autophagy, but these targets are involved in vital cellular processes. It seems natural to assume that the interference with vital functions results in a series of events that finally trigger the apoptotic cascade [66], but this is not true for all cases. In case of saporin, the induction of apoptosis also occurs before protein synthesis inhibition takes place [67]. Apart from this, in numerous reports there is a discrepancy in the exact mechanism in case of similar parameters studied. While in some cell lines apoptosis was indicated the same could not be confirmed in others. This also implies that the cellular response to targeted toxins is a multifaceted complex mechanism. Therefore, the choice of the toxin is a factor that must be given optimal thought in the design of targeted toxins.

Drug Delivery Technologies Employed in Targeted Toxin Therapy

Carrier-based drug delivery systems have been widely exploited for the targeted delivery of toxins to the tumors. Commonly used drug targeting systems include nanoparticles, liposomes, virosomes, carbon nanotubes, microspheres, nanofibers amongst others [68-70]. A summary of the carrier and non-carrier based systems is shown in Fig. 3. Despite the difficulty in formulation and the need for a more thorough stability and interaction assessment, the use of a carrier-based approach has distinctive advantages. Carriers help in a more specific ligand attachment increasing the specificity for the delivery of cargo. Increasing the circulatory time as in case of liposomal nanocarriers was of advantage for the delivery of cholera toxin, and despite its utility for adjuvant effects, the strategy has potential for its application in tumor therapy as well. A classical utilization of the liposomal drug delivery system was the delivery of gelonin [71]. It was delivered to the cytoplasm of TLX5 lymphoma cells most effectively by phosphatidylserine vesicles. This formulation could also successfully inhibit the protein synthesis in XC cells (rat fibroblasts transformed by Rous sarcoma virus) and phytohemagglutinin-stimulated CBA mouse lymphocytes. Phosphatidylcholine could only show the transport facility after addition of cholesterol to the cells. Addition of mixed bovine brain gangliosides in the following order phosphatidylcholine/cholesterol/gang-liosides (5: 5: 1) escalated the effectiveness as well [71, 72]. Tumor targeted RIPs may take advantage of nanoparticulate drug delivery systems for intracellular targeting.

Fig. (3).

A classification chart summarizing the carrier or non-carrier based approaches for targeted tumor therapy. While targeted toxins fall under the noncarrier based approach, toxins may also be incorporated in inert carriers for better stability.

In the same context, a generation-4 polyamidoamine (PAMAM) dendrimer induced cellular uptake and intracellular release by facilitating the endocytic uptake of RIPs. The use of photochemical internalization (PCI) technology could increase the effectiveness of free RIPs and PAMAM-RIPs [73]. After PCI treatment, PAMAM-RIP facilitated internalization as well as nuclear entry. Albeit this being a negative outcome, the use of ER signaling could in turn be used to avoid this side effect and elicit a site specific response. The use of nanocarriers, liposomes, aptamers or dendrimeric structures may be helpful in the targeted delivery of the toxins. They surely facilitate the efficacy by either resulting in multivalence or providing a dual component delivery in a single system.

Efficacy Enhancers in Targeted Toxin Therapy

In the past decade, a number of strategies have been attempted to circumvent the problems associated with immunogenicity, vascular leak syndrome and other off-target effects that are associated with targeted toxin therapy. Conventionally, the use of certain chemicals has been employed, which led to an elevation of the endosomal pH, thereby protecting the toxin from lysosomal enzyme degradation. Another strategy involved the use of pore forming agents (Fig. 4). Use of these components certainly helped in improving the efficacy but their proof in preclinical and clinical studies is still limited [74-78]. The details for other compounds including organic and inorganic substances, synthetic peptides and compounds of natural origin are detailed in Table 3. In an interesting study, the anti-tumoral effects of anti-CD5 immunotoxins, which were constructed using a monoclonal antibody Fab fragment linked to native ricin A-chain or partially deglycosylated ricin A-chain, were examined in combination with the enhancer monensin conjugated to human serum albumin and injected intraperitoneally. In this case, 90% of the tumor cells were killed. This potentiating effect was observable even at a 5% monensin saturation level. The authors were able to successfully inhibit the effect by injecting the unconjugated antibody.

Fig. (4).

A mechanistic description of the three widely known phenomena for the endosomal escape of molecules. Past internalization the toxin release may be facilitated via membrane fusion, pore formation or proton sponge effect. Table 3 describes in detail all the compounds that facilitate these effects for improving the efficacy of targeted toxins.

Table 3.

A detailed list of all the efficacy enhancers employed in the improvement of toxin efficacy and enhancement of endosomal escape.

Efficacy Enhancers	Origin	Factor	Site of Action	Application	Ref.
Lysosomotropic amines
Ammonium chloride	Inorganic	6700	Endosomes	Immunotoxins (RTA)	[693, 694]
Methylamine	Organic	13,300	Endosomes	Immunotoxins (RTA)	[693]
Dimethylamine	Organic	3300	Endosomes	Immunotoxins (RTA)	[693]
Trimethylamine	Organic	80	Endosomes	Immunotoxins (RTA)	[693]
Amantadine	Organic	1180	Endosomes	Immunotoxins (RTA)	[693, 695]
Chloroquine	Organic	2500	Endosomes	Immunotoxins (RTA, Gel)	[693, 696]
Lipopolyamines	Organic	10 - 250	Endosomes	Immunotoxins (Sap)	[697]
β-Glycylphenyl-naphthylamide (GPN)	Organic	10	Endosomes	Immunotoxins (PE)	[698]
Quinacrine	Organic	15	Endosomes	immunotoxins (Gel)	[696]
Carboxylic ionophores
Monensin	Organic	50,000	Lysosomes	Immunotoxins (RTA, Gel)	[693, 696, 699]
Grisorixin	Organic	25,000	Lysosomes	Immunotoxins (RTA)	[693]
Lasalocid	Organic	33,000	Lysosomes	Immunotoxins (RTA)	[693]
Nigericin	Organic	6700	Lysosomes	Immunotoxins (RTA)	[693]
Calcium channel antagonists
Verapamil	Organic	170	Lysosomes or other vesicular compartments	Immunotoxins (RTA, PE, Gel)	[696, 698, 700]
Diltiazem	Organic	10, 40	Lysosomes or other vesicular compartments	Immunotoxins (PE)	[698]
Methoxyverapamil (D-600)	Organic	40	Lysosomes or other vesicular compartments	Immunotoxins (PE)	[698]
Varapamil analogues	Organic	2 - 70	Lysosomes or other vesicular compartments	Immunotoxins (RTA, PE)	[700]
Perhexiline	Organic	10 - 2000	Lysosomes or other vesicular compartments	Immunotoxins (RTA)	[701]
SR 33557	Organic	540	Lysosomes or other vesicular compartments	Immunotoxins (RTA)	[702]
SR 33287	Organic	620	Lysosomes or other vesicular compartments	Immunotoxins (RTA)	[702]
Organic polymers
Polyethylenimine (PEI)	Organic polymer	From no-effect to effect	Lysosomes	Gene transfection	[703]
Poly(amidoamine)s (PAAs)	Organic polymer	100	Endosomes and lysosomes	Toxins (RTA, Gel, Sap), Gene delivery	[704-706]
Poly(propylacrylic acid) (PAAP)	Organic polymer	Significant increase	Endosomes	Gene transfection	[707]
Fusogenic lipids
DOPE	Organic	Significant increase	Endosomes	Gene transfection, liposomes	[708]
CHEMS	Organic	Significant increase	Endosomes	siRNA delivery	[709]
Monoolein	Organic	Significant increase	Endosomes	DNA delivery, nanoparticles	[75]
Other organic compounds
Retinoic acid	Organic	10,000	Golgi apparatus	Immunotoxins (RTA)	[710]
Cyclosporin A	Organic	100	Vesicular compartments	Immunotoxins (RTA)	[711, 712]
Brefeldin-A	Organic	1000	Golgi apparatus	Immunotoxins (RTA)	[713]
Bryostatin 1	Organic	Significant increase	Cell signalling	Immunotoxins (PE)	[714]
Wortmannin	Organic	Significant increase	Endosomes and lysosomes	Immunotoxins (ETA, Sap, Gel)	[715]
Synthetic surfactants	Organic	Significant increase	Endosomes	Gene transfection, siRNA delivery, nanoparticles	[716, 717]
EHCO	Organic	Significant increase	Endosomes	siRNA delivery, nanoparticles	[718]
Viruses and virus peptides
Adenovirus	Adenovirus	10,000	Endosomes, lysosomes or other vesicular compartments	Immunotoxins (PE, RTA, Sap, Gel), gene delivery	[719-721]
Penton base protein (adenovirus capsid protein)	Adenovirus	100	Endosomes and lysosomes	Immunotoxins (PE, Gel)	[722, 723]
Minor capsid protein VI	Adenovirus	From no-effect to effect	Endosomes	Nanoparticles	[724, 725]
KFT25 (N-terminus of Protein G)	Vesicular stomatitis virus	10 - 20	Lysosomes or other vesicular compartments	Immunotoxins (RTA, Dia)	[309, 528]
HA2 (hemagglutinin HA-2)	Influenza virus	10 - 100	Endosomes	Immunotoxins (RTA, Sap), gene transfer	[82, 726, 727]
HA2 / poly (L-lysine) (PLL)	Influenza virus	Significant increase	Endosomes	Gene transfer	[728]
HA23	Influenza virus	4 - 5	Endosomes	Immunotoxins (RTA)	[729]
GALA	Synthetic peptide (HIV)	From no-effect to effect	Endosomes	Gene transfection, liposomes, nanoparticles	[726, 730, 731]
KALA	Synthetic peptide (HIV)	From no-effect to effect	Endosomes and other membranes	Gene transfection	[732]
KALA/polyethylenimine (PEI)	Synthetic peptide (HIV)	Significant increase	Endosomes and other membranes	Gene transfection	[733, 734]
INF-7	Influenza virus	100	Endosomes	Gene delivery, siRNA delivery, liposomes	[735-737]
Tat (transcriptional activator Tat protein)	HIV	3340	Endosomes	DNA delivery, PNA delivery, liposomes, nanoparticles	[738-740]
gp41	HIV	Significant increase	Endosomes	Gene delivery, siRNA delivery	[741]
gp41/polyethylenimine (PEI)	HIV	Significant increase	Endosomes	Gene delivery, siRNA delivery	[742]
L2 (minor capsid protein)	Papillomavirus	From no-effect to effect	Endosomes and other membranes	Proteins (GFP)	[743]
Major envelope protein (E)	West Nile virus	From no-effect to effect	Endosomes	Natural process	[744]
VP22 (structural protein VP22)	Herpes simplex virus	From no-effect to effect	Actin-mediated endosomes	DNA delivery, proteins (GFP)	[729]
Synthetic analogue of glycoprotein H (gpH)	Synthetic peptide (Herpes simplex virus)	30	Endosomes	Gene transfection, liposomes	[745]
PreS2-domain of hepatitis-B virus surface antigen (TLM)	Hepatitis-B virus	2 - 20	Endosomes or other vesicular compartments	Immunotoxins (Sap, Ang)	[600, 746, 747]
Bacterial peptides
Listeriolysin O (LLO)	Listeria monocytogenes	Significant increase	Endosomes	DNA delivery, liposomes	[748, 749]
Pneumococcal pneumolysin (PLO)	Pneumococcos	From no-effect to effect	Endosomes	Toxins (Granzyme B)	[750]
Streptococcal streptolysin O (SLO)	Streptococcos	From no-effect to effect	Endosomes	Toxins (Granzyme B)	[750]
T-domain of diphtheriatoxin (DT)	Corynebacterium diphtheria	From no-effect to effect	Endosomes	Immunotoxins (DT)	[751]
T-domain of diphtheria toxin (DT) / poly(ethylenimine) (PEI)	Corynebacterium diphtheria	Significant increase	Endosomes	Gene transfection	[752]
Domain II of Pseudomonas exotoxin A (ETA)	Pseudomonas aeruginosa	From no-effect to effect	Endosomes and trans-Golgi network	Immunotoxins (PE)	[753]
REDLK	Pseudomonas aeruginosa	From no-effect to effect	Endoplasmatic reticulum	Immunotoxins (PE)	[754]
Animal and human peptides
Penetratin (homeotic transcription protein Antennapedia, Antp)	Drosophila melanogaster	From no-effect to effect	Pinocytic and other vesicular compartments	PNA delivery	[755]
R6-Penetratin (with arginine residues)	Synthetic (Drosophila melanogaster)	5 - 10	Endosmes and other vesicular compartments	PNA delivery	[756]
EB1 (synthetic analog of penetratin)	Synthetic (Drosophila melanogaster)	Significant increase	Endosomes	siRNA delivery	[757]
hCT (9-32) (human calcitonin derived peptide 9-32)	Human	From no-effect to effect	Endosomes or other vesicular compartments	Natural process	[758, 759]
Fibroblast growth factor-1 (FGF-1) sequence	Human	From no-effect to effect	Endosomes	Natural process	[760]
Melittin	Bee venom	From no-effect to effect	Endosomes	Gene delivery	[726, 761]
Melittin/polyethylenimine (PEI)	Bee venom	Significant increase	Endosomes	Gene delivery, siRNA delivery	[762-764]
Human β3 integrin signal sequence	Human	From no-effect to effect	Endosomes	Natural process	[765]
Heavy chain of immunoglobulin G	Caiman crocodylus	Significant increase	Cell membrane	Liposomes	[766]
Transportan	Synthetic peptide (neuropeptide galanin + wasp venom peptide mastoparan)	From no-effect to effect	Endosomes or other vesicular compartments	Proteins (GFP, Strep)	[767]
Bovine prion protein (bPrPp)	Synthetic peptide (bobine prion)	From no-effect to effect	Cell membrane, macropinosomes	Nanoparticles	[768, 769]
KDEL	Signal sequence	100 - 1000	Endoplasmatic reticulum	Immunotoxins (RTA, PE)	[770, 771]
Animal and human proteins
α-Interferon (INF)	Human	Significant increase	Cell signalling	Immunotoxins (RTA)	[772]
Perforin	Human	From no-effect to effect	Early endosomes	Immunotoxins (GzmB)	[773, 774]
Rituximab	Mouse/human chimeric mAb	80	Cell signalling	Immunotoxins (Sap)	[587]
Plant saponins
Saponinum album	Gypsophila paniculata L.	2,500,000	Late endosomes and lysosomes	Immunotoxins (Sap)	[599, 775, 776]
SA-1641	Gypsophila paniculata L.	Significant increase	Late endosomes and lysosomes	Immunotoxins (Sap, Dia)	[83, 84]
SA-1657	Gypsophila paniculata L.	From no-effect to effect	Late endosomes and lysosomes	Immunotoxins (Sap)	[52]
Saponaria saponins	Saponaria officinalis L.	10, 000	Late endosomes and lysosomes	Immunotoxins (Sap)	[52]
SO-1861	Saponaria officinalis L.	1000	Late endosomes and lysosomes	Immunotoxins (Sap, Dia)	[52]
Quillaja saponins	Quillaja saponaria Mol.	1400	Late endosomes and lysosomes	Immunotoxins (Sap)	[775]
Plant proteins
Ricin B-chain	Ricinus communis L.	From no-effect to effect	Interalization/Cell signalling	Immunotoxins (RTA)	[777]
Ricin B-chain immunotoxin	Ricinus communis L.	2 - 4	Interalization/Cell signalling	Immunotoxins (RTA)	[778]
Ricin B chain (piggyback)	Ricinus communis L.	2 - 6	Interalization/Cell signalling	Immunotoxins (RTA)	[779]
Synthetic peptides
Polyarginines	Synthetic peptide	Significant increase	Late endosomes, Golgi apparatus and endoplasmatic reticulum	DNA delivery, siRNA delivery, proteins (GFP)	[780-782]
Polylysines	Synthetic peptide	Significant increase	Endosomes	Gene transfection	[783]
Histidine 10	Synthetic peptide	7000	Endosomes	Gene transfection	[784]
(R-Ahx-R)4	Synthetic peptide	From no-effect to effect	Late endosomes, Golgi apparatus and endoplasmatic reticulum	PNA delivery	[74, 785]
Poly(L-histidine)	Synthetic peptide	Significant increase	Endosomes	DNA delivery	[786, 787]
Sweet arrow peptide (SAP)	Synthetic peptide	Significant increase	Endosomes	Gene delivery, nanoparticles	[788]
Loligomer	Synthetic peptide	From no-effect to effect	Endosomes or other vesicular compartments	Peptide delivery, fluorescent probes	[789]
Amphiphilic model peptide	Synthetic peptide	From no-effect to effect	Endosomes or other vesicular compartments	Polar bioactive compounds	[790]
IRQ peptide	Synthetic peptide	Significant increase	Endosomes	siRNA delivery	[709]
43E peptide	Synthetic peptide	Significant increase	Endosomes and lysosomes	Gene transfection	[791]
pJVE	Synthetic peptide	2	Endosomes	Immunotoxins (Dia)	[309]
RAWA	Synthetic peptide	Significant increase	Endosomes and other membranes	Gene delivery	[792]
Nuclear localization signals	Synthetic peptide	150	Cytoplasmic entrapment, nuclear membrane	Gene transfection	[793]
SynB1	Synthetic peptide	6	Endosomes and other membranes	Peptide delivery	[78, 794]
Pep-1	Synthetic peptide	From no-effect to effect	Endosomes and other membranes	Peptide delivery, proteins (GFP, β-Gal)	[795]
Physicochemical techniques
Photochemical internalization	Technique	1000	Endosomes	Immunotoxins (Sap, Gel), gene transfection, liposomes, nanoparticles	[796-798]
Ultrasound	Technique	30	Endosomes	Gene delivery, liposomes	[799, 800]
Plasmonic nanobubbles	Technique	30	Endosomes	Nanoparticles	[801]
Magnetic nanoparticles	Technique	From no-effect to effect	Endosomes	Gene transfection, siRNA delivery, nanoparticles	[76, 802]

These results show that the therapeutic efficacy of immunotoxins can be very well improved by following a pre-defined and optimized therapeutic regime [79]. Monensin is one of the compounds with proven efficacy. In the recent past, an even higher synergy has been observed by the concomitant use of saponins and plant RIPs which is the basis for the next section.

Saponins in Targeted Toxin Therapy

In our research group, we have been extensively working with the use of certain structurally specific triterpenoids viz. saponins. These compounds have shown tremendous potential in enhancing the effectiveness of targeted toxins; mainly plant type I RIPs saporin and dianthin (Fig. 5) [80, 81]. Saponins are generally classified as triterpenoidal or steroidal, based on the aglycone backbone. In general, the saponins have a sugar chain attached at either the C-3 or C-17 position (monodesmosidic saponins), or on both positions (bisdesmosidic saponins). In recent studies, the concomitant use of saponins from Saponaria officinalis L. and Gypsophila paniculata L. has been successful in synergistically enhancing the toxicity of saporin-EGF and dianthin-EGF [30]. Evaluation of the molecular mechanism revealed that the toxin was internalized via receptor mediated internalization, thereafter the saponins (which were used at a concentration far below their membrane pore forming concentrations) lead to an enhanced endosomal escape of the toxin, which in turn resulted in apoptosis. The efficacy of saponins to facilitate rapid cell death, when administered in unison with the targeted toxins was further confirmed in a real-time cytotoxicity evaluation. Cell death was observed as a fall of the impedance signal (representing the number of living cells) within the first 12 h of incubation of the toxin and the saponins, while the toxin alone requires a 10,000-fold higher concentration to induce cell death after a period of nearly 48 h of incubation. It is pertinent to mention here that the saponins were used at a concentration that has no effect on its own [74-76, 82-84].

Fig. (5).

A schematic description for the efficacy enhancement of certain plant type I RIPs using triterpenoidal saponins. 1. The toxin reaches the cell 2. Internalization and formation of endosomal vesicles, 3. Maturation of the endosomal vesicles along with the entrapped toxin, 4. Late endosome formation. 5. The toxin undergoes lysosomal degradation and thus the toxic effect is not elicited. 6. Particular saponins accumulates inside the endo/lysosomes by unknown mechanisms. The presence of saponins faciliates the endo/lysosomal release of the toxin in a pH dependent manner. 7. The toxin induces cell death inside the cytosol via apoptosis.

The structural features of saponins that are highly desirable for their enhancing effects have been studied extensively. It is now established that bisdesmosidic triterpenoidal saponins, which have a gypsogenin or quillaic acid backbone with a glucuronic acid at C-3 position are most effective. Moreover, there are further specific structural and sugar chain requirements that lead to a relatively small number of saponins, which show effectiveness as synergistic enhancers. As already detailed, for exerting cytotoxicity, the release of toxin in the cytosol is a very important step. This process is however very feeble in case of internalized RIPs. Interestingly, Weng et al. demonstrated that saponins which are also biosynthesized by Saponaria officinalis L., can in a very specific manner facilitate the cytosolic transfer of toxin without affecting the plasma membrane integrity. This effect mainly takes place in late endosomes and lysosomes at a pH range between 4-5.5. A strong binding affinity for saponins with RIPs using surface plasmon resonance was also verified and the combination of the targeted toxin and saponin was validated for its effectiveness in vivo in a syngeneic mouse tumor model [85]. Although using saponins or for that matter any toxicity enhancer is a novel approach for improving the effectiveness of targeted toxins, there are certain limitations associated with this strategy more importantly from a clinical perspective. Any clinical application involving the use of multiple components is always a practical and a regulatory problem. This problem in case of saponins or other enhancers can only be circumvented by the use of a drug carrier system, which either encompasses the two components together or either of the two components form a part of the delivery matrix.

OUTLOOK AND CONCLUSION

The initial hope for immunotoxin-based therapy in the treatment of cancer was their perceived role as magic bullets functioning as a cure for solid tumors and blood borne malignancies [86]. Since the 1990s, clinical studies have clearly shown that the targeted toxin therapy works as a good supplement to the existing chemotherapeutic agents [87]. The combination of chemo- and immunotherapy is manifolds better than the effectiveness of either of them alone. This when accompanied with the combined use of enhancers would surely give the clinicians more advantage in treating the patients effectively. There are numerous facets to targeted toxin therapies and as summarized by Chandramohan et al. [88], the therapeutic success requires selective killing, absence of side effects or nonspecific toxicity, which should be further juxtaposed with successful delivery of the immunotoxin to tumor cells. Another important aspect for a clinical success is the designing of targeted toxin. This should be done in such a way that the production of anti-toxin antibodies is minimized in an ideal case. Most of the target receptors are ubiquitous and therefore, a bystander effect of the targeting strategy into normal tissues in most cases is unavoidable. It is in such instances that the use of a targeted toxin enhancer as described in Table 3 can certainly come to rescue. Since use of such strategy minimizes the dosage and therefore the accompanying side effects, the target specific cell killing is limited to the amount of toxin molecules available.

Another important aspect is the fact that most of the disseminated tumors are highly heterogeneous. This is further complicated by the variations in the receptor expression levels with progression of tumor into the different stages of growth [89]. It is in such cases that a “cocktail therapy” could be highly effective and beneficial [90]. In the past, the targeted toxins have shown limited success independently and it could be imagined that for a higher efficacy, a combination of a small drug with targeted toxins, administered concomitantly, may minimize the side effects of either of them, with an increased effectiveness. In addition, another strategy could be the use of a combination of two or more targeted toxins as shown by a joint application of anti-CD19 and anti-CD22 immunotoxins (Combotox) [91] or multiple receptor targeting by aptameric configurations of the toxins making them more specific to different receptor types, overexpressed in the tumor that is being targeted. The future for such an approach has tremendous potential with the recent advancement in high-throughput screening techniques and the growing importance of personalized medicine in case of tumor treatment.

Furthermore, development of bi-specific and tri-specific therapeutic antibodies would surely add to the armor of the clinicians handling targeted toxins in tumor therapy [92, 93]. It can be foreseen that a cargo of toxin, with multiple targeting ligands would be highly beneficial, not only in solid tumors but in case of disseminated and metastasized tumors as well. The future of targeted toxin therapies appears to be even more promising than within the previous decade; this is mainly ascribable to the tremendous advancements in the biotechnological tools and an unprecedented growth in the field of proteomics and genomics. The possibility of identification and procurement of inactive mutants and humanized or human toxins, which can be further modified as targeted toxins opens up new vistas for tumor treatment. Recombinant DNA and expression techniques have reached a stage where a mutated version once identified to be effective, can be expressed and obtained under good manufacturing conditions in a quick succession. Recent advancements in the antibody-based therapeutics include the generation of bi-specific antibodies and bio-mimicking antibodies, while retaining selectivity these antibodies reportedly have a better efficacy. This surely adds to the repertoire of molecular biologists for reducing the incubation time during the drug development process.

CONFLICT OF INTEREST

Declared none.