Methods for the Preparation of Protein−Oligonucleotide−Lipid Constructs

Bioconjugate Chem. 2006, 17, 451−458

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Methods for the Preparation of Protein-Oligonucleotide-Lipid Constructs Jennifer Takasaki, Sameersingh G. Raney, Ghania Chikh, Laura Sekirov, Irina Brodsky, Ying Tam, and Steven M. Ansell* Inex Pharmaceuticals Corporation, 100-8900 Glenlyon Parkway, Burnaby, BC, V5J 5J8, Canada. Received February 24, 2005; Revised Manuscript Received November 21, 2005

A mixture of ionizable cationic lipids, steric barrier lipids, and colipids is used to encapsulate oligonucleotide DNA in lipidic particles called SALP. This material is under development as an adjuvant for vaccines. Previously we have shown that coupling the antigen directly to the surface of SALP can lead to enhanced immunological responses in vivo. Two different methods for preparing ovalbumin-SALP were assessed in this work. Originally the conjugates were prepared by treating SALP containing a maleimide-derivatized lipid with thiolated ovalbumin, a method we refer to as active coupling. This reaction was found to be difficult to control and generally resulted in low coupling efficiencies. The issues relating to this approach were characterized. We have recently developed alternative techniques based on first coupling ovalbumin to a micelle and then incubating the resultant product with SALP, methods we refer to as passive coupling. We have shown that this method allows accurate control of the levels of protein associated SALP and does not suffer from surface saturation effects seen with the active coupling method that places maximum limits on the amount of protein that can be coupled to the SALP surface. The products from the passive coupling protocol are shown to have activity comparable to those derived from the active coupling protocol in investigations of in vivo immune responses.

INTRODUCTION As part of a program for the development of drug delivery systems, an efficient means of formulating oligonucleotides in lipidic particles (1, 2) has been developed. These particles were formed through a self-assembly process after addition of an ethanolic solution of lipids to a solution of the oligonucleotide in a citrate buffer at pH ∼4.0. The lipids used included cholesterol, a bilayer-forming phospholipid (typically DSPC or POPC), a protonatable amino lipid, and a steric barrier lipid. Addition of the lipid solution to the buffer resulted in protonation of the amino lipid and formation of bilayer structures. The anionic oligonucleotide associates with these charged bilayer structures as they assemble, resulting in the formation of lipidic particles entrapping the oligonucleotide. The steric barrier lipid, typically a PEG-lipid, acts to moderate the rate of particle formation, regulating growth and inhibiting aggregation of particles. After the initial dilution, the preparation is extruded through a high-pressure filtration device and the external buffer replaced with one at pH ∼7.0 (usually HBS). At this pH the amino lipids are neutral. The final product is a lipidic particle that is stable in solution, with physical and pharmacokinetic properties much like liposomes of similar size. We refer to these particles as SALP (stabilized antisense lipid particles).1 During the biological characterization of SALP, a number of interesting observations were made. First, during early studies aimed at the investigation of the role played by the steric barrier molecule’s relative exchangeability on pharmacokinetic behavior, we noted that certain formulations would be rapidly cleared in vivo when administered on a repeat dose schedule (3). This effect was only observed with formulations with (a) PEG-lipids that did not exchange rapidly out of the bilayer on administration in vivo and (b) more than a certain level of encapsulated oligonucleotide. Further studies demonstrated that the clearance behavior was due to an immune response to PEG, * To whom correspondence should be addressed. Phone (604) 7331231. E-mail: [email protected].

and that this immune response was induced by the encapsulated oligonucleotide. Other studies with these oligonucleotide-lipid particles demonstrated that they were capable of potent enhancement of the immune stimulation associated with the CpG motif in oligonucleotides (4). This sequence had been shown to cause activation of macrophages, dendritic cells and B-cells, and release of various cytokines (5). These observations led us to investigate the use of SALP as adjuvants for vaccines. Initially we mixed the SALP with an antigen and found that coupling the antigen directly to the surface of the particle resulted in an enhanced biological response.2 These initial formulations were prepared using the classical protein-liposome coupling technique (6), namely derivatizing the protein with SPDP and coupling the resultant product (after deprotection) to SALP which had a maleimide lipid included in its composition. From a production point of view, the process of coupling a thiolated protein to the surface of a liposome or lipid particle is fraught with difficulty. Coupling efficiency tends to be variable 1 Abbreviations: SALP, stabilized antisense lipid particles; DSPC, 1,2-distearoyl-sn-glycero-3-phosphocholine; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; PEG, poly(ethylene glycol); HBS, hEPES-buffered saline; DSPE-ATTA2-MPA, N-(N′-(N′′-(3′′′-maleimidopropionoyl)-14′′-amino-3′′,6′′,9′′,12′′-tetraoxatetradecanoyl)-14′amino-3′,6′,9′,12′-tetraoxatetradecanoyl)-1,2-distearoyl-sn-glycero-3phosphoethanolamine; ATTA, 14-amino-3,6,9,12-tetraoxatetradecanoic acid; OVA, ovalbumin; MePEGS-2000-mCer, 1-O-(4′-O-(ωmonomethoxypoly(ethylene glycol)2000)succinoyl)-N-myristoylsphingosine; MePEGS-2000-DMG, 3-O-(4′-O-(ω-monomethoxypoly(ethylene glycol)2000)succinoyl)-1,2-dimyristoyl-sn-glycerol; DSPE-MPB, N-4′-(4′′-maleimidophenyl)butanoyl-1,2-distearoyl-sn-glycero-3-phosphoethanolamine; chol, cholesterol; DODMA, N,N-dimethyl-2,3-dioleyloxypropylamine; OGP, octyl β-D-glucopyranoside. 2 Yuan, Z. N., Brodsky, I., Ansell S. M., Klimuk, S.K, and Semple, S. C. (2005) Systemic and mucosal immune responses induced by liposomes with surface-coupled antigen and high levels of encapsulated immunostimulatory oligonucleotides. Unpublished data.

10.1021/bc050052j CCC: $33.50 © 2006 American Chemical Society Published on Web 01/12/2006

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and aggregation is a frequent problem (7). To study and characterize the in vivo effects of these particles we required systems that were reproducible and controllable; consequently, we required more robust methods of preparing these materials. A number of groups in recent years have attempted to solve many of the original problems associated with conjugating proteins directly to liposome surfaces by moving the point of attachment from the liposome surface to the end of PEG tethers (16, 17). In theory this should allow efficient conjugation while still allowing a formulation to have a PEG coating on the surface. However, by moving the reactive radius outside the steric barrier domain many of the advantages of such barriers in terms of aggregation control are potentially lost. In addition to this problem there are a number of other reasons why these systems are unsuitable for use in our particles. First, if a PEG based active lipid is used with an active coupling protocol, most of the active lipid would not conjugate to a protein and would still be present as free PEG attached to the surface. Since these compounds have relatively nonexchangeable anchors by design they would remain associated with the particle in vivo sufficiently long to generate an immune response in their own right. This would limit the use of the particle as a generic platform for vaccines since the application of one formulation in a patient would generate an immune response to the platform and would presumable render any other formulations useless in that patient. A second potential negative of the PEG tether approach relates to the exchangeability of the lipid-protein molecule once attached to the surface. The association-dissociation of a lipidmacromolecule from a bilayer surface is determined in part by the increase in freedom of motion of the macromolecule. PEG generally has considerable freedom of motion in solution, whereas most proteins and oligosaccharides are relatively rigid. Consequently, the exchange rates of PEG-lipids are much more sensitive to constraints on their freedom of motion than most biological macromolecules. Introducing a PEG tether between a macromolecule and a lipid anchor is expected to adversely affect the retention of the macromolecule in vivo, and this effect should become worse as the length of the tether increases. Finally, we were concerned with the chemical stability of the maleimide functions used in the active lipids during processing. These compounds are susceptible to hydrolysis, forming ring-opened maleamic acids which are fairly unreactive in the coupling reaction. This is not normally a major concern when preparing research quantities of various formulations since they can be prepared fast enough, but will be an issue for large scale preparations where processing times are usually significantly longer than lab scale. We have recently characterized general methods for an alternative method of preparing similar constructs, wherein the protein is coupled to a micelle, and the micelle lipids exchanged into liposome bilayers.3 These methods were found to be reproducible and controllable. In this work we describe the adaptation of these micelle-based methods to the production of protein-SALP particles, demonstrate the advantages of the new method, and show that constructs prepared by the micelle method are at least as efficacious as those prepared by traditional methods.

EXPERIMENTAL PROCEDURES Materials. Ovalbumin (grade V), cholesterol, and OGP were obtained from Sigma. MePEGS-2000-DMG (14) and DSPEATTA2-MPA3 were synthesized in our laboratories as described elsewhere. DPSC was obtained from Northern Lipids Inc., Vancouver. SIINFEKL peptide was synthesized on contract by 3 Takasaki, J., and Ansell, S. M. (2005) Micelles as intermediates in the preparation of protein-liposome conjugates. Unpublished data.

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Commonwealth Biotechnologies Inc., Richmond, VA. The oligonucleotides used were synthesized on contract by Trilink Biotech, San Diego, CA. CD8-FITC and TCR-cychrome were obtained from BDPharMingen, San Diego. Phycoerythrincoupled H-2Kb-SIINFEKL tetramers were synthesized on contract by Beckman Coulter Immunomics, San Diego, CA. Synthesis of DODMA. DODMA was synthesized using an adaptation of a previously described method (15). Briefly, a solution of 3-(dimethylamino)-1,2-propanediol in benzene was treated with an excess of potassium hydride with stirring for 1 h under argon. Oleyl bromide (1.5 equiv) was then added and the solution refluxed for 3 h. After workup, the product was isolated from the crude reaction mixture by column chromatography. Preparation of SALP. Solutions containing 26 µmol of lipid in 0.4 mL of ethanol and 4 mg of oligonucleotide in 0.6 mL of citrate buffer were warmed to 60 °C. The ethanol solution was then slowly added to the buffer while vortexing. The resultant suspension was passed 10 times through 2 × 100 nm filters using a pressurized extruding device. The SALP was then passed down a Sepharose CL-4B column in HBS to remove external oligonucleotide and to exchange the buffer. The quantities used were adjusted in proportion when larger amounts of material were required. Preparation of Micelle-Ovalbumin Conjugates. Ovalbumin was dissolved in HBS at a concentration of 30 mg/mL. An aliquot of from a stock solution of 2-iminothiolane in HBS corresponding to 1-5 equiv (depending on the particular experiment) was added to the ovalbumin solution and the mixture stirred at room temperature for 0.5 h. The solution was used without further purification. Lipid composed of the DSPE-ATTA2-MPA and a micelleforming lipid (DSPE-ATTAn, where n ) 2, 4, 8, or OGP) were weighed and combined. The lipid was dissolved in 1 part of ethanol, with warming if required, and diluted sequentially with 1 part of HBS, with vortexing and warming, until a total of 9 parts HBS had been added. The total volume should was such that the final concentration of lipid was approximately 10 mM. Any residual solid materials were removed by filtration through 0.22 micron filters. An aliquot of freshly prepared thiolated ovalbumin was added to freshly prepared maleimide-micelles in the appropriate ratio. In most cases this corresponded to 3000 g protein/mol micelle lipid. The micelle-protein mixture was allowed to stir at room temperature overnight. It was then either used immediately without additional purification or stored at 5 °C for use at a later date. Incubation of Protein-Micelles with SALP. SALP was mixed with ovalbumin-micelle conjugates in proportions appropriate for the particular experiment. Typically the standard preparation used a 1:19 micelle/SALP lipid ratio, corresponding to an initial protein/total lipid ratio of 150 g/mol. The SALPmicelle mixtures were incubated in a water bath at 60 °C for 30 min and then passed down a Sepharose CL-4B column (0.8 mL of sample/25 mL bed volume) using HBS as the running buffer. Assays. Lipid was assayed by determining the phosphate content as previously described (8). Samples containing oligonucleotide were analyzed for lipid content after processing as follows. An aliquot of the formulation (usually 100-250 µL) was diluted with 750 µL CHCl3/MeOH (1:2 vol/vol) in a 13 × 100 mm tube and vortexed for 1-2 min. A few drops of additional methanol were added if the solution was still turbid. Water (250 µL) and CHCl3 (250 µL) were then added and the solution vortexed for 1-2 min. The sample was then centrifuged at 3000 rpm for 10 min. The upper phase was carefully removed. An aliquot of water/methanol (1:1: 300 µL) was then added

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and the solution vortexed for 1-2 min. The solution was centrifuged at 300 rpm for 5 min and the upper layer carefully removed. The organic fraction was washed with water/methanol a further two times in the same manner. The organic fraction was first dried down under a nitrogen stream and then under vacuum. The lipid assay was then carried out as previously described. The protein content of the final liposome-protein conjugates was determined using a modified micro-BCA assay as previously described (8), with negative controls being performed where possible to check for background interference. Particle size, where determined, was measured using a Nicomp 380 submicron particle sizer. Oligonucleotide content was determined by diluting a 20 µL sample with 250 µL of distilled water, 1200 µL of methanol, and 500 µL of chloroform and determining absorbance at 260 nm relative to a similarly treated sample of HBS buffer. The oligonucleotide concentration was calculated using the equation [oligo (mg/mL)] ) 3.41 × A260 Immunization Protocols. 6- to 8-week-old female C57BL6 (H-2b) and BalbC (H-2d) mice were purchased from Harlan (Indianapolis, IN). All animal studies were completed using protocols that were approved by the institution’s animal care committee, and the methods used are consistent with the current guidelines of the Canadian Council of Animal Care. Mice (C57BL6 for cellular response and BalbC for humoral response; five animals in each test group) were immunized by injection sc with 100 µL of HBS alone or containing either 66 µg of ovalbumin, 66 of µg ovalbumin mixed with 100 µg of free 6295PS, 66 µg of ovalbumin mixed with 100 µg of 6295PS encapsulated in SALP, 73 µg of ovalbumin passively coupled to SALP containing 100 µg of 6295PS or 60 µg of ovalbumin actively coupled to SALP containing 100 µg of 6295PS. Mice received injections on day 0 and 7 for investigation of immunological cellular responses. Spleens were harvested 7 days after the last immunization and a cell suspension was generated for the tetramer assay. Mice received injections on day 0 and 14 for investigation of the humoral response. Blood was recovered by cardiac puncture after 42 days, placed in EDTA tubes, and centrifuged (4 °C, 2000 rpm, 10 min) to obtain plasma. The pooled plasma was examined for antibody induction by ELISA. Tetramer Assay. Specific CD8 T cells raised against the SIINFEKL epitope were assessed using a modified tetramer assay (9) as described by the manufacturer (Beckman-Coulter, Immunomics, San Diego, CA). Briefly, this consisted of incubation of 2 × 106 spleen cells with 1 µL of phycoerythrincoupled H-2Kb-SIINFEKL-tetramers for 1 h at room temperature. Samples were then labeled with anti-mouse CD8-FITC and TCR-Cychrome (BD PharMingen, San Diego, CA) for 20 min at 4 °C. After two washes with PBS 2% FBS, samples were analyzed using FACSort and CellQuest software (BecktonDickinson). Humoral Immunogenicity Studies. Ovalbumin-specific mouse IgG was detected and quantified by end-point dilution ELISA assay on samples of plasma pooled together from individual animals. Briefly, a solid phase of ovalbumin (100 µL of 10 µg/mL per well, overnight at 4 °C) was used to capture ovalbumin specific antibody from plasma samples serially diluted with 2% BSA-PBS. The antibody was then detected by treatment with horseradish peroxidase-conjugated rabbit antimouse IgG (1:3000 in PBS-BSA-2%; 100 µL/well), followed by incubation with TMB liquid substrate system (200 µL/well, 30 min at room temperature, light protected). The reaction was stopped by addition of 100 µL/well of 0.5 M sulfuric acid and the absorbance measured at 450 nm on an Elisa plate reader. End point dilution titers were defined as the highest dilution of

ovalbumin specific antibodies detected in plasma that resulted in absorbance value (OD450) two times greater than that of naive animal, with a cut off value of 0.1.

RESULTS We have drawn a distinction between coupling protocols where the conjugation reaction takes place at the liposome surface (active coupling) or at some point remote from the liposome surface (passive coupling).3 The traditional method of producing protein-liposome conjugates is an active coupling process in which a protein is first thiolated and then incubated with liposomes containing maleimide-derivatized lipids. Our first preparations of protein-SALP constructs were made using an active coupling protocol. The model antigen used in these studies was ovalbumin. The thiolation of the ovalbumin in the initial studies was performed using the reagent SPDP in HBS at pH 7.4. Once the protein had been derivatized, the protecting group had to be removed using DTT at pH 4.4. The external buffer was then replaced with HBS at pH 7.4 in preparation for the conjugation reaction. This process involves a large number of steps, and in later work the thiolation process was greatly simplified by using the reagent 2-iminothiolane (2-IT).3 The original SALP formulation had been developed with 10 mol % of the steric barrier lipid MePEGS-2000-Mcer as part of the lipid composition. The high levels of steric barrier lipids in these formulations presented a problem, since it is known that PEG-lipids in the bilayer inhibit the liposome-protein reactions required for preparing the construct. Most earlier work in the field of liposome-protein constructs had relied on zero length cross-linker active lipids, such as DSPE-MPB (6). In our work we have used an alternative lipid, DSPE-ATTA2-MPA. DSPE-ATTA2-MPA has a maleimide cap which is less susceptible to hydrolysis than that in DSPE-MPB and a cross-linker roughly equivalent to a 500 MW PEG in size. This lipid was chosen since aliphatic maleimides are supposedly more stable in aqueous solution than aromatic versions and since the small tether should provide greater accessibility of the reactive function on the bilayer surface. Effect of PEG-Lipids on SALP Stability and Coupling Efficiency. Earlier work showed that the presence of PEGlipids on a liposome surface inhibits the lipid-protein reaction, causing saturation of the surface to occur at lower concentrations. This effect results in a steady fall off in conjugation efficiency as the surface concentration of PEG rises, bottoming out at roughly 6 mol % PEG (18). However, significant amounts of PEG are required in SALP formulations to stabilize the construct during formation and prevent aggregation. Since these two requirements are mutually exclusive, we needed to determine what the minimum amount of PEG for a stable formulation of SALP actually was. A formulation experiment was conducted in which SALP was prepared with varying amounts of PEGlipid in the composition. The stability of the final product was assessed by doing particle sizing and seeing which samples showed evidence of aggregation (Figure 1). All samples showed roughly equivalent encapsulation efficiencies (50-70%), suggesting that particle formation was equivalent in all cases. However, particle sizes were uniform until the PEG concentration dropped to ∼2 mol %, at which point the particle size started to increase. The conclusion was that the minimum required PEG concentration in a SALP formulation was ∼2%. Previous work had shown that the range of PEG concentration that allowed effective conjugation in liposomes was 0-5 mol %. The cross-linker we were using in the current studies (DSPEATTA2-MPA) was rather larger than the cross-linker used in the earlier work, so it was not immediately obvious to what extent the SALP PEG-lipid would inhibit the coupling reaction.

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Figure 1. The effect of steric barrier lipid concentration on the stability of SALP particles. SALP composed of DSPC/chol/DODAP/MePEGS2000-DMG ((25 + n):45:20:(10 - n); where n ) 0, 5, 7, 8, and 9) was prepared with 6295 oligonucleotide encapsulated at an initial oligo/ lipid ratio of 0.15 g/mol. Encapsulation efficiencies ranged from 50 to 70%. Physical stability is assessed by the size of the product as determined using a Nicomp.

Figure 2. The effect of steric barrier molecules on coupling efficiencies when conjugating ovalbumin to SALP using the active conjugation method. Ovalbumin derivatized with 5 equiv of SPDP was treated with SALP composed of DSPC/chol/DODAP/MePEGS-2000-DMG/DSPEATTA2-MPA ((34 - n):45:20:n:1; n ) 2, 4, 6%). Initial protein/lipid ratios were 100 g/mol (solid bars), 200 g/mol (hatched left bars) and 400 g/mol (open bars). A negative control, where SALP (n ) 2) was incubated with underivatized ovalbumin, was included (hatched right bars).

We designed an experiment to determine what the effect of the SALP PEG concentration on coupling efficiency was. Ovalbumin was thiolated with 5 equiv of SPDP and conjugated to SALP containing 2, 4 or 6 mol % MePEGS-2000-DMG. Protein aliquots corresponding to an initial protein/lipid ratio of 100, 200, and 400 g/mol were incubated with samples of each SALP formulation, respectively. In addition, a negative control was performed for each SALP formulation, where the SALP was incubated with nonthiolated ovalbumin at 100 g/mol and worked up as usual. The results can be seen in Figure 2. Clearly the coupling efficiency drops off dramatically as the SALP PEG concentration increases. Increasing the initial protein/lipid ratio has only a small effect on the overall amount of protein coupled to the SALP surface, presumably since the surface is approaching saturation under these conditions. The response seen in the negative controls is due to background interference in the protein assay used. The implication of the experiments presented in Figures 1 and 2 is that for active coupling protocols the competing requirements that the levels of PEG in SALP be high enough

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to inhibit aggregation but low enough to allow reasonable coupling efficiencies means that in general anyone using these sorts of coupling protocols has a very narrow range of PEG concentration to work with. Small uncertainties in the PEG concentration might have dramatic effects on the coupling efficiency. In addition, the surface saturation effect caused by the presence of the steric barrier molecules during coupling means that the amount of protein that can be conjugated to the surface is relatively small. These limitations mean that the active coupling protocol is unattractive from a production point of view. Effect of PEG-Lipids and Micelle-Forming Lipids on the Passive Conjugation Protocol. In a companion report we have described the characterization of a passive coupling technique in which the protein is first coupled to the surface of a micelle and then exchanged into liposome bilayers.3 This method has a number of advantages over active coupling when applied to SALP. First, since the coupling reaction is performed at the micelle surface, the presence of PEG-lipids in SALP will not interfere. It should be possible to obtain a range of reproducible levels of protein at the SALP surface since the protein-lipid conjugates can be titrated into the liposomes. The lack of interference from the SALP PEG-lipids would also mean that in principle it should be possible to obtain much higher levels of protein on the SALP surface, if required. The second advantage of the passive conjugation method is that it separates the SALP formulation step from the protein-lipid coupling step. When using the active conjugation method it is necessary to have both the SALP preparation and the protein thiolation process occurring simultaneously because of the potential for competing side reactions to quench the reactive functionality of the reagent. This presents potential logistical problems when scaling the process up since larger scale processes tend to take longer periods of time. With the passive conjugation method the two components can be prepared independently and combined at the appropriate time. We conducted a study looking at the effects of various micelle-forming lipids and different PEG-lipid levels in SALP when using the passive conjugation method. SALP was prepared with 2, 4, and 10% PEG-lipid as part of the composition. The 2% formulation represented the lower limit for physical stability as determined in Figure 1. The 10% formulation represented the “classical” SALP particle, whereas the 4% one was our guess at the “ideal” SALP formulation for passive conjugation methods. A POPC/chol liposome formulation was also included in the data set to serve as a control. Micelles were prepared using DSPE-ATTA2-MPA as the active lipid in a 1:4 ratio with the micelle-forming lipid. The micelle-forming lipids used in this study were DSPE-ATTA2, DSPE-ATTA4 and DSPEATTA8. The results are shown in Figure 3. DSPE-ATTA4 was found to be the best micelle-forming colipid in the group studied for Figure 3, similar to earlier work with POPC/chol liposomes.3 DSPE-ATTA2 generally gave somewhat lower coupling efficiencies; however, this is likely due to the tendency of DSPE-ATTA2-MPA/DSPE-ATTA2 micelles to show some thermotropic behavior at room temperature. The much larger DSPE-ATTA8 resulted in sharply lower coupling efficiencies. This is comparable to the results we had seen earlier with MePEGS-2000-DSPE liposomes and is likely a result of steric inhibition of the micelle-protein reaction. The SALP formulations generally resulted in slightly lower final protein/lipid ratios than regular control liposomes. The reason for this is not clear, but it might be related to the fact that SALP frequently forms multilamellar structures, which would result in a lower particle protein/lipid ratio. The most interesting aspect of this experiment was the observation that the PEG level in the SALP did not appear to affect the final protein/lipid ratio. This is sharp contrast to the active coupling method, where 10

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Figure 3. The effect of steric barrier molecules on coupling efficiencies when conjugating ovalbumin to SALP using the passive conjugation method. Ovalbumin was thiolated with 5 equiv of SPDP and conjugated to DSPE-ATTA2-MPA/DSPE-ATTAn (1:4) micelles in a 3000 g/mol protein/lipid ratio. DSPE-ATTAn used were as follows: n ) 2, hatched left bars; n ) 4, open bars; and n ) 8, hatched right bars. Micelles were incubated with DSPC/chol/DODAP/MePEGS-2000-DMG ((25 + n):45:20:(10 - n); n ) 0, 6, 8) SALP or POPC/chol (55:45) liposomes (“Ctrl” in the figure) at 150 g/mol protein/lipid. The SALP was prepared using 6295 PS oligonucleotide at a 0.15 oligo/lipid ratio.

mol % of PEG would effectively shut down the conjugation reaction. This raised an interesting question, since we knew that PEG was capable of preventing surface conjugation reactions by inhibiting access of the protein to the reactive surface. The most reasonable explanation of the difference was that once coupled to a lipid anchor, the protein-lipid conjugate would behave like any other steric barrier molecule and would compete for a place on the liposome surface with the PEG-lipids already there. Given enough time an equilibrium should be reached based on the thermodynamic states of the molecules at the surface or in solution. If this is true, then less exchangeable PEG-lipids should still inhibit exchange of the protein-lipid conjugates from micelles, while exchangeable analogues are competed off. To determine if this is the case we set up an experiment where protein-micelles were incubated with POPC/chol liposomes with varying amounts of MePEGS-2000-DSPE (non exchangeable) or MePEGS-2000-DMG (exchangeable). The results are shown in Figure 4. In this experiment PEG-lipids start to inhibit association of the protein-lipid conjugates with the liposomes when levels exceed 4 mol %. The effect is more pronounced with the nonexchangeable PEG-lipid but takes place with both species. Presumably SALP formulations, which contain MePEGS-2000DMG, do not anchor the PEG-lipid as well as a POPC/chol liposome since no such drop-off is observed at high PEG-lipid concentrations. It is likely however, that the effect may still take place in SALP formulations to a greater or lesser extent based depending on the conditions of the final formulation step. With that in mind it is advisable to use a 4 mol % PEG SALP formulation. Comparison between the Active and Passive Coupling Protocols. An additional experiment was performed to characterize the surface saturation effect when using the active coupling protocol. SALP containing 2 mol % MePEGS-2000DSPE and 1 mol % DSPE-ATTA2-MPA was coupled to ovalbumin which had been thiolated with 2 or 5 equiv of 2-IT. Each data set was repeated with initial protein/lipid ratios ranging from 50 to 300 g/mol. The same two thiolated ovalbumin solutions were used to prepare ovalbumin-micelle reagents, which in turn were used to prepare protein-SALP conjugates under our standard passive conjugation conditions.

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Figure 4. The effect of the exchangeability of PEG-lipids in liposomes on the passive coupling protocol. Ovalbumin was thiolated with 5 equiv of 2-IT and conjugated to DSPE-ATTA2-MPA/OGP (1:4) micelles in a 3000 g/mol protein/lipid ratio. Micelles were incubated with POPC/ chol/PEG-lipid (55 - n:45:n; n ) 0, 2, 4, 6, 10) liposomes at 150 g/mol protein/lipid. PEG-lipids used included MePEGS-2000-DSPE (open bars) and MePEGS-2000-DMG (hatched right bars). The solid bar is the 0% PEG-lipid sample.

Figure 5. Comparison of active and passive coupling protocols. Ovalbumin was thiolated with 2 or 5 equiv of 2-IT. Thiolated ovalbumin (2 equiv of 2-IT: open bars; 5 equiv of 2-IT: hatched right bars) was coupled to DSPC/chol/DODMA/MePEGS-2000-DMG/DSPE-ATTA2MPA (32:45:20:2:1) SALP at initial protein/lipid ratios of 50, 100, 150 and 300 g/mol. The same two thiolated ovalbumin solutions were coupled to DSPE-ATTA2-MPA/OGP (1:4) micelles in a 3000 g/mol protein/micelle lipid ratio. These micelles were incubated with DSPC/ chol/DODMA/MePEGS-2000-DMG (31:45:20:4) SALP at 150 g/mol protein/final lipid, with the final results shown as the solid bars. All SALP was prepared with 6295 PS oligonucleotide at 0.15 g/mol oligo/ lipid.

The results are shown in Figure 5. It is clear that saturation of the surface is occurring at relatively low levels, since increasing the initial thiolated protein concentration has minimal effects on the final surface protein concentration. In addition the maximum surface concentration is quite low. In contrast the passive conjugation samples showed much higher levels of bound protein. The passive conjugation efficiency is largely regulated by saturation of the micelle surface. This means that it is possible to prepare relatively uniform samples if an excess of initial protein is used. Since the SALP PEG concentration does not inhibit association of the protein-lipid with the SALP surface (unlike the reaction inhibition seen with active coupling protocols), it should be possible to accurately titrate a specific amount of protein into a SALP formulation. We conducted an experiment where an ovalbumin-micelle reagent was prepared and then titrated into a SALP formulation. The results are shown

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Figure 6. Titration of ovalbumin-micelles into SALP. Ovalbumin was thiolated with 5 equiv of 2-IT and coupled to DSPE-ATTA2-MPA/ OGP (1:4) micelles at 3000 g/mol protein/lipid. Portions of this ovalbumin-micelle solution corresponding to 2, 4, 6, 8 and 10% of the SALP lipids were added to DSPC/chol/DODMA/MePEGS-2000DMG (31:45:20:4) SALP prepared using 6295 PS oligonucleotide at 0.15 g/mol oligo/lipid.

in Figure 6. As expected, the final protein-lipid ratio shows a uniform rate of increase in the surface protein levels in proportion to the amount of the reagent added to the SALP. The implication of this experiment is that not only is it possible to prepare SALP with relatively high levels of protein on the surface, it is also feasible to prepare samples with predetermined levels. This aspect is very important if the mechanism of in vivo action is to be properly characterized. Stability and Reproducibility during Formulation. Analysis of the oligonucleotide content of the samples in Figure 6 before and after incorporation with the micelles indicated that the treatment did not cause loss of entrapped oligonucleotide. However, since the micelles are detergents, it is likely that at some point sufficient detergent will be present to disrupt the integrity of the SALP particles. We have not determined what this point actually is, at best we can say that treatment with up to 10 mol % micelles will not disrupt the SALP. It may be possible to go significantly higher than this, provided that excess micelle-forming lipid is removed through some means, such as by dialysis. As a final demonstration of the utility of the method we performed a reproducibility study using the passive coupling protocol. Three sets of protein-micelles were prepared independently and incubated with samples taken from a single batch of SALP (Figure 7). After workup the products were assayed for size, protein/lipid ratio, and oligonucleotide/lipid ratio. The three samples were reassayed after one week of storage at 5 °C. The purpose of the experiment was to demonstrate that material prepared according to standard conditions would yield consistent results. This turned out to be the case in terms of levels of bound protein. The specific conditions used for the coupling generally result in limited aggregation. This aggregation is caused by proteins on the bilayer surface that have been derivatized by more than one lipid anchor. If one of the three batches included protein which was derivatized to a greater or lesser degree than the other batches, it would be reflected in a change in size even though the levels of bound protein were the same. The size differences between the three data sets was minimal, indicating that the protein in all three data sets was largely in the same state. Storage of the samples for a week did not appear to adversely affect the integrity of the constructs. These results suggest that the method potentially has a high degree of reproducibility and result in relatively stabile constructs.

Figure 7. Reproducibility of the passive coupling protocol when preparing protein-SALP conjugates. Three separate ovalbumin solutions were thiolated with 5 equiv of 2-IT and coupled to three individual DSPE-ATTA2-MPA/OGP (1:4) micelle solutions at 3000 g/mol protein/ lipid. These three sets of solutions were incubated at 150 g/mol protein/ lipid with samples of a single DSPC/chol/DODMA/MePEGS-2000DMG (31:45:20:4) SALP batch prepared using 6295 PS oligonucleotide at 0.15 g/mol oligo/lipid. The samples were analyzed at the time of production and again after one week of storage at 5 °C.

The effects of the structure of the oligonucleotide was also studied. SALP was prepared using the following oligonucleotides: 6295 PS (5′-TAACGTTAGGGGCAT-3′; phosphothioate chemistry); 6295 PO (5′-TAACGTTAGGGGCAT-3′; phosphodiester chemistry); 5001 PS (5′-AACGTT-3′; phosphothioate chemistry); and 2006 PS (5′-TCGTCGTTTTGTCGTTTTCTCGTT-3′; phosphothioate chemistry). The structure of an oligonucleotide plays a role in the encapsulation efficiency, and a wide range was observed in this case (75%, 47%, 13%, and 27%, respectively). All four SALP formulations were treated with the same protein-micelle reagent (initial protein/lipid ration of 150 g/mol) as usual. All four formulations showed similar levels of conjugated protein, with minimal size changes compared to the original SALP controls. The results demonstrate that the coupling process produces similar products irrespective of the structure of the oligonucleotide used. In Vivo Immunological Behavior. In a companion report2 we have shown that coupling an antigen to the surface of SALP results in enhanced humoral responses in vivo compared to a sample where the antigen and SALP were mixed but not coupled. The material used in that study was conjugated using the active coupling protocol. While it is reasonable to presume that the antigen-SALP conjugate prepared by the passive conjugation protocol would be equivalent, we needed to demonstrate that this was indeed the case. An experiment was conducted to compare the basic in vivo cellular and humoral responses to treatment with ovalbumin-SALP prepared by the two methods.

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Protein−Oligonucleotide−Lipid Constructs

Figure 8. Assessment of cellular response induced against OVA antigen delivered coupled with SALP-6295 PS either with active or passive protocol. Mice were immunized sc at day 0 and 7 (see Materials and Methods) with either HBS, OVA protein (66 µg), OVA (66 µg) mixed with free 6295PS (100 µg), OVA (66 µg) mixed with 6295PS (100 µg) encapsulated in SALP, OVA (73 µg) passively coupled to SALP containing 6295PS (100 µg), or OVA (60 µg) actively coupled to SALP containing 6295PS (100 µg). Seven days after the last injection spleens were assessed for CD8 T cell induced against SIINFEKL epitope using the tetramers assay. Y-axis report % of CD8+/TCR+/ OVA-tet+ among gated lymphocytes.

C57BL6 mice were injected with various controls and ovalbumin-6295 SALP conjugates prepared by the active and passive techniques on days 0 and 7. The spleens were recovered after a further 7 days and the level of CD8 T cells specific for an epitope derived from ovalbumin (SIINFEKL) determined by flow cytometry. The results are shown in Figure 8. As expected, the negative controls (HBS, SALP, and ovalbumin individually) show similar background levels of response. Mixing free oligonucleotide with ovalbumin did not cause proliferation of specific T cells, whereas oligonucleotide encapsulated in SALP and mixed with ovalbumin did. A similar response was seen with ovalbumin-SALP conjugates prepared using the active conjugation method. A much stronger response was observed when the ovalbumin-SALP sample prepared by the passive conjugation protocol was tested. The reason for the difference is not clear at this point. All the samples were normalized to the oligonucleotide content on administration, and consequently protein and lipid content may not be entirely comparable. We plan to do more detailed studies with these systems to determine which formulation parameters affect efficacy and will report that data when it becomes available. In the meantime, however, for the purposes of this work we can state that the passive coupled conjugate is at least as effective as the active coupled material in the stimulation of SIINFEKL reactive T-cell proliferation. In a second in vivo experiment samples and controls were injected into BalbC mice to investigate humoral responses to the formulations. The treatment was repeated after two weeks and the blood from the animals recovered after a further four weeks. Plasma was recovered from the pooled blood and the level of ovalbumin specific IgG determined using a serial dilution ELISA assay. The results are shown in Figure 9. The negative controls (HBS and SALP) produced no response, as expected. Positive controls (ovalbumin, SALP + ovalbumin, and oligonucleotide + ovalbumin) generated similar responses.

Figure 9. The effect of coupling on humoral immunonogenicity. Plasma obtained six weeks upon SC prime-boost immunization with ovalbumin mixed (66 µg), actively (73 µg), or passively coupled (60 µg) with 100 µg of free or SALP encapsulated 6295PS ODN. IgG levels measured by end-point dilutions ELISA. Data points represent the Mean value from group of four animals.

The two ovalbumin-SALP conjugates (prepared by the active and passive conjugation methods, respectively) produced responses 2 orders of magnitude greater than the positive controls. This demonstrated the increased efficacy associated with coupled systems as opposed to mixed analogues. More importantly, it shows that the products derived from the two methods are more or less equivalent in terms of inducing a humoral response.

DISCUSSION Active coupling protocols have been widely used to produce protein-liposome conjugates. While these methods are relatively straightforward to use on lab scales, they suffer from a number of problems. These range from the lack of fine control over the reaction to logistical problems on scale-up. More recent work (10, 11) has shown that passive coupling protocols may greatly simplify the logistics of scaling a preparation up. Using these ideas as a lead we have demonstrated that, with proper selection of materials and appropriate experiment design, it is possible to prepare protein-liposome conjugates with a relatively high degree of precision using passive coupling protocols. In addition, the passive coupling protocols greatly simplify many of the workup processes involved in the production of the final conjugate. SALP type formulations are particularly troublesome to deal with when using the active coupling protocol since the formulation requires relatively large amounts of PEG lipid in order to stabilize the particle. This has two effects, namely that it is very difficult to reproduce a specific level of coupled protein, and the surface becomes saturated at relatively low levels. The passive coupling technique solves many of these problems since it separates the process of forming the protein-lipid conjugate from the process of forming the protein-particle conjugate. The initial coupling event in the passive conjugation process occurs at the surface of a micelle, which acts as a surrogate for the SALP surface. Since the micelle has a much smaller surface area than the final SALP particle, it is possible to saturate this surface with protein and then use the resultant protein-micelles as a reagent for preparing the final protein-SALP conjugate. By saturating the micelle surface we are effectively placing an upper limit on the amount of protein that can in principle

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associate with the micelles, and this fact allows us to produce micelles with reproducible protein/lipid ratios. A second advantage of using micelles in this manner is that once formed, the protein-lipid conjugate in the micelle essentially becomes yet another steric barrier lipid when exposed to SALP and consequently can compete for spots on the surface of the SALP with the endogenous PEG-lipids. Once there it would be subject to the same thermodynamic influences that determine exchange rates, namely the enthalpy component derived from association of the lipid anchor with bilayer lipids (drives association with the bilayer) and the entropy factor associated with an increase in freedom of motion of the macromolecule on dissociation into free solution (drives disassociation from the bilayer). The PEG-lipids used in SALP are based on an uncharged dimyristoyl lipid anchor (DMG) and as such dissociate from the bilayer very readily (12, 13). The protein-lipid conjugates, on the other hand, use DSPE as a lipid anchor. DSPE is known to provide a much stronger association with the bilayer than DMG. In addition, even though proteins are much larger than the PEG molecules used as steric barrier molecules, they are relatively rigid in free solution and consequently do not have the same entropy contributions that PEG-lipids do. The result of this is that these DSPE-protein conjugates should compete out the PEG-DMG on the SALP surface, producing much higher levels of protein on the surface than would be possible using active conjugation methods. The effect of constraining freedom of motion of macromolecules on liposome surfaces has been shown in work carried out a number of years ago by Silvius (12). He studied interbilayer exchange rates using fluorescent labeled PEG lipids which made up 0.5 mol % of the donor membranes. In that work it was noted that including 4% of unlabeled PEG-lipid significantly increased the off rate of the fluorescent label, thereby providing strong evidence for the effect of steric interactions between individual PEG molecules. Silvius also showed that rigid macromolecule-lipids, such as the protein transferrin and dextrans in the size range 10-70 kDa, had much lower off rates than PEG analogues (2 kDa and 5 kDa) even though they are considerably larger in size. Also, while the off rate for PEG lipids showed a clear relationship to polymer size, rigid polymers such as dextran did not. The data presented by Silvius is consistent with the arguments made in this work. By being able to control both the amount of protein associated with the micelles, and the amount of the protein-micelles titrated into SALP, we were able to demonstrate that it was possible to produce a series of protein-SALP samples with accurate protein/lipid ratios. In doing so we have opened the door for future studies investigating the role that the relative amounts of protein/lipid/oligonucleotide play in in vivo efficacy. From a production point of view this technology allows us to produce sophisticated formulations with an unparalleled degree of accuracy. The separation of the SALP formation, proteinlipid conjugate formation, and protein-SALP formulation into three distinct and independent steps greatly improves the logistics of scale-up. Finally, we have shown that the in vivo efficacy of the product produced by the passive coupling protocol is at least as good as that of the material produced by the active coupling process. Supporting Information Available: Experimental details. This material is available free of charge via the Internet at http:// pubs.acs.org.

LITERATURE CITED (1) Semple, S. C., Klimuk, S. K., Harasym, T. O., Dos Santos, N., Ansell, S. M., Wong, K. F., Maurer, N., Stark, H., Cullis, P. R., Hope, M. J., and Scherrer, P. (2001) Efficient encapsulation of

Takasaki et al. antisense oligonucleotides in lipid vesicles using ionizable aminolipids: formation of novel small multilamellar vesicle structures. Biochim. Biophys. Acta 1510, 152-166. (2) Maurer, N., Wong, K. F., Stark, H., Louie, L., McIntosh, D., Wong, T., Scherrer, P., Semple, S. C., and Cullis, P. R. (2001) Spontaneous entrapment of polynucleotides upon electrostatic interaction with ethanol-destabilized cationic liposomes. Biophys. J. 80, 2310-2326. (3) Semple, S. C., Harasym, T. O., Clow, K., Ansell, S. M., Klimuk, S. K., and Hope, M. J. (2004) Immunogenicity and rapid clearance of lipososmes containing polyethylene glycol-lipid conjugates and nucleic acid. J. Pharmacol. Exp. Ther. 312, 1020-1026. (4) Mui, B., Raney, S. G., Semple, S. C., and Hope, M. J. (2001) Immune stimulation by a CpG-containing oligodeoxynucleotide is enhanced when encapsulated and delivered in lipid particles. J. Pharmacol. Exp. Ther. 298, 1185-1192. (5) Krieg, A. M., Yi, A. K., and Hartman, G. (1999) Mechanisms and therapeutic applications of immune stimulatory CpG DNA. Pharmacol. Ther. 84, 113-120. (6) Loughrey, H. C., Choi, L. S., Cullis, P. R., and Bally, M. B. (1990) Optimized procedures for the coupling of proteins to liposomes. J. Immunol. Methods 132, 25-35. (7) Harasym, T. O., Tardi, P., Longman, S. A., Ansel, S. M., Bally, M. B., Cullis, P. R., and Choi, L. S. L. (1995) Poly (ethylene glycol)modified phospholipids prevent aggregation during covalent conjugation of proteins to liposomes. Bioconjugate Chem. 6, 187-194. (8) Ansell, S. M., Tardi, P. G., and Buchkowsky, S. S. (1996) 3-(2pyridyldithio)propioinc acid hydrazide as a cross linker in the formation of liposome-antibody conjugates. Bioconjugate Chem. 7, 490-496. (9) Gallimore, A., Glithero, A., Godkin, A., Tissot, A. C., Plunckthun, A., Elliott, T., Hengartner, H., and Zinkernagel, R. (1998) Induction and exhaustion of lymphocytic choriomeningitis virus-specific cytotoxic T lymphocytes visualized using soluble tetrameric major histocompatibility complex class I-peptide complexes. J. Exp. Med. 187, 1383-1393. (10) Iden, D. L., and Allen, T. M. (2001) In vitro and in vivo comparison of immunolipososmes made by conventional coupling techniques with those made by a new post-insertion approach. Biochim. Biophys. Acta 1513, 207-216. (11) Supra, P., and Allen, T. M. (2004) Improved outcome when B-cell lymphoma is treated with combinations of immunoliposomal anticancer drugs targeted to both CD19 and CD20 epitopes. Clin Cancer Res. 10, 2530-257. (12) Silvius, J. R., and Zuckermann, M. J. (1993) Interbilayer transferr of phospholipids-anchored macromolecules via monomer diffusion. Biochemistry 32, 3153-3161. (13) Rex, S., Zuckerman, M. J., Lafleur, M., and Silvius, J. R. (1998) Experimental and monte carlo simulation studies of the thermodynamics of polyethylene glycol chains grafted to lipid bilayers. Biophys. J. 75, 2900-2914. (14) Ambegia, E., Ansell, S., Cullis, P., Heyes, J., Palmer, L., and MacLachlan, I. (2005) Stabilized plasmid-lipid particles containing PEG-diacylglycerols exhibit extended circulation lifetimes and tumor selective gene expression. Biochim. Biophys. Acta 1669, 155-163. (15) Eppstein, D. A., Felgner, P. L., Gadek, T. R., Jones, G. H., and Roman, R. N. (1990) N[ω,(ω-1)-dialkyloxy]- and N-[ω,(ω-1)dialkenyloxy]-alk-yl-N,N,N-tetrasubstituted ammonium lipids and uses therefore. US Patent 4,897,355. (16) Hansen, C. B., Kao, G. Y., Moase, E. H., Zalipsky, S., and Allen, T. (1995) Attachment of antibodies to sterically stabilized liposomes: evaluation, comparison and optimization of coupling procedures. Biochim. Biophys. Acta 1239, 133-144. (17) Kirpotin, D., Park, J. W., Hong, K., Zalipsky, S., Li, W. L., Carter, P., Benz, C. C., and Papahadjopoulos D. (1997) Sterically stabilized anti-HER2 immunoliposomes: design and targeting to human breast cancer cells in vitro. Biochemistry 36, 66-75. (18) Ansell, S. M., Harasym, T. O., Tardi, P. G., Buchkowsky, S. S., Bally, M. B., and Cullis P. R. (2000) Antibody conjugation methods for active targeting of liposomes. In Drug Targeting: Strategies, Principles and Applications (Francis, G. E., and Delgado, C., Eds.) pp 51-68, Chapt. 4, Humana Press, Totowa, NJ. BC050052J