3. Targeted Toxins as Components of Nanoagents

Despite the successful use of immunotoxins, immunotherapy strategies are still expensive, mainly due to the complicated preparation process. Immunotoxins can also stimulate the host immune system and trigger the production of neutralizing antibodies. Intravenous administration of targeted protein toxins may be characterized by poor pharmacokinetic profiles in addition to non-specific distribution in tissues and organs of the body and can cause serious side effects including systemic toxicity. Besides, the penetration of anticancer drugs into tumor tissues is usually low and the high doses of drugs are required for treatment ^[18][19][69,70]. The use of nanocarriers, especially the targeted ones, for delivering toxins to tumor foci may improve the pharmacokinetics and pharmacodynamics of agents, control drug release, improve the specificity, increase internalization and intracellular delivery, and reduce systemic toxicity ^[20][71]. Nanocarriers can facilitate selective accumulation in tumors via the enhanced permeability and retention (EPR) effect and active cellular uptake ^[21][72]. Among various nanoscale drug carriers, liposomes, polymeric nanoparticles and noble metal nanoparticles have demonstrated the greatest potential in clinical application ^[22][23][24][73,74,75].

Liposomes have proven to be an efficient vehicle for delivering a high molecular weight neurotoxin botulinum toxin A to treat hypersensitive bladder and overactive bladder (OAB) without systemic injection ^[25][86].

Recently, a new method has been proposed for the preparation of small (80–90 nm) unilamellar antigen-targeted liposomes containing large amounts (thousands of protein molecules per liposome) of highly toxic PE40 ^[26][93] (Figure 2a). Efficient encapsulation of the proteins was achieved through electrostatic interaction between positively charged toxin proteins at pH lower than pI and negatively charged liposome membrane. The external surface of proteoliposomes were functionalized with covalently coupled DARPin_9-29 using “click chemistry” through a relatively long flexible linker. Functionalized proteoliposomes specifically bind to HER2-positive cells and after internalization cause cell death at subnanomolar concentrations ^[27][94].

Figure 2. (a) Small unilamellar targeted liposomes containing PE40 ^[26][93]; (b) Hybrid biofunctional nanocomplex based on radioactive 90Y bearing core-shell UCNP and functionalized with targeted toxin DARPin-PE40 ^[28][97].

4. Cytotoxic Mechanisms of Natural Toxins

The killing mechanisms of protein toxins can vary, but they differ from the mechanisms that are implemented in conventional chemotherapy [4], so an obtained resistance to chemotherapeutic agents does not affect the effectiveness of protein toxins. Furthermore, the mechanism complementation can provide a synergistic effect of combined therapy. In addition, protein toxins are not mutagens and should not accelerate tumor progression due to enhanced mutagenesis. They can be mass-produced cheaply in bacteria as homogeneous proteins ^[16][5].

Toxins of bacterial and plant origin commonly used as cytotoxic component in chimeric proteins in anticancer therapy are summarized in Table 1. The most toxic proteins include enzymes that inhibit translation at the elongation step. Unsurprisingly, most of them arise from natural toxins that have been effectively preselected by evolution.

RES cells inactivation
Macrophages blockade decreasing toxic nanoparticles uptake
[	67	][68][78,179]
Mechanism of Action	Details	Examples	References
eEF2 inactivation	ADP-ribosylates elongation factor 2 (eEF2) and halt protein synthesis at the elongation step	Pseudomonas exotoxin A (PE, ETA)	[29][30][62,106]
		Diphtheria toxin (DT)	[9][31][12,83]
Ribosome inactivation	N-glycosidase depurinates a critical adenine in 28S rRNA, which results in the inability of the ribosome to bind elongation factor 2, thereby blocking protein translation	Ricin	[32][33][34][63,107,108]
		Shiga toxin (Stx)	[35][30]
		Abrin	[36][37][38][109,110,111]
RNA degradation	Nonspecific RNA cleavage blocks protein synthesis and leads to apoptosis	Barnase	[39][40][112,113]
		Binase	[41][114]
Cell signaling disruption	The cleavages of the MAP kinase family members leading to their inactivation; uncontrolled conversion of ATP to cAMP	Anthrax toxin	[42][115]
Photoinduced ROS production	The proteins absorb exciting light and produce reactive oxygen species	KillerRed	[43][44][116,117]
		miniSOG	[45][6]
Direct apoptosis induction	Effector caspases cleavage	Granzyme B	[46][118]
Enhanced diffusion of anticancer drug	Vascular network modulation	Botulinum neurotoxin	[47][48][57,58]
	Pore formation for better intracellular delivery	Listeriolysin O	[49][50][68,85]
		Streptolysin-O	[51][52][119,120]

5. Reducing Protein Toxins Side Toxicity

The protein toxins high toxicity is one of main advantages of these molecules but at the same time it increases the risk and severity of side effects. The side toxicity of a protein can be based on a direct cell killing and inflammation induction ^[53][177]. The most common side effects caused by DT, PE, and ricin include vascular leak syndrome, hepatotoxicity, and kidney damage ^[54][55][56][84,121,122]. In addition, Shiga toxin is notorious for its ability to cause hemolytic uremic syndrome (HUS), potentially leading to life-threatening complications ^[57][58][123,124]. The production of neutralizing antibodies can also serve as a cause on side effects due to anaphylaxis reactions.

To date the number of strategies were developed to reduce protein drug off-target toxicity, the main tools are summarized in Table 2.

Strategy Used for Side Toxicity Reduction	Principle	References
Impairment of natural tropism	Removing the natural targeting domains of AB toxins	[59][35]
	Introduction of point mutations attenuating the target binding	[60][145]
Construction of miniaturized toxin variants	Deletion of protein parts not directly involved in toxin mechanism of action to reduce any non-specific interaction and immunogenicity	[61][62][63][52,53,178]
Tumor-specific activation of a toxin	The replacement of furin cleavage site to tumor-specific proteases cleavage sites (MMP, uPA)	[64][65][66][138,139,140]

The natural tropism of a toxin can sometimes be used to target a tumor, as we have already discussed for anthrax toxin and Shiga toxin, but for the majority of protein toxins the natural tropism provides an off-target activity. To reduce the unwanted effects it is desirable to impair the targeting moieties. It was first implemented for the toxins consisting of targeting and effector modules, which predisposes them to be used in the truncated form. The truncated forms of protein toxins were used in the second generation of immunotoxins, which helped to reduce their in vivo side toxicity retaining their efficiency ^[13][16]. The targeted proteins with truncated toxins were first acquired with the use of DT and ricin ^[13][16], then the promising specific toxicity was proven for PE40, the engineered ETA ^[69][70][180,181]. Further miniaturization of PE led to the remarkable success in reducing both its immunogenicity and side toxicity. The PE-fused antibodies and other targeting proteins efficiently kill cancer cells in vitro ^[27][63][71][94,178,182] and reduce or stop the growth of tumors of various origin in vivo ^[72][27][73][22,94,183]. However, PE is notorious for its high immunogenicity: PE is a bacterial protein that can induce antibody responses and has a considerable side toxicity ^[74][75][184,185]. Although PE-based agents can be used successfully in combination with immunosuppressive chemotherapy [184] or in the treatment of hematologic malignances ^[76][77][186,187], the production of neutralizing antibodies reduces the efficiency of the PE-based therapy in patients with intact immune system and increases the probability of hypersensitivity reactions. The removal of domain II leads to a decrease in immunogenicity and, at the same time, reduces the protein degradation in the lysosomes. In addition, it helps to reduce off-target side toxicity in animal models ^[78][188]. Further investigation of PE helped to map the immunodominant epitopes of the catalytic domain and make them less visible to immune cells by deletions and point mutations ^[79][45]. The resulting toxin variants demonstrate high anti-cancer activity comparable to the activity of the initial variants of PE40 and PE38 and have decreased side toxicity and are less immunogenic ^[61][62][63][52,53,178].

For the anthrax toxin the introduction of point mutations impairing natural targets binding has proven to be effective: a double mutation in domain 4 of protective antigen (PA) led to the ablation of the protein native receptor-binding function. The resulting mPA fuse with EFG in a complex with LFN-DTA efficiently inhibited protein synthesis in EGFR-positive A431 cells in vitro (IC50 = 10 pM) not affecting the protein synthesis of CHO-K1 cells lacking EGFR. This variant was also used to target cells expressing HER2 ^[80][81][146,189], and both EGFR and carcinoembryonic antigen ^[82][190]. Still, the tumor-killing activity and side toxicity of these proteins in vivo are yet to be investigated.

In case of glycoproteins, the oligosaccharides involved in off-target binding can be chemically removed. The ricin oligosaccharides bind to CD206 mannose receptor on macrophages ^[83][84][38,46], and interact with glycosylated IgA and IgM ^[85][47]. The circulation time and anti-tumor activity of ricin-based immunotoxins can be increased by chemical deglycosylation of the toxin ^[86][87][39,48], but unfortunately, these forms are more toxic to mice than the glycosylated ones ^[86][39].

Another promising strategy relies on tumor-specific activation of a toxin that requires proteolytic cleavage for toxin functioning. Several toxins, namely DT, PE, and ricin are digested in endosomes by furin protease thus releasing active protein fragments. By means of gene engineering the furin cleavage site can be replaced by the sequences recognized by the proteases that are upregulated in tumors. This strategy was realized for anthrax toxin protective antigen (PA): it was obtained in matrix metalloproteinase-dependent and urokinase plasminogen activator-dependent variants ^[64][65][138,139] which were selectively activated by tumor cells expressing respective proteases. The MMP-activated PA in combination with anthrax toxin lethal factor efficiently treated melanoma xenografts, and lung and colon carcinoma xenografts irrespective of the B-RAF status, targeting not only tumor cells, but also tumor vasculature ^[88][137]. This engineered toxin was less toxic than wild-type LT to mice because of the limited expression of MMPs by normal cells and also displayed lower immunogenicity compared with the wild-type toxin. The systemically administered toxin produced greater anti-tumor effects than wild-type LT toward human xenograft tumors. Both types of activated PA molecules were used to obtain dual-activity dependent delivery system based on PA variants that can only form octamers after activation by both of the tumor-selective proteases, uPa and MMPs. This complex agent completely stopped tumor growth in mice and its components were well tolerated in higher doses, than the wild-type PA and LT ^[66][140].

The most recent strategy for prevention of toxic agents intake by macrophages is based not on a toxin modifications, but on a transient reticuloengothelial (RES) cells inactivation. It can be achieved either by injection if blocking nanoparticles ^[68][179] or by enhanced clearance of erythrocytes caused by anti-erythrocyte antibodies ^[67][78]. These methods were successfully used to prolong nanotherapeutic agents circulation time and can be possibly applied for toxin-based therapy.

Cancer treatment has been revolutionized due to antigen-targeting drugs that specifically deliver a cytotoxic component to cancer cells, and advances in genetic engineering and biotechnology, making it possible to produce any fusion proteins needed. Potent cytotoxic components include enzymatically active protein toxins based on plant or bacterial toxins. Here, we have summarized several decades of research devoted to targeting internalizing receptors of cancer cells with chimeric therapeutic molecules. The targeting approach can also be applied to drug carriers such as liposomes, polymers, and nanoparticles. The design of complex targeted agents or several drug application regimens that allow achieving a synergistic effect is also a promising area of anticancer therapy.