15 years of ATTEMPTS: a macromolecular drug delivery system based on the CPP-mediated intracellular drug delivery and antibody targeting.

COREL-07465; No of Pages 12 Journal of Controlled Release xxx (2014) xxx–xxx

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Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel

15 years of ATTEMPTS: A macromolecular drug delivery system based on the CPP-mediated intracellular drug delivery and antibody targeting Junxiao Ye a,b,1, Meong Cheol Shin c,1, Qiuling Liang b, Huining He b,⁎, Victor C. Yang c,d,⁎⁎ a Collaborative Innovation Center of Chemical Science and Chemical Engineering, and State Key Laboratory of Chemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China b Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics (Theranostics), School of Pharmacy, Tianjin Medical University, Tianjin 300070, PR China c Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor 48109-1065, USA d Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, and College of Medicine or College of Pharmacy, Seoul National University, Seoul, South Korea

a r t i c l e

i n f o

Article history: Received 30 September 2014 Received in revised form 18 November 2014 Accepted 1 December 2014 Available online xxxx Keywords: ATTEMPTS CPP-mediated intracellular drug delivery Antibody targeting Prodrug

a b s t r a c t Traditionally, any drug intended for combating the tumor would distribute profoundly to other organs and tissues as lack of targeting specificity, thus resulting in limited therapeutic effects toward the tumor but severe drug-induced toxic side effects. To prevail over this obstacle of drug-induced systemic toxicity, a novel approach termed “ATTEMPTS” (antibody targeted triggered electrically modified prodrug type strategy) was designed, which directly introduces both of the targeting and prodrug features onto the protein drugs. The ATTEMPTS system is composed of the antibody targeting component consisting of antibodies linked with heparin, and the cell penetrating peptide (CPP) modified drug component. The two components mentioned above self-assembled into a tight complex via the charge to charge interaction between the anionic heparin and cationic CPP. Once accumulated at the targeting site, the CPP modified drug is released from the blockage by a second triggering agent, while remaining inactive in the circulation during tumor targeting thus aborting its effect on normal tissues. We utilized the heparin-induced inhibition on the cell-penetrating activity of CPP to create the prodrug feature, and subsequently the protamine-induced reversal of heparin inhibition to resume cell transduction of the protein drug via the CPP function. Our approach is the first known system to overcome this selectivity issue, enabling CPP-mediated cellular drug delivery to be practically applicable clinically. In this review, we thoroughly discussed the historical and novel progress of the “ATTEMPTS” system. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Current anti-cancer drug therapies utilizing the traditional small molecular agents would result in negative therapeutic efficacy by three hurdles: lack of selectivity of cancer cells over normal cells, which would introduce the toxic side effect toward normal tissues [1, 2]; the second limitation lies in the rapid clearance of these watersoluble small molecular agents from the bloodstream and the last but the largest obstacle is the drug resistance commonly exhibited by tumor cells, which leads to the reduced drug accumulation [3–6]. Therefore, the traditional anti-cancer agents tend to localize in normal tissues rather than in tumors, thereby rendering higher toxic effects.

⁎ Correspondence to: H. He, School of Pharmacy, Tianjin Medical University, Tianjin 300070, China. ⁎⁎ Correspondence to: V.C. Yang, Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor 48109-1065, USA. E-mail addresses: [email protected] (H. He), [email protected] (V.C. Yang). 1 These authors contributed equally.

Macromolecular agents, like protein, antibody, enzymes and nucleic acid drugs, possess several attributes such as high specificity, high solubility, optimum activity under physiological conditions, and a repetitive reaction mechanism over small molecular agents [7]. However, despite these superior efficiency, the success oncological application of these agents is beset by several limitations, such as the proteolytic degradation property, the inability of these macromolecular agents to cross cell membranes and immunogenicity. Thus, only few of these macromolecular agents have currently been approved for clinical use [1,7,8]. In the past 10–20 years, approaches have been developed to enhance the anti-cancer efficiency with fewer side effects based on the constant progress accomplished in modern science and nanotechnology [9–14]. Among all these approaches, the molecular target drug delivery method and the prodrug strategy drew much attention. The target drug delivery became prevalent, attributing to the least toxic but promising therapeutic effects. This tendency, somewhat fashionable, is now rather problematic caused by the great genetic diversification during the process of genetic alteration in human cancer [15]. Prodrug was consisted of the grafting group and the active drug, which forms an inactive formulation. In general, a well-thought-out

http://dx.doi.org/10.1016/j.jconrel.2014.12.002 0168-3659/© 2014 Elsevier B.V. All rights reserved.

Please cite this article as: J. Ye, et al., 15 years of ATTEMPTS: A macromolecular drug delivery system based on the CPP-mediated intracellular drug delivery and antibody targeting, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.12.002

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J. Ye et al. / Journal of Controlled Release xxx (2014) xxx–xxx

prodrug strategy could achieve tissue-specific actions and reduce the undesired toxic effects, as the prodrug was designed according to the difference between the target environment and the abnormal physicochemical properties, like pH, temperature, over-expressed enzymes, receptors, and transporters etc. Considering that the common mechanism of the resistance to chemotherapy is the inability of drug transport across the cell membranes, transport moiety is especially critical in the case of anticancer therapy for protein drug [16], and various prodrug strategies were developed to achieve the selectivity issue [16–21]. Except for Prodrug strategy, various approaches based on carriers, like antibodies [22], tumor homing peptides [23–25], various nanoparticles [26–31], red blood cells [31–33], or even small molecule ligands [34, 35] have been utilized in the purpose of directing the macromolecular drugs only to the cancer cells [36,37]. Our strategy, therefore, combines both of the attributes of prodrug and target drug delivery methods into a single delivery system, expecting to deliver the macromolecular drugs to specific tissue targets with minimal toxicity. Once accumulating at the site of target tissue, the prodrug would be converted to its active form at the target site, for example the tumor tissue, while keeping inactive during targeting. One of the famous applications termed “ADEPT” (Antibody Directed Enzyme Prodrug Therapy) based on a specific enzymatic conversion of the prodrug into active form at the target tissue was designed abiding by the similar principle with us [1]. However, the ADEPT system is designed toward small and hydrophobic drugs (e.g. doxorubicin), which is not suitable for macromolecular agents like protein etc. Protein drugs, for example enzymes, possess multiple functional groups making it difficult to create a single prodrug from an enzyme while keeping its activity; hence, developing an effective delivery system for macromolecular agents is both an imperative and beckoning endeavor [7]. We developed another approach termed “ATTEMPTS”(Antibody Targeted Triggered Electrically Modified Prodrug Type Strategy) [1,2,7,

38–40], which directly introduces the prodrug feature onto the protein drugs, like t-PA and toxin, while after the accumulation at the targeting site, the drug is released from the blockage by a second triggering agent [41]. This system is based on the reversible masking/demasking between the cationic cell penetrating peptide (CPP) and the anionic heparin, where the clinically approved cationic protamine is used as the trigger agent [42–44]. As known, CPPs tend to bind to the anionic constituents of the cell surface, e.g. phospholipids and glycosaminoglycans, mediating delivery for the macromolecular drugs without any selection. A simple yet effective way to control the “Trojan horse” character of the CPPs is reversibly masking the cationic CPPs with anionic materials e.g. heparin and hyaluronic acid [36,37,45]. In our designed ATTEMPTS system, binding the anionic heparin to CPP would offer critical advantages: (1) it would inhibit the trans-membrane activity of CPP during the targeting process, thereby prohibiting the complex from entering normal cells; (2) without positive charge the CPP modified drug can keep away from the degradation by plasma trypsin-like proteases in circulation; and (3) it would offer a better targeting function of the antibody as the CPP is completely masked. The in vitro and in vivo feasibility of this strategy was proved in our studies. In this article, we thoroughly summarize the development of this ATTEMPTS system. 2. The design and development of the ATTEMPTS 2.1. The concept of ATTEMPTS The CPPs modified “ATTEMPTS” system as illustrated in Fig. 1, this system is composed of two components: (i) the anionic targeting part: an antibody-heparin conjugate, (ii) the anionic effector component: the CPP modified drug, and the two components formed a plasma-stable tight complex between the targeting component and the effecter component by a charge–charge interaction. Once the

Fig. 1. Illustration of CPP-modified ATTEMPTS system [36].



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complex was formed the positive charge of the CPPs is neutralized by the heparin, and this would improve the target efficiency of the antibody and plasma stability of CPP modified protein drugs against endogenous proteases. In this strategy, the system works in two steps: Firstly, following i.v. administration, the antibody-guided complexes would be accumulated at the tumor site while sparing interaction with normal tissue through antibody targeting. Secondly, a clinical heparin antidote protamine sulfate is systemically injected as a triggering agent to dissociate the CPP-Drug part from its Target-Hep counterpart, after the complex reaching the target site. As the protamine held higher heparin-binding affinity than CPP, the CPP-Drug part will be relieved from the complex and the CPP-Drug conjugate would enter into the tumor cells attributing to the trans-membrane activity of the CPP [2,7,37,46–48]. 2.2. The evolution of the “ATTEMPTS” strategy The “ATTEMPTS” system was first utilized for the target delivery of the tissue specific plasminogen activators (tPA) without potential bleeding risk [1,2,7,8,39,46,49–53]. The feasibility of this strategy was successfully demonstrated in both in vitro and in vivo studies and this first confirmed the administration of an enzyme drug in an inactive prodrug to the target site with the help of the antibody. However, it is not easy to select an appropriate cationic CPP for the strategy, as known that the antithrombin III in circulation also possesses high affinity to heparin, therefore the candidate CPP must bind to the heparin stronger than that of the antithrombin III, but with lower affinity than protamine. The first cationic peptide used in ATTEMPTS system is R7, and in further studies the most widely used CPP–TAT is also admitted. Another peptide applied is low molecular weight protamine (LMWP), which is derived from natural protamine by enzymatic digestion in our lab [48,54–58]. LMWP is proven possessing a similar cell penetrating ability as TAT and widely used for the intracellular drug delivery in various ways [33,54,59–61]. More interestingly, comparing to other CPPs like TAT which is derived from viral sources or rely solely on chemical synthesis, LMWP is obtained from a clinical drug and can be easily manufactured scaled-up to more than 10 grams per week in mass quantities [48]. LMWP could play a dual role in ATTEMPTS system: first, as a cationic species binding to the heparin antibody component; and after dissociation, the LMWP could display cellpenetrating ability to overcome various biobarriers via “the Trojan horse approach” [2]. Specifically, when it comes to cancer chemotherapy, antibodies (Ab) have been well recognized as an attractive targeting component, as the antibodies can selectively bind to the specific over-expressed antigens on tumor cell surfaces [22,62,63]. The first antibody used in the ATTEMPTS system is IgG, however the target ability of IgG is not ideal. Another typical example of the over-expressed cancer antigen is the carcinoembryonic antigen (CEA), a GPI-linked highly glycosylated cell surface protein (MW: ~ 200 kDa) [22,64,65], and the CEA is reported over-expressed in various human cancers including lung cancer, breast cancer, ovarian cancer, colon cancer and pancreatic cancers et al. [66,67], but minimally expressed in normal tissues [67]. CEA antigen is now recognized as an attractive target for the target drug delivery,

Fig. 2. Schematic illustrates the conjugation between heparin and antifibrin IgG [40,50].

owing to the distinctive expression profile [68–70]. A novel strategy for effective yet safe colorectal cancer therapy was reported recently by linking a CPP-modified gelonin toxin to a heparin-conjugated anti-CEA mAb (i.e. T84.66) via reversible electrostatic interaction. In vitro characterization displayed a high retention of the anti-cancer activity of TAT-gelonin as well as the CEA binding affinity of T84.66-Hep [22]. Finally, the evolution of the “ATTEMPTS” system was summarized in Table 1. 3. The Formation of the ATTEMPTS The ATTEMPTS system is formed via the charge to charge interaction between the anionic heparin on the targeting components and cationic CPP on the modified drug component. In order to further demonstrate the formation of the ATTEMPTS system, the preparation of the targeting-heparin component and the CPP modified drug components was summarized in the following parts, which is the key technology of the ATTEMPTS system.

Table 1 The evolution of the “ATTEMPTS” system. Complex

Azure-A-modified trypsin R7-t-PA/IgG-Hep TAT-ASNase/Hep TAT-gelonin/T84.66-Hep

CPP-Drug component

Target component (Ab-Hep)

Model drug

Cationic part

Antibody

Anionic part

trypsin t-PA ASNase gelonin

Azure A R7 TAT TAT

– IgG – T84.66

– Heparin Heparin Heparin

Trigger agent

Application

Reference

Protamine Protamine Protamine Protamine

Evaluate the in vitro feasibility of the ATTEMPTS system Treatment for thrombosis-induced cardiovascular diseases Diffused tumor treatment CEA over-expressed colorectal cancer therapy

[1,7,52] [1,8,38,46,53] [41,71] [22,72]


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3.1. The preparation of target-heparin component 3.1.1. Preparation and purification of the IgG-heparin conjugate (IgG-Hep) Nonspecific mouse IgG-heparin conjugate (IgG-Hep) was prepared as illustrated in Fig. 2. To prepare the antibody-heparin conjugate, antifebrin IgG was first oxidized with NaIO4 sodium acetate buffer, and the unreacted reagents were removed. Secondly, the heparin was partially degraded by nitrous acid depolymerization. Finally, the adipic acid hydrazide-activated IgG was mixed together with the degraded heparin followed by a Sephadex G-100 column and the protamineSepharose column purification.

3.1.2. Chemical conjugation and purification of T84.66-heparin (T84.66-Hep) T84.66, a murine anti-CEA monoclonal antibody (mAb) produced following the procedures described by Urva et al. [64], was conjugated with heparin via a thioether bond utilizing a heterobifunctional polyethylene glycol (NH2-PEG-MAL, 3.5 kDa) as the cross-linker as depicted in Fig. 3 [22]. Firstly, T84.66 was derivatized with thiol groups by the Traut's reagent [73]. Second, the thiol reactive maleimide groups were introduced to the heparin through NH2-PEG-MAL and EDC, and the heparin-PEG-MAL was purified by an anion exchange column. The amounts of heparin-PEG-MAL in the eluent were quantified by azure A [74] and barium iodide assay [75]. Third, the prepared heparin-PEGMAL was slowly added to thiol-activated T84.66 solution at room temperature overnight. After the reaction, the unreacted T84.66-SH was removed away from the T84.66-Hep on an anion exchange column. The chemical synthesis method was illustrated in Fig. 3. In addition, the amounts of both T84.66 and heparin were quantified by measuring the optical density at 280 nm (OD280) and azure A assay [74]. However,

the unreacted heparin and heparin-PEG-MAL were further removed from T84.66-Hep by ultrafiltration. 3.2. The preparation of CPP-Drug component 3.2.1. CPP-Drug conjugate based on chemical modification 3.2.1.1. Azure-A-modified trypsin. Trypsin is first selected as a model enzyme to evaluate the in vitro feasibility of the developed “ATTEMPTS” system, as most of the clinical significant proteases in circulation are trypsin-like enzymes for example the coagulation factors [1]. The Azure-A dye, which also possesses a positively charged quaternary ammonium center and can bind to heparin, was chosen as the model cation to modify trypsin. In addition, coupling of azure-A to trypsin can lead to visual and quantified monitor easily by a spectrophotometer, as azure A is a blue colored dye that absorbs visible light at 620 nm [1,40]. Carbodiimide (EDC) was selected as the activating agent to minimize both of the intra- and inter-molecular crosslinking during the coupling process. Azure-A was then conjugated to EDAC-activated trypsin, which was further purified using a Sephadex G-25 column. The result demonstrated that the azure-A-modified trypsin retained 80% of the original trypsin activity. These results suggested that incorporation of azure-A to trypsin did not seem to alter much of trypsin activity [1]. 3.2.1.2. Peptide (Arg) Cys-modified t-PA. One of the thrombolytic agents tPA is then selected as the model enzyme drugs to test the “ATTEMPTS” approach as they are the most widely used protease drug currently. A polycationic peptide consisting of seven arginine residues poly(Arg)7 (R7) is used as the cationic peptide conjugated to t-PA. The R7 peptide yields the appropriate heparin-binding strength that is required by the “ATTEMPTS” system, which holds stronger binding affinity than

Fig. 3. Scheme of the chemical synthesis of T84.66-Hep using a heterobifunctional PEG as the cross-linker [22].



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the mt-PA showed a significantly enhanced heparin-binding affinnity than the native t-PA, but mt-PA held the similar fibrin-binding ability and similar plasminogen activation kinetics to the native t-PA in the study [41–46]. It is worth noting that the “ATTEMPTS” system offers advantages for enzyme drug delivery in the form of the antibodyenzyme conjugates.

Fig. 4. Schematic diagram of the synthesis of modified tPA using tPA and (Arg) Cys [7].

the AT III but weaker than the triggering agent protamine. The synthesized R7 peptide is then linked to t-PA by using N-succinimidyl 3-(2pyridyldithio) propionate (SPDP) as the heterobifunctional crosslinking reagent. The amino groups on t-PA are firstly activated by SPDP, and next the SPDP-activated t-PA is conjugate to the SH-group on the cystein residue of the R7 peptide to produce the R7 modified t-PA (mt-PA) with a clearable S–S bond (Fig. 4). Characterization of

3.2.1.3. Chemical conjugation of recombinant gelonin with LMWP. Gelonin (~30-kDa), a plant derived toxin, belongs to the ribosome inactivating protein (RIP) family with superior efficiency in inhibiting protein synthesis. Traditionally, the plant toxin gelonin is reported to inactive the ribosomes by the cleavage of the adenine group at a specific position (A-4324) in the conserved sarcin/ricin loop of 28S ribosomal RNA with at an exceedingly low bioavailable drug concentrations [72,76]. However, the clinical application of gelonin is still limited by its poor cell uptake ability. Chemical conjugation of rGel with LMWP is accomplished using Traut's reagent and a heterobifunctional PEG (NHS-PEG-PDP, 2 kDa) as the cross-linker. The NHS group on one side of the PEG chain is amine reactive while the PDP group at the other end is thiol reactive. The conjugation scheme is shown in Fig. 5. Briefly, thiol groups are first introduced to rGel by incubation with Traut's reagent for 1 h RT. Unreacted Traut's reagent is removed by ultrafiltration. Next, the amine group on the LMWP peptide was reacted with NHS-PEGPDP for 4 h at RT, introducing LMWP with the thiol-reactive PDP group. After that the prepared thiolated-rGel (rGel-SH) is added to the LMWP-PEG-PDP solution slowly at an excess molar ratio of LMWP-PEG-PDP, incubating overnight at 4 °C. The final LMWP modified gelonin (cLMWP-gelonin) product is further purified by using the heparin column and elution with a salt gradient. Any unreacted LMWP and LMWP-PEG-PDP which might be present in the cLMWPgelonin peak fraction was further removed by centrifugal filtration [76].

Fig. 5. Scheme of gelonin-LMWP chemical conjugation via a disulfide bond using heterobifunctional PEG as the cross-linker [76].


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3.2.2. CPP-Drug developed by biological approach Strategies are attempted to develop the CPP-modified drug, for example the chemical modified methods mentioned. However, it is still not easy to conjugate the CPP to the protein drug, especially the enzyme drugs; the bioactivity of the enzyme is still somewhat reduced during the chemical modification and purification process, not to mention the economic feasibility of commercial native enzyme or toxin. Therefore, the biological approach by using Escherichia coli (E. coli) et al., which produces a CPP and drug fusion protein in large-scale production, is really essential to our ATTEMPTS system. 3.2.2.1. Preparation of the recombinant TAT–gelonin fusion toxin. A recombinant chimeric TAT–gelonin fusion toxin (TAT-Gel) using the genetic engineering method was successfully prepared in our lab. The in vitro functionality of the TAT-Gel was evaluated by utilizing the rabbit cellular assays and reticulocyte lysate. Results demonstrated that the TAT-Gel reserved the similar inhibitory profiles in luciferase translation as the recombinant gelonin, suggesting that incorporation of the TAT sequence had no influence on the inherent activity of gelonin in recombinant TAT-Gel fusion protein [72]. Furthermore, in order to assess the plausibility of regulating the cell uptake for the recombinant TAT-Gel, both in vitro and in vivo studies were carried out by binding TAT-Gel to anionic heparin via electrostatic interaction, and its protamine-induced release was also studied same to the ATTEMPTS system mentioned above (Fig. 6A). For in vitro studies, FITC-labeled TAT-Gel was added in rat plasma at 37 °C within or without the addition of the trigger agent protamine and the drug release

efficiency was monitored by confocal microscopic images [22,72]. As shown in Fig. 6, the majority (80%) of the TAT-Gel could bind stably onto the surface of the heparin, however, after the addition of protamine, 75% of TAT-Gel was instantly released in 30 min (Fig. 6B) [72] while, the in vivo feasibility was evaluated by using an LS174T s.c. xenograft tumor mouse model; once the average tumor size reached 100 mm3 about 16 days after tumor implantation at day 0, mice were treated twice with PBS, TAT-Gel, TAT-Gel/Hep, “TAT-Gel/Hep + Pro”, or protamine by intra-tumor injection at days 16 and 22 [72]. Significant inhibition was observed in the TAT-Gel treated group comparing to the PBS treated one, but there was no statistically significant difference between the TAT-Gel/Hep treated mice and the control group (see Fig. 6C and D). Moreover, mice treated with TAT-Gel/Hep + Pro also displayed substantial tumor suppression just several days after tumor implantation, indicating the feasibility of protamine triggered reversal of heparin masking. Overall, both of the in vitro and in vivo results clearly demonstrated the feasibility of protamine triggered delivery of the TAT-Gel. The hypothesis that the cell transduction of TAT-Gel could be effectively inhibited by heparin masking while protamine is able to reverse this heparin-induced block was confirmed. 3.2.2.2. Preparation of the recombinant LMWP-gelonin fusion protein. Another recombinant CPP-toxin fusion protein produced in our lab was LMWP-gelonin (rG-L) by using E. coli. The rG-L was expressed in the form of thioredoxin with 6×His tag at N-terminal (rTRX-G-L), and after the Ni-NTA resin purification procedures, the rTRX-G-L fusion

Fig. 6. Heparin/protamine modulation of TAT-Gel delivery. A: Schematic illustration of regulating TAT-Gel cell transduction via reversible masking and demasking of TAT on the TAT-Gel by anionic heparin and cationic protamine. B: Confocal microscopic images of LS174T cells treated with TAT-Gel, TAT-Gel/Hep or “TAT-Gel/Hep + Pro.” C, D: In vivo evaluation of heparin/ protamine regulation on TAT-Gel cell transduction using LS174T s.c. xenograft tumor mouse model. C: Tumor growth profiles. D: Average tumor sizes at day 38 when the average tumor volume of PBS-treated mice reached 2000 mm3 [72].



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Fig. 7. Anti-tumor effects of recombinant gelonin (rGel), gelonin-LMWP chemical conjugate (cG-L) and recombinant gelonin-LMWP chimera (rG-L) against (A) CT26, (B) LS174T, (C) 9 L and (D) PC-3 cell lines. Cells were plated onto 96well plates (104 cells/well) and cytotoxicity was measured using the XTT assay (N = 3). Both cG-L and rG-L displayed significantly higher cytotoxicity against all of the tested cancer cell lines than that of rGel, confirming the event of LMWP-mediated uptake in tumor cells [76].

Fig. 8. Representative fluorescence images of LS174T s.c. xenograft tumor bearing nude mice after i.v. administration of (A) TAT-gelonin-B4, (B) TAT-gelonin-B4/nIgG-Hep or (C) TATgelonin-B4/T84.66-Hep-C5. Tumors in the mice images are indicated by red circles and arrows. Specifically, after administration of TAT-gelonin-B4/T84.66-Hep-C5 complex, the in vivo behaviors of TAT-gelonin-B4 (left image of C) and T84.66-Hep-C5 (right image of C) were simultaneously monitored from the same mice along the time (TAT-gelonin-B4: Dylight 775-B4 labeled chimeric TAT-gelonin fusion protein, nIgG-Hep: nonspecific IgG-heparin conjugate, T84.66-Hep-C5: Dylight 679-C5 labeled T84.66-heparin conjugate) [22]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


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Fig. 9. In vivo evaluation of the therapeutic efficacy of T84.66-Hep-based tumor targeting of TAT-gelonin using LS174T s.c. xenograft tumor mouse model. (A) Tumor volume profiles; (B) Body weight changes of the mice [22].

protein was then incubated with TEV protease in order to remove the thioredoxin-6×His tag to form rG-L according to the vendor's protocol [76]. Both of the in vitro and in vivo inhibition of protein translation by rG-L was assessed. Comparing with the chemical modified LMWPgelonin (cG-L), rG-L exhibited no difference in in vitro cytotoxicity studies. However, a significantly improved cytotoxicity for rG-L was found when compared to the recombinant gelonin group (see Fig. 7), with the IC50 values being 120-fold lower than that of recombinant gelonin group (r-Gel) [76]. Moreover, significant inhibition of tumor growth was observed with rG-L compared to the recombinant gelonin group at 10-fold higher doses in vivo. This finding indicated the extraordinary potency of the rG-L, and CPP mediated the cell uptake of the fusion protein [60]. Overall, this study demonstrated the great potential of utilizing recombinant CPP-toxin fusion protein, considering its homogeneity, batch-to-batch manufacturing consistency, and the possibility for mass production to satisfy the need of large quantities. 4. Application of the “ATTEMPTS” system in solid tumor treatment: CPP mediated toxin delivery for colorectal cancer therapy Colorectal cancer is one of the most commonly diagnosed cancers, and the second leading cause of cancer related deaths for both men and women in the USA [77]. Moreover, it is difficult to treat those metastasized cancers with currently approved small anticancer drugs due to the lack of targeting and low therapeutic efficacy of the drugs, as well as the toxic effects caused by the non-selective drug action on normal tissues [78,79]. As mentioned before, gelonin is a member of N-glycosidase family, and it inhibits protein synthesis via the cleavage

of a single adenine residue (A4324) in the 28S ribosomal RNA [80]. The potency of gelonin to inhibit protein translation is so high that even a single gelonin molecule, assuming to be able to access the target ribosome, can kill one tumor cell [81]. Based on the description of the ATTEMPTS system, a combined antibody targeting and CPP-mediated intracellular toxin delivery for colorectal cancer therapy was developed, which linked a CPP-modified gelonin toxin to the heparin-conjugated anti-CEA mAb (T84.66) via reversible electrostatic interaction like other model drugs in the ATTEMPTS system [22]. Briefly, a chimeric TAT-gelonin (TAT-Gel) fusion protein as mentioned in Section 3.2.2.1 was genetically engineered, and the TAT-Gel exhibited remarkably enhanced anti-cancer efficiency against human colorectal cancer cells, with the IC50 values orders of magnitude lower than that of the unmodified gelonin [22]. A chemically synthesized conjugate of heparin and a murine anti-CEA antibody T84.66 as mentioned in Section 3.1.2 (also termed T84.66-Hep) was found able to bind strong enough specifically to CEA over-expressed on LS174T colorectal cancer cells. The binding affinity and specificity of T84.66-Hep to CEA were examined by two methods: a cell binding assay with high CEA expression LS174T cell and low expression HCT116 cell; and the ELISA measurement. Both of the cell binding assay and ELISA results demonstrated that, T84.66-Hep maintained the similar specificity and affinity toward CEA as the T84.66 without modification, thus being capable for the targeting delivery to the CEA over-expressed cancer cells. In vivo imaging study was conducted by using LS174T s.c. xenograft tumor mouse model, to access the feasibility of T84.66-mediated tumor targeting of the TAT-gelonin/T84.66-Hep complex (Fig. 8). As can be seen, due to the EPR effect via prolonged residence in circulation the TAT-gel complex with the IgG-heparin conjugate could still yield an enhanced tumor accumulation (Fig. 8B), but the most significant enhancement in tumor accumulation of TAT-gel was observed on mice treated with TAT-gelonin/T84.66-Hep (Fig. 8C) [22]. Compared to the mice administration of TAT-gelonin alone, a selective and significantly augmented (58-fold) delivery of TAT-gelonin to the tumor target was observed in the groups treated with the TAT-gelonin/T84.66-Hep. Preliminary in vivo studies suggested that the body weight seen in the TAT-gelonin-treated mice and TAT-gelonin/T84.66-Hep-treated mice experienced a statistically insignificant loss of body weight after the 3rd drug treatment, comparing to the PBS-treated control mice (Fig. 9) [22]. Overall, via specific binding to CEA expressed on the tumor cells, T84.66-Hep was able to selectively deliver TAT-gelonin to the CEA over-expressed tumor target. At the same time, significant therapeutic efficacy on the treatment of CEA-expressed colorectal tumor was achieved by the combination of TAT-mediated intracellular delivery of gelonin and T84.66-Hep induced tumor targeting. More importantly, the toxic side effects resulting from the non-selective action of TATgelonin on normal tissues were significantly reduced [22]. In short, this study demonstrated a novel and efficient strategy to deliver highly potent toxins such as gelonin selectively to tumor for effective colorectal cancer therapy. 5. Application of the “ATTEMPTS” system in diffused cancer therapy: CPP mediated asparaginase delivery for the treatment of acute lymphoblastic leukemia Presented herein is a modified version of the established “ATTEMPTS” approach applied in the delivery of asparaginase (ASNase) to the diffused tumor like acute lymphoblastic leukemia, which integrates the CPPs (TAT) into a heparin/protamine-regulated drug delivery system [41]. ASNase is an enzyme drug approved by the FDA for the induction of remission in patients with acute lymphoblastic leukemia, and ASNase is now widely used in the clinical treatment [71,82–86]. Researches show that the CPP modified ASNase had the ability to enter into MOLT-4 cells and elicit the cytotoxic effects [71].



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Fig. 10. A, B: Flow cytometry analysis of FITC-TAT-ASNase conjugate. (A) ASNase and TAT ASNase were FITC-labeled and incubated with MOLT-4 cells. (B) FITC-TAT-ASNase was incubated with heparin or heparin and protamine in MOLT-4 cells. (C) Survival curve for DBA/2 mouse bearing L5178Y mouse lymphoma cells. Each pool of 700,000 L5178Y cells was incubated with 6 IU of either TAT-ASNase (open triangle) or RPMI-1640 solution (positive control; diamond) for 2 h, followed by washing 3 times with 50% FBS in RPMI. The DBA/2 mice of 6 weeks old were then injected intraperitoneally with one of these three treated tumor cell samples (700,000 cells per sample). Mouse survival times were recorded [41].

To examine whether the heparin could inhibit TAT-mediated intracellular delivery of ASNase and the addition of the protamine could trigger the inhibition, the TAT-ASNase conjugate was treated with heparin to form a complex, and this complex was incubated with the MOLT-4 cells with or without the appearance of the protamine (Fig. 10A and B), it can be seen clearly that the CPP on the TAT-ASNase could be masked by heparin and triggered by the addition of protamine. Further in vivo findings using ASNase on DBA/2 mouse bearing L5178Y mouse lymphoma cells were able to validate the efficacy of this ATTEMPTS system (Fig. 10C). Anyway, the chemically constructed TAT-ASNase could also be regulated on and off by forming the complex with and the addition of protamine as a trigger agent [2,41]. The preliminary animal studies were done to assess the in vivo activity of the ATTEMPTS system to ASNase therapy. The drug delivery system herein is less different from the traditional ATTEMPTS system. The TAT-ASNase formed the complex with the heparin without an antibody targeting component in hand during the whole course. We purposely adopted the implanting ASNase-encapsulated tumor cells into the animals to biomimic the targeting events of the targeting component [71]. The findings showed that animals inoculated with L5178Y cells treated with TAT-ASNase reserved an extended survival rate 13% higher than that of the L5178Y cells untreated with the ASNase. Furthermore, the TAT-ASNase treated mice also exhibited a significantly improved hematological and liver histological status than the control groups (see Fig. 11) [71]. Meaningfully, this modified ATTEMPTS system therefore presents a new avenue of treatment of various types of cancers and other diseases with macromolecular drugs. In vitro characterization and a preliminary proof-of-concept animal investigation that demonstrates the feasibility of this CPP-mediated ASNase therapeutic system are subsequently described.

6. Discussion and the future development for this system Most of the failure in chemotherapy is attributed to the lack of targeting specificity for the drugs. In this way, any drug intended for combating the tumor would distribute profoundly to both of the normal organs and tissues, resulting in limited therapeutic effects toward the tumor but severe drug-induced toxic side effects to the normal tissue. To prevail over this obstacle of drug-induced systemic toxicity, a strategy was designed by incorporating a prodrug feature into a targeted DDS, termed as ATTEMPTS system, so that the drug will remain inactive in the circulation during tumor targeting thus aborting its effect on normal tissues. After accumulating at the tumor site, the drug is then converted to its active form selectively at the tumor target, thereby causing destruction only to tumor cells. In the previous system, we utilized a heparin-induced inhibition on the cell-penetrating activity of CPP to create the prodrug feature, and subsequently the protamine-induced reversal of heparin inhibition to resume cell transduction of the protein drug via the CPP function. The approach is the first known system to overcome this selectivity issue, enabling CPP-mediated cellular drug delivery to be practically applicable clinically. Other than the antibody targeted “ATTEMPTS” system, another drug delivery system based on the same masking and demasking strategy is also developed in our lab. The only difference between these two systems is the targeting component, and the targeting part herein is magnetic nanoparticles instead of the antibodies in the “ATTEMPTS” system. Similar to the “ATTEMPTS” system, the magnetic iron oxide nanoparticle (also termed as MION) is modied with heparin, termed as MION-Hep, and the negative MION-Hep interacts with the positive CPP-Drug to form a complex. Once the complex accumulates at the tumor tissue, especialy for the brain tumor, the trigger agent protamine is administrated, and the drug is


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Fig. 11. Liver metastases of L5178Y. A: Negative control (i.e., mice were injected with RPMI-1640 solution only). B: Positive control (i.e., mice were injected with L5178Y cells preincubated with PBS buffer). C: ASNase-treated group (i.e., mice were injected with L5178Y cells preincubated with ASNase). D: TAT-ASNase-treated group (i.e., mice were injected with L5178Y cells pre-incubated with TAT-ASNase). Liver cells were fixed in 10% formalin and stained with hematoxylineosin [71].

released from the MION to enter into the tumor cell. Excitingly, this drug delivery strategy has gained preliminary success in the animal model [28,87–90]. However, the previous systems rely on heparin-induced inhibition on the CPP to yield prodrug protection and protamine-induced reversal of heparin inhibition to reactivate the prodrug in alleviating drug's cytotoxicity on normal tissues. Success of these systems is thus hinged on the timing of protamine dosing relative to the dosing of the drug complex. If the dosing time for protamine is inappropriate, then a portion of the activated drug would linger in the circulation, triggering untoward systemic toxicity. Hence, this prodrug strategy would be, at best, to alleviate but not eliminate drug-induced toxicity. Thus, the most ideal strategy would be to create a direct tumor-selective prodrug conversion or a physical method triggered conversion so that the drug remains absolutely inactive in the circulation during targeting, but is then selectively activated only on the tumor surface. For example, magnetic field [91], electric field [92], temperature [93], osmotic pressure, ultrasound [94], light [95], ionic strength, pH [96], glucose and so on [34,97–102]. All of these studies offered a promising direction for the triggered delivery of macromolecular agents. Thus, more future work should be done to attempt the physical stimulation to further modify our “ATTEMPTS” system. In a word, during the past 15 years, the macromolecular drug delivery terms as “ATTEMPS” went through from “infancy” to its “adult age”, and we have obtained certain success in the cancer therapy at animal models, like the colorectal cancer and acute lymphoblastic leukemia mentioned in this article. Not to mention about the limitations for this strategy, the “ATTEMPTS” presented herein is still an impactful method for the target delivery of the macromolecular drug like enzyme and

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