ARTICLE
`
`doi:10.1016/j.ymthe.2005.09.014
`
`Hypersensitivity and Loss of Disease Site Targeting Caused
`by Antibody Responses to PEGylated Liposomes
`
`Adam Judge, Kevin McClintock, Janet R. Phelps, and Ian MacLachlan*
`
`Protiva Biotherapeutics, 100-3480 Gilmore Way, Burnaby, BC, Canada V5G 4Y1
`
`*To whom correspondence and reprint requests should be addressed. Fax: +1 604 630 5103. E-mail: ian@protivabio.com.
`
`Available online 7 November 2005
`
`The systemic application of nucleic acid drugs requires delivery systems that overcome the poor
`pharmacokinetics,
`limited biodistribution, and inefficient uptake of nucleic acids. PEGylated
`liposomes show considerable promise because of their intrinsic ability to accumulate at disease
`sites and facilitate transfection of target cells. Unlike many viral vectors, PEGylated liposomes are
`generally considered to be nonimmunogenic. We have developed a PEGylated liposome for the
`systemic administration of plasmid DNA that achieves high levels of selective gene expression at
`distal tumor sites. Here we report that the in vivo efficacy and safety of these systems can be severely
`compromised following repeat administration. This phenomenon is characterized by a loss of
`disease site targeting, accelerated clearance from the blood, and acute hypersensitivity. These
`effects are fully attributable to a surprisingly robust, long-lived antibody response generated against
`polyethylene glycol (PEG) that results from the strong adjuvant effect of the plasmid payload.
`Importantly, immunogenicity may be substantially reduced by modifying the alkyl chain of the PEG–
`lipid conjugate, thereby allowing successful repeat dosing of the modified plasmid formulations
`without adverse side effects. Immunogenicity is a relevant concern for a number of nonviral delivery
`systems given the potent immunostimulatory properties of many nucleic acid drugs.
`
`Key Words: non-viral vectors, immunogenicity, nucleic acid-based drugs, liposomes,
`antibody responses, polyethylene glycol, hypersensitive reactions
`
`INTRODUCTION
`Liposomes are an attractive drug delivery system for a
`diverse array of therapeutic agents due to their relatively
`high stability in the blood and intrinsic ability to
`extravasate into tissues with increased vascular perme-
`ability, such as solid tumors and sites of inflammation
`[1,2]. This so-called bpassive disease site targetingQ can be
`facilitated by the incorporation of polyethylene glycol
`(PEG) into liposomes to provide a steric barrier against
`opsonization and clearance by the reticuloendothelial
`system (RES) [3,4]. These attributes have been exploited
`in the field of oncology by a number of liposomal
`chemotherapeutic [2,5] and scintigraphic agents [1].
`PEGylated liposomes also show significant potential for
`developing nucleic acids as therapeutic agents, partic-
`ularly in applications requiring systemic administration.
`Lipid encapsulation of RNA or DNA provides protection
`from intravascular nuclease degradation, passive target-
`ing to disease sites and can enhance the intracellular
`delivery of nucleic acids that are otherwise poorly taken
`up by cells [6].
`
`Administration of many nucleic acids can cause
`activation of the mammalian immune system, leading
`to the release of interferons and proinflammatory cyto-
`kines. In the case of DNA,
`immune stimulation is
`triggered primarily by the recognition of unmethylated
`CpG sequence motifs by Toll-like receptor-9 (TLR9) [7]
`located within the endosomal compartment of antigen-
`presenting cells (APC), including B cells [8,9]. Similar
`immune recognition pathways are also activated by
`exogenous single [10,11] and double-stranded RNA [12]
`through TLR7/8 and TLR3, respectively. In this context,
`we [13] and others [14,15] have recently reported that
`synthetic siRNA, under development as a therapeutic
`mediator of RNA interference, can also induce potent
`immune stimulation. These immune responses elicited
`by nucleic acids can be greatly potentiated by the use of
`delivery vehicles that facilitate cellular uptake [13,16].
`Although the immunomodulatory effects of CpG DNA
`are now being harnessed therapeutically in oncology and
`allergy applications [17], in many cases immune activa-
`tion represents an additional hurdle to drug development
`
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`doi:10.1016/j.ymthe.2005.09.014
`
`ARTICLE
`
`due to the significant toxicities associated with excessive
`cytokine release and the potential for the drug carrier to
`be rendered immunogenic.
`It has long been recognized that liposomes can act as
`immunological adjuvants as a result of their particulate
`nature, efficient uptake by APC, and ability to crosslink
`surface receptors [18], and this property is enhanced
`when immunostimulatory agents such as CpG DNA are
`incorporated into the liposomes [19,20]. This has been
`exploited in the design of liposomal vaccines that
`generate strong antibody (Ab) responses against weakly
`immunogenic antigens grafted onto the liposome sur-
`face. It is therefore unsurprising that immunogenicity has
`proven to be a major obstacle in developing receptor-
`targeted liposomes that incorporate antibodies, peptides,
`or receptor ligands on their surface to enhance target cell
`uptake [21–23]. The addition of a PEG coating to these
`liposomes typically has a minor effect on reducing their
`immunogenicity [19,21,23].
`We have developed stable plasmid lipid particles (SPLP)
`as a nonviral systemic vector for the expression of
`therapeutic pDNA at disease sites such as tumors and sites
`of inflammation [24,25]. SPLP consist of a PEGylated
`liposome that fully encapsulates a single copy of plasmid
`DNA, thereby conferring protection from nuclease degra-
`dation and extended blood circulation times following
`systemic administration [24,25]. Here we report that the in
`vivo efficacy and safety of these systems can be severely
`compromised following repeat administration due to a
`surprisingly robust Ab response against PEG that arises
`from the primary administration. Importantly, the immu-
`nogenicity of the PEGylated liposomes can be significantly
`reduced by modification of the PEG–lipid component,
`allowing for the safe and effective re-administration of the
`formulated pDNA. Our findings raise important concerns
`regarding the potential immunogenicity of delivery
`vehicles currently under consideration for use with
`immunostimulatory nucleic acid-based drugs, including
`pDNA, siRNA, and antisense oligodeoxynucleotides.
`
`RESULTS AND DISCUSSION
`Disease Site Targeting and Blood Clearance of
`PEGylated Liposomes
`To demonstrate the utility of these systems for the delivery
`of nucleic acids to distal tumor sites, we encapsulated a
`CMV–luciferase reporter plasmid into SPLP containing 10
`mol% PEG-S-DSG (Luc-SPLP). A single intravenous (iv)
`administration of Luc-SPLP (5 mg/kg pDNA) into A/J mice
`bearing subcutaneous Neuro2a tumors on the hind flank
`resulted in significant reporter gene expression at the
`distal tumor site 48 h after administration (Fig. 1A).
`Transgene expression within other, nontarget organs
`including the liver, lungs, spleen, kidney, and heart was
`low (Fig. 1A). We obtained similar results in a CT26 liver
`metastases model in Balb/C mice (not shown).
`
`FIG. 1. Repeat administration of PEGylated liposomes is associated with loss of
`tumor-targeted transgene expression and accelerated blood clearance. (A)
`Luciferase expression in the tumor and nontarget tissues 48 h after a single iv
`administration of Luc-SPLP (100 Ag pDNA) containing PEG-S-DSG in Neuro2a
`tumor-bearing A/J mice. Transgene expression is expressed as pg luciferase/g
`tissue. Data represent means F SD; n = 5 mice. (B) Luciferase expression
`following repeat administration of Luc-SPLP. Mice were treated with Luc-SPLP
`4 to 15 days prior to a second treatment with Luc-SPLP (see key for treatment
`intervals). Luc expression was determined 48 h after second treatment. Data
`are presented as in (A). (C) Biodistribution of SPLP 1 h after first or second iv
`administration. ICR mice were treated with unlabeled SPLP containing 100 Ag
`pDNA. 7 days later, naRve (first dose) or pretreated animals (second dose)
`received 3H-labeled SPLP (100 Ag pDNA). Blood and major tissues were
`collected 1 h after radiolabeled SPLP administration and specific activity was
`determined. Values are expressed as percentage of
`injected dose/tissue
`(means + SD; n = 4 mice).
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`doi:10.1016/j.ymthe.2005.09.014
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`that changes in gene expression pattern after multiple
`SPLP administrations are likely attributable to rapid
`elimination of the PEGylated liposomes from the blood
`and their redistribution to the liver.
`
`Anti-PEG Antibodies Can Be Generated in Response to
`PEGylated Liposomes
`The loss of tumor targeting and altered biodistribution in
`multidose studies with SPLP suggested an immune-medi-
`ated clearance mechanism. To examine this possibility, we
`developed a modified ELISA [27] to detect antibodies
`
`To examine whether tumor-selective transgene expres-
`sion was maintained following multiple treatments, we
`subjected mice to a subsequent administration of Luc-
`SPLP at increasing time intervals after the initial treat-
`ment. A second administration of Luc-SPLP given 4 days
`after the first resulted in Luc expression within the tumor
`that was comparable to that observed after a single
`treatment (Fig. 1B). However, when the interval between
`injections was extended to 6 days or greater, tumor
`transfection resulting from the second SPLP treatment
`was significantly reduced. Loss of transgene expression in
`the tumor after multidosing was accompanied by
`increased expression in the liver, suggesting that the
`pharmacokinetic or biodistribution profile of the second
`SPLP dose was adversely affected (Fig. 1B). Changes in
`tumor growth rate or other tumor-specific changes
`induced by the first SPLP treatment were unlikely to be
`the cause of reduced transgene expression since expres-
`sion was also attenuated following injection intervals of
`11 days or greater, which required initial SPLP admin-
`istration to occur prior to tumor seeding. SPLP treatment
`had no effect on tumor growth rates (data not shown).
`To determine if the dramatic change in gene expres-
`sion profiles was caused by altered blood clearance or
`biodistribution of the second SPLP treatment, we incor-
`porated a nonexchangeable radiolabeled lipid marker
`[26] into SPLP. One hour after a single iv injection of
`radiolabeled SPLP 80% of the injected dose remained
`within the blood of naive mice (Fig. 1C), consistent with
`an expected blood circulation half-life of 12–14 h for
`liposomes containing PEG-C18 lipids such as PEG-S-DSG
`[24,25]. Less than 10% of the labeled liposomes had
`accumulated in the livers of these animals over the first
`hour after administration (Fig. 1C). By contrast, in mice
`treated with SPLP 7 days earlier, only 25% of the second
`SPLP dose remained in the blood 1 h after iv admin-
`istration. This was accompanied by significant accumu-
`lation of SPLP in the liver and, to a lesser extent, the
`spleen, implying involvement of the RES in the accel-
`erated blood clearance (Fig. 1C). These results indicate
`
`FIG. 2. PEGylated liposomes can induce long-lived antibody responses against
`PEG. (A) DSG-SPLP induces IgM and IgG antibodies reactive against PEG-S-
`DSG. ICR mice were treated with either PBS (vehicle control; dashed lines) or
`DSG-SPLP containing 100 Ag pDNA (solid lines). Serum IgM and IgG antibodies
`reactive against PEG-S-DSG (anti-PEG Ab) were measured 7 days later by a
`modified ELISA. Pooled serum (n = 4 mice) was assayed in duplicate by serial
`dilution. Values represent mean ODs of three individual assays F SD. Similar
`results were obtained in C57BL/6J and C3H/HeN mice. (B) Anti-PEG Ab
`responses are induced by low doses of SPLP. Mice were treated with SPLP at
`100, 20, or 2 Ag pDNA (1500–30 Ag total lipid, 600–12 Ag PEG-S-DSG) or
`empty liposomes of identical composition (equivalent to SPLP dose of 100 Ag
`pDNA). Total anti-PEG IgG (H+L) levels were assessed in serum 7 days later. Data
`are OD values from serum diluted 1:100 from individual animals in each group.
`(C) Duration of the anti-PEG IgM and IgG response. ICR mice were treated with
`SPLP (100 Ag) on day 0. Mice were test bled at the indicated times after
`treatment up to day 28. Anti-PEG IgM and IgG levels are expressed as mean OD
`values from pooled serum samples (n = 4) diluted 1:100 at each time point.
`
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`ARTICLE
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`against the lipid components of PEGylated liposomes.
`Seven days after a single iv injection of SPLP we detected
`significant levels of IgM and IgG reactive against the PEG-
`S-DSG component of SPLP (anti-PEG Ab) in the serum of
`treated mice (Fig. 2A). These Ab were reactive against other
`PEG-conjugated lipids and nonreactive against the native,
`unconjugated lipid, suggesting that the antigenic epitope
`was the PEG moiety itself rather than the lipid anchor. We
`did not detect increased Ab reactivities against the other
`three lipid components of SPLP (not shown). Anti-PEG Ab
`were generated by relatively low doses of liposomal pDNA.
`Mice treated with 0.1 mg/kg SPLP (approximately 2 Ag
`pDNA, 30 Ag total lipid, 12 Ag PEG-S-DSG) developed
`significant levels of anti-PEG Ab 7 days after administra-
`tion (Fig. 2B). However, treatment with empty liposomes
`demonstrated that the generation of anti-PEG Ab to SPLP
`was entirely dependent on the encapsulated pDNA within
`the liposome (Fig. 2B). We determined serum levels of
`anti-PEG IgM and IgG over 4 weeks after a single SPLP
`treatment. Anti-PEG IgM levels peaked 7 days after SPLP
`administration and then declined rapidly to reach near
`pretreatment levels by day 14 (Fig. 2C). In contrast, anti-
`PEG IgG increased for 20 days after treatment and
`remained elevated through day 28.
`These data demonstrate that the encapsulation of
`immunostimulatory pDNA within SPLP is sufficient to
`render the PEGylated delivery vehicle immunogenic. This
`manifests as a surprisingly robust, long-lived humoral
`immune response to PEG that is sufficient to cause
`accelerated blood clearance and loss of disease site target-
`ing upon subsequent re-administration. Liposomes incor-
`
`porating immunostimulatory molecules are known to act
`as potent adjuvants that can promote Ab responses against
`weakly immunogenic antigens [19,20], including lipids
`[27,28], displayed on the outer surface of the liposome.
`Reports have also shown that Ab responses against PEG
`can be raised when PEGylated proteins are used in
`conjunction with aggressive immunization regimens
`[29,30]. Therefore, despite PEG being typically regarded
`as nonimmunogenic, it is clear from the current study, as
`implied in the recent report by Semple et al. [31], that PEG
`itself can act as an antigenic epitope in a drug formulation
`when presented in the context of a strong adjuvant such as
`a liposome containing an immunostimulatory payload.
`Since pDNA can act as a polyclonal B cell activator [7–
`9], we examined the B cell proliferative response to SPLP in
`vivo to determine if the production of anti-PEG Ab is part
`of a generalized polyclonal Ab response. When we assessed
`proliferation by flow-cytometric analysis of bromodeoxy-
`uridine (BrdU) incorporation [32], we observed only a
`small increase in the proportion of IgM+ B cells prolifera-
`ting in the spleen following SPLP administration (Fig. 3A)
`compared to cells recovered from PBS (Fig. 3B) or empty
`liposome-treated control mice (Fig. 3C). Within the IgM+
`population, however, a distinct subset of plasmablasts was
`expanded specifically in SPLP-treated mice. These cells
`represented up to 3% of the total IgM+ population and
`were defined by their expression of the plasma cell marker
`syndecan-1 [33] and incorporation of BrdU over the 3 days
`following SPLP treatment (Fig. 3A). The majority of
`plasmablasts exhibited reduced expression of the B cell
`marker B220 (not shown).
`
`FIG. 3. B cell proliferation and differentiation
`in response to PEGylated liposomes. Mice
`were treated with (A) SPLP, (B) PBS, or (C)
`empty PEGylated liposomes and fed drink-
`ing water containing bromodeoxyuridine
`(BrdU) continually for 3 days to label pro-
`liferating cells. Four-color staining of spleen
`cell suspensions for flow cytometry identi-
`fied IgM+ B cells incorporating BrdU (left) or
`expressing the plasma cell marker syndecan-
`1 (Syn-1; middle) and gated Syn-1+, IgM+
`cells that had incorporated BrdU (right). The
`IgM+, Syn-1+, BrdU+ plasma-
`majority of
`blasts in SPLP-treated animals had down-
`regulated the B cell marker B220 (not
`shown). Plots are representative of spleens
`from three to six mice in each group assayed
`in two separate experiments. Values in
`upper right quadrant represent percentage
`IgM+ splenocytes.
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`anaphylactic response to PEGylated liposomes are sum-
`marized in Table 1.
`In the mouse, one mechanism of anaphylaxis
`involves PAF [42]. This reaction can be initiated by the
`formation of IgG containing immune complexes in the
`blood that trigger excess PAF release from Fcg receptor-
`expressing cells [42]. To test if this mechanism was
`responsible for the anaphylactic reaction to PEGylated
`liposomes, we treated SPLP-sensitized mice with PAF
`receptor antagonists immediately prior to a second
`administration of SPLP. Prophylactic treatment with
`PAF antagonist CV6209 or CV3988 inhibited the ana-
`phylactic reaction to PEGylated liposomes at challenge
`doses that otherwise proved fatal in control mice. This
`suggests that the acute toxicities following repeat admin-
`istration of PEGylated liposomes are due to the systemic
`release of PAF, triggered by immune complex formation
`involving anti-PEG Ab. Hypersensitivity in this model
`was not associated with excessive systemic cytokine
`release or significant elevations in plasma histamine or
`complement activation products (not shown).
`Although PEGylated liposomes have not been reported
`to be immunogenic in humans, their iv administration is
`associated with hypersensitive infusion reactions in a
`substantial number of patients [43,44]. Unlike the reac-
`tions described here, these clinical events occur upon first
`exposure to the liposome and have been correlated with
`the activation of complement [45,46] and a rapid redis-
`tribution of the liposomes from the blood to the liver and
`spleen [43]. Naturally occurring Abs against lipid compo-
`nents have been implicated in this hypersensitive
`response [46] and it cannot be discounted that this may
`include preexisting anti-PEG Ab in some patients. In this
`regard, preliminary analyses of sera from healthy volun-
`teers have identified low levels of anti-PEG reactivity in
`certain donors (A.J., unpublished data), a finding consis-
`tent with previous reports of naturally occurring anti-PEG
`IgM in a proportion of human subjects [47,48]. It appears
`therefore that the human B cell repertoire can generate
`anti-PEG Ab and suggests that the immunogenicity of
`PEGylated liposomes may become a clinically relevant
`
`TABLE 1: Characteristics of the hypersensitive response
`following repeat challenge with SPLP containing PEG-S-DSG
`
`Analysis of the early B cell response indicates that the
`generation of Abs against PEG likely reflects the selective
`activation and differentiation of a small subset of B cells
`rather than polyclonal B cell activation triggered by the
`nonspecific uptake of pDNA. We therefore envisage a
`multistep model leading to the production of anti-PEG
`Ab: first, liposome binding and crosslinking of surface
`immunoglobulin (sIg) on PEG-reactive B cells; second,
`internalization of the payload and activation of B-cell-
`stimulatory pathways such as TLR9 by the pDNA [9]; and
`third, the induction of cytokines from accessory cells that
`support maturation of the nascent Ab response. These
`signals have been shown to act in concert on B cells to
`generate Ab responses that are independent of T cell help
`[9,34,35]. Although not observed with our formulations,
`we do not exclude the possibility that PEGylated lip-
`osomes containing nonstimulatory payloads may also be
`weakly immunogenic. This may become apparent if the
`liposomes can more effectively crosslink sIg on B cells, a
`scenario analogous to thymus-independent type II Ab
`responses characterized by polymeric antigens with
`multiple repeating epitopes [36]. The efficiency of sIg
`crosslinking will likely be influenced by liposome size,
`composition, bilayer fluidity, and epitope density on the
`liposomal surface. Several groups have demonstrated the
`accelerated blood clearance of empty PEGylated lip-
`osomes in both rodents [37–40] and primates [37]
`following repeated administration. These phenomena
`have been attributed to unidentified soluble serum
`factors [37,40] and enhanced phagocytic activity of the
`RES [38]. However, it would be interesting to reexamine
`such models using appropriate assays to determine if Ab
`responses against the PEG or lipid components may be
`responsible.
`
`Anti-PEG Antibodies Trigger Platelet-Activating Factor
`(PAF)-Dependent Hypersensitive Reactions
`A single treatment with SPLP containing PEG-S-DSG was
`sufficient to induce hypersensitivity in mice that man-
`ifested as acute toxicity upon subsequent treatment.
`Symptoms typically developed 5–10 min after re-admin-
`istration and included lethargy, facial puffing, vasodila-
`tion, labored respiration, and significant mortality rates at
`higher challenge doses. These symptoms appeared typical
`of an Ab-mediated anaphylactic reaction [41]. Establish-
`ment of hypersensitivity required dosing intervals of at
`least 6 days and was achieved at SPLP doses as low as 2 Ag
`pDNA (~30 Ag total lipid), correlating with the develop-
`ment of anti-PEG Ab response in SPLP-treated animals. As
`such, we still observed hypersensitivity at dosing intervals
`greater than 28 days in animals with established anti-PEG
`IgG responses (Fig. 2). In contrast, priming mice with
`empty liposomes did not induce anti-PEG Ab (Fig. 2B) or
`hypersensitivity to subsequent SPLP treatment, although
`empty liposomes could trigger the anaphylactic reaction
`in animals presensitized with SPLP. Features of the
`
`Second dose (Day 7)
`SPLP 100 Ag
`SPLP 100 Ag
`SPLP 100 Ag
`SPLP 20 Ag
`SPLP 100 Ag
`Empty liposomes
`SPLP + PAF antagonists
`
`First dose (Day 0)
`SPLP 100 Ag
`SPLP 20 Ag
`SPLP 2 Ag
`SPLP 100 Ag
`Empty liposomes
`SPLP 100 Ag
`SPLP 100 Ag
`Mice were treated on day 0 (first dose) and day 7 (second dose) with DSG-SPLP or empty
`liposomes at 100, 20, or 2 Ag pDNA or the equivalent lipid dose. Hypersensitive reactions
`were scored according to the severity of symptoms 10–60 min after the second dose as
`described under Materials and Methods.
`
`Reaction
`
`Moderate–severe
`Moderate–severe
`Moderate–severe
`Mild
`No reaction
`Moderate–severe
`No reaction
`
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`concern as these nucleic acid drug delivery vehicles are
`advanced into human studies.
`
`Modified Liposomes Containing Diffusible PEG–Lipids
`Are Less Immunogenic
`Liposomes can be engineered in which the PEG–lipid
`dissociates more rapidly from the lipid bilayer upon in vivo
`administration by using a PEG–lipid with shorter alkyl
`chain length [25,49]. Since the Ab response to SPLP was
`directed against the PEG component and required close
`association with the immunostimulatory pDNA, we
`speculated that the use of more rapidly diffusible PEG–
`lipids may limit the opportunity for anti-PEG Ab
`responses to develop. To test this, we constructed a series
`of SPLP with similar lipid composition containing one of
`PEG-S-DSG, PEG-S-DPG, or PEG-S-DMG (C18, C16, or C14
`alkyl chain length, respectively). Treatment of mice with
`SPLP containing PEG-S-DMG (DMG-SPLP) induced peak
`anti-PEG IgM titers on day 7 that were approximately 10-
`fold lower than the response in DSG-SPLP-treated mice
`(Fig. 4A). Consistent with earlier results (Fig. 2C), the weak
`IgM response to DMG-SPLP was transient and had fully
`resolved within 14 days after treatment. Significantly,
`anti-PEG IgG could not be detected after treatment with
`DMG-SPLP at any time point (Fig. 4B). Antibody responses
`to the other lipid components in the formulation were
`also undetectable (not shown). In contrast, anti-PEG IgM
`and IgG responses were comparable in DSG-SPLP- and
`DPG-SPLP-treated mice (Figs. 4A and 4B), indicating that
`reducing the alkyl chain length of the PEG–lipid from C18
`to C16 had minimal effect on the immunogenicity of the
`lipid vesicle.
`Correlating with the significant reduction in immu-
`nogenicity, mice treated with DMG-SPLP showed no
`symptoms of anaphylaxis, or other overt signs of toxicity,
`when subsequently treated with PEGylated liposomes,
`even when the second dose comprised an immunogenic
`PEG–lipid formulation such as DSG-SPLP (Table 2). As
`predicted by the PEG Ab response, pretreatment with
`either DSG-SPLP or DPG-SPLP resulted in anaphylaxis
`when mice were rechallenged with any one of the
`PEGylated liposome formulations (Table 2).
`
`Maintenance of Tumor Targeting When Multidosing
`with DMG-SPLP
`To address whether DMG-SPLP was still able to deliver
`pDNA systemically to disease sites, we compared the
`ability of the modified liposomes to target the expression
`of pDNA to distal Neuro2a tumors. High-level transgene
`expression in the tumor was achieved after a single iv
`administration of DMG-SPLP (Fig. 4C), which was reduced
`approximately 2-fold compared to DSG-SPLP. This slight
`decrease in gene expression correlated with an equivalent
`reduction in DMG-SPLP accumulation at the tumor site
`(not shown). Strikingly, however, equivalent levels of
`tumor transgene expression were achieved following
`
`repeat administration of the same DMG-SPLP formulation
`7 days later without any changes to transgene expression
`in nontarget tissues, including the liver. This was in
`contrast to the properties of liposomes containing PEG–
`lipids with longer alkyl chains in which tumor transgene
`expression following repeat administration was reduced
`70 to 85% in DPG-SPLP- and DSG-SPLP-treated mice,
`respectively. As in earlier studies, the loss of tumor
`transfection with immunogenic liposomes was coinci-
`dent with increased gene expression in the liver (Fig. 4C).
`Successful re-administration of DMG-SPLP correlated with
`a 10-fold reduction in anti-PEG IgM titers, determined
`immediately prior to the second administration of SPLP
`(Fig. 4D). Unlike a number of other mouse strains
`examined, A/J mice used in this syngeneic tumor model
`failed to mount a significant IgG response to any of the
`SPLP formulations tested at this dose. This finding
`accounted for the observation that hypersensitivity and
`accelerated blood clearance were transient phenomena in
`this particular mouse strain and related to the duration of
`the anti-PEG IgM response (not shown).
`Radiolabeled formulations were used to assess the
`blood clearance rates of the PEG-S-DMG-modified lip-
`osomes upon repeat administration. Equivalent amounts
`of DMG-SPLP were present in the blood 15 min after the
`first or second iv injection given 7 days apart (Fig. 4E).
`After 1 h, only a minor increase in the rate of clearance of
`the second dose was observed, possibly reflecting the low
`anti-PEG IgM titers that were detected in the serum 7 days
`after initial treatment (Figs. 4A and 4D). By contrast, a
`significant proportion of the second dose of either DPG-
`SPLP or DSG-SPLP was cleared from the blood within 15
`min of administration (43 and 56%, respectively) and
`blood clearance of these immunogenic formulations was
`almost complete by 1 h (88 and 90% of first dose
`respectively; Fig. 4E).
`Taken together, these results demonstrate that the
`immunogenicity of PEGylated liposomes containing
`pDNA can be greatly reduced by using alternative PEG–
`lipids that diffuse more readily from the lipid bilayer
`upon administration. By substantially eliminating the Ab
`response to PEG, these modified liposomes can be safely
`re-administered to mice while maintaining the effective
`delivery of the pDNA payload to distal tumor sites.
`Administration of these modified liposomes was still
`associated with substantial cytokine induction (not
`shown),
`indicating that the reduced immunogenicity
`was not due to abrogation of the immunostimulatory
`activity of the pDNA payload. Instead, these findings
`support our hypothesis that robust Ab responses to PEG
`require the close physical association of the PEG–lipid
`with the pDNA and are driven by the specific binding and
`internalization of the PEGylated liposome containing
`stimulatory pDNA by PEG-reactive B cells. An alternative
`approach therefore to reduce carrier immunogenicity
`may be the development of less immunostimulatory
`
`MOLECULAR THERAPY Vol. 13, No. 2, February 2006
`Copyright C The American Society of Gene Therapy
`
`333
`
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`

`ARTICLE
`
`doi:10.1016/j.ymthe.2005.09.014
`
`FIG. 4. PEGylated liposomes can be modified to be less immunogenic and maintain disease-site targeting after multiple administrations. (A and B) Anti-PEG
`antibody responses to SPLP containing PEG–lipids with shorter alkyl chain lengths. (A) Anti-PEG IgM and (B) IgG titers in mice 7 days after administration of PBS
`or SPLP (50 Ag) containing PEG-S-DSG (C18 alkyl chain length), PEG-S-DPG (C16), or PEG-S-DMG (C14); n = 4 mice per group assayed as pooled sera.
`Qualitatively similar results were obtained in three separate experiments. (C) Luciferase expression in the tumor and liver 48 h after administration of one or two
`doses (50 Ag pDNA) of modified SPLP (as in A and B). Neuro2a tumor-bearing animals were either naRve (first dose) or pretreated with the same SPLP formulation
`7 days earlier (second dose). Transgene expression is expressed as pg luciferase/g tissue (means + SD; n = 5). (D) Anti-PEG IgM titers in A/J mice from (C)
`immediately prior to receiving a second treatment of SPLP. Values are mean ODs from pooled serum samples (n = 5 per group). (E) Blood clearance of a second
`treatment with SPLP containing PEG-S-DSG, DPG, or DMG administered 7 days after initial treatment. Mice were treated with unlabeled SPLP (100 Ag pDNA) 7
`days prior to receiving 3H-labeled SPLP. Values are expressed as the amount of radiolabel (% of single dose values) remaining in the blood 15 and 60 min after
`administration (means + SD; n = 4).
`
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`MOLECULAR THERAPY Vol. 13, No. 2, February 2006
`Copyright C The American Society of Gene Therapy
`
`

`

`doi:10.1016/j.ymthe.2005.09.014
`
`ARTICLE
`
`TABLE 2: Absence of a hypersensitive response following
`repeat challenge with SPLP containing PEG-S-DMG
`
`First SPLP dose
`(Day 0)
`
`Second SPLP dose
`(Day 7)
`
`Reaction (n)
`
`PEG-S-DSG
`PEG-S-DPG
`
`PEG-S-DSG
`PEG-S-DPG
`
`Moderate (4/9), severe (5/9)
`Mild (1/9), moderate (4/9),
`severe (4/9)
`No reaction (13/13)
`PEG-S-DMG
`PEG-S-DMG
`Mild (1/4), moderate (3/4)
`PEG-S-DMG
`PEG-S-DSG
`No reaction (13/13)
`PEG-S-DSG
`PEG-S-DMG
`Mice were treated on day 0 (first dose) and day 7 (second dose) with 100 Ag pDNA in SPLP
`containing PEG-S-DSG, PEG-S-DPG, or PEG-S-DMG. Hypersensitive reactions were scored
`according to the severity of symptoms 10–60 min after the second dose as described under
`Materials and Methods. Parentheses indicate number of mice reacting per group. Data are
`from two separate experiments.
`
`nucleic acids such as CpG-free pDNA and antisense oligos
`or synthetic siRNA with minimal capacity to activate
`cytokine responses [13].
`The potential
`for a drug or its excipient to be
`immunogenic is a serious concern in drug development
`since the establishment of an Ab response can severely
`compromise both the safety and the efficacy of a drug.
`This has hampered the development of certain drug
`classes,
`including protein-based therapeutics such as
`monoclonal Abs and viral vectors that contain immuno-
`genic components. The present study highlights that the
`potential for nonviral vectors to be immunogenic also
`becomes a concern when these systems are used for the
`delivery of immunostimulatory agents, such as pDNA,
`that can act as potent immune adjuvants. We have
`recently demonstrated that certain synthetic siRNAs can
`induce potent immune stimulation in vivo [13] and have
`found that this can also drive the production of a strong
`anti-PEG Ab response when these molecules are encapsu-
`lated in PEGylated liposomes containing C18 PEG-lipids
`(A.J., unpublished dat