Formulation optimization of an ephrin A2 targeted immunoliposome encapsulating reversibly modified taxane prodrugs

Article abstract of DOI:10.1016/j.jconrel.2019.08.006

Ephrin A2 targeted immunoliposomes incorporating pH-sensitive taxane prodrugs were developed for sustained delivery of active drug to solid tumors. Here we describe the systematic formulation development and characterization of these immunoliposomes. We s

Full text of DOI:10.1016/j.jconrel.2019.08.006

Accepted Manuscript
Formulation optimization of an ephrin A2 targeted
immunoliposome encapsulating reversibly modified taxane
prodrugs
Zhaohua Richard Huang, Suresh Kumar Tipparaju, Dmitri B.
Kirpotin, Christine Pien, Tad Kornaga, Charles O. Noble,
Alexander Koshkaryev, Jimmy Tran, Walid S. Kamoun, Daryl C.
Drummond
PII:
S0168-3659(19)30470-5
DOI:
Reference:
https://doi.org/10.1016/j.jconrel.2019.08.006
COREL 9893
To appear in:
Journal of Controlled Release
Received date:
Revised date:
Accepted date:
4 May 2019
1 August 2019
7 August 2019
Please cite this article as: Z.R. Huang, S.K. Tipparaju, D.B. Kirpotin, et al., Formulation
optimization of an ephrin A2 targeted immunoliposome encapsulating reversibly modified
taxane prodrugs, Journal of Controlled Release, https://doi.org/10.1016/
j.jconrel.2019.08.006
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ACCEPTED MANUSCRIPT
Formulation Optimization ofan Ephrin A2 Targeted Immunoliposome
Encapsulating Reversibly Modified Taxane Prodrugs
Zhaohua Richard Huang, Suresh Kumar Tipparaju, Dmitri B Kirpotin, Christine Pien,
Tad Kornaga, Charles O Noble, Alexander Koshkaryev, Jimmy Tran, Walid S.
Kamoun, Daryl C Drummond*
Merrimack Pharmaceuticals, Inc., One Broadway, 14th Floor, Cambridge, MA 02142,
USA
*Correspo
nding
authors:
Daryl
Drummond
, Ph.D.
Merrimack Pharmaceuticals
One Broadway, 14th Floor
Cambridge, MA 02142, USA
Phone: +1-617-803-8887
E-mail: ddrummond@merrimack.com
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Abstract
Ephrin A2 targeted immunoliposomes incorporating pH-sensitive taxane prodrugs were
developed for sustained delivery of active drug to solid tumors. Here we describe the systematic
formulation development and characterization of these immunoliposomes. We synthesized both
paclitaxel and docetaxel prodrugs to formulate as ephrin A2-targeted liposomes stabilized in the
aqueous core with sucroseoctasulfate (SOS). The optimized lipid formulation was comprised of egg-
sphingomyelin, cholesterol, and polyethylene glycol distearoyl glycerol (PEG-DSG). The
formulations examined had a high efficiency of prodrug encapsulation (as high as 114 mole% taxane
per mole phospholipid) and subsequent stability (> 3 years at 2-8 ºC). The taxane prodrug was
stabilized with extraliposomal citric acid and subsequently loaded into liposomes containing a
gradient of SOS, resulting in highly stable SOS-drug complexes being formed inside the liposome.
The internal prodrug and SOS concentrations were optimized for their impact on in vivo drug release
and drug degradation. Cryo-electron microscope images revealed dense prodrug-SOS complex in the
aqueous core of the immunoliposomes. Ephrin A2-targeted taxane liposomes exhibited sub-
nanomolar (0.69 nM) apparent equilibrium dissociation constant toward the extracellular domain of
the ephrin A2 receptor, long circulation half-life (8-12 h) in mouse plasma, a release rate dependent
on intraliposomal drug concentration and stable long-term storage. At an equitoxic dose of 50 mg
taxane/kg, ephrin A2-targeted liposomal prodrug showed greater antitumor activity than 10 mg/kg
of docetaxel in A549 non-small cell lung, as well as MDA-MB-436 and SUM149 triple negative
breast cancer xenograft models. The lead molecule entered a Phase I clinical trial in patients with
solid tumors (NCT03076372).
Keywords: ephrin A2, taxane, liposome, tumor targeting, prodrug, drug loading
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1. Introduction
Paclitaxel and docetaxel are widely used for the treatment of solid tumors, including breast,
gastric, pancreatic, prostate, and non-small cell lung cancers [1-3]. Like other chemotherapeutics,
taxanes also demonstrate significant toxicity in normal tissues because of non-selective cell killing.
The patient’s quality of life is significantly impacted by taxane-related side effects, including
neutropenia, anemia, neuropathy, fatigue, and thrombocytopenia [4]. It is desirable to improve the
treatment outcome by minimizing the exposure of normal tissues to taxanes while increasing the
delivery of therapeutic concentrations of drug in the tumor. Nanoparticles have been widely explored
as the vehicle to deliver more drug to the tumor based on the EPR effect [5, 6]. The challenges
presented by the original emulsion-formulation of paclitaxel has led to several approved nanoparticle
formulations of taxanes, including albumin-bound nab-paclitaxel (Abraxane™, Celgene) [7];
polymer micelle-encapsulated paclitaxel (Genexol-PM™, Samyang Biopharm) [8]; liposome-
formulated (Lipusu™, Luye Pharma); and vitamin A micelle formulation of paclitaxel (Paclical™,
Oasmia Pharmaceutical) [9]. These formulations were approved primarily based on improvement of
patient safety and tolerance by removing the toxic solubilizing agent Cremophor® EL from the
formulation. The efficacy improvement results over Cremphore® EL emulsion of paclitaxel
(Taxol™, Bristol-Myers Squibb), however, are mixed, and the stability of the resulting particles is
often insufficient to take advantage of either the EPR effect or molecular targeting imparted by the
antibody [10-14].
Liposome technology is a viable approach to improve formulations for chemotherapeutics,
exemplified by the FDA approved liposomal oncology products such as PEGylated liposomal
doxorubicin (Doxil™, Janssen Pharmaceuticals) [15]; liposomal daunorubicin (Daunoxome™,
Galen Pharmaceuticals); cytarabine liposome injection (DepotCyt™, Pacira Pharmaceuticals) [16];
liposomal vincristine (Marqibo™, Talon Therapeutics) [17]; irinotecan liposome injection
(Onivyde™, Ipsen Bioscience) [18], and liposomal daunorubicin and cytarabine (Vyxeos™, Jazz
Pharmaceuticals) [19]. Despite the significant progress in the liposome field, there are currently no
approved immunoliposomes on the market. Immunoliposomes offer substantial advantages
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compared with antibody drug conjugates (ADC) by encapsulating the active drug stably inside a lipid
nanocarrier conjugated indirectly to an antibody fragment on the surface of the nanoparticle [20-23].
Stabilizing the drug inside the liposome is a critical quality attribute that allows targeting of solid
tumors due to the enhanced permeability and retention effect (EPR) [5], as well as due to molecular
targeting of the cell surface receptor, in this case ephrin A2 (EphA2) [23, 24]. We have advanced an
ephrin A2-targeted taxane immunoliposome into phase I clinical trials for patients with solid tumors
(NCT03076372). The systematic studies of biodistribution, tolerability and pharmacology of the final
clinical formulation in multiple animal models were published recently [25]. In this report, we focus
on the formulation optimization to achieve the requisite stability for both passive targeting related to
the size of the liposome, as well as active targeting resulting from the conjugated antibody fragments,
as part of the successful design of a highly active immunoliposome.
The design of the ephrin A2 targeted taxane liposome is illustrated in Figure 1. Anti-ephrin A2 single
chain fragment (scFv) is used as the targeting moiety and attached to the liposome surface through 1,2-
distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) with a polyethylene glycol 2000 (PEG 2000)
spacer. Ephrin A2 was selected, primarily due to its broad overexpression in cancers where taxanes
have demonstrated activity [26-29], as well as its ability to be internalized [30]. In normal tissues,
liposomes have limited access to ephrin A2 receptors due to restrictions in tissue penetration related to
the particle size. Taxanes are highly hydrophobic drugs and are, thus, encapsulated in most nanocarriers
through hydrophobic interactions. One major drawback with such an approach is the relative fast
dissociation of taxane from the carrier upon entering the blood stream. The nanocarrier and taxane
typically show two distinct pharmacokinetic profiles: a long circulating carrier, and a fast eliminating
drug [31-33]. The proposed advantage of the nano drug delivery system is insufficiently realized in
such systems. Certain base-labile prodrugs were initially described by Mathew et al. and shown to be
stable under acidic conditions, but hydrolyzed to the active taxane at neutral pH or under basic
conditions [34]. Others have also reported the liposome formulation of different weakly basic or acidic
derivatives of taxanes [35, 36]. Encapsulating taxane prodrugs inside the aqueous core of the liposome
with a trapping agent enables the extended plasma half-life for the prodrug. The released prodrug,
however, needs to be converted to the active taxane to exert anti-cancer activity. We have synthesized
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three taxane prodrugs (compounds 1, 2, and 3) for further evaluation.
In order to translate a research stage immunoliposome drug candidate to the clinical phase,
significant formulation and process development efforts are needed to manufacture GMP quality drug
product with reasonable storage stability for all major components including active pharmaceutical
ingredient, lipids, and the antibody. Here, we describe our optimization of the final prodrug liposomal
formulation for efficiency of drug encapsulation, chemical stability of both the prodrug and bilayer-
forming phospholipid components, and drug concentration dependent in vivo release. Additionally, we
report the binding kinetics of the scFv, scFv-PEG-DSPE, and the immunoliposome. We also
demonstrate significant preclinical activity with several prototype formulations in xenograft models.
Figure 1 Schematic illustration of immunoliposome of pH-sensitive taxanes. From outside to inside: a) Ephrin A2 antibody
as the active targeting ligand inserted into the lipid bilayer as a lipid-scFv conjugate: scFv-PEG-DSPE; b) Polyethylene
glycol distearoyl glycerol (PEG-DSG) for extended circulation time and sphingomyelin/cholesterol/PEG-DSG lipid matrix
for stability; c) SOS as loading aid for efficient and stable encapsulation of taxane prodrugs. Particle diameter is around 110
nm for passive targeting.
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2. Materials and methods
2.1. Materials
Paclitaxel and docetaxel were obtained from Haosun Pharmaceuticals (Hubei, China). Egg-
sphingomyelin and PEG-DSG were from NOF Corporation (Tokyo, Japan). High purity cholesterol
was from Dishman (Middlesex, NJ). 1,2-Distearoyl-sn-glycero-3-phosphatidylcholine (DSPC) and
1,2-dioctadecyl-sn-glycero-3-phosphatidylcholine (DODPC) were from Avanti Polar Lipids
(Alabaster, USA). Anti-ephrin A2 scFv and the lipid conjugate scFv-PEG-DSPE were prepared in
house as described previously [37]. Triethylamine sucroseoctasulfate (TEA-SOS) was manufactured
in house as previously reported [38]. 2'-O-(4-(N,N-Diethylamino) propanoyl)-paclitaxel (DPX) was
synthesized as previously described [34]. [3H] cholesteryl hexadecyl ether was from Perkin Elmer
(Waltham, USA). Biacore assay related reagents and supplies were purchased from GE Healthcare
(Pittsburgh, PA). All other general reagents were purchased from common suppliers.
2.2. Synthesis of taxane prodrugs
The synthesis of taxane prodrugs 1-3 involved two chemical transformations (Figure 2a). The N-
dialkylamino acid side chains were prepared from ethyl-4-bromobutyrate by treatment with
appropriate dialkylamine, followed by ester hydrolysis using 10 M HCl. Esterification of the 2'-
hydroxyl on paclitaxel or docetaxel with corresponding N-dialkylamino acid side chain was carried
out using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as a coupling agent and 4-
(dimethylamino) pyridine (DMAP) as base and catalyst.
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Figure 2 2a) Synthesis of pH-sensitive taxane prodrugs. Taxanes were modified at the 2′-hydroxy group through the ester
bond with N-dialkylamino acids. 2b) Hydrolysis of prodrug 1 was measured over time at two different pHs, 2.5 and 7.5.
Synthesis of N-dialkylamino acid:
General synthesis of N-dialkylamino acid involved stirring dialkyl amine (4 equivalents) with
ethyl-4-bromobutyrate (1 equivalent) in ethyl acetate for 24 h at room temperature. After completion
of reaction (monitored by TLC and visualized with iodine vapor), the reaction mixture was washed
with water, sodium bicarbonate, and brine. The organic layer was separated and dried on sodium
sulfate, filtered, and evaporated. The crude oil was purified by flash column chromatography using a
Reveleris™ flash system on a silica gel cartridge, using 3-15% MeOH in CHCl3. Purified 4-N-
dialkylamino ester was taken in 10 M HCl (10 equivalents) and heated at reflux at 110 C for 8 h. The
reaction mixture was then allowed to cool to room temperature and subsequently concentrated under
vacuum until an oily residue remained. The remaining residue was re-dissolved in distilled water, and
the concentration process repeated twice to remove excess HCl to give an off-white solid. The solid
was dissolved in hot glacial acetic acid (4 mL) with the addition of a small quantity of water (~ 0.5
mL). The solution was placed at room temperature for 3 h and then transferred to a refrigerator (2-8
C) for 16 h. The solid obtained was triturated and washed three times with 20 mL ether, and the
supernatant ether was discarded. The solid obtained was dried in high vacuum and then used for further
reaction.
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Synthesis of taxane prodrugs 1-3:
Paclitaxel or docetaxel (1 equivalent), 4-dialkylamino butyric acid hydrochloride (1.2 equivalent),
EDC.HCl (2 equivalent), and DMAP (1.5 equivalent) were all weighed into a round-bottomed flask
under argon. To this, anhydrous dichloromethane (DCM) (20 volume equivalents) was added at room
temperature under argon and stirred for 18-24 h. The reaction was monitored by HPLC. The reaction
was stopped upon completion. The reaction mixture was diluted with 30 mL of 1:3 mixture of
DCM/MeOH and washed with 20 mL of 1 N aqueous HCl. The aqueous layer was washed twice with
a mixture of 30 mL DCM and 5 mL MeOH. The combined organic portions were dried on sodium
sulfate, filtered, and evaporated to give a white solid. The solid was dissolved in chloroform and
directly loaded on a Reveleris™ silica gel cartridge pre-equilibrated with CHCl3, and flash column
chromatography was performed using 0–15% MeOH/CHCl3. Fractions were pooled together, 0.5 mL
of 0.05 M HCl in 2-propanol was added and evaporated under 35 ºC to give white solids.
1
Prodrug 1: 2'-O-(4-(N,N-Dimethylamino) butanoyl)-docetaxel hydrochloride. H NMR (300
MHz, CDCl3+ (CD3)2SO): δ = 8.02 (d, 2H), 7.60–7.42 (m, 6H), 7.38–7.25 (m, 4H), 7.24–7.12 (m,
1H), 6.92 (d, 1H), 6.40–5.88 (m, 1H), 5.53 (d, 1H), 5.35–5.21 (m, 1H), 5.20–5.08 (m, 2H), 4.85 (d,
1H), 4.74 (d, 1H), 4.26 (s, 1H), 4.13 (dd, 3H), 3.74 (d, 1H), 3.56 (s, 1H), 3.16–2.74 (m, 3H), 2.64–
2.48 (m, 7H), 2.49–2.00 (m, 5H), 1.84–1.68 (m, 4H), 1.62 (m, 3H), 1.30 (s, 9H), 1.07 (s, 3H), 1.02 (s,
3H) ppm. MS (ESI) m/z: 921.4 [M]+
Prodrug 2: 2'-O-(4-(N,N-Diethylamino) butanoyl)-docetaxel hydrochloride. 1H NMR (300 MHz,
CDCl3): δ = 8.11 (d, 2H), 7.65–7.57 (m, 1H), 7.56–7.46 (m, 2H), 7.45–7.29 (m, 5H), 6.29–6.12 (m,
1H), 5.99 (d, 1H), 5.68 (d, 1H), 5.58–5.40 (m, 1H), 5.31 (d, 1H), 5.24 (s, 1H), 4.96 (d, 1H), 4.38–4.08
(m, 5H), 3.91 (d, 1H), 3.20–2.30 (m, 3H), 2.70–2.52 (m, 2H), 2.50–2.42 (m, 2H), 2.25–2.05 (m, 3H),
1.94 (s, 3H), 1.92–1.78 (m, 2H), 1.75 (s, 3H), 1.50–1.35 (m, 9H), 1.32 (s, 9H), 1.30–1.20 (m, 6H),
1.12 (s, 3H) ppm. MS (ESI) m/z: 949.4 [M]+
Prodrug 3: 2'-O-(4-(N,N-Diethylamino) butanoyl)-paclitaxel hydrochloride. 1H NMR (300 MHz,
CDCl3): δ = 8.20-8.11 (m, 3H), 8.00 (d, 2H), 7.70-7.35 (m, 10H), 7.25-7.21 (m, 1H), 6.28 (s, 1H),
6.07 (t, 1H), 5.91 (dd, 1H), 5.65 (d, 1H), 5.50 (d, 1H), 4.96 (d, 1H), 4.47-4.40 (m, 1H), 4.31 (d, 1H),
4.19-4.11 (m, 3H), 3.76 (d, 1H), 3.20-2.90 (m, 3H), 2.79-2.63 (m, 1H), 2.60-2.42 (m, 7H), 2.42-2.22
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(m, 1H), 2.20 (s, 3H), 2.19-2.00 (m, 1H), 1.92-1.80 (m, 4H), 1.66 (s, 3H), 1.43 (t, 6H), 1.30-1.21 (m,
6H), 1.11 (s, 3H) ppm. MS (ESI) m/z: 995.4 [M]+
2.3. Preparation of ephrin A2-targeted taxane liposomes
Ephrin A2-targeted taxane liposomes were generally prepared in three steps: liposome extrusion,
drug loading, and ligand insertion. First, the lipids of the given formulation were dissolved in ethanol
(400-800 mM phospholipids ) at 60-70 ºC and added into the TEA-SOS solution (0.6 N to 1.1 N, pH
6.0 -7.0) at 60-70 ºC with stirring to obtain multilamellar vesicles (MLVs) (overall ethanol volume is
less than 15% of the total volume). The MLVs were then extruded through a stack of polycarbonate
membranes twice (0.1 µm × 6 plus one 0.2 µm on top) at 500 psi using a Lipex™ extruder to give
small unilamellar vesicles (SUVs). Second, the SUVs were diafiltrated against water using a 500 KD
MWCO polysulfone ether (PES) hollow fiber module to remove ethanol and external TEA-SOS. The
drug was dissolved in 8% PEG 400 (3-5 mg/mL) at pH 2.8-3.5, mixed with citrate buffer (20-30 mM
final citrate) and the diafiltrated SUVs to pH 5.0-5.8, osmolality balanced with dextrose to
approximately 500 mOsm, heated up and incubated at 70 ºC for 30 minutes before chilling to 2-30 ºC.
Last, the targeting ligand scFv-PEG-DSPE solution (1.0-2.0 mg/mL) was mixed with the drug loaded
liposome at 5 g scFv per mol lipid, heated up and incubated at 60 ºC for 30 minutes before chilling to
2-30 ºC. The mixture was then diafiltrated against formulation buffer, and sterile filtered to give the
final ephrin A2-targeted taxane liposomes. The non-targeted taxane liposomes were prepared similarly
by skipping the ligand insertion step. Drug purity and strength were determined by HPLC using a
Phenomenex RP C18 polar column with a 30% -68% B gradient (mobile phase A: 0.1% TFA in water,
mobile phase B: acetonitrile). Free taxanes (paclitaxel and docetaxel) were used as HPLC standards.
All taxane prodrug concentrations were calculated as the molar equivalent of the corresponding
taxanes and used throughout the paper in the expression of drug to lipid ratio and drug dosage.
Phospholipid concentrations were measured by Bartlett’s phosphate assay with slight modification
[39]. Drug to lipid ratios (D/L) are expressed as grams of drug per mole of phospholipids (g/mole).
Alternatively, the mole% drug per mole of phospholipids can be obtained by dividing the g drug/mol
PL value by the molecular weight of the taxane prodrug being evaluated, and multiply by 100.
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Specifically, we use a docetaxel molecular weight of 807.879 g/mol for prodrugs 1 and 2, and a
paclitaxel molecular weight of 853.907 g/mol for prodrug 3.
2.4. Preparation of tritium-labelled liposomes
The tritium-labelled liposomes were prepared by a similar method described for the cold
liposomes, except that a trace amount of [3H] cholesteryl hexadecyl ether (1µCi/µmol phospholipids)
was added to the lipids before extrusion. [3H] Cholesteryl hexadecyl ether was received as a solution
in toluene. An appropriate amount of solution was transferred to a clean glass vial. Toluene was
evaporated with a nitrogen stream in the fume hood. The residual toluene was removed under high
vacuum. Then the lipids were added into the same vial, dissolved in ethanol, and hydrated in TEA-
SOS solution. The scale was much smaller for the hot liposome preparation. The TEA-SOS gradient
was generated on a Sepharose® 4 fast flow column. The drug loading procedure was similar to that of
cold liposomes. Loading mixture was purified on the PD-10 G-25 columns, pooled, concentrated, and
sterilized for in vivo pharmacokinetics studies. The phospholipid concentration was correlated with
radioactivity. The radioactivity was measured on a 1450 MicroBeta TriLux microplate scintillation
and luminescence counter (Perkin Elmer, Waltham, USA).
2.5. Size measurements
The size and size distribution of nanoparticles were determined by dynamic light scattering
(DLS) using a Zetasizer Nano ZS90 instrument (Malvern Instrument Ltd., UK). Samples were diluted
100 times in water for size measurements.
2.6. Cryo transmission electron microscope imaging of taxane liposomes
Cryo electron microscopy (Cryo-EM) was performed using an FEI Tecnai T12 electron microscope,
operating at 120 k eV equipped with an FEI Eagle 4k × 4k CCD camera. Diluted liposome samples
(0.1 mM phosphate) were preserved in vitrified ice supported by holey carbon films on 400-mesh
copper grids. Each sample was prepared by applying a 3 μL drop of liposome suspension to a cleaned
grid, blotting away with filter paper, and immediately proceeding with vitrification in liquid ethane.
Vitreous ice grids were transferred into the electron microscope using a cryo stage that maintains the
grids at a temperature below -170 °C. Images of each grid were acquired at multiple scales to assess
the overall distribution of the specimen. After identifying potentially suitable target areas for imaging
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at lower magnifications, pairs of high magnification images were acquired at nominal magnifications
of 52,000x (0.21 nm/pixel) and 21,000x (0.50 nm/pixel). The images were acquired at a nominal
underfocus of -4 µm (52,000x) and -5 µm (21,000x) and electron doses of ~6-9 e/Å2.
2.7. Stability study of taxane liposomes
Non-GMP liposome samples were aliquoted into HPLC vials and stored at 4 ºC for regular
storage stability studies, and at 30 ºC for accelerated stability studies. Samples were analyzed at
given time points for monitored parameters, such as size, lipid and drug concentration, and purity.
For GMP stability study, samples were aliquoted in sterile bottles and tested for a larger set of pre-
specified parameters at planned time points.
2.8. Surface plasmon resonance binding assay
The binding kinetics of the anti-ephrin A2 scFv, scFv-PEG-DSPE and the ephrin A2 targeted
liposomes were measured by surface plasmon resonance (SPR) method on a Biacore 3000 machine.
The ephrin A2 extracellular domain was conjugated on the surface of the CM5 chip through EDC-
NHS chemistry with a target SPR response of 250 RU. HBS-P was used as the running buffer with a
flow rate of 30 µL/min. All kinetics samples were measured with 3 min association and 5 min
dissociation time. Different surface regeneration conditions were used: a) for scFv, 10 mM glycine
pH 2.0, 30 s; b) for scFv-PEG-DSPE, 10 mM glycine pH 2.0, 30 s first, then 0.5% SDS, 30s; c) for
liposome, 3.0 M MgCl2, 30 s. For each sample, five series of concentrations were run in duplicate
plus the blank. The raw data were double referenced and analyzed with BiaEvaluation software using
1:1 binding model and global fitting.
2.9. Pharmacokinetics study of taxane liposomes
The careand treatment of experimental animals were in accordance with Institutional Animal Care
and Use Committee (IACUC) guidelines of Merrimack Pharmaceuticals, Protocol MAP004. Female
Swiss Webster mice aged 6-8 weeks were obtained from Charles River Laboratories (Wilmington,
MA). Three mice per group were dosed with tritium-labeled taxane liposomes for each formulation.
Blood samples (30-60 µL) were collected into lithium heparin coated tubes through saphenous vein
bleeding at time points specified as: 5 min, 1.5 h, 4 h, 8 h, and 24 h. At the 48 h time point,
approximately 400 µL blood was collected through terminal cardiac bleeding. Blood samples were
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immediately centrifuged (within 10 min) at 12,000 rpm for 5 min at 4 °C. Plasma samples were then
removed and stabilized with 200 mM pH 2.5 phosphate buffer with a dilution factor no less than 3.
The stabilized samples were split for radioactivity counting and drug concentration measurement by
HPLC. The concentration over time data for lipids, taxanes, and drug lipid ratio were analyzed by
Phoenix software using an NCA model.
2.10. Pharmacology study of ephrin A2-targeted taxane liposome
Experiments were performed according to approved IACUC guidelines of Merrimack
Pharmaceuticals, Protocol MAP013. Female Ncr nude mice were obtained from Charles River
Laboratories (Wilmington, MA), at 6 – 8 weeks of age. Human non-small cell lung cancer A549 cell
line, breast cancer cell lines MDA-MB-436 and SUM149 were used to establish the mouse xenograft
models. Each mouse was inoculated with 5×106 cells subcutaneously. Animals were randomized, and
ten animals were treated in each group, unless otherwise indicated. Ephrin-A2 targeted liposome
formulation of compound 2 (Liposome 2) was administered intravenously at 50 mg/kg when tumors
reached an average volume of 150-200 mm3 for all three models. For the MDA-MB-436 model, 12.5
mg/kg and 25 mg/kg doses were added in a dose-response study. The treatment was continued for a
total of four weekly doses. Docetaxel (Taxotere™) was administered at 10 mg/kg as the positive
control arm. Saline (0.9% w/v NaCl) was used as the negative control. Tumor volumes were measured
bi-weekly during the dosing cycle, then weekly until tumors reached 2000 mm3 or animals were in
poor general health. Animals were sacrificed prior to surpassing the maximum tumor volume. The
percent tumor growth was calculated by dividing tumor volume change at the time of measurement by
the starting volume. The statistical significance of differences between treatment groups was
determined by the Student's t test (for two groups) in GraphPad Prism 7. Differences with P < 0.05
were considered significant.
3. Results
3.1. Synthesis and characterization of taxane prodrugs
Taxane prodrugs were synthesized by esterification of the phenylpropranoate 2'-hydroxyl group with
dialkylamino acid using well established EDC/DMAP coupling chemistry. Selective esterification at
the 2'-hydroxyl group was achieved by appropriate stoichiometry and monitoring of the reaction by
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HPLC. In this report, we have synthesized two docetaxel prodrugs (compound 1 and 2) and one
paclitaxel prodrug (compound 3) for formulation development (Figure 2a). All compounds were
purified by flash chromatography and characterized by NMR, MS, and HPLC. The hydrolysis profiles
of these prodrugs at different pH and conditions were determined. Compound 1 has a representative
hydrolysis profile (Figure 2b) demonstrating rapid conversion to docetaxel at pH 7.5 while remaining
stable at pH 2.5. It should be noted that the hydrochloride salt of the prodrugs had relatively low
solubility in water and the selected candidates were later converted to the mesylate salt for large scale
manufacturing.
3.2. Liposome formulation of taxane prodrugs
Lipid composition is critical for the desired liposome properties. Traditional liposome
formulations use diacyl phosphatidylcholine and are stable at pH 6.5 for long term storage. However,
compounds 1, 2, and 3 are unstable at pH 6.5 by design and require lipids that are stable at low pH.
At the very early stage of formulation development, we formulated a prototype taxane prodrug (2'-
O-(4-(N,N-diethylamino) propanoyl)-paclitaxel, DPX) with three different phospholipids (PLs):
DSPC (traditional ester linked-phospholipid), DODPC (ether linked-phospholipid  more resistant
to acid hydrolysis), and E-SM (sphingosine backed phospholipid  also more resistant to acid
hydrolysis), at a molar ratio of 3:2:0.3 for PL/Cholesterol/PEG-DSG. Lipid stability was evaluated
by monitoring the percentage of lysophospholipid generated during storage of the liposomes at pH
5.5 at 4 °C. The structures of the lipids and the nine month stability plot are shown in Figure 3. Both
DODPC and E-SM liposomes demonstrated negligible generation of lyso-PC during storage, while
DSPC liposomes generated significant amount of lyso-PC in 6 months (> 20%). Clearly, DSPC
hydrolyzed rapidly under acidic conditions and did not meet our formulation stability requirement.
Eventually, we chose E-SM over DODPC, as a result of the more readily available commercial supply
of GMP grade lipid, but either phospholipid has the desired properties to be utilized in a pharmaceutical
grade formulation of these taxane prodrugs.
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Figure 3 Lipids storage stability. Liposome formulation: phospholipids/cholesterol/PEG-DSG 3/2/0.3 molar ratio. Storage
conditions: 2-8 C, pH 5.5. Lyso-phospholipids were monitored as the main degradation products of the liposomal membrane.
Left panel: percent of lysoPC formation over time, 1) solid circle: DSPC; 2) solid square: DODPC; 3) solid triangle: E-SM.
Right Panel: Chemical structures of DSPC, E-SM, DODPC and LysoPC.
The drug loading process is illustrated in Figure 4. We systematically evaluated the impact of PEG-
lipids on the loading efficiency at various drug/lipid ratios for our lead compounds. The results (Table
1) indicated that PEG-DSG allowed high loading efficiencies over a broad range of drug-lipid ratios
for all three compounds while the inclusion of PEG-DSPE limited the acceptable formulations to those
with drug-lipid ratios less than 300 g/mol. The results strongly support the use of PEG-DSG in
formulations of these taxane prodrugs.
Figure 4 Loading of taxane prodrugs into liposomes by chemical equilibrium shift. Taxane prodrugs complex with weak
polyanions such as citrate outside the liposome first, then cross the lipid bilayer as the free base at elevated temperature and
appropriate pH and form a stable complex with SOS inside the liposome.
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Table 1. Impact of PEG-lipids on drug loading
PEG-Lipids
PEG-DSPE
Loading
Taxane
PEG-DSG
Loading
Prodrug
D/L
D/L
(g/mol)
(%)
(g/mol)
(%)
141
289
95.0
97.9
153
N/A
95.4
N/A
463
744
842
1200
98.7
89.5
93.8
A
457
27.6
N/A
N/A
N/A
1
N/A
N/A
N/A
167
201
100.0
95.0
175
220
98.3
98.6
359
412
93.9
99.3
N/A
400
N/A
A
2
3
525
623
925
99.6
100.6
64.4
563
N/A
900
A
N/A
A
633
805
949
1200
96.1
96.3
95.5
A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
A: aggregation, typically less than 10% loading efficiency.
We also evaluated the impact of PEG-DSG concentration in the membrane and TEA-SOS
concentration on drug loading, using compound 2, with the aim of maximizing the PEG-DSG density
on the surface used to increase circulation lifetimes of the liposome. In addition, the ability to utilize
high concentrations of intraliposomal SOS enables high drug loading capacity of the liposome, and
thus lower doses of liposomal excipients, while allowing for a higher residual pH gradient following
drug loading that stabilizes the drug chemically in the prodrug form. The results (Table 2) showed
that with 0.6 N TEA-SOS, the loading efficiency was reduced from 90-to 36.7 % when PEG-DSG
increased from 4 to 6 mol% of the total phospholipid. When 1.1 N TEA-SOS was used as the trapping
agent, the loading efficiency remained high (89.7%) between 4 and 7 mol% PEG-DSG and dropped to
67.2% with 8 mol% PEG-DSG. The drug loading results from compound 2 formulations demonstrated
that loading was dependent on PEG-DSG concentration, but that higher concentrations of the
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intraliposomal trapping agent (i.e., 1.1 N TEA-SOS) facilitated efficient loading at higher concentrations
of PEG-DSG.
Based on the results of lipid stability, influence of PEG-lipids type and density, and TEA-SOS
concentration, a final liposome formulation of E-SM/cholesterol/PEG-DSG at 1:0.67:0.06 molar ratio
was chosen with 1.1 N TEA-SOS as the trapping agent. A ligand density of 5 g of scFv per mole of
phospholipid was used for the liposome formulation, based on previous study of the ephrin A2 scFv-
PEG-DSPE (unpublished observations). The scFv-PEG-DSPE insertion efficiency was nearly
quantitative.
Table 2. Effect of TEA-SOS concentration and PEG-DSG density on drug loading
TEA-SOS Conc. (N)
PEG-DSG (mol %)
Loading Efficiency (%)
4
5
90.0
76.2
0.6
0.6
6
4
5
6
7
8
36.7
91.6
91.0
96.2
89.7
67.2
0.6
1.1
1.1
1.1
1.1
1.1
3.3. Particle size and morphology of taxane liposomes
The particle size and polydispersity of taxane liposomes were measured by dynamic light
scattering. The drug loaded taxane liposomes typically have a particle size around 110 nm with a
polydispersity index (PDI) less than 0.1. The morphology of both empty liposomes (Figure 5, panel
A) and the ones loaded with prodrug 2 at 450 g/mol D/L ratio (Figure 5, panel B) were visualized
by cryo-EM. For both empty and drug-loaded liposomes, the predominant particles were spherical
and unilamellar, having diameters in the range of 50-170 nm. While the empty liposomes had a
slightly denser interior than the surrounding buffer, the loaded liposomes showed a much denser
intraliposomal area, with a clear boundary, consistent with the drug being present in the form of an
insoluble salt with sucroseoctasulfate.
3.4. Storage stability of taxane liposomes
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Liposome formulations of prodrug 1, 2, and 3 at various drug-lipid ratios were evaluated for their
storage stability at 4 °C for 12 months, and at 30 °C for 3 months. No significant change in particle
size or polydispersity were observed. Hydrolysis of the prodrug was used as the primary stability
indicator for the formulation. The results are summarized in Table 3. For prodrug 1 and 2, all
formulations exhibited suitable storage stability at 4 °C, with less than 3% prodrug hydrolyzed after
12 months. The drug-to-lipid (D/L) ratio resulted in little impact on hydrolysis at 4 °C. The
formulations hydrolyzed faster under accelerated conditions (30 °C), but the rate of hydrolysis was
slower at higher D/L ratios. Liposomes of prodrug 3 were less stable than those of prodrug 1 and 2,
and demonstrated improved stability at 4 °C for formulations with higher D/L ratios.
Figure 5 Cryo electron microscope images of liposomes. A) Empty liposome without taxane prodrug, B) Ephrin A2-targeted
liposome with taxane prodrug 2 loaded at D/L ratio 450 g/mol.
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3.5. Binding kinetics of ephrin A2-targeted taxane liposomes
The binding kinetics towards the ephrin A2 extracellular domain were determined utilizing an
SPR method and a Biacore 3000 instrument (GE Healthcare; Marlborough, MA). The sensorgrams
of scFv (Panel A), scFv-PEG-DSPE (Panel B), and the ephrin A2 targeted liposome (Panel C) are
shown in Figure 6. The equilibrium dissociation constants (KD) were obtained by global fitting of the
experimental data using a Langmuir 1:1 binding model (Table 4). The scFv had an initial KD value of
6.92 nM, which approximately tripled to 21.6 nM when it was conjugated to PEG-DSPE, but
significantly decreased to 0.69 nM when the conjugate was inserted in the outer leaflet of the liposome
membrane.
Table 3. Storage stability of selected liposome formulations
Liposome Storage Temperature
Taxane
4 C
30 C
Time (month) Hydrolysis (%)
Prodrug
Time (month)
Hydrolysis (%)
D/L (g/mol)
D/L (g/mol)
283
134
0
283
0
457
0
134
0
457
0
0
3
0
0
1
0.48
0.77
2.18
0.54
1.08
0.53
0.8
2.14
1
2
3
6.53
11.12
13.31
4.96
8.12
4.14
6.32
7.45
6
12
2.36
D/L (g/mol)
409
10.27
D/L (g/mol)
409
191
0
529
0
191
0
529
0
0
3
0
0
0
1
2
3
2
3
0.21
0.37
2.23
0.58
0.47
0.79
0.66
2.82
4.73
8.66
11.48
3.56
6.33
3.17
4.73
5.58
6
12
2.73
D/L (g/mol)
359
7.36
D/L (g/mol)
359
166
0
525
0
166
0
525
0
0
3
0
0
1
2
3
0
3.45
3.86
17.7
1.03
2.93
4.52
1.39
2.91
6.66
44.7
52.4
56.4
23.57
45.10
52.68
25.73
44.09
49.02
6
12
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Figure 6 Binding kinetics by surface plasmon resonance. Sensorgrams with global fitting for different analytes: A) Ephrin A2
scFv; B) Ephrin A2 scFv-PEG-DSPE; C) Ephrin A2-targeted liposomal prodrug 2.
Table 4. Summary of binding kinetics
Analyte
ka (M-1. S-1) kd (S-1) Rmax KD (nM)
(RU)
7.38E+05
7.90E+04
1.21E+05
5.11E-03 22
1.71E-03 23
8.44E-05 332
6.92
21.6
0.69
scFv
scFv-PEG-DSPE
Eph A2 Liposome
3.6. Pharmacokinetics and in vivo release of taxane liposomes
The pharmacokinetics of liposomal taxane prodrugs were evaluated in mice by monitoring both
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the prodrug and lipid concentrations over a time course of 48 hours. The in vivo release rate was
obtained by fitting the D/L ratio over time data. PK profiles of liposomal prodrug 1 are shown in
Figure 7 by using percent injected dose (ID%) as the Y axis to normalize drug, lipid and D/L over
time. These PK profiles were typical of the formulations screened, and generally showed the
circulation half-life of 8-12 hours for the drug and lipids, and the resulting slow release of drug from
the liposomes. We also studied the PK of liposomal taxanes formulated at various D/L ratios. The
drug release rates are summarized in Table 5. The general trend is, the higher D/L (intraliposomal
drug concentration), the slower in vivo release rate, although the absolute half-life values vary for
different liposomal prodrugs. When the release rate was too slow at very high D/L ratios, it was
difficult to achieve a meaningful fitting of the data, due to the exceedingly slow release of the drug
relative to the time course for which the drug clearance could be reliably captured using the HPLC
method employed in these studies.
Figure 7 Pharmacokinetics profiles of liposomal prodrug 1. Liposome formulation: E-SM/Cholesterol/PEG-DSG 3/2/0.24
with a drug-lipid ratio of 300 g/mole. Semi-log graph of percent injected dose over time were plotted, 1) solid triangle:
drug/lipid; 2) solid square: lipids; 3) solid circle: drug.
3.7. Pharmacology of ephrin A2-targeted liposome of prodrug 2
As a proof of concept, we evaluated the antitumor effect of the ephrin A2 targeted liposomal
prodrug 2 (liposome 2) in three different cell line derived xenograft models: a human non-small cell
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lung adenocarcinoma model (A549) and two triple negative breast cancers MDA-MB-436 and
SUM149. The polysorbate formulation of docetaxel at 10 mg/kg, was used as the positive control.
The equitoxic dose of liposome 2 (50 mg/kg) was used as the treatment arm, which is approximately
50% of the maximum tolerated dose. In all three models, liposome 2 demonstrated superior antitumor
activity than docetaxel, as evidenced by the clear separation of the tumor growth curves (P < 0.01)
(Figure 8). Clear dose-dependent responses were observed in MDA-MB-436 model. Liposome 2 at
12.5 mg/kg demonstrated similar tumor growth rate to that of 10 mg/kg docetaxel. At equitoxic dose
(50 mg/kg), liposome 2 resulted in tumor regression. Additionally, multiple tumor-free animals were
observed only in the liposome 2 treatment group at the end of the study while tumor regrowth
occurred on all animals treated with docetaxel. Similar efficacy was also observed for liposomal
prodrug 1 (data not shown). The enhanced efficacy of liposome 2 may be explained by prolonged
exposure of tumor cells to taxanes slowly released from liposomes after accumulation in solid tumors.
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A 5 4 9
4 0 0
A
3 0 0
2 0 0
1 0 0
0
- 1 0 0
0
2 0
4 0
6 0
T im e ( D a y )
S U M 1 4 9
2 5 0 0
2 0 0 0
1 5 0 0
1 0 0 0
5 0 0
B
0
- 5 0 0
0
2 0
4 0
6 0
T im e ( D a y )
M D A - M B - 4 3 6
8 0 0
6 0 0
4 0 0
2 0 0
0
C
0
2 0
4 0
6 0
T im e ( D a y )
Figure 8 Efficacy of ephrin A2-targeted liposomal prodrug 2 in mice models. Tumor growth curves for saline (solid circle),
docetaxel (10 mg/kg, solid triangle) and liposome 2 (50 mg/kg, equitoxic dose, solid square) were shown. A) Human lung
cancer cell line A549; B) Human triple negative breast cancer cell line SUM149 C) Dose dependent efficacy in MDA-MB-436
mouse xenograft model, empty circle: 12.5 mg/kg liposome 2, solid diamond: 25 mg/kg liposome 2.
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4. Discussion
4.1. Design of taxane prodrugs
To design taxane prodrugs suitable for liposome formulation by active loading, we applied two
major strategies: 1) a weak base side chain to increase the hydrophilicity of the prodrug and to enable
trapping in the aqueous core of the liposome; 2) a linkage that is stable during formulation process and
storage but cleaves in the physiological environment to allow conversion of the prodrug to the active
taxane metabolite. We chose an ester bond as the linkage at the phenylpropanoate 2'-hydroxyl group
of the taxane (Figure 2). Unlike the esterase-dependent linkage, release of the taxanes at physiological
pH is not subject to the variation of enzyme level among patients and enables more consistent prodrug
conversion to the active taxane. Taxane prodrugs with pH-labile linkers have been described previously
by both us and others [34, 35, 40]. However, the pH-sensitive property presents challenges for
stability during drug loading, processing, and storage. We overcame the pH-sensitivity hurdle by
processing and storing prodrugs and liposomes at relatively low pH (pH 5.0-5.8).
4.2. Engineering of antibody-targeted immunoliposomes
The targetability and stability of immunoliposomes can be impacted by a range of parameters
related to the final formulation and process for drug encapsulation. These include, but are not limited
to, the drug encapsulation method employed, the salt used for trapping the drug stably in the interior,
the lipid composition of the membrane, lipopolymer chemical structure and density in the membrane,
and, finally, the drug encapsulation rate within the liposomes. Prodrugs 1, 2, 3, amphiphilic by
design, also appear to behave like detergents. The traditional methods of direct encapsulation, reverse
phase evaporation, and remote loading do not work for these compounds since they can readily lyse
the lipid bilayer. One strategy is to load the prodrug at very low concentration to prevent micelle
formation and the damage to the lipid bilayer during drug loading. However, this is not scalable or
practical for manufacturing. To tackle this problem, we used polyanion counter ions, such as citrate,
that form a temporary and relatively weak complex with the prodrug to limit the free prodrug
concentration in the solution (Figure 4). When free prodrugs cross the lipid bilayer, they form stable
complex with SOS inside the liposome. The weak extraliposomal complexes, mostly insoluble,
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dissociate continuously to release more prodrug until the loading is complete. The drug loading
process can be interpreted as a chemical equilibrium shift from an extraliposomal weak prodrug
complex to an intraliposomal stable prodrug complex, mediated by the free prodrug crossing the
lipid bilayer.
The pH-sensitive property of the prodrugs also requires a lipid matrix that can have long-term
stability under acidic storage conditions. We have compared the traditional diacyl phospholipid,
DSPC, with more stable lipids such as sphingomyelin and DODPC for their storage stability at pH
5.5, when used as a major component in the liposome formulation of taxane prodrug DPX (Figure
3). Both DODPC and E-SM were remarkably stable due to the superior stability of the ether bond
and amide bond over the ester bond (e.g., a significant amount of DSPC hydrolyzed). Considering
the availability of GMP quality raw material, we chose E-SM as one of the key components of the
lipid matrix. Cholesterol is known to condense the lipid bilayer, thus it was selected as another
desired component of our formulation.
PEGylation has been shown to impact both drug loading efficiency and stability of liposome in
the circulation for certain vinca alkaloids [41]. We have evaluated two types of PEG lipids: the
negatively charged PEG-DSPE and the neutral PEG-DSG. The minor structural difference between
PEG-DSPE and PEG-DSG may have little impact on drugs that can be easily loaded into liposomes.
However, our lead compounds are more demanding on formulation due to their detergent-like property.
The drug loading results summarized in Table 1 clearly support the use of PEG-DSG in our
formulations. Presumably, the negatively charged PEG-DSPE can interact with partially positively
charged prodrugs and negatively impact the loading of the respective prodrugs.
Additionally, we found that the drug loading efficiency was negatively impacted once the
percentage of PEG-DSG in the liposome formulation was increased above certain membrane densities,
depending on the concentration of TEA-SOS used (Table 2). We have observed a concentration
dependence previously for vinorelbine [41]. Although the exact mechanism is not clear, it may be
related to the detergent-like property of the prodrug. The lipid bilayer can accommodate certain number
of detergent-like molecules such as PEG-DSG and the taxane prodrugs. During drug loading at
elevated temperature, the lipid bilayer is perturbed by the crossing prodrug and is liable to leakage.
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When the initial amount of PEG-DSG in the formulation is high, the lipid bilayer is less tolerant of the
transient accumulation of the prodrug in the bilayer. If too much prodrug is accumulated in the bilayer
due to the slow loading rate, the membrane can be disrupted leading to the leakage of SOS. Leaked
SOS can in turn precipitate the unloaded extraliposomal prodrug, resulting in poor loading efficiency.
The high SOS gradient from 1.1 N TEA-SOS enables fast and efficient drug loading. Ultimately, the
formulation was determined by the physicochemical property of the prodrug, the drug loading
efficiency, and the percentage of PEG-DSG needed to meet the circulation half-life requirement. No
significant change of circulation half-life was observed when PEG-DSG molar amount in the
formulation was reduced from 8% to 6% (unpublished data).
The targeting moiety is an integral part of immunoliposome design. The engineering of the ephrin
A2 ligand has been extensively studied in a separate report [37]. The scFv has been screened and
selected with the desired biophysical properties including: 1) specific binding to ephrin A2 with
suitable affinity, 2) ability to mediate liposome internalization, and 3) adequate thermal stability to
allow manufacturing.
4.3. Stability of liposomes
Acceptable storage stability is important for successful translation of liposomal drug to clinical
use. This is even more challenging when the taxane prodrugs are pH-sensitive and detergent-like.
We took a holistic approach by applying multiple features to the formulation simultaneously: a) a
stable lipid matrix comprised of E-SM/cholesterol/PEG-DSG; b) a strong SOS-drug complex to
minimize drug release during storage; c) an excessive internal SOS gradient to maintain low pH in
the aqueous core of liposome; and d) a mild acidic extraliposomal buffer (pH 5.5) and high ionic
strength (250 mM NaCl). Promising stability results were obtained for some of the formulations
evaluated (Table 3). The further optimized final formulation of prodrug 2 demonstrated a superior
stability profile after more than three years storage (ongoing stability studies) showing consistent
particle size, purity, strength, and binding activity.
4.4. Avidity effect of Immunoliposomes
The avidity effect is one of the advantages of immunoliposomes over ADCs. When multiple
ephA2 scFvs were attached on the liposome surface through the lipid anchor, their apparent
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equilibrium dissociation constant (KD as determined by SPR) changed from the initial 6.92 nM to
0.69 nM (Figure 6, Table 4). Such a dramatic increase of the apparent affinity may be explained by
the changes of instant on and off rates. Although the binding pocket of the scFv remains the same in
different formats and should have the same activity, its interaction with the ephrin A2 extracellular
domain can be affected by the environment. Free scFv, with a relatively low molecular weight (25
K Dalton), had a fast on-rate (7.38×105 M-1. S-1) and a fast off-rate (5.11×10-3 S-1). The liposome
showed approximately six times slower on-rate (1.21×105 M-1. S-1) than the free scFv, presumably
due to the steric hindrance of the liposome, but also a very slow off-rate (8.44×10-5 S-1) which was
likely due to the avidity effect from the multiple ligands on the liposome surface. Such an avidity
effect of immunoliposome allows the use of antibodies of moderate initial affinity to exert high
avidity as the immunoliposome format. The slow off-rate allows effective cellular uptake of ephrin
A2-targeted taxane liposomes at very low concentrations, which may be beneficial for targeting
metastatic tumor cells.
4.5. Concentration-dependent drug release
We have previously shown that increasing intraliposomal drug concentrations, using the same
trapping agent, can reduce the drug release rate from the same liposomes using liposomal vinorelbine
and liposomal irinotecan [24, 41]. Others have also observed this in liposomal vincristine
formulations loaded by the magnesium sulfate/ionophore technique [42]. As summarized in Table
5, all our liposome formulations showed long retention of encapsulated drug while in circulation.
The concentration-dependent (or D/L) drug release feature was observed for all three prodrugs. This
was consistent with their in vitro stability profiles observed at 30 C (Table 3). With this relationship
understood, we can tune the in vivo drug release rate of these liposomes by adjusting the D/L in the
formulation.
Table 5. Drug/lipid ratio-dependent in vivo drug release
Taxane
Nominal D/L
(g/mol)
In vivo release half-life*
(h)
Prodrug
300
600
150
223
312
47
1
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300
450
600
200
400
800
76
101
2
3
Data not fittable
110
305
Data not fittable
*: Average pharmacokinetics data from three mice were fitted with non-
compartment analysis (NCA) model using Phoenix WinNonlin.
4.6. High drug carrying capacity of the immunoliposomes
Typically, taxane drugs were incorporated in the liposome bilayer with very limited loading capacity
(3-4 mole %). Here, we demonstrated that high mole percentage of taxane prodrugs (as high as 114
mole % per mole phospholipid) can be stably encapsulated inside immunoliposomes through systematic
formulation development. The optimized immunoliposome has approximately 15 scFv on each
liposome and carries more than 30,000 taxane molecules per liposome. In contrast, ADCs typically
have a drug antibody ratio around 4 and no more than 8. Compared with ADCs, immunoliposomes
provide far higher drug carrying capacity and deserve further development efforts for clinical
translation.
5. Summary
In this study, we have developed ephrin A2-targeted liposome formulations for pH-sensitive
taxane prodrugs of which the optimized formulation comprised lipids matrix of E-
SM/cholesterol/PEG-DSG at 1/0.67/0.06 molar ratio and ephrin A2 scFv-PEG-DSPE at 5 g per mole
phospholipid. The lipid composition was chosen based on the storage stability of the primary bilayer-
forming phospholipid in the prototype taxane liposomes and the impact of both the PEG-
phospholipid chemical structure and density on loading of the taxane prodrugs. We created a process
to efficiently load detergent-like taxane prodrugs into the liposome by shifting the chemical
equilibrium from the extraliposomal weak prodrug complex to the stable intraliposomal prodrug-
SOS complex. The cryo-EM images confirmed the formation of dense complex inside the liposome.
We also determined the equilibrium dissociation constant for ephrin A2 scFv, the scFv-PEG-DSPE,
and the ephrin A2-targeted taxane liposome. With approximately 15 scFvs per liposome, the ephrin
A2-targeted liposome exhibited strong avidity effect and a very slow apparent dissociation rate. The
taxane liposomes also demonstrated good long-term storage stability, long circulating
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pharmacokinetics, and the rate of drug release that was dependent on the intraliposomal drug
concentration. In the preclinical mouse models, ephrin A2-targeted taxane liposomes demonstrated
strong antitumor activity against the human lung cell A549 and the triple negative breast cancer
xenograft models MDA-MB-436 and SUM149. After further preclinical development, the ephrin
A2-targeted liposome 2 has advanced to a Phase I clinical trial in patients with solid tumors
(NCT03076372).
Acknowledgment
s
The authors would like to thank NanoImaging Services for cryo-EM services, Murat Cirit for early
PK/PD modeling efforts on taxane liposomes, and the Merrimack protein group for ephrin A2 scFv
development.
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