Liver Gene Transfer of Interkeukin-15 Constructs That Become Part of Circulating High Density Lipoproteins for Immunotherapy

Apolipoprotein A-I (Apo A-I) is a major component of high density lipoproteins (HDL) that transport cholesterol in circulation. We have constructed an expression plasmid encoding a chimeric molecule encompassing interleukin-15 (IL-15) and Apo A-I (pApo-hIL15) that was tested by hydrodynamic injections into mice and was co-administered with a plasmid encoding the sushi domain of IL-15Rα (pSushi) in order to enhance IL-15 trans-presentation and thereby bioactivity. The pharmacokinetics of the Apo A-I chimeric protein were much longer than non-stabilized IL-15 and its bioactivity was enhanced in combination with IL-15Rα Sushi. Importantly, the APO-IL-15 fusion protein was incorporated in part into circulating HDL. Liver gene transfer of these constructs increased NK and memory-phenotype CD8 lymphocyte numbers in peripheral blood, spleen and liver as a result of proliferation documented by CFSE dilution and BrdU incorporation. Moreover, the gene transfer procedure partly rescued the NK and memory T-cell deficiency observed in IL-15Rα−/− mice. pApo-hIL15+ pSushi gene transfer to the liver showed a modest therapeutic activity against subcutaneously transplanted MC38 colon carcinoma tumors, that was more evident when tumors were set up as liver metastases. The improved pharmacokinetic profile and the strong biological activity of APO-IL-15 fusion protein holds promise for further development in combination with other immunotherapies.


Introduction
There is much interest in the development of interleukin-15 for immunotherapy [1,2]. This is because it inhibits activation induced T cell death [3], homeostaticaly increases lymphocyte numbers [4,5], and up-regulates the function of NK cells [6,7,8,9] and IKDC (interferon-producing killer dendritic cells) [10]. IL-15 is more a costimulatory molecule than a soluble cytokine in the sense that it is physiologically trans-presented [11] as a cell surface complex which is non-covalently attached with high affinity to IL-15Ra [8,9,11,12].
The region of IL-15Ra involved in transpresentation of IL-15 has been identified as the sushi domain (so named because of structural resemblance with the roll shape of a popular Japanese dish) [13,14,15]. The binding of IL-15 to the sushi domain of IL-15Ra is believed to orient the molecule and improves the interaction with the IL-2Rb/IL-2Rc signaling receptors [13,15].
Indeed, IL-15 coupled to the sushi domain has been engineered to increase bioactivity [15].
For cancer immunotherapy, IL-15 has created great expectations based on mouse data from injections of the soluble cytokine [1] or gene transfer [16,17,18]. However, the most striking effects of IL-15 are observed in combinatorial immunotherapeutic strategies such as those with adoptive T cell transfer [19] or vaccines [20,21].
The pharmacokinetic profile of IL-15 as a soluble molecule is not favourable since such a small protein undergoes rapid renal clearance. To stabilize the molecule and provide trans-presentation, IL-15Ra-Fc chimeric proteins are conjugated ex vivo to IL-15. The resulting complexes are much more bioactive and exert more potent immunotherapeutic effects [22,23,24].
GMP-grade IL-15 has been tested in non-human primates mainly showing expanding effects on CD8 memory T cells and NK cells [25,26]. Most effects are transient and cease following cytokine withdrawal [26] thus offering a promising overall safety profile. Accordingly, phase I trials have begun (NCT01021059).
In mice, the immunotherapeutic effects of IL-15 against tumors are dependent on NK and CD8 T cells [2,9,27]. Importantly, the effects of IL-15 are very different from those of IL-2, although both cytokines share similar receptors and attain similar effects on lymphocyte cultures [2]. For instance, IL-15 inhibits activationinduced cell death in vivo, whilst IL-2 induces the contrary [2]. Furthermore, the phenotypes of IL-15 2/2 [28] and IL-15Ra 2/2 mice [29] show that IL-15 is absolutely required for NK and NKT cell ontogeny as well as for homeostatic maintenance of CD8 memory T cells. On the contrary, IL-2 2/2 mice show a totally unrelated phenotype with autoimmune inflammatory bowel disease [30].
Lipids are transported in plasma wrapped in apolipoproteins that coalesce forming lipoparticles. Apo A-I is a major constituent of high density lipoproteins (HDL), whose main function is to gather cholesterol from the tissues and take it back to the liver [31]. High plasma concentrations of HDL are linked to lower risk of cardiovascular events [31]. Apo A-I protein is an HDL constituent synthesized and secreted by hepatocytes and interacts with the surface scavenger receptor BI (SR-BI) broadly distributed in the organism [32].
Because of these features, Apo A-I has been used to stabilize interferon a to achieve sustained levels of the cytokine with a safer profile [33]. In these constructions, the cytokine is supposed to be facing the hydrophilic side, popping from the hydrophobic core of the lipoprotein.
In this study, IL-15 was fused to Apo A-I and gene-transferred to the liver by hydrodynamic injections as previously performed with several cytokine genes [34] including IL-15 [35]. We show that this chimeric gene encompassing IL-15 leads to more sustained serum concentrations than the concentrations achieved with non-fused IL-15. Furthermore, we co-transferred to the hepatocytes the sushi domain of IL-15Ra and the resulting complex showed enhanced bioactivity and as a monotherapy shows partial efficacy against colon carcinomas derived from the MC38 cell line.

Ethics Statement
All animal procedures were conducted under Navarra Government recommendations and the protocol was approved by the Committee on the Ethics of Animal Research of the University of Navarra (study approval number 060/10). All surgery was performed under anesthesia, and all efforts were made to minimize suffering.

Plasmids
The human interleukin-15 expression plasmid (pVkL/IL-15IRESneo), which carries a modified cDNA encoding for hIL-15 protein was produced and purified as described [35,38] and will be called phIL15.
For sushi domain construction, complete mIL15Ra was cloned into the BamHI and NotI sites of the vector pcDNA TM 3.1 (Invitrogen, Carfsbed, CA) (pIL15Ra). The C-terminal part was removed by PCR (Uprim. TAA AGA GGG CCC TAT TCT ATA GTG and Lprim.GGG GTC TCT GAT GCA CTT GAG) and subsequent ligation of the product. The transgene in the expression plasmid was verified by sequencing. We will refer to this as pSushi. Apolipoprotein A-I plasmid (apoa1, GeneID:11806) was produced and purified as described [33] and will be called pApo.

Apolipoprotein A-I and Interleukin 15 Gene Fusion Designs
PCR amplification was carried out using the sense primer FwATGmApoA1 (59-ATGAAAGCTGTGGTGCTGGC-39) on pApo as a template, and the antisense primer RvAscImApoA1 (59-GGCGCGCCCTGGGCAG-TCAGAGTCTCGC-39), introducing the restriction site for the AscI enzyme (GGCGCGCCC) in the 39 end of apolipoprotein A-I gene, eliminating the stop codon and including a GAP peptide as linker of the two coded proteins. mApoA-I-AscI purified cDNA was cloned in the expression vector pcDNA TM 3.1/V5-His TOPOH TA (Invitrogen, Carfsbed, CA), which we will call pCMV-mApoA1-AscI.
To carry out the gene fusion, plasmid pCMV-mApoA1-AscI was digested with the AscI/NotI enzymes (New England Biolabs). The ligation was performed with the open plasmid pCMV-mApoA1-AscI and the AscI-hIL15-NotI insert in a 1:3 (vector:insert) ratio using T4 DNA ligase High Concentration and 2X Rapid Ligation Buffer (Promega, Wl). The resulting 6669nucleotide plasmid will hereinafter be called pApo-hIL15. All plasmids were confirmed by sequencing the cloned genes.
For the fusion of mouse albumin with hIL15, the plasmid pApo-hIL15 was digested with AscI/XhoI and the insert containing the hIL15 was cloned into a the plasmid pALF [39] after removal of the sequence of IFN by AscI/XhoI digestion.

Hydrodynamic Injections and ELISA
C57BL/6 or IL-15Ra 2/2 mice received an intravenous injection (tail vein) of 10 mg of plasmid in a volume of 100 ml kg 21 using a 27-G needle at a rate of 0.4 ml s 21 , as described [40].

RT-PCR and Analysis of hIL15 and mSushi Expresion
Total RNA from mice livers was isolated from samples using TRI reagent (Sigma, Dorset, UK). The concentration and purity of samples were determined by absorbance at 260 and 280 nm with background correction at 320 nm in a spectrophotometer. RNA was treated with DNase I and retrotranscribed to cDNA with MMLV RT in the presence of RNase OUT (all the reagents Invitrogen, Carlsbad, CA, USA). The reaction was incubated for 1 hour at 37uC, denatured for 1 minute at 95uC and taken to 4uC. The samples were used immediately for PCR or stored at 220uC.
Murine actin was used to standardize gene expression. The mRNA values were represented by the formula 2D Ct , where DCt indicates the difference in the threshold cycle between mActin and the target genes (all reagents from BioRad, Hercules, CA, USA). Isolation of HDLs by differential ultracentrifugation in sodium bromide gradient. 24 hours after hydrodynamic delivery of pApo, pApo-hIL15 or pApo-hIL15+ pSushi plasmids, HDLs were isolated from plasma samples as described by Rodriguez-Sureda et al [41]. Electrophoresis and immunoblotting against Apo A-I were performed as described [33].

IL-15 Bioactivity Assay
CTLL-2 cells were plated in a 96-well plate at 10 4 cells/well and 1:10 diluted serum obtained 24 h after hydrodynamic injections was added. Each serum sample was serially diluted 1:2 twelve times. The plates were incubated for 2 days and subsequently the CTLL-2 cells were pulsed with 1 mCi of tritiated thymidine ([ 3 H]TdR) 8 h before [ 3 H]TdR incorporation was measured using a Topcount liquid scintillation counter (Packard).

Isolation of Mononuclear Cells from Spleen and Livers
Spleens and livers were incubated in collagenase and DNase (Roche) for 15 min at 37uC, mechanically disrupted and passed through a 70 mm nylon mesh filter (BD Falcon, BD Biosciences). Dissociated cells from livers were centrifuged with Percoll ß (GE Healthcare, Chalfont St Giles, UK) at 35% (500 g, 10 min, 20uC) making a gradient in order to eliminate parenchymal cells. Erythrocytes were lysed with ACK buffer. For the study of CD8 T and NK cell proliferation, splenocytes were enriched in DX5 + or CD8 + cells using immunomagnetic beads according to the manufacturer's protocol (Miltenyi Biotec, Bergisch Gladbach, Germany).

CFSE Labeling of Cells, Adoptive Transfer and BrdU Assessment of Proliferation in vivo
Enriched DX5 + or CD8 + cells were washed twice with phosphate-buffered saline and resuspended at 10 7 cells ml 21 in 5 mM CFSE (Sigma-Aldrich) for 15 min at room temperature. 3610 5 -5610 5 stained cells were transferred to mice by the retro orbital sinus. The proliferation of adoptively transferred NK and CD8 T lymphocytes was analyzed 72 h after the transfer by CFSE dilution using a FACSCalibur cytometer. For BrdU incorporation in vivo and detection by flow cytometry, we used the FITC BrdU flow kit (BD Biosciences) according to manufacturer instructions. Intraperitoneal injections of BrdU were provided three times every 24 h with doses of 1.5 mg.

In vivo Tumor Growth
For the subcutaneous models, C57Bl/6 mice received a s.c. injection of MC38 cells (5610 5 per mouse). On the indicated days, mice were treated with a hydrodynamic injection with the plasmids. Tumor diameters were measured using an electronic caliper every 2 to 4 days, and tumor size was determined by multiplying perpendicular diameters.
For the intrasplenic tumor model, C57BL/6 mice were anesthetized and a small incision in peritoneum was performed to access the spleen. MC38 cells were injected in the spleen (5610 5 per mouse) in 50 mL of PBS. On day 1, mice were treated with the corresponding plasmids by hydrodynamic injection. Mice were observed daily and were sacrificed when one of the mice presented impaired mobility. Following sacrifice, tumor nodules in spleen and liver metastases were counted and measured. Mice were weighed as well as various excised organs upon necropsy.

Fusion Proteins Encompassing IL-15 and Apo A-I are more Stable in Plasma and Partly become Complexed with HDL
IL-15 renal clearance is rapid due to the small molecular size of the protein. In order to stabilize IL-15 in circulation, constructions that encompassed Apo A-I lipoprotein and human IL-15 were prepared. Figure S1 represents the expression plasmids in which the indicated genes are placed under the transcriptional control of the CMV promoter. The sushi domain of IL-15Ra was also cloned in a similar expression cassette.
C57BL/6 mice were injected by hydrodynamic infusions with the expression plasmids as single agents or in combinations to achieve expression of the encoded proteins in the liver [35]. Since Apo A-I is naturally biosynthesized in the liver, local production of the secretable transgene products was expected to reach circula-tion. IL-15Ra sushi domain has been reported to conjugate with IL-15 non-covalently during protein processing [13,42]. Figure 1A shows the time courses of the serum concentrations of human IL-15 following hydrodynamic injections of the plasmids. As can be seen, conjugation to Apo A-I dramatically extends the pharmacokinetic profile and reaches much higher concentrations of the transgene product than non-fused IL-15. Liver mRNA from the expression plasmids was quantified by RT-PCR and found to correlate with peak protein expression (figure 1B). In addition, pSushi RNA was also detected by RT-PCR in similar samples, when pSushi was co-administered (figure S2).
Differential centrifugation of the plasma of the mice to separate the lipid fractions indicated that immunoreactive IL-15 by ELISA was observed both in high density lipoproteins (HDL) and in lipidfree plasma upon liver gene co-transfer of APO-IL-15 and IL-15Ra sushi domain, or upon a single hydrodynamic injection of the APO-IL-15-encoding plasmid ( figure 1C). This indicates that the chimeric protein circulates in serum both free and complexed to HDL. The absence of HDL in the lipid free plasma fractions was checked by ELISA quantification of Apo A-I (inset to figure 1C).
These data were confirmed using HDL fraction and lipid-free plasma to test IL-15 bioactivity on CTLL2 cells. As can be seen in figure 1D both fractions contained bioactivity, but a comparison on nmol/mL basis indicates that the HDL complexed fraction is slightly more bioactive. Given the fact that IL-15 is detected both in the HDL and the lipid free plasma fractions ( figure 1C and D), we performed experiments to see if IL-15 were released from HDL upon overnight incubation at 37uC in PBS. We found that almost none of the HDL-complexed IL-15 was released into the buffer (data not shown).
In figure 2A, an immunoblot of the HDL fractions of the mice was probed with an anti-Apo A-I polyclonal Ab that showed a shift in molecular weight from the abundant non-complexed Apo A-I when the APO-IL-15 sequences had been transferred by hydrodynamic injection. This finding further indicates that APO-IL-15 was incorporated in part into circulating HDL. Furthermore, we developed similar blots with an anti-IL15 antibody that also showed a compatible molecular weight for the fusion form of IL-15 in the corresponding HDL fractions from mice in which pApo-hIL15 plasmids had been hydrodynamically injected ( figure 2B).
In summary, IL-15 in covalent conjugation with Apo A-I complexes with HDL and extends its half-life. In addition, gene transfer of the sushi domain of IL-15Ra enhances the bioactivity of the cytokine.
One of the intended functions of Apo A-I is to increase molecular weight ( figure 2A and B) is such a way that it surpasses the renal filtration threshold and hence extends half-life. This can be achieved to a similar extent with other fusion patners, such as albumin (figure S3), even though the biodistribution of the lipidcomplexed IL-15 could be different. Another important question was if HDL from mice receiving plasmids encoding Apo-hIL15 preserved their anti-inflammatory and cholesterol transporting functions. As can be seen in figure S4, such HDL fractions preserved their ability to counteract TNFa induction of VCAM on mouse endothelial cells and promote the efflux of cholesterol from 3T3 cells.
Importantly, mice which received pApo-hIL15+ pSushi showed a marked increase of CD8b + T cells, CD44 high CD8 + memory T cells and NK cells in the spleen if analyzed five days following gene transfer. This finding as shown in figure 3A indicates that cotransfer with the sushi domain increases in vivo bioactivity. A time course (figure S5A) of an independent series of experiments indicates that the peak of the effects on immune system cells takes place around four days after the hydrodynamic injection of the plasmids. As previously reported [42], the IL-15Ra sushi domain also enhances the activity of IL-15 without Apo A-I. Of note, pSushi transference lacked by itself any kind of detectable effects (data not shown).
Liver lymphocytes followed a similar trend with a dramatic accumulation of memory CD8 T lymphocytes and NK cells, as shown in figure 3B. Indeed, the time course was similar with a peak around day 4 after plasmid injection (figure S5B). Sequential follow-up of peripheral blood mononuclear lymphocytes in treated groups of mice showed also these changes ( figure S5C).
When testing the natural killer cytotoxic activity of spleen cells harvested three days following treatment with the pApo-hIL15+ pSushi in comparison to that of mice treated with pApo A-I, a clear increase in activity against YAC-1 target cells was observed (figure S6).

Gene Co-transfer of APO-IL-15 and IL-15Ra Sushi Domain Induces the Proliferation of CD8 + and NK Lymphocytes
Accumulation of CD8 memory T cells and NK cells could be the result of enhanced proliferation. To study this possibility, CFSE-labeled NK and CD8 T cells were infused into mice one day following hydrodynamic injection of control plasmids or the pApo-hIL-15+ pSushi combination. As is shown in figure 4, most adoptively transferred cells undergo at least one cycle of proliferation in response to gene transfer of pApo-hIL15+ pSushi. Therefore, proliferation contributes at least in part to accumulation of such lymphocytes. To evaluate the induction proliferation on endogenous CD8 + T cells and NK cells, we performed experiments in which the mice were treated with BrdU and splenocytes stained for incorporation of this compound into the DNA of each gated lymphocyte subset. As it can be seen in figure  S7, there was a clear stimulation of a proliferative response in both endogenous lymphocyte subpopulations upon treatment with pApo-hIL15+ pSushi.
Gene Co-transfer of APO-IL-15 and IL-15Ra Sushi Domain Partially Rescues the NK and CD8 Phenotype of IL-15Ra 2/ 2 Mice IL-15 2/2 and IL-15Ra 2/2 mice are virtually devoid of NK and NKT cells and experience a drastic reduction in CD8 memory T cells [28,29]. We intended to see if pApo-hIL-15+ pSushi liver gene transfer could modify the phenotype of IL-15Ra 2/2 mice. Figure 5 shows the normal proportions of NK and CD8 cells in the liver of WT C57Bl/6 mice. IL-15Ra 2/2 mice, even if transduced with pApo control plasmid four days before, showed negligible levels of NK (CD3 2 NK1.1 + ) and NKT (CD3 + NK1.1 + ) cells. Transfer of phIL-15 and pApo-hIL15 only marginally increased these numbers. However, the combination of pApo-hIL15+ pSushi enhanced the number of NK cells, but not as much as NKT cells. These results were reproducible in the spleen of three individual mice per group as shown in figure 5B. Moreover, we have extended these results to IL-15Ra2/2 mice in B6129S2 background using five mice per group as shown in figure S8A and B. In this background, the recovery of CD8 T cells with a memory phenotype was remarkable and NK cells, at least partially recovered in most cases, were identified as We performed these experiments also with a plasmid encoding a full length pIL-15Ra instead of only the sushi domain. As can be seen in figure S8C, pSushi was superior to pIL-15Ra indicating that a sequence upstream the sushi domain somehow interferes with trans-presentation as suggested by previous reports [14,42].

Therapeutic Effects of Liver Gene Co-transfer with APO-IL-15 and IL-15Ra Sushi Domain on Subcutaneously and Intrasplenically Injected MC38 Colon Cancer Cells
Based on the observation of biological activity, we performed a series of experiments in mice grafted for six days with subcutaneous MC38-derived colon carcinomas. As can be seen in figure 6, a combination of pApo-hIL15+ pSushi resulted in 7 out of 26 complete rejections with an overall delay in tumor growth when compared to control groups. pApo-hIL15 cured 3 out of 14 animals while phIL15+ pSushi only 1 in 10. In the case of pApo A- I, one mouse rejected a tumor that could be related to the recently described non immune anti-tumor actions of Apo A-I [43]. Therapeutic effects resulted in extended survival as shown in figure 6B.
Since our gene transfers are phenomena confined to some extent to the liver, it was conceivable that if MC38 were injected into the spleen to metastize to the liver via the portal vein, a more prominent therapeutic effect could then appear. Indeed, if mice that had been gene transferred on day 1 were euthanized on day 19 after tumor cell inoculation, the number of observable liver metastases was drastically reduced by all the plasmids encoding IL-15, but not by the pApo A-I expression plasmid. In the case of pApo-hIL15+ pSushi, 5 out of 6 mice were free of metastatic lesions on the liver surface ( figure 7). Moreover, observations of the spleen tumors (measured as spleen weight/mouse weight) indicated that pApo-hIL15+ pSushi also markedly retarded the tumor at the primary site of malignant cell inoculation (figure 7B with inset photographs of representative spleen tumors). The pApo-hIL15 (without pSushi) and phIL15+ pSushi (without fusion to Apo A-I) showed a more partial effect on the splenic tumors as well. The role of the cellular immune system in these antitumor effects was confirmed because selective depletions with antibodies against CD8 b , CD4 or NK cells hampered the therapeutic effects on the liver metastases (data not shown).
As a whole, these APO-IL-15 chimeric constructions transiently expressed by the liver give rise to therapeutic effects, which become candidates for improvement by means of combinatorial strategies [44].

Discussion
This study intended to generate more stable and bioactive forms of IL-15 by incorporation into lipoproteins. Hydrodynamic injections offer a versatile technical opportunity to test these new agents without the need for cumbersome protein bio-production and purification procedures [45,46]. In our case, the advantage was obvious because we are able to achieve the synthesis of our protein in the actual organ which produces HDL. Even though liver gene transfer faces obstacles for clinical translation, it is certainly a clinical alternative to be considered since it might be feasible and cost/effective [18,46].
The idea of using Apo A-I fusions was originally used by our group to stabilize interferon a [33]. In the case of IL-15, this idea is  Figure S7 shows proliferative effects on endogenous lymphocytes studied by BrdU intake. doi:10.1371/journal.pone.0052370.g004 likely to be more suitable since IL-15 is trans-presented [12,22]. In essence, trans-presentation can be conceived to involve cell surface lipid bilayers and IL-15Ra [11,42,47]. Both conditions could be mimicked to some extent by the surface of an HDL lipoparticle, if IL-15Ra is complexed there to IL-15. Therefore, IL-15 could not only be stabilized but also become more bioactive, taking advantage in this way of its physiological mechanisms of action that depend on trans-presentation. It is of note that APO-IL-15 complexes only in part to HDL, so the free protein and the particle absorbed forms coexist in circulation, and maybe in equilibrium, although once complexed APO-IL-15 tends to remain attached to HDL at body temperature.
A series of studies had reported that the sushi domain region of IL-15Ra was required for trans-presentation and that the isolated sushi domain performs even more actively than the whole IL-15Ra protein for this cooperative function [13,14,15], hence giving a reason for our choice of this protein domain. Our results indicate that the complex pApo-hIL15:pSushi domain is stable and bioactive on NK and T cells in vivo.
The effects of gene transfer were able to correct in part the immune phenotype of IL-15Ra 2/2 mice which have reduced CD8 + memory-phenotype T lymphocytes and virtually no NK or NKT cells. These findings underscore the biological potency of the constructions.
Our strategy has readily observed biological effects. However, direct side by side comparisons to other complexes of IL-15 such as the IL-15Ra-Fc conjugates [19,21] or other previously reported constructs linking IL-15 to IL-15Ra [42] need to be carried out both in terms of pharmacokinetics and pharmacodynamics experiments. Indeed, our data with pAlbumin-hIL15 suggest that this fusion partner stabilizes IL-15 pharmacokinetics to a comparable extent, albeit the biodistribution and biological propierties may be quiet different.
Future research is required to optimize liver gene transfer and comparisons with the corresponding recombinant proteins are a must to ascertain the translational potential of these strategies. Using gene therapy and the liver to create a factory of proteins that complex into HDL has the advantage of using the natural routes of biosynthesis. However, the alternative of exogenously provided recombinant proteins is deemed more feasible for translation. In any case hydrodynamic gene transfers, although so far not translatable to humans, have provided important information on the pharmacokinetics and bioactivity of these new constructions. Indeed, although recombinant fusion proteins are our preferred approach to be translated into the clinic, we are further exploring the potential of liver gene transfer with gutless adenoviral vectors.
Interestingly, Apo A-I fusion cytokines incorporated to HDL do not impair the anti-inflammatory an cholesterol transporting functions of the HDL fractions, at least at the relative quantities generated in our studies. This has important implications for the overall strategy since as a result these agents are unlikely to result in increased cardiovascular risk.
IL-15 is being tested in patients as a monotherapy for melanoma and renal cell carcinoma in a dose escalating clinical trial (NCT01021059). However the use of IL-15 is more attractive when combined to adoptive T cell therapy [19], vaccines [20,21] or immunostimulatory mAb [48]. We are currently testing various combinatorial approaches that include our APO-IL-15 constructs.
In a sense, the antitumor effects observed under our conditions of treatment are less efficacious than what we had predicted based on the intense proliferative effects on NK and T cells. However, MC38 is a difficult-to-treat tumor model [49] and therefore, reaching more than 25% curative efficacy in monotherapy against 6-day established MC38 tumors is a remarkable achievement, which would be, for instance, comparable to what agonist anti-CD137 mAb attain in the same context [50]. The experiments in mice bearing liver MC38 tumors are illustrative of the local potency of the approach against metastatic disease and are conceivably associated to the fact of in situ activation of intrahepatic lymphocytes. However we have observed that these numeric increases of lymphocytes at such organ do not correlate with brighter expression of effector or activation-denoting molecules (data not shown). Therefore, the provision in combinatorial immunotherapies of therapeutic tools to enhance tumoricidal activity on cell per cell basis is guiding our next steps (MC Ochoa et al. unpublished results). In any case, it is of note that unresectable liver metastases of colon cancer constitute a major unmet therapeutic need and that our results show evidence for therapeutic efficacy in difficult to treat mouse models.
All in all, we have constructed chimeric IL-15 variants that are incorporated at least in part into HDL. In our experiments, we took advantage of IL-15 trans-presentation by the sushi domain of IL-15Ra to attain stronger bioactivity. Partial antitumor efficacy as a monotherapy strongly advocates for further development with combinatorial approaches, both in terms of adding other immunotherapeutic agents and in terms of further engineering the cytokine transgene products to be built into HDL. Figure S1 Schematic representation of the plasmids used for hydrodynamic injections. pcDNA3.1-based expression plasmids were engineered to encode for cassettes of expression encompassing Apo A-I lipoprotein, Apo A-I fused to human IL-15 and the sushi domain of IL-15Ra. Human IL-15 was cloned in a pIRES1neo-based plasmid (TIF) Figure S2 Expression of Sushi mRNA in the liver of mice receiving hydrodynamic injections with pApo-hIL15 and the plamid encoding the IL-15Ra sushi domain. Mice received 10 mg of pApo-hIL15 or pApo-hIL15+ pSushi by hydrodynamic injection and 12 h following treatment, livers were harvested and expression of Sushi mRNA was studied by quantitative RT-PCR normalized to b-actin. Data from 2 mice per group are presented. (TIF) Figure S3 Similar kinetics of hIL-15 serum concentrations in mice hydrodynamically gene-transferred with pAlbumin-hIL15 and pApo-hIL15. C56Bl/6 mice received a hydrodynamic injection with 10 mg of a construction coding for a protein encompassing albumin and hIL-15 or pApo-hIL15. Sera were obtained at the indicated times and hIL-15 concentrations were measured by ELISA. (TIF) Figure 7. Antitumor effects following the transfer of IL-15 constructions against MC38 tumors metastatic to the liver. Mice injected intraesplenically with 5x10 5 MC38 cells upon laparotomy were treated by hydrodynamic injections of the indicated plasmids one day later. On day 19 mice were euthanized and (A) liver surface was inspected to count macroscopic metastases observed in the surface of the liver under magnifying lens and data are represented on individual basis (*** indicates p,0.0001 between pApo-hIL15+ pSushi and the rest of the groups in Bonferroni test following one-way ANOVA test). (B) The weights of the spleens that also grafted tumors were recorded. Representative images of spleen tumors in the indicated groups are provided. The experiment shown is representative of two similarly performed (* indicates p,0.05 between pApo-hIL15+ pSushi and pApo groups in a t-student test). doi:10.1371/journal.pone.0052370.g007 Figure S4 HDL-fractions from pApo-hIL15 injected mice preserve their normal physiological functions. (A) The endothelial Py4-1 cell line was cultured in presence or absence of TNF-a for 4 h and later incubated or not overnight with HDL from mice injected with pApo or pApo-hIL15. Expression of VCAM was examined by flow cytometry. (B) 3T3 L1 cells were loaded by incubation with 3H-cholesterol and then HDLs from mice treated with pApo, pApo-hIL15 or pApo-hIL15+ pSushi were added to the culture. Cholesterol efflux was measured as 3H cpms in the culture supernatans. (TIF) Figure S5 Percentage of CD8 and NK cells in spleen, liver and blood increases in response to liver gene cotransfer of Apo-IL-15 and IL-15Ra sushi domain. Time course analyses of cell suspensions from spleen (A), liver (B) and blood (C) defining the percentages of lymphocyte subsets at the indicated time points (* indicates p,0.05 and **p,0.01 between pApo-hIL15+ pSushi and phIL15+ pSushi group in a Bonferroni test following two-way ANOVA test with p,0.0001). This experiment is representative of two performed with 2-3 mice for each time point. In (B) mice were sequentially followed. (TIF) Figure S6 Apo-IL-15 and IL-15Ra sushi domain gene transfer to the liver increases NK activity in the spleen. Natural cytotoxicity of spleen cells against the classical NK target Yac-1 at the indicated E:T ratios. Effector cells were obtained from mice treated 3 days earlier with the indicated plasmids or plamid combination. Data are from one representative experiment out of two performed. Each experiment was performed with two mice per group and rendered similar results. (TIF) Figure S7 Apo-IL-15 and IL-15Ra sushi domain gene transfer to the liver induces the proliferation CD8 and NK lymphocytes measured by BrdU incorporation. Mice were gene-transferred by hydrodynamic injections of the indicated plasmids. Subsequently three daily ip doses of BrdU were provided. Incorporation of BrdU to DNA was measured at 72 h since the gene transference by immunostaining and flowcytometry.Gating strategy (A and C) and data of an experiment with two mice per group (B and D) are showed. (TIF) Figure S8 NK and CD8 memory-phenotype T cells augment in IL15Ra2/2 mice on a B6129S background upon combined liver gene transfer of pApo-IL-15 and pSushi. (A and B) B6129S WT and syngenic IL15Ra2/2 mice were treated by hydrodynamic injections of the indicated plasmids 3 times every 7 days. At day 25 mononuclear leukocytes from the spleen were immunostained. Dots represent individual data from a single experiment with five mice per group. (C) Represents a separate experiment as in A/B that compares pSushi with a full length IL15Ra version upon coinjection with pApo-hIL15 to rescue the NK and CD8 phenotype of IL-15Ra2/2 mice. (TIF)