Patent Application
20130210896
Brain is a common location for tumor metastasis. Approximately one third of patients with systemic cancer ultimately develop central nervous system (CNS) involvement. This is in part due to the blood-brain barrier (BBB) that prevents distribution of chemotherapeutic drugs into interstitial space and brain's “immune-privileged” status that blocks penetration of inflammatory cells into the CNS. Recent work, however, has demonstrated that CNS immunosurveillance does indeed take place both in healthy and inflammatory CNS conditions (1). Even in experimental gliomas, activated T cells have been shown to find their targets in the brain (2). These latter observations have stimulated the development of vaccine therapies against brain tumors. However, despite induction of systemic immunity, immunosuppressive tumor microenvironment itself may attenuate the host anti-tumor responses (3-4). This is highlighted in recent trials that have demonstrated poor CNS response to systemic immunotherapies (2, 5).
Local immunosuppressive tumor milieu, caused by low levels of MHC class I expression (5), production of immunosuppressive factors (6), and scarcity of antigen presenting cells have all been considered to account for poor immune responsiveness of brain tumors (3, 8 and 22). One strategy to overcome this local barrier is through activation of the innate immune system. Cells that comprise this system (e.g. microglia, macrophages, monocytes, natural killer (NK), and dendritic cells) express pattern-recognition receptors that collectively recognize macromolecules that are broadly expressed by micro-organisms. Among these, activation of toll-like receptors (TLRs) has been shown to enhance phagocytosis, promote secretion of T helper type 1 (Th1) cytokines, and mediate leukocyte recruitment to infected tissues (6-8). Accordingly, agonists such as CpG oligodeoxynucleotides (CpG or CpG ODNs) that bind TLR9 have been evaluated as cancer vaccine adjuvants and have shown some efficacy in inducing adaptive and antigen-specific cellular anti-tumor immune responses (9). However, early-stage clinical trials in patients with melanoma and gliomas have been less promising (6, 10-11). Furthermore, high doses of CpG may be toxic due to exacerbation of brain edema. Thus, there is a need to provide a novel CpG conjugation to improve CpG delivery into inflammatory cells associated with brain tumors (e.g. gliomas and metastatic brain tumor).
One aspect of the invention relates to a method for improving CpG delivery into brain tumor associated inflammatory cells in a subject comprising administering a therapeutically effective amount of nanoparticle (NANO, e.g. carbon nanotube (CNT), gold, iron, silica, organic polymers, or carbon nanomaterials (e.g. fullerenes, rapheme, nanohorns, nanodiamond, etc.)) conjugated CpG (NANO-CpG) or a pharmaceutical composition thereof to the subject. In one embodiment, the brain tumor is gliomas. In another embodiment, the brain tumor is metastatic brain tumor.
Another aspect of the invention relates to a method for treating and/or preventing a brain tumor in a subject comprising administering a therapeutically effective amount of NANO-CpG or a pharmaceutical composition thereof to the subject.
In one embodiment, the brain tumor is gliomas. In another embodiment, the brain tumor is a metastatic brain tumor.
Another aspect of the invention relates to a composition comprising
Another aspect of the invention relates to a type II NANO-CpG prepared by a method comprising modifying a NANO with a NANO modifying agent (NANOMA) conjugated CpG (NANOMA-CpG).
Another aspect of the invention relates to an aqueous NANO-CpG-II dispersion.
One aspect of the invention relates to a method comprising administering a therapeutically effective amount of a nanoparticle (NANO) conjugated CpG (NANO-CpG) or a pharmaceutical composition thereof to a subject, wherein the NANO-CpG administration improves the CpG delivery into brain tumor associated inflammatory cells in the subject compared to an administration of the free CpG at a same dose.
Examples of NANO include, without limitation, carbon nanotubes (CNTs), gold, iron, silica, organic polymers, carbon nanomaterials (e.g. fullerenes, graphene, nanohorns, nanodiamond, and any combinations thereof), and any combinations thereof.
As used herein, the term “therapeutically effective amount” means any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder. The term also includes within its scope amounts effective to enhance normal physiological function.
CNTs are allotropes of carbon with a cylindrical nanostructure (9). Examples of CNTs include, without limitation, single-walled CNTs (SWCNTs), double-walled CNTs (DWCNTs), triple-walled CNTs (TWCNTs), multi-walled CNTS (MWCNTs) and combinations thereof.
CpG or CpG ODN, is a short single-stranded synthetic DNA molecule that contains a cytosine “C” followed by a guanine “G”. The “p” refers to a phosphodiester backbone of DNA
or a modified phosphorothioate backbone in a modified DNA
As used herein the term “conjugate” means to connect two compounds through formation of one or multiple covalent and/or non-covalent interactions. In certain embodiments, the one or multiple covalent or non-covalent interactions may be cleavable at a physiological condition of a subject. Examples of such cleavable interactions include, without limitation, cleavable covalent bonds such as bisulfide, ether, ester, amide, thio-ether, thio-ester, carbonate, carbamate, phosphate, and oxime bonds, and non-covalent interactions between DSPE and nanoparticles (e.g. CNTs).
Nanoparticles (e.g. CNTs) and CpG can be modified respectively to comprise functional groups that can form one or more covalent bonds together or can facilitate such conjugation. Such modifications can be through covalent and/or non-covalent interactions. Examples of the functional groups include, without limitation, carbonyl, carboxylic acid, acid, amine, hydroxyl, alkoxyl, and thio groups, including protonated and deprotonated forms thereof.
For example, CNTs can be modified into soluble functionalized CNTs (fCNTs) (10-12). A CpG can be modified to be a thiolated CpG (sCpG).
In certain embodiments, one or more linkers L is used to connect the NANO (e.g. CNT) and CpG. Examples of L include, without limitation, crosslinking agents that can connect the NANO (e.g. CNT) and CpG.
As used herein, a NANO-CpG conjugate may also be referred as NANO-CpG or NANO/CpG; a CNT-CpG conjugate may also be referred as CNT-CpG or CNT/CpG; and a SWCNT-CpG conjugate may also be referred as SWCNT-CpG or SWCNT/CpG.
A NANO-CpG used in the methods disclosed herein may be a type I NANO-CpG (NANO-CpG-I), a type II NANO-CpG (NANO-CpG-II), or a combination thereof.
The NANO-CpG-I is prepared by conjugating a CpG and/or a modified CpG to a modified NANO with or without L. The interactions among the CpG, modified CpG, L, and the modified NANO can be covalent and/or non-covalent interactions.
The NANO-CpG-II is prepared by providing a first CpG conjugate that can conjugate to an unmodified NANO, and then conjugating the first CpG conjugate with a NANO (modified or unmodified) to provide the NANO-CpG-II. The interactions between the first CpG conjugate and the NANO can be covalent and/or non-covalent interactions. The first CpG conjugate can be fully characterized before introduced to the NANO, thus the NANO-CpG-II is prepared with an improved characterization and reproducibility compared to the NANO-CpG-I.
In certain embodiments, the NANO-CpG-I is prepared by a method comprising the following steps:
1-a) optionally modifying a NANO to provide a NANO modified with at least a first free functional group (modified NANO);
1-b) optionally modifying a CpG to provide a CpG modified with at least a second free functional group (modified CpG); and
1-c) conjugating the modified NANO and the modified CpG to provide the NANO-CpG-I, optionally via a linker L though covalent and/or non-covalent interactions. In certain embodiments, the first and the second free function groups may be the same or different. In certain embodiments, the NANO is a CNT (e.g. a SWCNT).
In certain embodiments, the NANO-CpG-II is prepared by a method comprising the following steps:
2-a) optionally providing a NANOMA that can modify a NANO with at least a first free functional group;
2-b) optionally providing a CpG modified with at least a second free functional group (modified CpG);
2-c) optionally conjugating the NANOMA and the modified CpG to provide a NANOMA-CpG conjugate (NANOMA-CpG), optionally via a linker L though covalent and/or non-covalent interactions; and
2-d) modifying the NANO with the NANOMA-CpG to provide the NANO-CpG-II. In certain embodiments, the first and the second free function groups may be the same or different.
In certain embodiment, an aqueous NANO-CpG-II dispersion is prepared by a method comprising Steps 2-a), 2-b), 2-c) and 2-d), wherein:
Step 2-d) further comprising sonicating an aqueous mixture of NANO, NANOMA-CpG, and NANOMA.
The amount of the NANOMA needed to provide an aqueous NANO-CpG-II dispersion depends on the method used in step 2-d), and the concentrations and/or characteristics of the NANOMA, NANO and/or NANOMA-CpG (e,g. surface charges, stabilities, NANO aggregation properties). In one example, the NANO concentration is about 0.5 mg/mL or higher, about 1 mg/mL or higher, or about 2 mg/mL or higher. Optionally surfactants such as sodium dodecyl sulfate (SDS) can be used in this step to facilitate dispersal. In certain embodiments, the additional NANOMA facilitates the NANO dispersion and improve the dispersal stability, unexpectedly.
In one embodiment, the amount of additional NANOMA is at least about 1 molar eq., at least about 1.5 molar eq., or at least about 2 molar eq. of that of NANOMA-CpG. Without being bound by any specific mechanism, as it has been reported that sufficient surface charge can allow for stable dispersal of single-walled CNT (SWCNT) bundles in water (75). Thus, when CNTMA comprises one or more charged groups, these charges may be introduced to the surface of the CNT and facilitate the stable CNT dispersal.
In another embodiment, the weight ratio of NANO:NANOMA-CpG is at least about 1:10, at least about 1:5, at least about 1:2, at least about 1:1, at least about 2:1, or at least about 5:1.
In another embodiment, the CNT is a SWCNT, the CNTMA is Lipid-PEG-NH2 as shown below:
wherein:
R1 is Ca alkyl;
R2 is Cb alkyl;
a and b are the same or different and are independently selected from the group consisting of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30; and
n=30, 31 . . . 44, 45, 46 . . . or 100.
A method described by Liu et al. (18) can be used to modify the SWCNT with Lipid-PEG-NH2 to provide SWCNT-Lipid-PEG-NH2 (Scheme 1):
wherein the modification involves non-covalent interactions.
In another embodiment, the CpG has a sequence of SEQ ID: NO. 1 (5′-TGA CTG TAA CGT TCG AGA TGA-3′) or SEQ ID: NO. 2 (5′-TAA ACG TTA TAA CGT TAT GAO GTC AT-3′).
A CpG can be thiolated to provide a sCpG as described by Rosi et al. (12). SWCNT-Lipid-PEG-NH2 and sCpG can be conjugated via a linker L as described by Kam et al. (19). In a preferred example, the sequence of a thiolated CpG is a thiolated sCpG having a sequence of SEQ ID: NO. 3 (5′-TGACTGTAACGTTCGAGATGA-3′) or SEQ ID: NO. 4: (5′-TAAACGTTATAACGTTATGACGTCAT-3′). The linker L is Sulfo-LC-SPDP (S-LC-SPDP) as shown below, SWCNT-Lipid-PEG-NH2 and sCpG are conjugated through disulfide bonds to provide SWCNT-Lipid-PEG-LC-SPDP-CpG-I, which is a CNT-CpG-I (Scheme 2).
wherein the modification involves non-covalent interactions.
In one example, a=b=16, and n=45.
In another embodiment, SWCNT-Lipid-PEG-LC-SPDP-CpG-II that is a CNT-CpG-II is prepared by first reacting Lipid-PEG-NH2 with a sCpG and a linker L (S-LC-SPDP) to provide Lipid-PEG-LC-SPDP-CpG (Scheme 3):
Then a SWCNT is modified with Lipid-PEG-LC-SPDP-CpG in the presence of Lipid-PEG-NH2 (e.g. about 2 molar eq. of that of Lipid-PEG-LC-SPDP-CpG) to provide SWCNT-Lipid-PEG-LC-SPDP-CpG-II that is a CNT-CpG-II (Scheme 4):
wherein the modification involves non-covalent interactions.
Examples of inflammatory cells include, without limitation, microglia and macrophages (MG/MP), NK cells, and dendritic cells (DC). Examples of inflammatory cells associated with brain tumors include, without limitation, tumor-associated MG/MP, NK cells, and DC.
A NANO-CpG can be administered to a subject by a parenteral systematic administration, and preferably an intracerebral administration.
A pharmaceutical composition of a NANO-CpG comprises the NANO-CpG and a pharmaceutically acceptable carrier.
Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil or injectable organic esters, or emulsions such as oil/water emulsions or a triglyceride emulsion, various types of wetting agents, tablets, coated tablets and capsules. A suitable pharmaceutically acceptable carrier may be selected taking into account the chosen mode of administration.
A pharmaceutically acceptable carrier can also contain physiologically acceptable compounds that act, for example, to stabilize or to increase the absorption of the conjugate. Such physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. One skilled in the art will know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration of the composition.
In one embodiment, the pharmaceutical carrier may be a liquid and the pharmaceutical composition would be in the form of a solution. In another equally preferred embodiment, the pharmaceutically acceptable carrier is a solid and the pharmaceutical composition is in the form of a powder or tablet.
A solid carrier can include one or more substances which may also act as flavoring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, binders or table-disintegrating agents, it can also be an encapsulating material. In powders, the carrier is a finely divided solid that is in admixture with the finely divided active ingredient. In tablets, the active-ingredient is mixed with a carrier having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The powders and tablets may contain up to 99% of the active ingredient. Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.
Besides containing an effective amount of the NANO-CpG described herein the pharmaceutical compositions may also include suitable diluents, preservatives, solubilizers, emulsifiers, adjuvant and/or carriers.
The compound can be administered in the form of a sterile solution or suspension containing other solutes or suspending agents, for example, enough saline or glucose to make the solution isotonic, bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 80 (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like.
Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular compound in use, the severity of the disease state, drug combination(s), reaction sensitivities, and response to therapy. Additional factors depending on the particular subject being treated, including the general health of the subject, the age, weight, gender and diet of the subject, and time and frequency of administration, will result in a need to adjust dosages. Administration of the NANO-CpG or pharmaceutical composition thereof may be effected continuously or intermittently. In any treatment regimen, the NANO-CpG or pharmaceutical composition may be administered to a patient either singly or in a cocktail containing other therapeutic agents, compositions, or the like, including, but not limited to, other chemotherapies, radiation therapy, tolerance-inducing agents, potentiators and side-effect relieving agents. Preferred potentiators include temozolomide, ipilimumab (or other immune modulators), monensin, ammonium chloride, perhexyline, verapamil, amantadine, and chloroquine. All of these agents are administered in generally-accepted efficacious dose ranges such as those disclosed in the Physician's Desk Reference, 41st Ed., Publisher Edward R. Barnhart, N.J. (1987), which is incorporated herein by reference.
In the treatment, an appropriate dosage level will generally be about 0.001 to 10 mg per kg subject body weight per day that can be administered in single or multiple doses. Preferably, the dosage level will be about 0.005 to about 25 mg/kg, per day; more preferably about 0.01 to about 10 mg/kg per day; and even more preferably about 0.05 to about 1 mg/kg per day.
The frequency of dosing will depend upon the pharmacokinetic parameters of the NANO-CpG (e.g. CNT-CpG) in the formulation used. Typically, a composition is administered until a dosage is reached that achieves the desired effect. The composition may therefore be administered as a single dose, or as multiple doses (at the same or different concentrations/dosages) over time, or as a continuous infusion. Further refinement of the appropriate dosage is routinely made. Appropriate dosages may be ascertained through use of appropriate dose-response data. For example, long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half-life and clearance rate of the particular formulation.
In one embodiment, intracerebral administration of a therapeutically effective amount of a NANO-CpG (e.g. CNT-CpG) or a pharmaceutical composition thereof showed an unexpected enhancement in the CpG uptake by tumor-associated leukocytes (See, e.g. Example 3).
Another aspect of the invention relates to a method for treating and/or preventing a brain tumor in a subject comprising administering a therapeutically effective amount of a NANO-CpG (e.g. CNT-CpG) or a pharmaceutical composition thereof to the subject. In one embodiment, the brain tumor is gliomas. In another embodiment, the brain tumor is metastatic brain tumor. Examples of the NANO-CpG include, without limitation, the NANO-CpGs described supra. Unless otherwise specified, NANO-CpG can comprise NANO-CpG-I, NANO-CpG-II or a combination thereof.
As used herein, the term “treating” means curing, alleviating, inhibiting, or preventing. The term “treat” as used herein means cure, alleviate, inhibit, or prevent. The term “treatment” as used herein means cure, alleviation, inhibition or prevention.
In one embodiment, the method disclosed herein provides an immunotherapy for brain cancer.
In another embodiment, a subject treated with the NANO-CpG composition disclosed herein or a pharmaceutical composition thereof developed immunity to brain tumor.
In another embodiment, intracerebral administration of a therapeutically effective amount of a NANO-CpG (e.g. CNT-CpG) or a pharmaceutical composition thereof unexpectedly resulted in eradication of intracranial (i.c.) gliomas in glioma-bearing mice (e.g. Example 7) and abrogated the growth of subcutaneous (s.c.) tumors in subjects bearing both i.c. and s.c. melanomas. The therapeutically effective amount of the NANO-CpG or a pharmaceutical composition thereof was unexpectedly low. The survived subjects also exhibited durable tumor-free remission and were protected from tumor rechallenge, showing induction of systemic antitumor immunity (e.g. Example 18). Thus, the intracerebral CpG therapy with NANO delivery system could be applicable to the treatment of systemic immunogenic tumors with brain metastasis.
Another aspect of the invention relates to a composition comprising
In one embodiment, the conjugation between the NANOMA and CpG is optionally through a linker L.
In another embodiment, the composition consists essentially of:
In another embodiment, the NANO is a CNT.
In another embodiment, the CNT is a SWCNT.
In another embodiment, the composition further comprises the NANOMA.
The amount of additional NANOMA is at least about 1 molar eq., at least about 1.5 molar eq., or at least about 2 molar eq. of that of NANOMA-CpG.
In another embodiment, the NANO-CpG is SWCNT-Lipid-PEG-LC-SPDP-CpG and the NANOMA is Lipid-PEG-NH2 as described supra.
In another embodiment, the composition treats or prevents brain cancer.
Another aspect of the invention relates to a NANO-CpG-II as described supra, prepared by a method comprising modifying a NANO with a NANOMA-CpG.
In one embodiment, the NANO-CpG-II is a type II CNT-CpG (CNT-CpG-II).
In another embodiment, the CNT-CpG-II is a SWCNT-Lipid-PEG-LC-SPDP-CpG prepared by a method comprising the following steps:
2-a0) optionally providing a Lipid-PEG-NH2 that can modify a SWCNT with at least a NH2 group;
2-b0) optionally providing a sCpG;
2-c0) optionally reacting Lipid-PEG-NH2 with the sCpG and an optional linker L (e.g. S-LC-SPDP) to provide a Lipid-PEG-L-CpG (e.g. Lipid-PEG-LC-SPDP-CpG) according to Scheme 3; and
2-d0) modifying the SWCNT with the Lipid-PEG-L-CpG in the presence of Lipid-PEG-NH2 to provide the SWCNT-Lipid-PEG-LC-SPDP-CpG (CNT-CpG-II, Scheme 4), wherein R1, R2, and n are defined the same as supra.
In certain embodiments, R1, R2 are C16 alkyl groups, and n=45.
In certain embodiments, step 2-d0) further comprising sonicating an aqueous mixture of SWCNTs, Lipid-PEG-L-CpG and Lipid-PEG-NH2 to provide the SWCNT-CpG-II.
Another aspect of the invention relates to an aqueous NANO-CpG-II dispersion as described supra.
The amount of the NANOMA needed to provide an aqueous NANO-CpG-II dispersion depends on the method used in step 2-d), and the concentrations and/or characteristics of the NANOMA, NANO and/or NANOMA-CpG (e,g. surface charges, stabilities, NANO aggregation properties). In one example, the NANO concentration is about 0.5 mg/mL or higher, about 1 mg/mL or higher, or about 2 mg/mL or higher. Optionally surfactants such as sodium dodecyl sulfate (SDS) can be used in this step to facilitate dispersal. In certain embodiments, the additional NANOMA facilitates the NANO dispersion and improve the dispersal stability, unexpectedly.
In one embodiment, the amount of the additional NANOMA is at least about 1 molar eq., at least about 1.5 molar eq., or at least about 2 molar eq. of that of NANOMA-CpG. Without being bound by any specific mechanism, as it has been reported that sufficient surface charge can allow for stable dispersal of single-walled CNT (SWCNT) bundles in water (75). Thus, when CNTMA comprises one or more charged groups, these charges may be introduced to the surface of the CNT and facilitate the stable CNT dispersal.
In another embodiment, the weight ratio of NANO:NANOMA-CpG is at least about 1:10, at least about 1:5, at least about 1:2, at least about 1:1, at least about 2:1, or at least about 5:1.
In another embodiment, the aqueous NANO-CpG-II dispersion is an aqueous CNT-CpG-II dispersion.
In another embodiment, the aqueous CNT-CpG-II dispersion is an aqueous SWCNT-Lipid-PEG-LC-SPDP-CpG dispersion prepared by a method comprising Steps 2-a0), 2-b0), 2-c0) and 2-d0), wherein:
Step 2-d0) further comprising sonicating an aqueous mixture of Lipid-PEG-LC-SPDP-CpG, Lipid-PEG-NH2 and the SWCNT. The suitable amount of the Lipid-PEG-NH2 presented in Step 2-d0) should be sufficient to provide a substantially stable dispersion of the CNT-CpG-II at a desired concentration. When the concentration of the CNT-CpG-II is at least about 1 mg/mL, the amount of Lipid-PEG-NH2 is at least about 1 molar eq. of that of Lipid-PEG-LC-SPDP-CpG, at least about 2 molar eq. of that of Lipid-PEG-LC-SPDP-CpG, at least about 3 molar eq. of that of Lipid-PEG-LC-SPDP-CpG, or from about 1 molar eq. to about 3 molar eq. of that of Lipid-PEG-LC-SPDP-CpG. When the concentration of the CNT is higher, the amount of Lipid-PEG-NH2 needed also increases. A person of ordinary skill in the art would know how to determine the suitable amount of Lipid-PEG-NH2 by characterizing the CNT-CpG-II prepared using different ratio among CNT, Lipid-PEG-NH2, and Lipid-PEG-LC-SPDP-CpG. For an aqueous CNT-CpG-II dispersion having about 1 mg/mL CNT, the preferred weight ration between CNT and Lipid-PEG-LC-SPDP-CpG is at least about 1:10, at least about 1:5, at least about 1:2, at least about 1:1, at least about 2:1, at least about 5:1, or at least about 10:1.
In certain embodiments, the aqueous CNT-CpG-II dispersion as described supra is unexpectedly more stable than SDS-dispersed SWCNT at an unexpectedly high concentration (see, Example 16, wherein sonicating the SWCNT (1 mg/mL) with a 2:1 molar ratio of Lipid-PEG-NH2 to Lipid-Peg-LC-SPDP-CpG provided an aqueous dispersion of CNT-CpG-II that was stable for over four months, with no change in activity). While a stable aqueous dispersion of the type I CNT-CpG (CNT-CpG-I)has a concentration of not more than 0.25 mg/mL. Thus, the CNT-CpG-II prepared according to the methods disclosed herein provides an unexpected more stable aqueous dispersion of CNT-CpG conjugate at an unexpected high concentration.
Furthermore, the production of the CNT-CpG-II is better characterized than that of the CNT-CpG-I, and the product of the CNT-CpG-II is better characterized than that of the CNT-CpG-I. In the preparation of the CNT-CpG-I, the final step is the conjugation of the modified CpG with the modified CNT via the linker L. The modified CNT may only react with L (CNT-L) and does not conjugate with CpG to provide the desired CNT-CpG. The amounts of CNT-L and CNT-CpG cannot be characterized. Furthermore, CNT may absorb unreacted modified CpG and/or linker L, which cannot be characterized either. Thus, it is difficult to provide a reasonable characterization of the final product. To the contrary, in the preparation of the CNT-CpG-II, the final reaction is the modification of the CNT with the CNTMA-CpG in the presence of CNTMA. The CNT is modified with one or more CNTMA-CpG and/or CNTMA, which can be better characterized. Thus, the CNT-CpG-II prepared according to the methods disclosed herein provides an unexpected better aqueous dispersion of CNT-CpG conjugate at an unexpected high concentration with a better characterization and reproducibility.
A thiolated CpG (SEQ ID NO:5: sCpG: 5′-TGACTGTAACGTTCGAGATGA-3′) and thiolated control oligodeoxynucleotides (sODN: 5′-TGACTGTAAGGTTAGAGATGA-3′, SEQ ID:NO. 6) were constructed as described by Rosi et al. (12) and labeled with Cy5.5 (Lumiprobe, LLC). Anti-NK1.1 (clone PK136) was purchased from eBioscience (eBioscience Inc., San Diego, Calif.). Anti-CD8 Ab (clone H35) was purified as previously described (13). Control normal mouse IgG was purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology Inc., Santa Cruz, Calif.). All flow Abs (i.e. CD11b, CD45, CD11c, CD8, and NK1.1) and isotype controls were purchased from BD Biosciences (San Jose, Calif.) or eBiosciences (San Diego, Calif.).
Cell Lines
eGFP and luciferase-expressing GL261 glioma cell lines (GL261.gfp and GL261.luc) were generated as described before (14-15). Primary bone marrow-derived monocytes (BMM) were harvested from normal C57BL/6 or CX3CR1GFP mice. After washing the bone marrow with cold PBS, cells were isolated and collected with Cell Strainer (BD Biosciences, San Jose, Calif.). The isolated BMM were then cultured in L929-conditioned DMEM medium. Red blood cells and other non-adherent cells were removed by changing the culture medium in 24 hours. Cultures with more than 90% CD11b+ purity (as assessed by FACS) were used for experiments.
Single-Walled Carbon Nanotubes Construction and Functionalization
Single-walled carbon nanotubes (SWCNTs or CNTs) measuring 200-400 nm in length were generated and characterized by electron microscopy as described before (16-17). CNT functionalization was performed using methods described by Liu et al. (18). Briefly, hipco CNTs were sonicated extensively (1 hour) in a solution of 1,2-distearoyl-Sn-glycero-3-phosphoethanolamine-N-[amino (polyethylene glycol) 2000] (PEG) (Avanti Polar Lipids, Alabama). The supernatant solution of PEG-CNT was collected after centrifuge at 24,000 g for 6 hours. After removal of the excess PEG molecules by Amicon centrifugal filter device (100 kDa), functionalized PEG-CNTs were conjugated with Sulfo-LC-SPDP (Thermo Fisher Scientific Inc., USA) for 1 hour at room temperature. After removal of the excess Sulfo-LC-SPDP by Amicon centrifugal filter device (100 kDa) (Millipore, Billerica, Mass.), the CNT conjugates were quantified by SpectraMax M2 (Sunnyvale, Calif., USA) spectrometer with a weight extinction coefficient of 0.0465 l mg−1 cm−1 at 808 nm. CNTs were then conjugated with sCpGs or sODNs through a cleavable disulfide bond at 4° C. for 24 hours. Free sCpGs were then separated from the solution by Amicon centrifugal filter device (100 kDa) (Millipore, Billerica, Mass.) and measured with NanoDrop 1000 Spectrophotometer (Thermo Scientific). The CNT-bound sCpGs were quantified by subtracting the unbound sCpG from the total sCpG added prior to the conjugation reaction.
Before the CNTs were used as drug carriers, they were functionalized using a modified noncovalent technique reported by Kam et al. and linked to the CpG (19).
Real-Time PCR (RT-PCR)
BMM (5×104 cells/well in 24-well plates) were incubated with sCpG (5 pg/well), CNT-sCpG (CNT 2.5 μg-sCpG 5 μg/well), blank CNT (2.5 μg/well), or CNT+sCPG mixture. At various times, cells were collected and the total RNA was generated using the Trizol system (Invitrogen Carlsbad, Calif.) followed by double DNase treatment and column purification using the Qiagen RNeasy Clean-up Protocol. Real-time PCR was performed in a TaqMan 5700 Sequence Detection System (Applied Biosystems, Foster City, Calif.) as described previously (25). The PCR conditions were optimized such that a minimum of 10,000 fold range could be detected for each primer.
Cytokine Multiplex Analysis
Supernatants from PCR samples were analyzed for 20 cytokines using mouse Cytokine twenty-Plex Antibody Bead Kit (Invitrogen, Camarillo, Calif.) as per the manufacturer's protocol. Assay plates were analyzed by a Bioplex HTF Luminex reader (Bio-Rad Laboratories, Inc., Hercules, Calif.) instrument. Cytokine concentrations were calculated using Bio-Plex Manager 3.1 software with a five parameter curve-fitting algorithm applied for standard curve calculations for duplicate samples. Data from detectable cytokines and chemokines were compared between each group.
In Vitro NF-κB Assay:
RAW MP cells (RAW-Blue™) stably transfected with a reporter construct expressing a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factors NF-κB and AP-1 (InvivoGen) were used to measure TLR-9 activation. Upon TLR stimulation, RAW-Blue™ cells induce the activation of NF-κB and AP-1, and subsequently the secretion of SEAP. SEAP activity will be evaluated after treatments with: saline (control), S-CpG (free thiolated CpG), scrambled oligodeoxynucleotide sequence (ODN control), blank nPs, or nPs loaded with either S-CpG (nP-CpG) or scrambled sequence (nP control).
Tumor Implantation, Treatment, and Imaging
All animals were housed and handled in accordance to the guidelines of City of Hope Institutional Animal Care and Use Committee (IACUC). Intracranial tumor implantation was performed as described previously (20). GL261.luc or GL261.gfp cells were harvested by trypsinization, counted, and resuspended in PBS. Female C57BL/6 or CX3CR1GFP mice that express EGFP under control of the endogenous Cx3cr1 locus (Jackson Laboratory, Bar Harbor, Me.) weighing 15-25 g were anesthetized by intraperitoneal (i.p.) administration of ketamine (132 mg/kg) and xylazine (8.8 mg/kg), and immobilized in a stereotactic head frame. Through a small burr hole, 3 μl of PBS containing 1×105 tumor cells was injected unilaterally as described before (20).
Four days after i.c. tumor implantation, mice received one i.t. injection of PBS (control, 10 μL), free sCpG (5 μg/10 μL PBS), blank CNT (2.5 μg) mixed with free sCpG (CNT+sCpG; 5 μg sCpG/10 μL PBS), or sCpG conjugated to CNT (CNT-sCpG; 2.5 μg CNT/5 μg sCpG/10 μL PBS) through the initial burr hole. Tumor growth was assessed by Xenogen IVIS In Vivo Imaging System (Xenogen, Palo Alto, Calif.) as previously described (14).
NK and CD8 depletion studies were carried out as described (14). In these experiments mice were injected with anti-CD8, anti-NK1.1, or control IgG (200 pg/mouse, i.p.) mAb one day prior to tumor implantation and each i.t. injection. Leukocyte depletion was confirmed with FACS analysis of peripheral blood (14). For tumor rechallenge experiments, naive or GL261-bearing mice that had survived for at least three months after the initial CNT-sCpG treatment and were tumor-free by imaging, were re-challenged with i.c. GL261 (1×105 cells).
In Vivo Uptake and Biodistribution Studies
Tumor-bearing w.t. or CX3CR1GFP mice were injected with CNT bound to Cy5.5-labeled sCpG (CNT-sCpG5.5, 2.5 μg CNT/5 μg sCpG/10 μL PBS) or free sCpG5.5 (5 μg sCpG/10 μL PBS). Tumors were harvested at various time intervals and evaluated by flow cytometry as described below. For imaging, frozen brain sections were embedded in O.C.T. (Tissue-Tek) and 10 μm sections were cut using cryostat (Leica Microsystem Inc., Bannockburn, Ill.). Sections were mounted in Vectashield mounting medium containing 4060-diamidino-2-phenylindole (DAPI) (Vector, Burlingame, Calif.). Images were obtained by AX-70 fluorescent microscopy (Leica Microsystems Inc., Bannockburn, Ill.) and were prepared by Zeiss LSM Image Browser software.
Chromium Release Cytotoxicity Assay
Cytotoxicity against B16.F10 melanoma cells was determined using a standard 51Cr release assay (16). Briefly, effector cells were derived from spleens of B16.F10-bearing C57BL6 mice (n=4) treated with either i.c. or s.c. CNTCpG. CNT-CpG injections were given 3 times, 4 days after initial tumor implantation, and every subsequent 3 days. Mice were sacrificed 48 hours after the final treatment and splenocytes (effectors) were harvested and co-incubated with irradiated (30,000 rad) B16.F10 cells for 7 days. Effectors were then co-incubated for 6 hours with 5,000 51Cr-loaded B16.F10 targets in 96-well plates at ratios of 100:1, 20:1, and 4:1 (in triplicate). Radioactivity released into the supernatant was measured using a Cobra Quantum gamma counter (PerkinElmer). Percent specific lysis was calculated as: (experimental release−spontaneous release)/(maximum release−spontaneous release)×100%.
Flow Cytometry Analysis
Tumors and blood samples were harvested and examined by flow cytometry as described previously (14). Cell suspensions from brain tissue were forced through a 40 μm filter. Blood samples were incubated in Gey's buffer (pH 7.2) for 10 min. Freshly-prepared samples were resuspended in 0.1 M PBS containing 1% FBS and 2 mM EDTA and incubated with FcγIII/IIR-specific Ab to block nonspecific binding. Samples were then stained with different combinations Abs or isotype controls for 1 h at 4° C. and analyzed by a CyAn fluorescence cell sorter (BDIS, San Jose, Calif.). Inflammatory cells were gated and separated from the rest of sorted cells base on forward vs. side-scatter analysis and their staining characteristics. FlowJo 8.4.7 software (Tree Star, Inc., Ashland, Oreg.) was used for data analysis and the proportion of each cell type was measured as percent of total inflammatory cells. Glioma MPs were gated as CD11b+/CD45high and MG as CD11b+/CD45low based on previously-described phenotype characterization (21).
G-MDSC have a phenotype of CD11b1Ly6G1Ly6Clow, whereas MMDSC have a phenotype of CD11b1Ly6G_Ly6Chigh [23, 24].
Statistical Analysis
Statistical comparison in all different experimental conditions was performed with the prism software using two-way analysis of variance (ANOVA) or Student's t-test. Survival was plotted using a Kaplan-Meir survival curve and statistical significance was determined by the Log-rank (Mantel-Cox) test. A P value of less than 0.05 was considered significant.
CNT conjugation capacity plateaued at 1-2 μg sCpG per pg CNT, which was consistent with other oligonucleotides (24). To evaluate sCpG uptake in vitro, BMM derived from w.t. or CX3CR1GFP mice were incubated with free sCpG5.5 (5 μg/mL) or CNT-sCpG5.5 (2.5 μg CNT-5 μg sCpG5.5/mL). The sCpG5.5 uptake was visualized by fluorescent microscopy and quantified by flow cytometry. Dot plots demonstrated that CNT-sCpG uptake by BMM was more efficient than update of free sCpG5.5 (
CNT-sCpG (2.5 μg CNT-5 μg CpG/well), sCpG (5 μg/well), or CpG (5 pg/well) were incubated with BMM (5×104 cells/well in 24-well plates) and the cytokine expression was evaluated by qRT-PCR (
BMM (5×104 cells/well in 24-well plates) were incubated with sCpG (5 pg/well), CNT-sCpG (CNT 2.5 μg-sCpG 5 μg/well), blank CNT (2.5 μg/well), or CNT/sCpG mixture (CNT+sCpG) and cytokine/chemokine expression was evaluated by qRT-PCR and ELISA. The data were representative of two separate experiments (
The thiolation process that was employed to link CpG with CNT appeared to suppress CpG pro-inflammatory function (
To evaluate CNT-sCpG uptake in vivo, two different i.c. GL261 glioma models were used. To assess uptake by tumor-associated inflammatory cells, GL261 cells were implanted into CX3CR1GFP mice that express eGFP under control of the endogenous Cx3cr1 locus. Although in these transgenic mice eGFP is expressed in MG, MPs, and other myeloid-derived cells, the flow cytometry studies (not shown) have indicated that the majority (more than 70%) of eGFP-expressing cells in this glioma model are MPs (CD11b+, CD45high) and MGs (CD11b+, CD45low) based on previously-described phenotype characterization (26). In the second model, GL261.egfp cells were i.c. implanted into w.t. mice in order to measure sCpG uptake by tumor cells. Four (GL261 model) or ten days (slower-growing GL261.egfp model) after i.c. tumor implantation, the tumors were injected with CNT-sCpG (2.5 μg CNT/5 μg sCpG/10 μL PBS) or sCpG, (5 μg/10 μL PBS) and tumor GFP cells were sorted and examined for Cy5.5 uptake at various time points (the left panels of
Consistent with the in vitro experiments, CNT conjugations enhanced sCpG uptake by tumor-associated inflammatory cells, and to a lesser (non-significant) degree, by gliomas. Interestingly, the representative fluorescent micrographs of brains of the treated mice showed that both sCpG5.5 particles and sCpG5.5+ MG/MP persisted in the brains of these animals for more than seven days after the initial injection (
To further evaluate CNT-sCpG+ cell types, i.c. GL261 tumors in w.t. mice were injected with PBS (control), CNT-sCpG5.5, free sCpG5.5 or CNT/sCpG5.5 mixture at the same concentration as in
As expected, CNT conjugation promoted CpG uptake by MG/MP, NK, and DC cell (but not CD8 cells, not shown) (FIGS. 9 and 10A-10C). Although 20-30% of tumor leukocytes internalized CNT-sCpG5.5 particles within two days, the actual proportion of cells that were sCpG5.5+ locally was probably much higher because cells from the entire tumor (and not just the injection site) were analyzed in these experiments (
Also, to assess which cell types were the prominent carriers of CNT-sCpG, and to check if the decline in sCpG5.5+ cells in the CNT-sCpG5.5 group at four days was due to the migration of these cells away from the injection site or trafficking of circulating sCpG5.5-negative cells into the tumor, the total number of each cell type for all treatment groups were measured. Neither sCpG nor sCpG/CNT treatments induced a significant change in tumor leukocyte infiltration (not shown). However, in the CNT-sCpG-treated mice, total and Cy5.5+ cells rapidly increased within two days of injection before stabilizing (
To evaluate CNT-sCpG immunotherapy, mice bearing four day-old i.c. GL261.luc gliomas were given a single intratumoral (i.t.) injection of PBS (control), free sCpG (5 μg/10 μL PBS), CNT+sCpG (2.5 μg CNT+5 μg sCpG/10 μL PBS), or CNT-sCpG (2.5 μg CNT−5 μg sCpG/10 μL PBS). The CNT/sCpG dose combination was selected based on the CNT conjugation capacity of sCpG (discussed above) and the observation indicating CpG to effectively eradicate gliomas at this dose (i.e. 5 μg), albeit after multiple injections (21). Intracranial tumor burden was assessed by biophotonic imaging of mice at 3, 7, and 21 days after tumor injection (
Mice bearing four day-old i.c. GL261.luc gliomas were given a single i.t. injection of PBS (control), free sCpG (5 μg/10 μL PBS), blank CNT (2.5 μg), CNT-sODN (2.5 μg CNT/5 μg sODN/10 μL PBS) or CNT-sCpG (2.5 μg CNT/5 μg CpG/10 μL PBS). Kaplan-Meier analysis was used to assess the survival for the treated mice (
As seen in the previous in vivo studies (21), a single low-dose injection of free CpG had no anti-tumor effect (
The exact mechanism for this improved sCpG therapy with CNT-conjugated delivery is unclear but may have been due to, without being bound to a specific mechanism, 1) higher sCpG uptake by tumor-associated TLR9 inflammatory cells (such as MG/MP), 2) slower intra- or extra-cellular sCpG release and clearance by CNTs (depot effect), and/or 3) migration of CNT-carrying tumor inflammatory cells into lymphoid organs where other immune cells are activated.
In order to investigate inflammatory cellular responses to CNT-sCpG therapy, mice bearing 4 day-old i.c. tumors were treated with PBS, sCpG, CNT-sCpG or CpG-sODN of the same doses as in Example 4, and the local and systemic inflammatory changes were analyzed by FACS (
Although all agents increased CNS MG/MP, CD8, and NK cell responses, only CNT-sCpG caused a significantly higher infiltration of MG/MP over free sCpG. Furthermore, only CNT-sCpG-treated mice demonstrated a sustained elevation of circulating NK cells. These observations suggested, without being bound to a specific mechanism, that both MG/MP and NK cells played a role in the CNT-sCpG anti-tumor response. To better characterize which cell types were responsible for tumor rejection, leukocyte depletion studies were performed next in Example 6.
Naïve mice were depleted of CD8 or NK cells by intraperitoneal (i.p.) injection of depleting doses of mAbs specific to CD8 or NK (αCD8 or αNK) or treated with control IgG (200 μg/injection) one day prior to both tumor implantation and CNT-sCpG (2.5 μg CNT/5 μg CpG/10 μL PBS) treatments. Intracranial tumor burden was assessed by biophotonic imaging at day 4, 7, 14, and 21 post tumor implantation (
To determine if CNT-sCpG treatment induced immunity against gliomas, CNT-sCpG-treated GL261-bearing mice that had survived for at least three months, along with normal naïve mice, were rechallenged with an i.c. injection of GL261 glioma(1×105). Intracranial tumor burden was assessed by biophotonic imaging at 4 h, days 1, 4, and 7 after the tumor implantations (
Before animal experiments, CNT-CpG activity was examined using an in vitro NF-κB assay as described supra (
To confirm whether i.c. CNT-CpG elicited a systemic antitumor response, mice bearing both i.c. and s.c. B16F10-luc melanomas were treated twice (7 and 11 days (arrows) after initial tumor implantations) with i.c. CNT-CpG (2.5 μg CNT/5 μg CpG/10 μL, ) or i.c. CNT (2.5 μg/10 μL, ▪) that were functionalized only with PL-PEG (
When administered i.c., CNT-CpG (but not blank CNT) inhibited the growth of not only i.c., but also, s.c. melanomas (
To evaluate the impact of injection site on CNT-CpG efficacy, a similar experiment as described supra in this example was performed except that the animals were treated with either s.c. or i.c. CNT-CpG three injections (5, 8, and 12 days) after tumor implantation (
Similar to the previous experiments, i.c. CNT-CpG (but not blank CNT) inhibited the growth of both i.c. and s.c. melanomas (
Because CNT-CpG biodistribution directly affected its function, CNT-CpG clearance in both i.c. and s.c. tumors were compared in Example 10.
To study the potential differences in CNT-CpG uptake and retention in tumors, mice bearing both i.c. and s.c. B16-luc melaomas (green) were injected i.t. with Cy5.5-labeled (red) CpG5.5 or CNT-CpG5.5 into the four-day old i.c. or s.c. tumors (indicated by arrows,
Representative mice from each group were shown in
In another experiment similar to the experiment described supra in this example, the brains were harvested, sectioned and imaged with fluorescent microscopy (
Without being bound to a specific mechanism, inflammatory cells (e.g. MG, MP, and NK cells) appeared to be the main carriers of CNT-CpG particles in experimental gliomas (see, Example 3,
In order to investigate the cellular responses to the CNT-sCpG therapies, mice bearing 4 day-old i.c. and s.c. B16F10 melanomas received i.t. treatment of PBS or CNT-CpG (2.5 μg CNT/5 μg CpG), and the tumors were analyzed by FACS after 24 hours (
To confirm whether the local cellular responses to CNT-CpG correlated with the systemic antitumor activities, splenocytes of treated animals in the previous experiment were harvested and tested for tumor cytotoxicity. i.t. CNT-CpG elicited a stronger ex vivo anti-tumor response when injected into i.c. melanomas than into s.c. tumors (
CD11b+ cells (MG and MPs) were shown to be prominent carriers of CNT-CpG in experimental gliomas (e.g. Example 3,
Mice bearing seven-day old i.c., s.c., or both i.c. and s.c. tumors were examined for TLR9 expression (
Immunotherapy is an attractive treatment modality for immunogenic tumors such as melanomas. Although various approaches for melanoma immunotherapy are been pursued with mixed results, recent findings from a phase III randomized trial of CTLA-1 blocking agent has validated the efficacy of this approach. One limitation for effective melanoma immunotherapy, however, may be the high frequency of brain metastasis which may occur in 10-30% of patients. Although activated T cells can penetrate intracranial tumors, clinical studies have demonstrated less than 50% of CNS metastasis to respond to systemic adoptive immunotherapies in melanoma patients. Thus, there is a need for improved targeting of metastatic brain tumor. While examining the role of CpG immunotherapy with nanoparticles, it was noted that a systemic antitumor response in experimental gliomas (Example 7). Furthermore, i.c. CNT-CpG not only inhibited the growth of brain melanomas, but also had a robust remote inhibitory effect in untreated s.c. tumors (Examples 9-12). These observations, which were unexpected, imply that i.c. CNT-CpG therapy may be applied to the treatment of not only gliomas, but also metastatic brain tumors.
Conjugation of CNT to CpG enhanced CpG immune responses in glioma models. Several features may be unique for CNT-CpG immunotherapy in the brain. First, biodistribution of CNT-CpG and CpG appeared to be different in i.c. tumors than s.c. melanomas. Whereas all of CNT-CpG (and CpG) was cleared from s.c. tumors, some CpG was retained even a week after i.c. injections. Second, CNT-CpG distribution around tumors was different than free CpG. Local MG actively uptook CNT and CpG complexes and since these cells did not migrate out of CNS, their retention and distribution around i.c. tumors may have accounted for a stronger antitumor response when injected into i.c. tumors.
The value of IT i.c. vaccine approaches to enhance systemic therapies has been suggested before but none of these studies evaluated the utility of this approach for systemic immune.
Human relevance: There has been a disparity in CpG immunotherapy results between mice and humans. Initial therapies in mice have shown excellent results, but these findings could not be duplicated in human. This disparately has been attributed to variation in TLR expression. In mice a number of cells (NK, etc.) express high levels of TLR9, but in humans, most TLR expression is localized to DC cells. TLR9, however, has also been shown to express by MG in humans, thus the strategy described herein may still be applicable to human trials. In humans, prDC are most prevalent TLR9-expressing cells in peripheral circulation.
Toxicity has been raised as a potential limitation of CNT biomedical application. While CNTs can be biodegraded through enzymatic catalysis (16), pristine (non-modified/non-functionalized) CNTs have been shown to persist for several months (12) raising concerns of potential negative health effects. fCNTs studied herein were non toxic after i.c. injections in these short-term experiments, thereby supporting their utilization in cancer immunotherapy.
In summary, CNT delivery system was shown to significantly enhance CpG immunotherapy, eradicate i.c. gliomas at low doses, and induce immunity against tumor rechallenge without inducing toxicity. Similar method may be used for CNT-based treatments for malignant brain tumors.
Furthermore, a person of ordinary skill in the art would know that other types/combinations of CNTs (e.g. other SWCNTs, double-walled CNTs, multi-walled CNTs, and combinations thereof) and/or other NANO described herein (e.g. gold, iron, silica, organic polymers, or carbon nanomaterials (e.g. fullerenes, rapheme, nanohorns, nanodiamond, etc.)) may also be used to provide other NANO delivery systems that are similar to the CNT delivery system disclosed herein, and can be used in the methods disclosed herein.
As used in examples 13-17, unless otherwise specified, SWCNT/CpG, SWCNT-CpG, CNT/CpG, CNT-CpG and CNT or SWCNT CpG conjugates are type II NANO-CpG.
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt) (Lipid-PEG) was purchased from Avanti Polar Lipids, Inc. Dithiotheritol (DTT) was purchased from VWR. Dithiolated CpG oligonucleotides (5-HO-C6-SS-C6-TAAACGTTATAACGTTATGACGTCAT-3, SEQ ID NO: 15) (RSS-CpG) and Cy5.5 labeled CpG oligonucleotides (5-HO-C6-SS-C6-TAAACGTTATAACGTTATGACGTCAT-C6-Cy5.5-3′, SEQ ID NO: 16) (RSS-CpG-Cy5.5) were provided by the DNA core facility at Beckman Research Institute at City of Hope. Sulfosuccinimidyl 6-(3′-[2-pyridyldithio]-propionamido)hexanoate (Sulfo-LC-SPDP) was purchased from ProteoChem.
Preparation of Lipid-PEG-LC-SPDP-CpG
First, 337 mg Lipid-PEG-NH2 and 52.7 mg Sulfo-LC-SPDP were added to 10 mL distilled water and stirred for 2 hours at room temperature to form Lipid-PEG-LC-SPDP. In the mean time, 50 mg RSS-CpG and 5 mL of 10 mM DTT were added into 15 mL distilled water and stirred for 2 hours at room temperature. DTT treated RSS-CpG was then purified using nap-25 column (GE health care). Concentration of the freshly prepared HS-CpG was determined with NanoDrop 1000 Spectrophotometer (Thermo Scientific). Then, excess Lipid-PEG-LC-SPDP was added to the freshly prepared HS-CpG. The reaction was kept at 60° C. for at least 2 hours. The UV absorbance at 345 nm for the byproduct free 2-mercaptopyridine was monitored by removing 10 μL aliquots from the reaction. When the intensity of this peak stopped increasing the reaction was judged to be complete. To purify the CpG containing polymer, isopropanol was added (by volume, 3× the volume of the solution) and the resulting mixture was stored at −20° C. overnight, then centrifuged at 15,000 rpm for 45 minutes. The obtained pellet was collected and redissolved in water, dialyzed to remove isopropanol, and lyophilized. The proton NMR (1H NMR) spectra was obtained on a VARIAN NMR system/400-MR using deuterated dimethyl sulfoxide as solvents (
Ultrasonication
All ultrasonication in this example was performed using a QSONICA Sonicator Q700 (QSONICA, Newtown, Conn., USA) equipped with a cup horn cooled with running water from the sink. Samples were loaded in 2.0 mL eppendorfs. The sonicator was set at amplitude 80 with pulse-on for 15 seconds followed by pulse-off for 15 seconds to avoid overheating.
Preparation of SWCNT-CpG
First, Lipid-PEG-LC-SPDP-CpG prepared above was dissolved in filtered (0.2 μm) distilled water. The concentration of CpG was 1.2 μg/μL, which was monitored with NanoDrop 1000 Spectrophotometer by observing the absorbance at 260 nm (Extinction coefficient: 21130) (Thermo Scientific). Then 2 mg SWCNT was added into 2 mL of the prepared solution and ultrasonication was applied for 3 hours as described above. Then 4 mg of Lipid-PEG-NH2 was added to the system and another 3 hours ultrasonication was applied. The dispersion was then dialyzed for 24 hours (15,000 Mw cut-off regenerated cellulose bag); water was changed every 2 hours in the first 8 hours. 200 μL of the final product was treated with DTT (50 mM, 200 μL) for 4 hours at room temperature to cleave the CpG, followed by ultracentrifugation at 15000 rpm for 1 hour to remove SWCNTs. The resultant free CpG in the supernatant was quantified by the UV absorbance at 260 nm (Extinction coefficient: 21130) to determine the final concentration of CpG in the SWCNTs/CpG conjugate (
Zeta Potential Measurements
The zeta potential of the dilute suspensions (0.01 wt %) was measured with a Zeta potential analyzer (Zetapals, Brookhaven, N.Y., USA). Each sample was ultrasonicated for 1 h prior to analysis. The ionic strength was maintained at 10−3 M using NaCl.
Transmission Electron Microscopy
SWCNT dispersions were imaged using a FEI Tecnai 12 TEM equipped with a Gatan Ultrascan 2K CCD camera at an accelerating voltage of 120 kV.
The synthesis and purification of Lipid-PEG-LC-SPDP-CpG was monitored by 1H NMR in deuterated dimethylsulfoxide (DMSO) (
Delivery of therapeutics directly into the brain was constrained by the volume of liquid that can be injected because the brain has little free space and exerts a back pressure during injection. Thus, it would be beneficial to prepare SWCNTs/CpG as concentrated as possible. SWCNTs were known to be difficult to disperse at high concentration in aqueous solutions, with common surfactants such as sodium dodecyl sulfate (SDS) generally providing concentrations generally less than 0.01 mg/mL (20, 21).
Preparation of a SWCNT concentration of 1 mg/mL was attempted by sonicating the Lipid-PEG-LC-SPDP-CpG and SWCNTs together in an eppendorf tube for 3 hours. Surprisingly, nearly all of the SWCNTs settled to the bottom of the eppendorf tube after the sonication. Transmission electron microscopy (TEM) revealed that under these conditions only large aggregates of SWCNTs were formed (
To facilitate the dispersion of SWCNTs in aqueous solutions, SWCNTs were sonicated with various ratios of Lipid-PEG-NH2: Lipid-PEG-LC-SPDP-CpG. A 1:1 ratio still produced large, unstable aggregates (
The SWCNT-CpG-II prepared according to Examples 13-14 was tested for in vitro activity. RAW macrophage cells were treated with SWCNT-CpG-II, Lipid-PEG-LC-SPDP-CpG and RSS-CpG, respectively. The macrophage cells (RAW-Blue™) were stably transfected with a reporter construct that allowed for the measurement of NF-κB activation, as described supra. One day after SWCNT-CpG-II, free CpG, Lipid-PEG-LC-SPDP-CpG or PBS buffer was added to RAW-Blue cells, the NF-κB activity was measured using Quanti-Blue according to the manufacturer's instruction (InvivoGen).
In order to avoid activity related to changes in the media, no more than 1% by volume SWCNT-CpG-II solution was added to the cells in media. The samples were ultrasonicated for 1 hour prior to each test. All data were normalized relative to the NF-κB activity of RSS-CpG at a concentration 0.01 μg/μL, ***: P<0.001, and **: P<0.01. As shown in
This in vitro activity assay was then used to optimize the ratio of SWCNTs to the coating mixture (2:1 Lipid-PEG-NH2/Lipid-PEG-LC-SPDP-CpG) (
It has been reported that SDS-dispersed SWCNTs are not stable during prolonged storage (23). As a comparison, 1 mg of SWCNTs was dispersed in 1 mL of 2:1 Lipid-PEG-NH2/Lipid-PEG-LC-SPDP-CpG ([CpG]=1 mg/mL) or SDS alone (3 mg/mL). After 1 month of storage at 4° C., there was no obvious change for SWCNT-CpG dispersion (CpG,
To further investigate how SWCNT conjugation improved the immunostimulatory response of CpG, human peripheral blood mononuclear cells (PBMCs) were incubated with SWCNT-CpG-II (1 mg/mL per well), free CpG (1 mg/mL per well) or PBS, and cytokine expression was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR).
Human PBMCs Isolation
PBMCs from healthy adult donors at City of Hope were isolated by Ficoll density gradient centrifugation. Experiments with human materials were performed according to protocols approved by the institutional review committee. The purity of freshly isolated CD14+ monocytes was more than 80% as analyzed by flow cytometry. Monocytes were cultured in 12-well plates with RPMI 1640 medium containing 10% FBS. M-CSF and GM-CSF were purchased from PeproTech and used at a final concentration of 33 ng/ml. Cytokines were added to cultures every 3 days.
At various times, cells were collected and total RNA was isolated using the Trizol system (Invitrogen) followed by double DNase treatment and column purification using the Qiagen RNeasy Clean-up Protocol. Real-time PCR was performed in a TaqMan 5700 Sequence Detection System (Applied Biosystems) as described previously (25). PCR conditions were optimized such that a minimum of 10.000-fold range could be detected for each primer. GAPDH: 5′-TGCACCACCAACTGCTTAGC-3′ (SEQ ID NO: 17) and 3′-GGCATGGACTGTGGTCATGAG-5′ (SEQ ID NO: 18). TNFα: 5′-CCTGCCCCAATCCCTTTATT-3′ (SEQ ID NO: 19) and 3′-CCCTAAGCCCCCAATTCTCT-5′(SEQ ID NO: 20). IL-16: 5′-CCCTAAACAGATGAAGTGCTCCTT-3′ (SEQ ID NO: 21) and 3′-GTAGCTGGATGCCGCCAT-5′ (SEQ ID NO: 22). IL-12 (3: 5′-TGGAGTGCCAGGAGGACAGT-3′ (SEQ ID NO: 23) and 3′-TCTTGGGTGGGTCAGGTTTG-5′(SEQ ID NO: 24).
At the same low doses (1 mg/mL per well), a negligible response was observed for free CpG treatment while a significant upregulation of pro-inflammatory cytokines (including IL-1β, IL-12β, TNF-α and IL6 mRNA) was seen for SWCNT-CpG-II treatment, peaking at 12 hours after the administration (
One possibility of the dramatic difference in the cytokine expression of the free CpG treated and SWCNT-CpG-II treated PBMCs may be that SWCNT-CpG-II delivered more CpG into the cells, while another possibility may be that SWCNT-CpG-II altered the manner in which the cells interacted with CpG.
Lipid-PEG-NH2 was labeled with Alexa Fluor 555 (AF555) to produce Lipid-PEG-AF555. RSSCpG was prepared labeled with Cy5.5 such that the final tri-block polymer was Lipid-PEG-LC-SPDP-CpG5.5. It was envisioned that the Lipid-Peg-AF555 would remain bound to the SWCNTs throughout the cell treatment, while the Cy5.5 would track the location of the CpG as the disulfide bond linking it to the construct was cleaved and the CpG was processed by the cells.
Confocal Microscopy Imaging
PBMCs were plated in 33 mm2 dishes in 1 mL media and AF555-SWCNT/CpG5.5, CpG5.5 or Lipid-PEG-AF555 were added. At various times intervals (4, 24 and 48 hours) the cells were washed three times with PBS and imaged by confocal microscopy. HeNe 678 nm and 543 nm lasers were used to excite Cy5.5 and AF555, respectively.
When human PBMCs were cultured in the presence of a mixture of free Lipid-PEG-AF555 (AF555 was false colored in red) and Lipid-PEG-CpG5.5 (Cy5.5 was false colored in blue) both polymers were quickly taken up by human PBMCs and remained in the cytoplasm without being consumed for at least 48 hours (
Tumor Implantation, Treatment and Tumor Rechallenge
All animals were housed and handled according to the guidelines of City of Hope Institutional Animal Care and Use Committee. Intracranial tumor implantation was done as described previously (24). GL261.luc cells were harvested by trypsinization, counted, and resuspended in PBS. Female C57BL/6 mice weight 15 to 25 g were anesthetized by intraperotenial administration of ketamine (132 mg/kg) and xylazine (8.8 mg/kg) and immobilized in a stereostactic head frame. Through a small burr hold, 3 μL of PBS containing 1×105 tumor cells were injected unilaterally at the coronal suture, 1 mm laceral to the midline, and 3 mm deep into the frontal lobes, using a Hamilton syringe (Fisher Scientific). Four days after intracranial implantation of tumor cells, PBS (control, 5 μL), CpG (5 μg/5 μL PBS) and SWCNTs/CpG (5 μg SWCNT/5 μg CpG/5 μL PBS) were administered through the initial burr hole aiming to target the tumor site (5 mice per group). For tumor rechallenge experiments, naive or GL261-bearing mice that had survived for at least 3 months after the initial SWCNT/CpG treatment were rechallenged with a subcutaneous injection of GL261 (1×106 cells). Tumor growth was monitored every other day for 15 days using calipers.
SWCNT-CpG-II prepared following the optimized protocol described in Examples 13-15 was evaluated for the treatment of a murine model of glioma. Mice bearing 4-day-old intracranial GL261.luc gliomas were given a single intratumoral injection of PBS (control), free RSS-CpG (5 mg CpG/10 mL PBS) and SWCNT-CpG-II (5 mg SWCNT/5 mg CpG/10 mL PBS). The mice were then monitored for up to 90 days, and mice that became ill were humanely euthanized. The brains of all euthanized mice were evaluated to determine if the glioma was the likely cause of illness. As shown in
Thus, the mice that had survived 90 days after treatment with SWCNT-CpG were injected with GL261 cells subcutaneously in their flank, as were a new set of control mice. Caliper measurements showed that while the subcutaneous tumors grew quickly in the control mice, the mice previously treated with SWCNT-CpG-II had no detectable tumors remaining after 15 days (
The following references are incorporated herein by reference:
This application claims priority to U.S. Application No. 61/557,843, filed Nov. 9, 2011, which is incorporated herein by reference.
This work was supported by R21CA131765-01A2, James S. McDonnell Foundation (BB), ThinkCure Foundation (to BB and DJD), and P01-CA030206 and CA077544 (DJD). The City of Hope Flow Cytometry Core was equipped in part through funding provided by ONR N00014-02-1 0958, DOD 1435-04-03GT-73134, and NSF DBI-9970143.
| Number | Date | Country | |
|---|---|---|---|
| 61557843 | Nov 2011 | US |
