A Novel Immunoliposome Technology for Enhancing the Activity of

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A Novel Immunoliposome Technology for Enhancing the Activity of Agonistic Antibody against Tumor Necrosis Factor Receptor Superfamily Takako Niwa, Yuji Kasuya, Yukie Suzuki, Kimihisa Ichikawa, Hiroko Yoshida, Akiko Kurimoto, Kento Tanaka, and Koji Morita Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b01167 • Publication Date (Web): 12 Apr 2018 Downloaded from http://pubs.acs.org on April 12, 2018

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Molecular Pharmaceutics

A Novel Immunoliposome Technology for Enhancing the Activity of Agonistic Antibody against Tumor Necrosis Factor Receptor Superfamily Takako Niwa, Yuji Kasuya, Yukie Suzuki, Kimihisa Ichikawa, Hiroko Yoshida, Akiko Kurimoto, Kento Tanaka and Koji Morita*

Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan *Phone: +81-3-3492-3131. Fax: +81-3-5740-3643. E-mail: [email protected]

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Abstract Graphic

Abstract We have developed a technology for efficiently enhancing the anti-cancer apoptosis-inducing activity of agonistic antibodies against the tumor necrosis factor receptor (TNFR) superfamily by the formation of immunoliposomes. To induce apoptosis in cancer cells, agonistic antibodies to the TNFR superfamily normally need cross-linking by internal immune effector cells via the Fc region after binding to receptors on the cell membrane. To develop apoptosis-inducing antibodies that do not require the support of cross-linking by immune cells, we prepared immunoliposomes conjugated with TRA-8, an agonistic antibody against death receptor 5 (DR5), with various densities of antibody on the liposome surface, and evaluated their activities. The TRA-8 immunoliposomes exhibited apoptosis-inducing activity against various DR5-positive human carcinoma cells at a significantly lower concentration without cross-linking than the original TRA-8 and its natural ligand (TRAIL). The activity of the immunoliposomes was correlated with the density of antibodies on the surface. As the antibody component, not only full-length antibody but also Fab′ fragment could be used and the TRA-8 Fab′ immunoliposomes also showed exceedingly high activity compared with the parental antibody, namely, TRA-8. Moreover, cytotoxicity of TRA-8 full-length or Fab′ immunoliposome against normal cells such as human primary hepatocytes was lower than that for TRAIL. Enhanced activity was also observed for immunoliposomes conjugated with other apoptosis-inducing antibodies against other receptors of the TNFR superfamily, such as death

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receptor 4 (DR4) and Fas. Thus, immunoliposomes are promising as a new modality that could exhibit significant activity at a low dose, for cost-effective application of an antibody fragment and with stable efficacy independent of the intratumoral environment of patients as a TNF superfamily agonistic therapy.

Keywords immunoliposome, multivalent, clustering effect, TNFR superfamily, antibody fragment, apoptosis

Abbreviations DR4: death receptor 4, DR5: death receptor 5, TRAIL: TNF-related apoptosis-inducing ligand, TNFR: tumor necrosis factor receptor, DISC: death-inducing signaling complex, FADD: Fas-associated protein with death domain, NK: natural killer

1. Introduction Immunoliposomes are functional liposomes that have target-cell-binding specificity based on antibody affinity. In most studies, antibody conjugation to liposomes has been applied to deliver small-molecule drugs efficiently to the target tissue in an active-targeting manner. However, cytotoxic activity of immunoliposomes without encapsulating any cytotoxic drugs was recently reported using antibodies that inhibit cell membrane receptors1-3. For example, trastuzumab- or rituximab-conjugated immunoliposomes without any cytotoxic drugs demonstrated higher cytotoxic activity than the respective intact antibodies. This enhanced activity was explained by the multivalent effect realized by the multiple antibodies immobilized on one liposome. The tumor necrosis factor receptor (TNFR) superfamily is an attractive target for cancer therapy. This is

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particularly true for death receptor 4 (DR4) and death receptor 5 (DR5), which are members of this superfamily that are not expressed in normal tissues but are expressed specifically in tumors. By binding to endogenous TNF-related apoptosis-inducing ligand (TRAIL), DR4 and DR5 form homotrimer or heterotrimer, which induces the recruitment of a death-inducing signaling complex (DISC) including Fas-associated protein with death domain (FADD) and procaspase 8 on the cytoplasmic domains. This signal transduction subsequently induces a pro-apoptotic signal through the activation of caspase 8 in cancer cells4. DR4 and DR5 are promising targets for cancer therapy and various agonistic antibodies against death receptors are under development in the clinical stage, but none has been released on the market5, 6. In this context, there is a strong need for technologies that functionalize and/or reinforce cancer therapy using anti-TNFR antibody. In this study, we examined the potential of immunoliposomes as a technology for enhancing the activity of agonistic antibodies against members of the TNFR superfamily. As a model antibody for immunoliposomes, we used TRA-8 which is a humanized anti-human DR5 antibody, can inhibit the proliferation of a variety of TRAIL-sensitive tumor cells and induce apoptosis via a caspase-dependent pathway both in vitro and in vivo7-10, to prepare TRA-8-conjugated immunoliposomes (hereinafter referred to as TRA-8 immunoliposomes). The supposed mechanism for enhancing the apoptosis-inducing activity of TRA-8 immunoliposomes is shown in Fig. 1. TRAIL, an endogenously secreted trimeric ligand for DR4 and DR5, promotes the trimerization of these receptors, which results in the intracellular recruitment of the DISC complex, leading to caspase 8 activation and cell death. In contrast, TRA-8 requires cross-linking to induce receptor trimerization and apoptosis by effector cells such as natural killer (NK) cells and macrophages, which recognize the Fc domain of antibodies and promote clustering. If multiple copies of TRA-8 are flexibly immobilized on a liposome, the resulting immunoliposome may demonstrate apoptosis-inducing function by itself without any effector cells. There are several expected advantages of

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TRA-8 immunoliposomes. First, the multimerization of antibodies would enhance their binding affinity and biological activity11. Second, the efficacy would be independent of intratumoral effector cells in patients. This approach is expected to be beneficial for patients with a deteriorated immune system as a result of treatment with chemotherapeutic drugs. Third, because the Fc domain that is indispensable for cross-linking is not necessarily required, antibody fragments can instead be applied, which could confer benefits with respect to cost-effective manufacturability and lower immunogenicity12. Given these potential benefits, TRA-8 immunoliposomes were investigated in this study.

2. Experimental Section 2.1. Materials 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine

(DPPC),

1,2-dioleoyl-sn-glycero-3-phosphocholine

(DOPC), and poly(ethylene glycol) succinyl distearoylphosphatidylethanolamine attached with a maleimide group at the end of polyethylene glycol (DSPE-PEG-Mal, the average molecular weight of PEG: 2000 or 3400) were purchased from NOF Corporation (Tokyo, Japan). Egg yolk lecithin was purchased from kewpie Corporation (Tokyo, Japan). Cholesterol was purchased from Sigma-Aldrich (St. Louis, MO, USA). Immobilized pepsin and Traut’s reagent (2-iminothiolane) were purchased from Thermo Fisher Scientific Inc. (Rockford, IL, USA). CBQCA protein quantitation kit was purchased from Life Technologies (Carlsbad, CA, USA). Choline quantitation kit, dithiothreitol (DTT), cysteine, and

β-mercaptoethanol were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). CellTiter-Glo

Luminescent

Cell

Viability

Assay

kit

and

Z-VAD-FMK

(carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone) were purchased from Promega Corporation (Madison, WI, USA). An agonistic anti-human DR4 monoclonal antibody, 2E12, was kindly provided by Tong Zhou and Robert P. Kimberly from the University of Alabama at Birmingham13. An

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agonistic anti-human DR5 monoclonal antibody, TRA-87, and a mouse anti-human/mouse Fas monoclonal antibody, HFE7A14, were prepared as described previously. Goat affinity-purified antibody to human IgG Fc was purchased from MP Biomedicals, LLC (Santa Ana, CA, USA). TRAIL was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). PE-conjugated F(ab′)2 fragment goat anti-human IgG was purchased from Jackson ImmunoResearch (West Grove, PA, USA). 7-AAD was purchased from BD Pharmingen (Franklin Lakes, NJ, USA).

2.2. Cells and cell culture Jurkat, COLO205, WiDr, A2058, BxPC-3, A375, MDA-MB-231, DLD-1, Lovo, A549, and Alexander cells were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA). 293F cells were purchased from Thermo Fisher Scientific Inc. Human primary hepatocytes were purchased from Life Technologies and a medium set for hepatocytes was purchased from Biopredic International (Saint-Grégoire, France). All other cells were cultured in accordance with the recommended protocol of the supplier.

2.3. Preparation of TRA-8 Fab′ fragment For preparation of the TRA-8 Fab′ fragment, 10 mg/mL TRA-8 in 20 mM acetate buffer (pH 4.5) was treated with immobilized pepsin (1/8 volume relative to the TRA-8 solution) at 37°C for 8.5 h. F(ab′)2 fragment was purified by removing other peptide fragments and full-length TRA-8 from the reaction mixture by ion-exchange chromatography [column: Resource S (GE Healthcare Inc. (Chicago, IL, USA)), solution A: 50 mM citrate buffer, pH 4.0, solution B: 50 mM citrate buffer, 1 M NaCl, pH 4.0, linear gradient: 15%–40%] to obtain the F(ab′)2 fragment. The citrate buffer was replaced with HEPES buffer (20 mM HEPES, 150 mM NaCl, pH 7.4) by ultrafiltration procedures using the LabScale TFF system (Merck

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Millipore Inc. (Billerica, MA, USA)) equipped with a polyethersulfone membrane [Pellicon XL, Biomax 50 (molecular cut-off: 50,000); Merck Millipore Inc.]. To obtain the TRA-8 Fab′ fragment, 2.5 mg/mL F(ab′)2 fragment in HEPES buffer was reduced in the presence of 40 mM dithiothreitol at room temperature for 30 min or 10 mM cysteine at room temperature for 90 min. The reducing agent was removed by gel filtration purification (PD-10 desalting column; GE Healthcare Inc.) and the TRA-8 Fab′ fragment fraction was collected in HEPES buffer.

2.4. Preparation of sulfhydryl-modified full-length antibody To introduce sulfhydryl groups on a lysine residue of the antibody, 4 mM Traut’s reagent in DMSO was added to 6 mg/mL full-length antibody in 20 mM HEPES, 150 mM NaCl, and 2 mM EDTA (pH 8.0) at a molar ratio of antibody:Traut’s reagent of 1:4–1:16, followed by reaction at room temperature for 90 min. Subsequently, Traut’s reagent was removed by gel filtration chromatography (NAP-5 desalting column; GE Healthcare Inc.) with HEPES buffer to obtain sulfhydryl-modified full-length antibody.

2.5. Preparation of immunoliposome Liposomes composed of phosphatidylcholine, cholesterol (Chol), and DSPE-PEG-Mal (molar ratio 100:66:1) were prepared by a thin-film15 or ethanol-injection16, 17 method. Subsequently, this liposome dispersion in HEPES buffer was repeatedly extruded through a polycarbonate membrane with a pore size of 50–100 nm using an extruder (Avanti Polar Lipids, Inc. (Alabaster, AL, USA)) to produce liposomes with a controlled particle size16, 18. For the preparation of Fab′ immunoliposomes, Fab′ was conjugated via a sulfhydryl group of the hinge cysteine residue. For the preparation of full-length immunoliposomes, the antibody was conjugated via the primary amine of a lysine residue with introduced sulfhydryl groups. The antibody and the liposome dispersion were mixed at various ratios to induce reaction of the sulfhydryl

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group of the antibody with the terminal maleimide group of the PEG chain on the liposome at room temperature overnight. Unbound maleimide groups were inactivated by the addition of β-mercaptoethanol (10 equivalents to DSPE-PEG-Mal) followed by subsequent reaction at room temperature for 30 min. Immunoliposomes were purified with HEPES buffer by the removal of unreacted antibody by size exclusion chromatography (HiLoad Superdex 200 16/60 prep grade; GE Healthcare Inc.).

2.6. Immunoliposome characterization The mean diameter of immunoliposomes was determined by dynamic light scattering (Nicomp 370; Particle Sizing Systems, Inc. (Port Richey, FL, USA)). Liposomal phosphatidylcholine concentration and the amount of antibodies conjugated to liposome were determined with a choline quantitation assay kit and CBQCA protein assay kit, respectively, in accordance with the manufacturers’ protocols. The number of antibodies on the liposomes was calculated from the mean particle diameter, lipid concentration, and antibody concentration of the immunoliposomes19. This calculation was based on the following assumptions: [I] The liposome is unilamellar. [II] The area occupied by one molecule of phosphatidylcholine on the liposome surface is 72 Å2 and that of cholesterol is 19 Å2 20. In the case of liposomes composed of phosphatidylcholine:cholesterol= : (molar ratio), (I) the concentration of liposomal particles and (II) the number of antibodies on one liposome were calculated by the following equations:

(I) Concentration of liposomal particles (particles⁄L) = (II) Number of antibodies on one liposome =

y x c×6.02×1023 × 0.72× x+y +0.19× x+y

2×4×π×(1/2×d)2

a×6.02×1023 a×2×4×π×(1/2×d)2 = Concentration of liposomal particles c× 0.72× x +0.19× y

x+y x+y

a: antibody concentration (mol⁄L), c: total lipid concentration (mol⁄L) x: molar ratio of phospholipid, y: molar ratio of cholesterol, d: particle radius (nm)

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2.7. In vitro apoptosis-inducing activities of immunoliposome Cells were seeded at 1×104 per well in a 96-well plate. Immunoliposomes and antibodies with secondary anti-human IgG Fc or its Fab′ fragment were added to the cells at various antibody concentrations. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO2 for 72 h in the presence or absence of apoptosis inhibition reagent, Z-VAD. Cell viability was estimated by measuring the ATP level using the CellTiter-Glo Luminescent Cell Viability Assay kit, in accordance with the manufacturer’s protocol.

2.8. Immunoliposome hepatotoxicity Freezing-preserved human primary hepatocytes were supplemented with thawing medium. After thawing, the hepatocytes were washed with thawing medium and suspended in seeding medium. At that stage, their viability was determined to be 95%. Hepatocytes were then seeded at 3.5×104 cells per well in a collagen-coated 96-well plate. After incubation for 4 h, the seeding medium was changed to incubation medium and hepatocytes were further incubated overnight. Immunoliposomes, TRA-8 with secondary anti-human IgG Fc (TRA-8 with cross-linker), or TRAIL was added to the hepatocytes at various protein concentrations. After 6 h of incubation, cell viability was estimated by measuring the ATP level.

2.9. Immunoliposome binding affinity 293F cells were seeded at 2.5×105 per well in 5% FCS, 0.04% NaN3, and PBS in a 96-well plate. TRA-8 immunoliposomes or TRA-8 with a cross-linker were added to the cells at various concentrations. The cells were incubated at 4°C for 1 h. After the cells had been washed three times in 5% FCS, 0.04% NaN3, and PBS, PE-conjugated F(ab′)2 fragment goat anti-human IgG was added, followed by incubation at 4°C for 30 min. Cells were again washed three times and resuspended in 5% FCS, 0.04% NaN3, and PBS. 7-AAD

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was added and then cells were analyzed by flow cytometry (Becton Dickinson) to detect cell-bound antibody on the immunoliposomes. Kd was calculated as described previously21. In brief, the inverse of the determined fluorescence intensity was plotted as a function of the inverse of drug concentration to determine Kd by the Lineweaver–Burk method22. Kd values were determined by the following equation (III). This equation was based on the assumption that the bound drug concentration is much lower than the total drug concentration. Fmax was calculated from the plot.

(III)

1 1 Kd 1 = + × F Fmax Fmax Ct F: fluorescence units, Ct: concentration of immunoliposome or antibody

2.10. Statistical analysis Analyses were performed using the software of EXSUS ver.8.1 (CAC Croit, Tokyo, Japan) based on SAS release 9.2 (SAS Institute Japan, Tokyo, Japan). The relative potency and their 95% confidence interval (CI) were analyzed by parallel line analysis. In general, the difference in the potency was considered to be significant if the 95% CI of relative potency excluded 1. The 50% growth inhibitory concentration (IC50) was analyzed by sigmoid Emax model.

3. Results 3.1. Preparation and characterization of TRA-8 immunoliposome The scheme for the preparation of TRA-8 immunoliposomes is shown in Fig. 2. Liposomes composed of a fixed lipid composition ratio (DPPC:Chol:DSPE-PEG-maleimide=100:66:1) were first prepared by a thin-film or ethanol-injection method. Then, antibody was conjugated to the maleimide group on the liposome surface. In the immobilization process, different synthetic chemistry was applied depending on the antibody format (full length or Fab′). For the preparation of Fab′ immunoliposomes, Fab′ was

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conjugated via the sulfhydryl group of the hinge cysteine residue site-specifically. For the preparation of full-length immunoliposomes, conjugation with the sulfhydryl group of the reduced cysteine disulfide in the hinge region of the antibody was first conducted, but resulted in a very low yield, probably due to steric hindrance (Fig. 3). Thus, an alternative method was conducted; that is, sulfhydryl groups were partially introduced to the primary amines of lysine residues of the antibody by Traut’s reagent, followed by conjugation to the liposome using the same chemistry as that for Fab′. To investigate the effect of immobilization density of the antibody on the liposome, a series of TRA-8 immunoliposomes with different antibody densities were prepared. Briefly, antibody and the liposome (DPPC:Chol:DSPE-PEG-maleimide=100:66:1) dispersion were mixed at various molar ratios of antibody and DSPE-PEG-Mal to prepare immunoliposomes. The molar ratio of mixing and the resultant density of antibody conjugated on the liposome are plotted in Fig. 3. The antibody density on the liposome was increased with the feed ratio of antibody to DSPE-PEG-Mal in the liposome. Fab′ fragment was immobilized approximately twice as much as the full-length antibody under the same reaction conditions. This difference in the number of immobilized antibodies was considered to be due to the difference in size of the areas occupied by Fab′ and full-length antibody on the liposome surface. The results of a series of TRA-8 full-length or Fab′ immunoliposomes with different antibody densities (0.006–0.009) are summarized in Table 1. On the surface of those immunoliposomes, 4–50 full-length antibodies or 14–72 Fab′ antibody molecules were immobilized.

3.2. Dependence of the apoptosis-inducing activity on TRA-8 immunoliposome structure First, in vitro cytotoxic activity of TRA-8 immunoliposomes (IL-A) was evaluated using DR5-positive human T-cell leukemia cells (Jurkat cells, Fig. 4). Intact TRA-8 demonstrated apoptosis-inducing activity with the cross-linking by the anti-human IgG Fc antibody (TRA-8 with cross-linker). Moreover, full-length

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TAR-8 immunoliposomes demonstrated potent cytotoxic activity even in the absence of the secondary antibody, as expected. Unexpectedly, the immunoliposomes exhibited their activity at a concentration about 100-fold lower than that of intact TRA-8 with cross-linker. TRA-8 immunoliposome was significantly superior to TRA-8 with cross-linker (The relative potency of IL-A to TRA-8 with cross-linker was 114 (95% CI: 69.1–176)). To confirm the caspase dependence of the cytotoxicity of TRA-8 immunoliposome, Z-VAD-FMK was used, which is a cell-permeant pan-caspase inhibitor that irreversibly binds to the catalytic site of caspase proteases and can inhibit the induction of apoptosis. In the presence of Z-VAD-FMK, the cytotoxic activity of the immunoliposomes was completely canceled. This suggests that the high cytotoxicity of immunoliposomes was caused through apoptosis via the caspase-dependent pathway. Next, a series of TRA-8 immunoliposomes (IL-B–D, IL-a–c) with different antibody types (full length or Fab′) and antibody densities (the number of antibodies on a liposome) were tested (Fig. 5). Generally, the apoptosis-inducing activity of immunoliposomes increased with the antibody density on the immunoliposomes. In the case of full-length antibody, although there was a statistically significant difference in the activity of IL-B–D (relative potency of IL-C and IL-D to IL-B was 1.81 (95% CI: 1.46–2.24) and 4.03 (95% CI: 3.27–4.96)), in general they all showed drastically enhanced activity. In the case of using the Fab′ fragment, which could not induce apoptosis by itself, Fab′ immunoliposomes also showed high potency to induce apoptosis (IL-a–c) as with full-length immunoliposomes. This indicates that the monovalent antibody fragment enables acceleration of the multimerization and clustering of DR5 receptor by the formation of immunoliposomes. Compared with Fab′ fragments, full-length antibody had significantly higher potency when immobilized on the liposomes in the case of a low antibody density (The relative potency of IL-B to IL-a was 5.26 (95% CI: 3.57–7.75). This indicated that full-length antibody (divalent recognition sites) on the liposomes promotes receptor

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clustering more efficiently than Fab′ fragment (monovalent recognition site). However, the maximum potency levels of full-length immunoliposomes (IL-D) and Fab′ immunoliposomes (IL-c) were similar and showed more than 100-fold higher activity than intact TRA-8. It was considered that the maximum efficacy was dependent on the maximum apoptosis signal in cancer cells and immunoliposomes of both types reached a plateau in this regard with increasing antibody density. Next, we examined the influence of the lipid composition of the liposomes on apoptosis-inducing activity from the perspective of the membrane fluidity of liposomes. To focus on the phase transition temperature (Tc) of PCs, three kinds of phospholipid, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, Tc=41°C), egg yolk lecithin (Tc=−15 to −5°C), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, Tc=−22°C), were selected23. We prepared TRA-8 Fab′ immunoliposomes using these three types of PCs at a fixed lipid composition (PCs:Chol:PEG-lipid=100:66:1). The antibody density of these immunoliposomes was adjusted to around 40 antibodies/liposome (antibody density: 0.065 mol% to total lipid) by controlling the reaction conditions in order to evaluate the influence of lipid composition directly. The activity of immunoliposomes with this antibody density doesn’t reach to its saturation against A2058 cells as shown in Table 2(b). It was thought that the fluidity of the lipid may affect on the activity because it is estimated that the antibodies on the immunoliposome are located at a sufficiently long distance beyond the size of PEG-chain-conjugated Fab′ (The detailed calculation was described in Supplementary Information 1.). Their apoptosis-inducing activity is shown in Fig. 6. Although statistical analysis means there was difference in the activity among immunoliposomes composed of DPPC, eggPC or DOPC (the relative potency of immunoliposome composed of eggPC and that of DOPC to that of DPPC was 1.53 (95% CI: 1.31–1.78) and 0.866 (95% CI: 0.796–0.943), it seems extremely similar. These results indicated that there was no distinct correlation between the membrane fluidity of liposomes and their efficacy.

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3.3. Apoptosis-inducing activity of TRA-8 immunoliposomes against various human carcinomas and toxicity against human primary hepatocytes The apoptosis-inducing activity of TRA-8 Fab′ immunoliposomes against various DR5-positive human carcinoma cells was also examined. TRA-8 Fab′ immunoliposomes showed potent efficacy against various types of human carcinoma compared with intact antibody (TRA-8 with cross-linker) (Table 2). In each human carcinoma, the apoptosis-inducing activity increased with the number of antibodies on the liposomes. Against BxPC-3, A375, and LoVo cell lines, which were not sensitive to TRA-8 with a cross-linker, TRA-8 Fab′ immunoliposomes demonstrated cytotoxic activity. TRA-8 immunoliposomes were not effective against MDA-MB-231 and A549 cell lines, even though they were DR5-positive cells. The lack of such a correlation between DR5 expression and efficacy was already reported in intact TRA-88. It was suggested that detectable DR5 expression is essential for the induction of apoptosis and other factors also regulate it. DR5 is expressed on almost all tumor cells and is an attractive target for cancer therapy. However, as DR5 is also expressed on human hepatocytes, concern about the risk of hepatic toxicity should be examined24. We examined the hepatotoxicity of immunoliposomes and TRAIL, which can strongly induce apoptosis in human carcinoma cells. As shown in Fig. 7, TRA-8 immunoliposomes caused cytotoxicity to human

primary

hepatocytes

in

an

antibody-density-dependent

manner.

However,

even

for

immunoliposomes with the highest antibody density (IL-f), the cytotoxicity against hepatocytes was approximately 35-fold lower than that for TRAIL. In contrast, the efficacy of immunoliposomes against human carcinoma was higher than that for TRAIL, as shown in Fig. 5. Furthermore, a variety of human carcinoma cell lines were more sensitive to TRA-8 immunoliposomes than hepatocytes were (Table 2b). These results suggest that TRA-8 immunoliposomes are effective for treating cancer while having acceptable side effects on hepatocytes.

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3.4. Mechanism of action of immunoliposome As the mechanism behind the enhanced apoptotic activity of immunoliposomes, two mechanisms are proposed: increased binding affinity by multivalent antibody (avidity effect) and efficient receptor clustering after binding. However, it is not easy to distinguish the respective contributions of these two effects. Chiu et al. reported that antibody-dependent cytotoxic activity was enhanced by the construction of immunoliposomes using trastuzumab (anti-HER2 antibody) and rituximab (anti-CD20 antibody). In these cases, antibodies are not agonistic and the clustering effect makes no contribution to immunoliposome activity. The extent of activity enhancement by the immunoliposome construct was only 25-fold1, 3. In contrast, in the case of TRA-8, significantly higher enhancement (more than 100-fold) was observed. Such a large difference in the extent of activity enhancement implied that the clustering effect worked effectively in TRA-8 immunoliposomes. We analyzed the correlation between the avidity and efficacy of TRA-8 immunoliposomes. The binding affinity and apoptosis-inducing activity of immunoliposomes against 293F cells were examined (Table 3). More than carcinoma cells, 293F cells express a sufficient amount of DR5 antigen to detect cell-bound immunoliposomes by flow cytometry; therefore, we used 293F cells as a model for a binding assay. TRA-8 full-length immunoliposomes efficiently induced the apoptosis-inducing activity against 293F cells and the activity of full-length immunoliposomes with even a few antibodies/liposome reached close to saturation. Therefore we examined factors contributing to the enhancement of activity using Fab′ immunoliposomes. Both the Kd value and the IC50 (50% growth inhibitory concentration) of TRA-8 Fab′ immunoliposomes were correlated with the antibody density of immunoliposomes. Although the binding affinity of Fab′ immunoliposomes was slightly inferior to that for TRA-8 with cross-linker, the apoptosis-inducing activity was similar or superior to that of TRA-8 with cross-linker (17 and 70 antibodies on the liposome vs. TRA-8

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with cross-linker). This indicates that the clustering effect also contributes to enhancing the activity of TRA-8 immunoliposomes.

3.5. Application to TNFR superfamily We applied multivalent technology using immunoliposomes to another TRAIL receptor, death receptor 4 (DR4). DR4 also forms a homotrimer and requires clustering for subsequent apoptosis signal transduction, as well as DR525. Anti-human DR4 monoclonal antibody, 2E12, is an agonistic antibody and induces apoptosis. We prepared immunoliposomes of anti-DR4 antibody, 2E12, in the same manner as for TRA-8 immunoliposomes. The apoptosis-inducing activity of 2E12 immunoliposomes is shown in Fig. 8. 2E12 immunoliposomes had higher apoptosis-inducing activity than 2E12 with cross-linker. Even low-antibody-density immunoliposomes had higher potency than the antibody itself, and the efficacy increased with antibody density, just as observed for TRA-8 immunoliposomes. Fas, which is one of the TNFR family members, also has properties similar to those of DR4 and DR526. Enhancement of activity and dependence on the antibody density were also observed in immunoliposomes with an anti-Fas monoclonal antibody, HFE7A (Supplementary Figure 1). These observations suggest that our multivalent technology using immunoliposomes is applicable not only to a particular species of antibody but also to a variety of them used in cancer therapy for targeting the TNFR superfamily.

4. Discussion The intact agonistic antibody therapy requires two kinds of interactions: (I) interaction between antibody and antigen on the target cell, and (II) interaction between Fc domain of antibody and Fcγ receptor on effector cells, such as NK cells and macrophages. Namely, the intratumoral environment of a patient such as the presence of effector cells near the target cells and the expression level of Fcγ receptor on the effector

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cells affects the therapeutic effect27. However, immunoliposomes would exhibit efficacy independent of the above-mentioned patients’ condition because they do not require cross-linking by effector cells. In addition, the design, chemistry, manufacturing, and control of immunoliposomes have been well studied. For immunoliposomes, not only full-length antibodies but also antibody fragments can be applied. The advantage of antibody fragments is that they can be produced at lower cost than full-length antibodies using highly productive cells such as yeast instead of mammalian cells28. In the case of full-length antibodies, immunoliposomes express potent activity with several antibodies, so the amount of antibody on each liposome can be minimized. Since antibodies on liposomes may cause antigenicity, it is thus possible to minimize the influence of this on pharmacokinetics29. TRA-8 immunoliposomes efficiently induced apoptosis at a low dose against various human carcinomas. However, there were some cells for which TRA-8 immunoliposomes were not effective despite the expression of DR5. For example, the A549 cell line was resistant to both TRA-8 and TRA-8 immunoliposomes. This is reasonable given the previous finding that non-small cell lung cancer (NSCLC) such as A549 was resistant to TRAIL30. Cross-activation between the death receptor-initiated apoptosis pathway and the mitochondrion-initiated apoptosis pathway through Bid and caspase-9 is often required to induce NSCLC to undergo cell death. It was also pointed out that the overexpression of TRAIL decoy receptors (DcR1 and DcR2) caused resistance. TRA-8 immunoliposomes enable the activation of mainly the death receptor-initiated apoptosis pathway and are considered not to be effective in cases in which (1) the mitochondrial pathway is significant for apoptosis induction, (2) there is a resistant factor such as mutations in molecules related to the death receptor initiating pathway, and (3) decoy receptors are overexpressed. Some reports have described the hepatotoxicity of anti-DR5 antibody7, 8, 31, 32. TRA-8 does not induce apoptosis in normal hepatocytes or, even if it does, it is very weak. In addition, the efficacy of TRA-8 for

17



tumor cells is relatively weak. In contrast, TRAIL strongly induces apoptosis in both tumor cells and normal hepatocytes. TRA-8 immunoliposomes also exhibit hepatotoxicity. However, this is weaker than the hepatotoxicity of TRAIL and TRA-8 immunoliposomes can induce the apoptosis of tumor cells more effectively than TRAIL. Furthermore, TRA-8 immunoliposomes exhibit strong activity against tumor cell strains for which TRA-8 alone is not effective and can maximize the range of different cancers against which TRA-8 targeting can be used. TRA-8 immunoliposome showed much lower toxicity against hepatocytes than TRAIL in vitro as described above. TRAIL and TRA-8 immunoliposome may differ in mode and efficiency of multimerization of receptors. TRAIL naturally forms trimer and binds tightly to the DR5 trimer. An antibody on the immunoliposome is full-length (bivalent) or Fab′ fragment (monovalent), therefore, the multimerization is not as efficient and tight as a natural ligand. It might be considered that the expression of DR5 on hepatocytes is too low and that immunoliposomes cannot induce efficient multimerization of DR5 and subsequent apoptosis. In in vivo situations, it is reported that the particulate drug such as immunoliposome tends to accumulate to liver. For more accurate consideration, it is necessary to evaluate the pharmacokinetics of immunoliposome. Immunoliposome structure such as particle size, antibody format and antibody density should be optimized from the viewpoint of pharmacokinetics and tissue distribution. Recently, research on a similar concept to immunoliposome in which agonistic activity can be enhanced by particulate nanomedicine was reported via other modalities of HPMA copolymer33, 34, lipid- and polyion complex-based micelle35, and poly(glycolate) nanoparticles36. Regarding reports related to the TNFR superfamily, anti-DR5 antibody-conjugated poly(lactate-co-glycolate) (PLGA) nanoparticles and anti-DR5 antibody- or anti-Fas antibody-conjugated lipid- and polyion complex-based micelles were reported. Fay et al. reported that conatumumab (an agonistic anti-DR5 antibody)-coated PLGA nanoparticles potently enhanced apoptosis-inducing activity36. In this case, the extent of the activity enhancement was only a few

18


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fold compared with that of intact antibody. The constructs of PLGA nanoparticles were rigid and did not have a PEG-linker. The conatumumab-coated PLGA nanoparticles thus seemed to lack the mobility of antibody on the surface of PLGA to produce apoptosis-inducing activity effectively, compared with immunoliposomes. There was no clear correlation between the phase transition temperature of PCs, which is one of the components of immunoliposomes, and immunoliposome activity. In the presence of cholesterol, lipid membrane does not show a clear phase transition19, 37-39. As a reason for no correlation between the phase transition temperature and the activity of TRA-8 immunoliposome, it is considered that cholesterol obscured the phase transition and all immunoliposomes had sufficient membrane fluidity at 37°C. Furthermore, immunoliposomes had the advantage of being able to encapsulate both hydrophobic and hydrophilic chemotherapeutic agents, in contrast to PLGA nanoparticles and micelles. In this application, the activities of the encapsulated drug and of the multiple ligand molecules function in parallel. Substantial

knowledge

has

been

accumulated

regarding

drug

encapsulation

techniques

and

drug-encapsulating liposomes40. Several reports have also been published about the combination therapy of anti-DR5 antibody and chemotherapy8, 41-56. By combining these findings, synergistic effects of antibody multimerization technology and chemotherapy are expected. In addition, in the field of protein engineering, multivalent antibody technology has been developed. Multivalent peptide57, 58 and multivalent nanobody59 that mimic the activity of the natural ligand TRAIL were reported. These constructs also demonstrated efficient apoptosis-inducing activity in vitro. Among the various constructs mentioned above, multivalent nanobody was reported to show excellent results in vitro and in vivo. The construct was a tri-, tetra-, or pentamer of anti-DR5 VHH (heavy heavy variable domain) fragment antibody linked by a glycine-serine polypeptide. The multivalent nanobody was cleared more rapidly from the blood after intravenous administration than its parental IgG antibody. Nevertheless, it also presented superior potency in vivo compared with its parental IgG antibody. This means that continuous

19



apoptosis signal stimulation is not required to suppress tumor growth in vivo. Furthermore, the multivalent nanobody maintained potency under the depletion of NK cells and tumor-associated macrophages. These results indicate that the multivalent nanobody exhibited efficacy independent of the patient’s intratumoral environment. Immunoliposomes tend to be captured in liver and have a shorter t1/2 of plasma concentration

in vivo than conventional antibodies. However, according to the results for multivalent nanobody, constant exposure of the immunoliposomes in the blood may not be necessary to suppress tumor growth, and they may have significant potency in vivo, similar to that of multivalent nanobody. Increasing the exposure of immunoliposomes to the tumor tissue would lead to an enhanced in vivo therapeutic effect. It is important to optimize the antibody format and antibody density from the viewpoint of the accumulation in the tumor tissue. In addition, because immunoliposomes can attach to more antibodies than protein-engineered constructs, more potent anti-tumor activity would also be expected.

5. Conclusion Immunoliposomes carrying anti-TNFR superfamily antibody exhibited multivalent and clustering effects followed by significantly higher apoptosis-inducing activity compared with intact antibody. It was confirmed that the number of antibody molecules immobilized on each liposome was an important factor in determining the apoptosis-inducing activity of the immunoliposomes. Furthermore, it seems reasonable to assert that the clustering effect makes the main contribution to expression of the apoptosis-inducing activity of immunoliposomes. Immunoliposomes thus appear to be a promising new therapeutic modality for targeting members of the TNFR superfamily.

Acknowledgment The authors thank Tong Zhou and Robert P. Kimberly from the University of Alabama at Birmingham

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for providing anti-DR4 antibody (2E12).

Supporting Information Supplementary Figure 1. Apoptosis-inducing activity of HFE7A immunoliposomes against human carcinoma (Jurkat). Supplementary Information 1. The validation verification of the experiment to evaluate the influence of membrane fluidity in Figure 6.

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Table 1. Characterization of TRA-8 immunoliposomes TRA-8 immunoliposomes with three different antibody densities were prepared and characterized. Liposomes composed of DPPC:cholesterol:DSPE-PEG2000-maleimide at a ratio of 100:66:1 were prepared. Antibody and the liposome dispersion were mixed at various ratios. All immunoliposomes were prepared from the same liposome dispersion. (a) Full-length immunoliposome, (b) Fab′ fragment immunoliposome (a) Immunoliposome Antibody

Conjugation site

Particle size

Antibody density

Antibody number

(nm)

(mol% to total lipid)

/ liposome 35 *

Lipid composition (mol%)

code IL-A

full length

Lys

DPPC:Chol:DSPE-PEG2000-Mal=100:66:1

50-nm extrusion *

0.1110 *

IL-B

full length

Lys


79 ± 19

0.0061

4

IL-C

full length

Lys


84 ± 20

0.0293

27

IL-D

full length

Lys


92 ± 22

0.0717

50

Particle size

Antibody density

Antibody number

(nm)


/ liposome

* "Antibody density" and "antibody number/liposome" were calculated based on the assumption that the diameter of immunoliposome was 50 nm.

(b) Immunoliposome Antibody

Conjugation site


code IL-a

Fab′

Cys


74 ± 24

0.0148

14

IL-b

Fab′

Cys


74 ± 24

0.0523

48

IL-c

Fab′

Cys


81 ± 27

0.0884

72

Table 2. Apoptosis-inducing activity of TRA-8 immunoliposomes against various human carcinomas (a) TRA-8 Fab′ immunoliposomes with different antibody densities were prepared and characterized. (b) The cytotoxicity of TRA-8 Fab′ immunoliposomes against various human carcinomas was examined. Cells were treated with immunoliposomes or TRA-8 with cross-linker at various concentrations for 72 h. Then, cell viability was estimated by measuring the ATP level and IC50 (50% growth inhibitory concentration) was calculated. (a) Immunoliposome Antibody

Particle size

Antibody density

Antibody number

(nm)


/ liposome


code IL-d

Fab′

DPPC:Chol:DSPE-PEG 3400-Mal=100:66:1

73 ± 36

0.006

7

IL-e

Fab′


87 ± 40

0.050

62

29



IL-f


Fab′

Page 30 of 37

84 ± 42

0.103

127

(b) IC50 (ng/ml) Cell line

Origin

TRA-8 immunoliposome

TRA-8 with cross-linker

IL-d

IL-e

IL-f

Jurkat

T lymphoma

17

8

1000

101

39

A375

Melanoma

>1000

>1000

68

23

MDA-MB-231

Mammary

>1000

>1000

>1000

>1000

DLD-1

Colorectal

335

300

4.5

2.3

LoVo

Colorectal

>1000

>1000

37

8.1

A549

Lung

>1000

>1000

>1000

>1000

Table 3. Apoptosis-inducing activity and binding affinity of TRA-8 immunoliposomes The correlations of apoptosis-inducing activity and binding affinity of immunoliposomes with different antibody densities were analyzed. To evaluate the apoptosis-inducing activity, 293F cells were treated with immunoliposomes, TRA-8 with cross-linker, or TRA-8 alone for 24 h. Then, cell viability was estimated by measuring the ATP level and IC50 (50% growth inhibitory concentration) was calculated. To evaluate the binding affinity, 293F cells were treated with immunoliposomes, TRA-8 with cross-linker, or TRA-8 alone at 4°C for 1 h. Then, PE-conjugated F(ab′)2 fragment goat anti-human IgG was added and incubated at 4°C for 30 min. After washing, cells were analyzed by flow cytometry and Kd was determined by the Lineweaver–Burk method.

IC50 Antibody density

Antibody number

IC50


/ liposome

(ng/mL)

Kd Kd

relative to TRA-8

relative to TRA-8 (nM)

with cross-linker

with cross-linker

0.007

5

103.6

39.3

12.9

17.4

0.022

17

3.18

1.2

2.12

2.86

0.094

70

0.96

0.4

0.97

1.3

-

-

2.63

1.0

0.74

1.0

TRA-8 Fab′ immunoliposome

TRA-8 with cross-linker

30


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Figure 1. Concept of TRA-8 immunoliposomes. (a) TRAIL, a natural ligand of DR4/DR5, binds to these receptors and promotes the recruitment of the DISC complex. Subsequently, it induces the apoptosis signaling pathway. (b) TRA-8, an anti-DR5 antibody, itself cannot effectively recruit the DISC complex. (C) Only when FcγR-expressing effector cells cross-link TRA-8 can it trigger the recruitment of the DISC complex leading to the caspase cascade and cell death. (d) TRA-8 immunoliposomes can mimic the effector cell function.

Figure 2. Scheme of immunoliposome preparation. Immunoliposomes were prepared by different methods for each antibody format.

Figure 3. Dependence of antibody density on the feed ratio in the reaction mixture. Various immunoliposomes were prepared under different reaction conditions for each antibody format. Liposomes with a fixed lipid composition (DPPC:cholesterol:DSPE-PEG-maleimide=100:66:1) were prepared. Antibody and the liposome dispersion were mixed at various molar ratios of antibody:DSPE-PEG-Mal to react the sulfhydryl group of the antibody with the terminal maleimide group of the PEG chain on the liposomes. Full-length antibody conjugated at the hinge cysteine (open diamond), full-length antibody conjugated at lysine (open square), and Fab′ fragment conjugated at the hinge cysteine (closed diamond).

Figure 4. Apoptosis-inducing activity of TRA-8 immunoliposomes against human carcinoma (Jurkat). The apoptosis-inducing activity of TRA-8 immunoliposomes against Jurkat cells. Cells were treated with immunoliposomes or TRA-8 with cross-linker at various concentrations in the presence or absence of Z-VAD for 24 h. Then, cell viability was estimated by measuring the ATP level. Each data point represents

31



the mean value ± standard deviation (n=3). TRA-8 with cross-linker (open circle), TRA-8 with cross-linker and Z-VAD (closed circle), TRA-8 immunoliposome (IL-A) (open square), TRA-8 immunoliposome (IL-A), and Z-VAD (closed square).

Figure 5. Apoptosis-inducing activity of various TRA-8 immunoliposomes against human carcinoma (Jurkat). The apoptosis-inducing activity of TRA-8 immunoliposomes against Jurkat cells was examined. Cells were treated with immunoliposomes, TRA-8 with cross-linker, or TRAIL at various concentrations for 72 h. Then, cell viability was estimated by measuring the ATP level. (a) TRA-8 full-length immunoliposomes: TRA-8 with cross-linker (open circle), TRAIL (closed circle), IL-B (open square), IL-C (open diamond), IL-D (closed square). (b) TRA-8 Fab′ immunoliposomes: TRA-8 with cross-linker (open circle), TRAIL (closed circle), IL-a (open square), IL-b (open diamond), IL-c (closed square). Each data point represents the mean value ± standard deviation (n=3).

Figure 6. Apoptosis-inducing activity of TRA-8 Fab′ immunoliposomes against human carcinoma (A2058). The apoptosis-inducing activity of TRA-8 Fab′ immunoliposomes composed of different phospholipids against A2058 cells was examined. Cells were treated with immunoliposomes or TRA-8 with cross-linker at various concentrations for 72 h. Then, cell viability was estimated by measuring the ATP level (n=2). DPPC liposome (open diamond), eggPC liposome (open square), DOPC liposome (closed circle), TRA-8 with cross-linker (open circle). Each data point represents the mean value (n=2).

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Figure 7. Cytotoxicity of TRA-8 immunoliposomes against human primary hepatocytes. The cytotoxicity of TRA-8 immunoliposomes against human primary hepatocytes. Cells were treated with immunoliposomes or TRAIL at various concentrations for 6 h. Then, cell viability was estimated by measuring the ATP level. TRAIL (closed circle), IL-d (open diamond), IL-e (closed diamond), IL-f (open square). Each data point represents the mean value (n=2).

Figure 8. Apoptosis-inducing activity of 2E12 immunoliposomes against human carcinoma (Alexander). The apoptosis-inducing activity of 2E12 immunoliposomes was examined. Alexander cells were treated with immunoliposomes or antibody with cross-linker at various concentrations for 72 h. Then, cell viability was estimated by measuring the ATP level. 2E12 with cross-linker (open circle), 3 full-length antibodies/liposome (closed square), 12 full-length antibodies/liposome (closed circle), 25 full-length antibodies/liposome (open square), 63 full-length antibodies/liposome (open diamond). Each data point represents the mean value ± standard deviation (n=3).

33



Figure 1. Concept of TRA-8 immunoliposomes.

Figure 2. Scheme of immunoliposome preparation.

34


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Figure 3. Dependence of antibody density on the feed ratio in the reaction mixture.

Figure 4. Apoptosis-inducing activity of TRA-8 immunoliposomes against human carcinoma (Jurkat).

35



Figure 5. Apoptosis-inducing activity of various TRA-8 immunoliposomes against human carcinoma (Jurkat).

Figure 6. Apoptosis-inducing activity of TRA-8 Fab′ immunoliposomes against human carcinoma (A2058).

36


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Figure 7. Cytotoxicity of TRA-8 immunoliposomes against human primary hepatocytes.

Figure 8. Apoptosis-inducing activity of 2E12 immunoliposomes against human carcinoma (Alexander).

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