Injectable, thermogelling, prolonged-release hydrogel formulations for immunomodulatory oligonucleotides

Abstract

Some aspects are related to injectable compositions configured for intratumoral drug delivery. In an exemplary set of embodiments, the injectable composition includes a block copolymer (e.g., poly(lactide-co-glycolide-b-ethylene glycol-b-lactide-co- glycolide) (PLGA-PEG-PLGA)) configured such that the injectable composition is a liquid at room temperature and is a gel at 37 °C, and may therefore be injected into a subject and retained the location of injection for certain periods of time. Additionally, the injectable composition may include an immunomodulatory oligonucleotide present within the injectable composition at a concentration of greater than or equal to 0.001 mg / mL to facilitate an immune response for treating cancer. The injectable composition may further include a polycationic polymer and / or an ionizable lipid that form nanoparticles therein and facilitate uptake of the immunomodulatory oligonucleotides by cells of the subject in which the composition is injected.

Description

[0001] INJECTABLE, THERMOGELLING, PROLONGED-RELEASE HYDROGEL FORMULATIONS FOR IMMUNOMODULATORY OLIGONUCLEOTIDES

[0002] TECHNICAL FIELD

[0003] Injectable compositions, for example, for the delivery of immunomodulatory oligonucleotides are generally described.

[0004] BACKGROUND

[0005] New treatments such as immunomodulatory oligonucleotides for cancer therapy may increase the anti-tumoral immune response and thus increase checkpoint inhibitor response rate. However, certain immunomodulatory oligonucleotides, such as CpG-oligodeoxynucleotide (ODN)-based therapeutics, have not been FDA approved due to an associated toxicity of the CpG-ODNs. For instance, the CpG-ODNs are typically not retained at the tumor site, which leads to a lack of efficacy against the target and undesired off-target toxicities. Thus, such treatments are typically done over the course of 5-10 multiple intratumoral injections, ranging from daily to weekly injections, which are challenging to implement clinically and undesirable from a patient perspective. Additionally, CpG is a negatively charged macromolecule, which has a low cell uptake. However, certain receptors targeted by CpG-ODNS (e.g., toll-like receptor 9 , TLR9) are predominantly located intracellularly, and thus a lack of intracellular delivery of CpG-ODNs limits efficacy of the therapeutic.

[0006] Accordingly, improved systems and methods of the delivery of immunomodulatory oligonucleotides are needed.

[0007] SUMMARY

[0008] Injectable compositions, for example, for the delivery of immunomodulatory oligonucleotides are generally described. The subject matter of the present disclosure involves, in some cases, interrelated products, alternative solutions to a particular problem, and / or a plurality of different uses of one or more systems and / or articles.

[0009] Some aspects are generally related to compositions.

[0010] In some embodiments, the composition is an injectable composition configured for intratumoral drug delivery, the injectable composition comprising: poly(lactide-co-glycolide-b-ethylene glycol-b-lactide-co-glycolide) (PLGA-PEG-PLGA) having a lactic acid (LA) to glycolic acid (GA) ratio of greater than or equal to 1:1, wherein a number average molecular weight of each PLGA is greater than or equal to 500 and less than or equal to 2500 and a number average molecular weight of each PEG is greater than or equal to 500 and less than or equal to 2500, an immunomodulatory oligonucleotide present within the injectable composition at a concentration of greater than or equal to 0.001 mg / mL; and a polycationic polymer and / or a lipid, wherein the injectable composition is a liquid at room temperature and is a gel at 37 °C, and the immunomodulatory oligonucleotide exhibits an extended-release profile from the injectable composition over greater than or equal to 24 hours as measured in phosphate-buffered saline at 37 °C. In some such embodiments, the immunomodulatory oligonucleotide comprises an oligonucleotide selected from the group consisting of single- stranded DNA (ssDNA), double- stranded DNA (dsDNA), anti-sense oligonucleotides (ASO), single- stranded RNA (ssRNA), short-hairpin RNA (shRNA), short-interfering RNA (siRNA), non-coding RNA (ncRNA), and microRNA (miRNA).

[0011] In some embodiments, the composition is an injectable composition configured for intratumoral drug delivery, the injectable composition comprising: poly(lactide-co-glycolide-b-ethylene glycol-b-lactide-co-glycolide) (PLGA-PEG-PLGA) having a lactic acid (LA) to glycolic acid (GA) ratio of greater than or equal to 1:1, wherein a number average molecular weight of each PLGA is greater than or equal to 500 and less than or equal to 2500 and a number average molecular weight of the PEG is greater than or equal to 500 and less than or equal to 2500; an immunomodulatory oligonucleotide present within the injectable composition at a concentration of greater than or equal to 0.001 mg / mL; and a radio-opaque label, wherein the injectable composition is a liquid at room temperature and is a gel at 37 °C and the immunomodulatory oligonucleotide exhibits an extended-release profile from the injectable composition over greater than or equal to 24 hours as measured in phosphate-buffered saline at 37 °C.

[0012] In some embodiments, the composition is an injectable composition configured for intratumoral drug delivery, the injectable composition comprising: poly(lactide-co-glycolide-b-ethylene glycol-b-lactide-co-glycolide) (PLGA-PEG-PLGA) having a lactic acid (LA) to glycolic acid (GA) ratio of greater than or equal to 1:1, wherein a number average molecular weight of each PLGA is greater than or equal to 500 and less than orequal to 2500 and a number average molecular weight of the PEG is greater than or equal to 500 and less than or equal to 2500; a CpG oligodeoxynucleotide present within the injectable composition at a concentration of greater than or equal to 0.001 mg / mL; a polycationic polymer and / or a lipid; and a radio-opaque label, wherein the injectable composition is a liquid at room temperature and is a gel at 37 °C, and the CpG oligodeoxynucleotide exhibits an extended-release profile from the injectable composition over greater than or equal to 24 hours as measured in phosphate-buffered saline at 37 °C.

[0013] In some embodiments, the composition is an injectable composition configured for intratumoral delivery, the injectable composition comprising: poly(lactide-co-glycolide-b-ethylene glycol-b-lactide-co-glycolide) (PLGA-PEG-PLGA) having a lactic acid (LA) to glycolic acid (GA) ratio of greater than or equal to 1:1, wherein a number average molecular weight of each PLGA is greater than or equal to 500 and less than or equal to 2500 and a number average molecular weight of the PEG is greater than or equal to 500 and less than or equal to 2500; an immunomodulatory nucleic acid present within the injectable composition at a concentration of greater than or equal to 0.001 mg / mL; and one or more of a polycationic polymer, a lipid, and a radio-opaque label associated with the injectable composition, wherein the injectable composition is a liquid at room temperature and is a gel at 37 °C, and the immunomodulatory nucleic acid exhibits an extended-release profile from the injectable composition over greater than or equal to 24 hours as measured in phosphate-buffered saline at 37 °C. In some such embodiments, the immunomodulatory nucleic acid is an oligonucleotide. In some such embodiments, the immunomodulatory nucleic acid comprises natural and therapeutic forms of RNA, DNA, and / or hybrids thereof, the RNA, DNA, and / or hybrids thereof comprising one or more of double- stranded DNA (dsDNA), single- stranded DNA (ssDNA), anti-sense oligonucleotide (ASO), double- stranded RNA (dsRNA), single- stranded RNA (ssRNA), short-hairpin RNA (shRNA), short-interfering RNA (siRNA), non-coding RNA (ncRNA), long non-coding RNA (IncRNA), messenger RNA (mRNA), and microRNA (miRNA).

[0014] Some aspects are generally related to methods.

[0015] In some embodiments, the method is for treating a tumor. In some embodiments, the method of treating a tumor comprises injecting a composition intratumorally,peritumorally, into tumor draining lymph nodes, and / or near tumor draining lymph nodes wherein the composition is a liquid at room temperature and is a gel at 37 °C, wherein the composition comprises: poly(lactide-co-glycolide-b-ethylene glycol-b-lactide-co-glycolide) (PLGA-PEG-PLGA) having a lactic acid (LA) to glycolic acid (GA) ratio of greater than or equal to 1:1, wherein a number average molecular weight of each PLGA is greater than or equal to 500 and less than or equal to 2500 and a number average molecular weight of the PEG is greater than or equal to 500 and less than or equal to 2500, an immunomodulatory oligonucleotide present within the injectable composition at a concentration of greater than or equal to 0.001 mg / mL; and a polycationic polymer and / or a lipid; and determining the position of the composition during injection using one or more of ultrasound, CT, visual inspection, optical microscopy, and MRI.

[0016] Other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments of the disclosure when considered in conjunction with the accompanying figures. In cases where the present specification and a document incorporated by reference include conflicting and / or inconsistent disclosure, the present specification shall control.

[0017] BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Non-limiting embodiments of the present disclosure will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale unless otherwise indicated. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the disclosure shown where illustration is not necessary to allow those of ordinary skill in the art to understand the disclosure. In the figures:

[0019] FIG. 1A is a schematic diagram of an injectable composition, according to one set of embodiments;

[0020] FIG. IB is a schematic diagram of an injectable composition, according to one set of embodiments;

[0021] FIG. 2 is a schematic diagram of the injection of a CpG-ODN containing hydrogel, according to some embodiments;FIG. 3A are images of the gelling nature of a CpG-ODN containing solution, according to some embodiments;

[0022] FIG. 3B is a plot of release of CpG-ODN from the gel formed from the composition shown in FIG. 3A, according to some embodiments;

[0023] FIGS. 4-5 are plots showing the release profile of CpG-ODN from compositions, according to some embodiments;

[0024] FIG. 6 is a plot of signals obtained over time during an in vitro experiment, according to some embodiments;

[0025] FIG. 7 is a plot of signals obtained from various samples during an in vitro experiment, according to some embodiments;

[0026] FIG. 8 is a plot of signals obtained over time during an in vitro experiment, according to some embodiments;

[0027] FIGS.9A are images detailing the distribution of a signal from an in vivo transfection experiment, according to some embodiments;

[0028] FIG. 9B is a plot of signals obtained over time during an in vivo experiment, according to some embodiments; and

[0029] FIG. 10 is a plot of signals obtained over time during an in vivo experiment, according to some embodiments.

[0030] DETAILED DESCRIPTION

[0031] Some aspects are related to injectable compositions configured for intratumoral drug delivery. In an exemplary set of embodiments, the injectable composition includes a block copolymer (e.g., poly(lactide-co-glycolide-b-ethylene glycol-b-lactide-co-glycolide) (PLGA-PEG-PLGA)) configured such that the injectable composition is a liquid at room temperature and is a gel at 37 °C, and may therefore be injected into a subject and retained the location of injection for certain periods of time. Additionally, the injectable composition may include an immunomodulatory oligonucleotide present within the injectable composition at a concentration of greater than or equal to 0.001 mg / mL to facilitate an immune response for treating cancer. In some embodiments, injectable composition may include an immunomodulatory nucleic acid present within the injectable composition at a concentration of greater than or equal to 0.001 mg / mL to facilitate an immune response for treating cancer. The injectable composition may furtherinclude a polycationic polymer and / or an ionizable lipid that form nanoparticles therein and facilitate uptake of the immunomodulatory oligonucleotides by cells of the subject in which the composition is injected. Still other aspects are directed to related methods, kits, or the like.

[0032] Checkpoint inhibitors (anti-PDl, anti-CTLA4) have revolutionized cancer therapy, offering the possibility of durable responses and even cure to some patients with metastatic disease. However, the vast majority of patients do not respond to treatment. New emerging adjunctive treatments may increase the anti-tumoral immune response and thus increase checkpoint inhibitor response rate. One example is the use of intratumoral injection of immunotherapies at one site of metastatic disease to increase local recognition of tumor by the immune system. One possible drug type to trigger this effect and activate the local immune system includes synthetic CpG oligodeoxynucleotide (CpG-ODNs), as CpG motifs are common for human-foreign sources, such as bacterial or viral DNA. As these are common to human-foreign sources CpG motifs trigger cascaded immune cell activations. Accordingly, CpG-ODNs may be recognized by B cells, plasmacytoid dendritic cells (pDCs), and / or other immune cells, thereby upregulating immune activity and the innate immune system.

[0033] However, CpG-ODN-based therapeutics have not been FDA approved due to an associated toxicity of the CpG-ODNs. For instance, the CpG-ODNs are typically not retained at the tumor site, which leads to a lack of efficacy against the target and undesired off-target toxicities. Thus, such treatments are typically done over the course of 5-10 multiple intratumoral injections, ranging from daily to weekly injections, which are challenging to implement clinically and undesirable from a patient perspective.

[0034] Additionally, CpG is a negatively charged macromolecule, which is expected to have low cell uptake. However, certain receptors targeted by CpG-ODNS (e.g., toll-like receptor 9 , TLR9) are predominantly located intracellularly, and thus a lack of intracellular delivery of CpG-ODNs limits efficacy.

[0035] In view of the above, some aspects of this disclosure are related to compositions including injectable and thermogelling compositions that include immunomodulatory nucleic acids (e.g., CpG-ODNs), such that the nucleid acid may be injected intratumorally, and then retained at the site due to the gelling nature of the composition. This advantageously decreases off-target toxicity of the immunomodulatory nucleic acid--1-based therapeutics. Moreover, some of the compositions described herein include polymeric and / or lipid nanoparticles that encapsulate the immunomodulatory nucleic acids , and facilitate cellular uptake thereof. Such uptake by the cells advantageously increase the efficacy of immunomodulatory nucleic acids -based therapeutics, in accordance with some embodiments. Finally, some compositions described herein include radio-opaque labels, which facilitate guided injection and therefore targeted delivery of the immunomodulatory nucleic acids to certain locations internal to a subject.

[0036] In view of the above, the inventors have recognized and developed an injectable, thermoresponsive, locally resident and imageable viscous gel to contain and deliver a high concentration of immunomodulatory nucleic acids (e.g., CpG-ODNs). The term “subject," as used herein, refers to an individual organism such as a human or an animal. In some embodiments, the subject is a mammal (e.g., a human, a non-human primate, or a non-human mammal), a vertebrate, a laboratory animal, a domesticated animal, an agricultural animal, or a companion animal. In some embodiments, the subject is a human. In some embodiments, the subject is a rodent, a mouse, a rat, a hamster, a rabbit, a dog, a cat, a cow, a goat, a sheep, or a pig.

[0037] Accordingly, some aspects of the present disclosure are related to injectable compositions, for example, that are configured for drug delivery. In some such embodiments, the injectable compositions are configured for intratumoral delivery.

[0038] In some embodiments, the injectable composition comprises a polymer. In some embodiments, the polymer comprises polylactide (PLA), polyglycolide (PGA), polyethylene glycol (PEG), combinations thereof, and / or block copolymers thereof. For example, in an exemplary set of embodiments, the injectable composition comprises a block copolymer comprising poly(lactide-co-glycolide-b-ethylene glycol-b-lactide-co-glycolide) (PLGA-PEG-PLGA).

[0039] The composition may comprise the polymer in any of a variety of suitable amounts, in accordance with some embodiments. Advantageously, in some embodiments, the composition comprises a total polymer content such that the composition does not substantially form a gel at room temperature. In some cases, the composition comprises a polymer in an amount greater than 0 wt%, greater than or equal to 5 wt%, greater than or equal to 10 wt%, greater than or equal to 15 wt%, greater than or equal to 20 wt%, or greater than or equal to 25 wt% versus the total weight of thecomposition. In some cases, the composition comprises a polymer in an amount less than or equal to 30 wt%, less than or equal to 25 wt%, less than or equal to 20 wt%, less than or equal to 15 wt%, less than or equal to 10 wt%, or less than or equal to 5 wt% versus the total weight of the composition. Combinations of the foregoing ranges are possible (e.g., greater than 0 wt% and less than or equal to 30 wt%). Other ranges are also possible. In some cases, a balance of weight of the composition may comprise other components described herein and / or a solvent (e.g., water, a buffer, etc.).

[0040] In some embodiments, as illustrated in FIG. 1A, injectable composition 100 comprises block copolymer 110 and one or more active substances 120 (e.g., an immunomodulatory oligonucleotide). In some embodiments, block copolymer 110 comprises poly(lactide-co-glycolide-b-ethylene glycol-b-lactide-co-glycolide) (PLGA-PEG-PLGA). For example, in some embodiments, block copolymer 110 comprises PEG block 112 and PLGA blocks 114. Each PEG chain may be the same or different (e.g., having different molecular weights), optionally substituted. Each PLGA chain may be the same or different (e.g., having different molecular weights), and / or optionally substituted.

[0041] In some embodiments, active substance 120 is associated with block copolymer 110 (e.g., via hydrophobic interactions, hydrostatic interactions, via a bond, etc.). In some embodiments, the association is by thermodynamically favorable association between hydrophobic groups expelling water. In some embodiments, the active substance is associated with the block copolymer via formation of a bond, such as an ionic bond, a covalent bond, a hydrogen bond, Van der Waals interactions, and / or the like. The covalent bond may be, for example, carbon-carbon, carbon-oxygen, oxygensilicon, sulfur- sulfur, phosphorus-nitrogen, carbon-nitrogen, metal-oxygen, or other covalent bonds. The hydrogen bond may be, for example, between hydroxyl, amine, carboxyl, thiol, and / or similar functional groups. For example, in some embodiments, the block copolymer is functionalized (e.g., a PLGA group further comprises a functional group, a PEG group further comprises a functional group) such that the block copolymer is capable of forming a bond with the active substance (e.g., via a functional group). In other embodiments, the active substance is non-covalently associated with the block copolymer. In some embodiments, the active substance may be dispersed or encapsulated within a portion of the block copolymer e.g., by hydrophilic and / or hydrophobic forces.In some embodiments, the block copolymer of the injectable composition is configured to gel upon injection into a subject. For instance, in some embodiments, the block copolymer is a liquid at room temperature (e.g., greater than or equal to 20 °C, and / or less than or equal to 25 °C). In some embodiments, the liquid state of the block copolymer is desirable as it may facilitate injection into a subject. Following injection, in some embodiments, the injectable composition may gel due to an increase in temperature (e.g., due to a body temperature of the subject; at greater than or equal to 32 °C and less than or equal to 42 °C depending on the identity of the subject).

[0042] The injectable composition may include a micellar structure. In some embodiments, the block copolymer (e.g., PLGA-PEG-PLGA) forms a micellar structure in solution. In some embodiments, the micellar structure defines an internal volume and an external volume. In some embodiments, the micellar structure may include a hydrophobic interior and substantially hydrophilic exterior such that the micellar structure may be miscible and / or soluble with aqueous solutions while the interior may be hydrophobic such that active substances and / or portions thereof (e.g., hydrophobic moieties of an immunomodulatory nucleic acid such as a CpG-ODN) may be soluble at relatively high loadings, as described in more detail elsewhere herein. In some cases, active substances and / or portions thereof may be contained and / or associated with the internal volume of the micellar structure. In some embodiments, the association between the block copolymer and the active substance is retained by and / or in the block copolymer for long periods of time, e.g., for extended release internal to a subject.

[0043] According to some embodiments, a micellar structure of the injectable compositions comprises a block copolymer. In some embodiments, the micellar structure comprises poly(lactide-co-glycolide-b-ethylene glycol-b-lactide-co-glycolide) (PLGA-PEG-PLGA). The lactic acid (LA) to glycolic acid (GA) ratio in the PLGA blocks of the PLGA-PEG-PLGA block copolymer, in some embodiments, may impact the micellar structure and / or how the micellar structure encapsulates active substances, as described in more detail elsewhere herein. For example, LA includes a methyl group, and is accordingly more hydrophobic when compared to GA. Thus, by increasing the content of the LA in the PLGA blocks, the PLGA may become relatively more hydrophobic when compared to PLGA blocks with relatively less LA. While much of the disclosure herein is generally related to tri-block copolymers, those of ordinary skill in the art wouldunderstand that other multi-block copolymers of combinations of PLGA and PEG (e.g., of varying molecular weight) are also possible. For example, without wishing to be limited as such, in some embodiments, the composition may comprise a multiblock copolymer having a structure as in poly(lactide-b-glycolide-b-ethylene glycol-b-lactide-b-glycolide) (e.g., PLA-PGA-PEG-PLA-PGA, where the LA and GA of the PLGA block are blocks rather than being randomly interspersed). Other non-limiting examples include PLA-PGA-PEG-PGA-PLA and PLA-PGA-PLA-PEG-PLA-PGA-PLA, polycaprolactone (PCL)-PEG-PCL, and PGA-PLA-PGA-PEG-PGA-PLA-PGA. Without wishing to be bound by theory, in some embodiments, the distribution of the LA and GA within the PLGA block may also affect the thermoresponsive behavior of the injectable composition. It will be understood that, in some embodiments, while the injectable composition is configured to gel upon heating (e.g., upon injection into a subject), the composition may not be configured to form a micellar structure.

[0044] FIG. IB depicts a schematic diagram of an example micellar structure of an injectable composition. In this embodiment, an injectable composition 100 comprises micellar structure 102 comprising block copolymer 110. In some embodiments, block copolymer 110 is PLGA-PEG-PLGA, configured and arranged such that the PEG blocks 112 face a hydrophilic exterior (e.g., an aqueous solution, a bodily fluid, etc.) and the PLGA blocks 114 are oriented inwards towards a relatively more hydrophobic core of the micellar structure 102. The micellar structure 102 may, in some embodiments, encapsulate one or more active substances 120. In some cases, the relatively hydrophobic core of the micellar structure 102 is configured to encapsulate one or more active substances. In some embodiments, the micellar structure is configured to retain one or more active substances for a period of time, during which and / or after the one or more active substances may be released. For example, in some embodiments, the injectable composition is constructed and arranged to release the active substance (e.g., an immunomodulatory nucleic acid such as an immunomodulatory oligonucleotide) from the micellar structure(s) (e.g., upon delivery to a subject such as intratumoral injection). In some embodiments, the active substance is designed for release from the micellar structure. Such embodiments may be useful in the context of drug delivery. In some embodiments, the active substance may be bound (e.g., via one or more interactions and / or bonds such as a covalent bond, an ionic bond, etc. as described above) to themicellar structure. In other embodiments, the active substance is non-covalently associated with the micellar structure. In some embodiments, the active substance may be dispersed or encapsulated within the micellar structure by hydrophilic and / or hydrophobic forces. It will be understood that, in some embodiments, if the composition does not comprise a micellar structure when gelled, certain interactions (e.g., via one or more interactions and / or bonds such as a covalent bond, an ionic bond, etc. as described above) between an active substance and the composition may still be present to allow for long-term and localized delivery of the active substance.

[0045] In FIG. IB, the PLGA blocks are depicted to include a 2: 1 ratio of LA to GA groups. The micellar structure comprising the PLGA-PEG-PLGA, in accordance with some embodiments, may have any of a variety of suitable LA to GA ratios. In some embodiments, the LA to GA ratio is greater than or equal to 1:1, greater than or equal to 2:1, greater than or equal to 3: 1, greater than or equal to 4: 1, greater than or equal to 5: 1, greater than or equal to 6: 1, greater than or equal to 7: 1, greater than or equal to 8: 1, or greater than or equal to 9: 1. According to some embodiments, the LA to GA ratio is less than or equal to 10:1, less than or equal to 9:1, less than or equal to 8:1, less than or equal to 7:1, less than or equal to 6:1, less than or equal to 5:1, less than or equal to 4:1, less than or equal to 3:1, or less than or equal to 2:1. Combinations of the foregoing ranges are possible (e.g., greater than or equal to 1:1 and less than or equal to 10:1, greater than or equal to 1:1 and less than or equal to 5:1, greater than or equal to 1:1 and less than or equal to 2:1, greater than or equal to 3:1 and less than or equal to 5:1). Other ranges are also possible. In some embodiments, the LA to GA ratio in each of the PLGA blocks of the block copolymer (e.g., PLGA-PEG-PLGA) may, on average, be the same or different.

[0046] In some embodiments, the LA to GA ratio in the PLGA block of the copolymer (e.g., a PLGA-PEG-PLGA copolymer) impacts the size of micelles formed therefrom. For example, when the LA to GA ratio is relatively low (e.g., greater than or equal to 1 : 1 and less than or equal to 2:1), the PLGA block is relatively more hydrophilic.

[0047] Accordingly, the PLGA blocks may not pack as densely (e.g., see micellar structure in FIG. IB), resulting in relatively larger micelles with more water dispersed therein. The larger micelles may facilitate a high loading capacity of the immunomodulatory active substance. In contrast, when the LA to GA ratio is relatively high (e.g., greater than orequal to 3: 1 and less than or equal to 5: 1), the PLGA block is relatively more hydrophobic. In some such cases, the PLGA blocks may pack more densely leading to smaller micelles. In some embodiments, the smaller micelles have a smaller loading capacity of the immunomodulatory active substance than the larger micelles.

[0048] As described above, a micellar structure comprising PLGA-PEG-PLGA may be more or less densely packed based on the LA to GA ratio of the PLGA blocks, in accordance with some embodiments. Accordingly, in some embodiments, an average maximum dimension of the micellar structures in the injectable composition may be determined using dynamic light scattering. In some embodiments, an average maximum dimension of the micellar structures in the injectable composition is greater than or equal to 50 nm, greater than or equal to 60 nm, greater than or equal to 70 nm, greater than or equal to 80 nm, greater than or equal to 90 nm, greater than or equal to 100 nm, greater than or equal to 110 nm, greater than or equal to 120 nm, greater than or equal to 130 nm, greater than or equal to 140 nm, greater than or equal to 150 nm, greater than or equal to 160 nm, greater than or equal to 170 nm, greater than or equal to 180 nm, or greater than or equal to 190 nm. According to some embodiments, the average maximum dimension of the micellar structures in the injectable composition is less than or equal to 200 nm, less than or equal to 190 nm, less than or equal to 180 nm, less than or equal to 170 nm, less than or equal to 160 nm, less than or equal to 150 nm, less than or equal to 140 nm, less than or equal to 130 nm, less than or equal to 120 nm, less than or equal to 110 nm, less than or equal to 100 nm, less than or equal to 90 nm, less than or equal to 80 nm, or less than or equal to 70 nm. Combinations of the foregoing ranges are possible (e.g., greater than or equal to 50 nm and less than or equal to 200 nm, greater than or equal to 70 nm and less than or equal to 120 nm, greater than or equal to 70 nm and less than or equal to 90 nm). Other ranges are also possible.

[0049] In some embodiments, the molecular weight (e.g., size) of each block of the block copolymer may affect the resulting structure of the gel and / or micelles. In some embodiments, the molecular weight (e.g., number average molecular weight) of each PLGA block present in a PLGA-PEG-PLGA copolymer of the injectable composition is independently greater than or equal to 500, greater than or equal to 600, greater than or equal to 700, greater than or equal to 800, greater than or equal to 900, greater than or equal to 1000, greater than or equal to 1100, greater than or equal to 1200, greater than orequal to 1300, greater than or equal to 1400, greater than or equal to 1425, greater than or equal to 1450, greater than or equal to 1475, greater than or equal to 1500, greater than or equal to 1525, greater than or equal to 1550, greater than or equal to 1575, greater than or equal to 1600, greater than or equal to 1700, greater than or equal to 1800, or greater than or equal to 1900. In some embodiments, the molecular weight (e.g., number average molecular weight) of each PLGA block present in a PLGA-PEG-PLGA copolymer of the injectable composition is independently less than or equal to 2000, less than or equal to 1900, less than or equal to 1800, less than or equal to 1700, less than or equal to 1600, less than or equal to 1575, less than or equal to 1550, less than or equal to 1525, less than or equal to 1500, less than or equal to 1475, less than or equal to 1450, less than or equal to 1425, less than or equal to 1400, less than or equal to 1300, less than or equal to 1200, less than or equal to 1100, less than or equal to 1000, less than or equal to 900, less than or equal to 800, less than or equal to 700, or less than or equal to 600. Combinations of the foregoing ranges are possible (e.g., greater than or equal to 500 and less than or equal to 2000, greater than or equal to 1400 and less than or equal to 1600). Other ranges are also possible. Note that, in some embodiments, the molecular weight of each PLGA block is, on average, the same. In other embodiments, the molecular weight of each PLGA block is different. For example, a first PLGA of the PLGA-PEG-PLGA copolymer may have a first molecular weight of greater than or equal to 500 and less than or equal to 2000 (e.g., greater than or equal to 1400 and less than or equal to 1600) and a second PLGA of the PLGA-PEG-PLGA copolymer may have a second molecular weight of greater than or equal to 500 and less than or equal to 2000 (e.g., greater than or equal to 1400 and less than or equal to 1600), different than the first molecular weight.

[0050] In some embodiments, the molecular weight (e.g., number average molecular weight) of each PEG present in a PLGA-PEG-PLGA copolymer of the injectable composition is greater than or equal to 500, greater than or equal to 600, greater than or equal to 700, greater than or equal to 800, greater than or equal to 900, greater than or equal to 1000, greater than or equal to 1100, greater than or equal to 1200, greater than or equal to 1300, greater than or equal to 1400, greater than or equal to 1425, greater than or equal to 1450, greater than or equal to 1475, greater than or equal to 1500, greater than or equal to 1525, greater than or equal to 1550, greater than or equal to 1575, greater than or equal to 1600, greater than or equal to 1700, greater than or equal to 1800, or greater thanor equal to 1900. In some embodiments, the molecular weight (e.g., number average molecular weight) of the PEG present in a PLGA-PEG-PLGA copolymer of the injectable composition is less than or equal to 2000, less than or equal to 1900, less than or equal to 1800, less than or equal to 1700, less than or equal to 1600, less than or equal to 1575, less than or equal to 1550, less than or equal to 1525, less than or equal to 1500, less than or equal to 1475, less than or equal to 1450, less than or equal to 1425, less than or equal to 1400, less than or equal to 1300, less than or equal to 1200, less than or equal to 1100, less than or equal to 1000, less than or equal to 900, less than or equal to 800, less than or equal to 700, or less than or equal to 600. Combinations of the foregoing ranges are possible (e.g., greater than or equal to 500 and less than or equal to 2000, greater than or equal to 1400 and less than or equal to 1600). Other ranges are also possible.

[0051] As described above, in some embodiments, the injectable composition may be configured to be a liquid at room temperature and to be a gel at body temperature.

[0052] Advantageously, in some embodiments, the injectable composition remains a liquid at or around room temperature to facilitate injection. In some embodiments, upon heating, the injectable composition may undergo a solution-gelation transition (e.g., a sol-gel transition). Advantageously, in accordance with some embodiments, the sol-gel transition may occur at or around body temperature such that, following injection of the liquid injectable composition at a location internal to the subject, the solution may heat and gel to remain at the location of injection. Maintaining a location of the injectable composition via gelation, in some embodiments, may facilitate a localized extended release of a immunomodulatory nucleic acid of the injectable composition to the localized region within the subject. In some embodiments, a LA to GA ratio of greater than or equal to 1:1 and less than or equal to 10:1 may facilitate a sol-gel transition at such temperatures. In some embodiments, a LA to GA ratio of greater than or equal to 3:1 and less than or equal to 5:1 may facilitate a sol-gel transition at such temperatures. As noted elsewhere herein, in some embodiments, the thermoresponsive behavior (e.g., the sol-gel temperature) may also be affected by the distribution of the LA and GA within the PLGA blocks.

[0053] In some embodiments, the injectable composition remains a liquid at a temperature of greater than or equal to 18°C, greater than or equal to 19°C, greater thanor equal to 20°C, greater than or equal to 21 °C, greater than or equal to 22°C, greater than or equal to 23°C, greater than or equal to 24°C, greater than or equal to 25°C, greater than or equal to 26°C, greater than or equal to 27°C, greater than or equal to 28°C, or greater than or equal to 29°C. In some embodiments, the injectable composition remains a liquid at a temperature of less than or equal to 30°C, less than or equal to 29°C, less than or equal to 28°C, less than or equal to 27°C, less than or equal to 26°C, less than or equal to 25°C, less than or equal to 24°C, less than or equal to 23°C, less than or equal to 22°C, less than or equal to 21 °C, less than or equal to 20°C, or less than or equal to 19°C. Combinations of the foregoing ranges are possible (e.g., greater than or equal to 18°C and less than or equal to 30°C, greater than or equal to 20°C and less than or equal to 25°C). Other ranges are also possible, and may be dependent on the components of the injectable composition.

[0054] In some embodiments, the injectable composition may undergo a sol-gel transition at or around body temperature of a subject. It will be understood that the body temperature of the subject may depend on the identity of the subject. In some embodiments, the sol-gel transition may at least partially occur at a temperature of greater than or equal to 32°C, greater than or equal to 35°C, greater than or equal to 36°C, greater than or equal to 37°C, greater than or equal to 38°C, or greater than or equal to 39°C. In some embodiments, the sol-gel transition may at least partially occur at a temperature of less than or equal to 42°C, less than or equal to 40°C, less than or equal to 39°C, less than or equal to 38°C, less than or equal to 37°C, or less than or equal to 36°C. Combinations of the foregoing ranges are possible (e.g., greater than or equal to 32°C and less than or equal to 42°C, greater than or equal to 35°C and less than or equal to 40°C, greater than or equal to 36°C and less than or equal to 37°C). Other ranges are also possible.

[0055] Still, in some embodiments, the LA to GA ratio may be selected such that the PLGA-PEG-PLGA solution does not gel and, due to the interior of the micellar structures including a larger proportion of hydrophilic GA residues, burst release of the immunomodulatory nucleic acid from the micellar structure following injection may occur. In some embodiments, burst release may be chosen when, for example, extended release of the compounds contained within the micellar structure provides no benefits over burst release. To achieve a burst release, in some embodiments, a relatively lowerLA to GA ratio may be chosen (e.g., greater than or equal to 1 : 1 and less than or equal to 2:1).

[0056] In some embodiments, the injectable composition comprises an immunomodulatory nucleic acid. In an exemplary set of embodiments, the injectable composition includes an immunomodulatory oligonucleotide (e.g., a CPG-ODN). In another exemplary set of embodiments, the injective composition includes a natural, synthetic, and / or therapeutic forms of RNA, DNA, and hybrids thereof. Suitable nucleic acids and / or oligonucleotides are described in more detail, below.

[0057] As used herein, the term “immunomodulatory” is given its ordinary meaning in the art and generally refers to an agent that can be administered to a subject and elicits an immune response. Such an immune response may be induced to treat a disease, disorder, or other clinically recognized condition, or for prophylactic purposes, and, in such embodiments, has a clinically significant effect on the body of the subject to treat and / or prevent the disease, disorder, or condition. For example, in some embodiments, the immunomodulatory nucleic acid has a therapeutic effect that implicates the immune response of the subject. For illustrative purposes and without wishing to be limited to such, in some embodiments, the injectable composition comprises an immunomodulatory nucleic acid that is adapted and configured to trigger an immune response against a tumor into and / or near to which the composition is being injected. In some such embodiments, and without wishing to be bound by theory, the immunomodulatory nucleic acid induces the immune system to have an immune response (e.g., to seek and / or kill cells with tumor antigens). For example, immunomodulatory nucleic acids such as CpGs and other TLR agonists may be administered to trigger antigen presentation and, without wishing to be bound by theory, thereby leading to T effector responses. Other effects are also possible. In some embodiments, the immunomodulatory nucleic acid is non-coding and / or serve as targets that are implicated in immune response. In some embodiments, the immunomodulatory nucleic acid is coding and / or codes for one or more molecules and / or one or more proteins (and / or antigens) implicated in the immune response and / or that recruits immune cells / response (e.g., overexpression of tumor expressing antigens).

[0058] While much of the description below and herein is generally related to immunomodulatory oligonucleotides such as CpG-ODNs, such embodiments related tooligonucleotides are intended to be illustrative and those of ordinary skill in the art would be capable of selecting one or more nucleic acids for use in conjunction with the injectable compositions described herein. Non-limiting examples of suitable nucleic acids include known natural, synthetic, and / or therapeutic forms of RNA, DNA, and hybrids thereof. For example, in some embodiments, the immunomodulatory nucleic acid is selected from the group consisting of double- stranded DNA (dsDNA), singlestranded DNA (ssDNA), anti-sense oligonucleotide (ASO), double-stranded RNA (dsRNA), single- stranded RNA (ssRNA), short-hairpin RNA (shRNA), short-interfering RNA (siRNA), non-coding RNA (ncRNA), long non-coding RNA (IncRNA), messenger RNA (mRNA), microRNA (miRNA), and combinations thereof. For example, in some embodiments, the nucleic acid may be non-coding RNA or DNA (e.g., non-coding nucleic acids that generally work against targets that are implicated in immune response). In some embodiments, the nucleic acid may be coding RNA or DNA (e.g., coding for molecules implicated in the immune response and / or coding for one or more proteins (and / or antigens) that recruits immune cells and / or elicit an immune response (e.g., for overexpression of tumor expressing antigens)).

[0059] In some embodiments, the immunomodulatory oligonucleotide is configured to be retained by the injectable composition, e.g., at a location internal to the subject. For instance, in some embodiments, the immunomodulatory oligonucleotide is encapsulated by the PLGA-PEG-PLGA micellar structure (e.g., or other block copolymer structure), thereby at least partially retaining the immunomodulatory oligonucleotide. In some embodiments, the immunomodulatory oligonucleotide is at least partially retained by the injectable composition after injection (e.g., after gelling) such that the CPG-ODN exhibits an extended-release profile into the subject from the injectable composition following injection. In some embodiments, the extended-release profile extends over greater than or equal to 24 hours, greater than or equal to 2 days, greater than or equal to 3 days, greater than or equal to 4 days, greater than or equal to 5 days, greater than or equal 10 days and / or less than or equal to 15 days, less than or equal to 20 days, less than or equal to 25 days, or less than or equal to 30 days, as measured in phosphate-buffered saline at 37 °C. Accordingly, in some embodiments, bioactivity associated with the injectable composition extends over greater than or equal to 24 hours, greater than or equal to 2 days, greater than or equal to 3 days, greater than or equal to 4 days, greaterthan or equal to 5 days, greater than or equal 10 days and / or less than or equal to 15 days, less than or equal to 20 days, less than or equal to 25 days, or less than or equal to 30 days, as measured in phosphate-buffered saline at 37 °C.

[0060] In some embodiments, a micellar structure of the injectable composition may encapsulate an immunomodulatory oligonucleotide, e.g., a CpG-ODN. In some embodiments, due to the hydrophobic nature of the interior of the micellar structure (e.g., when the LA to GA ratio is greater than or equal to 1:1 and less than or equal to 10:1, when the LA to GA ratio is greater than or equal to 3: 1 and less than or equal to 5: 1), the immunomodulatory oligonucleotide may have relatively slow release kinetics from the interior of the micellar structure to the exterior of the micellar structure (e.g., into the subject). For instance, initially, the immunomodulatory oligonucleotide may only release from the micellar structure by diffusion. In some embodiments, the release rate of the immunomodulatory oligonucleotide may increase over time due to the degradation of the micellar structure, for instance, if the structure is biodegradable.

[0061] Due to the micellar structure retaining the immunomodulatory oligonucleotide, in some embodiments, the time over which the immunomodulatory oligonucleotide is released from the micellar structure may be relatively long. In some embodiments, the immunomodulatory oligonucleotide exhibits an extended-release profile from the injectable composition as measured in phosphate-buffered saline at 37 °C. For example, in some embodiments, the time over which at least 90 wt% of the immunomodulatory oligonucleotide is released from the micellar structure is greater than or equal to 24 hours, greater than or equal to 2 days, greater than or equal to 3 days, greater than or equal to 4 days, greater than or equal to 5 days, greater than or equal to 6 days, greater than or equal to 7 days, greater than or equal to 8 days, greater than or equal to 9 days, greater than or equal to 10 days, greater than or equal to 12 days, greater than or equal to 15 days, greater than or equal to 18 days, greater than or equal to 20 days, or greater than or equal to 25 days. In some embodiments, the time over which at least 90 wt% of the immunomodulatory oligonucleotide is released from the micellar structure is less than or equal to 30 days, less than or equal to 25 days, less than or equal to 20 days, less than or equal to 18 days, less than or equal to 15 days, less than or equal to 12 days, less than or equal to 10 days, less than or equal to 9 days, less than or equal to 8 days, less than or equal to 7 days, less than or equal to 6 days, less than or equal to 5 days, less than orequal to 4 days, less than or equal to 3 days, or less than or equal to 2 days.

[0062] Combinations of the foregoing ranges are possible (e.g., greater than or equal to 24 hours and less than or equal to 30 days). Other ranges are also possible. Note, however, in other embodiments, the release of the immunomodulatory oligonucleotide from the injectable composition may be relatively quick, e.g., as a burst release as described elsewhere herein.

[0063] In some embodiments, the immunomodulatory oligonucleotide is an oligonucleotide. In some embodiments, the immunomodulatory oligonucleotide is a CpG oligodeoxynucleotide (CpG ODN). According to some embodiments, the CpG ODN is Class A (type D) CpG ODN, Class B (type K) CpG ODN, Class C CpG ODN, or class P CpG-ODN. It will be understood that the immunomodulatory oligonucleotide may have a phosphothioate backbone or a phosphodiester backbone. Typically, a phosphothioate backbone is desirable, as it confers nuclease resistance. Without wishing to be bound by any particular theory, in some embodiments, the use of a phosphodiester backbone is facilitated due to the encapsulation of the immunomodulatory oligonucleotide within the gel and thus a lack of exposure to nucleases, relative to an unencapsulated immunomodulatory oligonucleotide. In some embodiments, the CpG ODN is selected from the group comprising ODN 1585, ODN 2216, ODN 2336, ODN 1018, ODN 1668, ODN 1826, ODN 2006, ODN 2007, ODN D-SL01, ODN 2395, ODN M362, ODN D-SL03, ODN 2243, ODN 2336, ODN 2395, ODN M362, ODN BW006 (ODN 684), ODN D-SL01, CMP-001, vidutolimod, Litenimod, and CpG 685 (GNKG-168).

[0064] The immunomodulatory oligonucleotide may be present within the injectable composition at any suitable concentration, in some embodiments. For example, in some embodiments, the concentration of the immunomodulatory oligonucleotide may be selected such that the immunomodulatory oligonucleotide may be released from the injectable composition at a therapeutically relevant level. In some embodiments, the immunomodulatory oligonucleotide is present within the injectable composition at a concentration of greater than or equal to 0.001 mg / mL, greater than or equal to 0.01 mg / mL, greater than or equal to 0.1 mg / mL, or greater than or equal to 1 mg / mL. In some embodiments, the immunomodulatory oligonucleotide is present within the injectable composition at a concentration of less than or equal to 10 mg / mL, less than or equal to 1 mg / mL, less than or equal to 0.1 mg / mL, or less than or equal to 0.01 mg / mL.Combinations of the foregoing ranges are possible (e.g., greater than or equal to 0.001 mg / mL and less than or equal to 10 mg / mL). Other ranges are also possible.

[0065] The immunomodulatory oligonucleotide comprises CpG-ODNs in some embodiments, which may be present within the injectable composition at any suitable concentration, according to some embodiments. For example, in some embodiments, the CpG-ODN is present within the injectable composition at a concentration of greater than or equal to 0.001 mg / mL, greater than or equal to 0.01 mg / mL, greater than or equal to 0.1 mg / mL, or greater than or equal to 1 mg / mL. In some embodiments, the CpG-ODN is present within the injectable composition at a concentration of less than or equal to 10 mg / mL, less than or equal to 1 mg / mL, less than or equal to 0.1 mg / mL, or less than or equal to 0.01 mg / mL. Combinations of the foregoing ranges are possible (e.g., greater than or equal to 0.001 mg / mL and less than or equal to 10 mg / mL). Other ranges are also possible.

[0066] In some embodiments, the injectable composition includes an polycationic polymer and / or a lipid. In some embodiments, the injectable composition includes an polycationic polymer . In some embodiments, the injectable composition includes an ionizable lipid. In some embodiments, the polycationic polymer and / or the lipid is configured to be retained by the injectable composition, e.g., at a location internal to the subject. For instance, in some embodiments, the polycationic polymer and / or an ionizable lipid is encapsulated by the PLGA-PEG-PLGA micellar structure (e.g., or other block copolymer structure), thereby at least partially retaining the polycationic polymer and / or an ionizable lipid.

[0067] In some embodiments, the composition comprises a polycationic polymer. In some embodiments, the polycationic polymer comprises a peptide (e.g., polylysine, poly ornithine), synthetic polymer (e.g., polyethyleneimine), polysaccharide (e.g., cyclodextrin, chitosan), natural polymer (e.g., histone, collagen), composite material (e.g., DEAE (diethylaminoethyl)-dextran), and / or thickening agent (e.g., HPMC

[0068] (hy droxypropyl methylcellulo se) ) .

[0069] In some embodiments, the composition comprises a lipid. In some embodiments, the composition comprises an ionizable lipid. For example, in some embodiments, the ionizable lipid comprises cKK-E12, C12-200, 503013, and / or DODAP. In some embodiments, the composition comprises a zwitterionic lipid. For example, in someembodiments, the zwitterionic lipid comprises DOPE, DSPC, POPE, DMPC, and / or DOPS. In some embodiments, the composition comprises a cationic lipid. For example, in some embodiments, the cationic lipid comprises DOTAP. Non-limiting examples of other possible lipids include SM102, MC3, and ALC0315. Other lipids are also possible. For example, in some embodiments, the lipid comprises a phospholipid. In some embodiments, the lipid comprises DPPC (l,2-dipalmitoyl-sn-glycero-3-phosphocholine). In some embodiments, the lipid comprises DSPC (l,2-distearoyl-sn-glycero-3-phosphocholine). In some embodiments, the lipid comprises a synthetic lipid. In some embodiments, the lipid comprises DMG-PEG2000 (l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000). In accordance with some embodiments, the composition may comprise two or more lipids. In some cases, the composition comprises two or more lipids, three or more lipids, four or more lipids, or five or more lipids and / or less than or equal to six lipids, less than or equal to seven lipids, or less than or equal to eight lipids. It will be understood that more than eight lipids are also possible, in some cases. In some cases, there may be a primary lipid, a helped lipid, a PEG lipid, and / or a small molecule lipid (e.g., cholesterol). Other lipids are also possible and those of ordinary skill in the art would be capable of selecting suitable lipids for use with the compositions described herein based upon the teachings of this specification.

[0070] In some embodiments, when multiple lipids are present, the lipids may be present in a ratio of the first lipid to second lipid of greater than or equal to 1:1, greater than or equal to 2:1, greater than or equal to 5:1, greater than or equal to 10:1, greater than or equal to 20:1, or greater than or equal to 50:1 and / or less than or equal to 75:1, less than or equal to or equal to 90:1, or less than or equal to 100:1. Combinations of the foregoing ranges are possible (e.g., greater than or equal to 1:1 and less than or equal to 100:1). Other ranges are also possible.

[0071] The composition comprises a lipid in any of a variety of suitable amounts, in accordance with some embodiments. In some cases, the composition comprises a total lipid content that is greater than 0 wt%, greater than or equal to 5 wt%, greater than or equal to 10 wt%, greater than or equal to 15 wt%, greater than or equal to 20 wt%, or greater than or equal to 25 wt% versus the total weight of the composition. In some cases the composition comprises a total lipid content that is less than or equal to 30 wt%, less than or equal to 25 wt%, less than or equal to 20 wt%, less than or equal to 15 wt%, lessthan or equal to 10 wt%, or less than or equal to 5 wt% versus the total weight of the composition. Combinations of the foregoing ranges are possible (e.g., greater than 0 wt% and less than or equal to 30 wt%). Other ranges are also possible. It will be understood that, when multiple lipids are present, the total lipid content corresponds to the total amount of all of the multiple lipids combined.

[0072] In some embodiments, the injectable compositions described herein are configured to form particulates upon injection. In some embodiments, the injectable compositions described herein are particulates. In some embodiments, the polycationic polymer and / or the lipid form nanoparticles encapsulating at least some of the immunomodulatory oligonucleotide of the composition. For example, in some embodiments, the presence of the polycationic polymer and / or an ionizable lipid may result in the formation of polymeric and / or lipid nanoparticles, respectively. In some such embodiments, the formation of polymeric and / or lipid nanoparticles may result when cholesterol, a PEG-lipid, arachidonic acid, oleic acid, myristic acid, and / or sodium lauryl sulfate are also present. Accordingly, in some embodiments, the injectable composition further comprises cholesterol and / or a PEG-lipid. In some embodiments, the injectable composition further comprises arachidonic acid, oleic acid, myristic acid, and / or sodium lauryl sulfate. It will be understood that nanoparticles have an average maximum lateral dimension of less than or equal to 1 micron and / or greater than or equal to 1 nm or greater than or equal to 10 nm. In some embodiments, the formation of polymeric and / or lipid nanoparticles may desirably contain and / or be associated with an immunomodulatory oligonucleotide that is injected with the injectable composition. The presence of nanoparticles that encapsulate the immunomodulatory oligonucleotide may advantageously facilitate delivery thereof into cells to facilitate therapeutic efficacy, in some cases. In some embodiments, where the composition is configured to form lipid and / or polymer nanoparticles, the nanoparticles may be configured to be retained by the composition at a location of injection, thereby facilitating long term and / or localized release of the nanoparticles and immunomodulatory oligonucleotide contained therein at or near the site of injection. In some embodiments, administration of the composition to a subject results in localized activity of the nucleic acid in the vicinity of the site of administration.In some embodiments, lipid nanoparticles may be formed that encapsulate the immunomodulatory oligonucleotide, whereafter the lipid nanoparticle may be at least partially contained within a thermogelling composition. In some embodiments, the composition comprises a polymer as described elsewhere herein (e.g., PLGA-PEG-PLGA) that is liquid a room temperature but gels when heated, lipid nanoparticles associated with the polymer, and an immunomodulatory oligonucleotide associated with the lipid nanoparticles. In some embodiments, an immunomodulatory oligonucleotide associated with the polymer is encapsulated within the lipid nanoparticles. In some embodiments, lipid nanoparticles associated with the polymer are encapsulated within the polymer.

[0073] According to some embodiments, the injectable composition comprises a radioopaque label. In some embodiments, a radio-opaque label may be encapsulated within a micellar structure of the injectable composition and, in some cases, may be coencapsulated along with an immunomodulatory oligonucleotide. The presence of the radio-opaque label may facilitate the visualization and thus targeted injection of the injectable composition, e.g., by injecting the injectable composition containing the radioopaque label during a computer tomography (CT) scan. The presence of both the radioopaque label and the immunomodulatory oligonucleotide, in some embodiments, may deleteriously impact the ability of the injectable composition to retain either the radioopaque label or the immunomodulatory oligonucleotide, which may result in a burst release of the radio-opaque label and the immunomodulatory oligonucleotide upon injection of the injectable composition. In some such embodiments, burst release may occur in the present of both the radio-opaque label and the immunomodulatory oligonucleotide without regard to the composition of the block polymer of the injectable gel. Advantageously, the compositions described herein are configured to avoid burst release, in accordance with some embodiments.

[0074] The radio-opaque label may be any of a variety of agents, in accordance with some embodiments. In some embodiments, the radio-opaque label is an iodinated contrast agent. In some embodiments, the radio-opaque label comprises a metal. Nonlimiting examples of radio-opaque labels include iopamidol, ioversol, iron, iron oxide (e.g., Fe2O3 and / or other iron oxides), iopromide, lipiodol (ethiodol), iodixanol, calcium, calcium salts (e.g., calcium chloride, calcium sulfate, etc.), barium, barium sulfate, zinc,gold, titanocene, iohexol, diatrizoate meglumine, diatrizoate sodium, ethiodized oil, and gadolinium (e.g., in free and / or chelated forms). Other labels are also possible. In some embodiments, the radio-opaque label may be ultrasound visible and / or magnetic resonance imaging (MRI) visible. For instance, in some embodiments, the radio-opaque label is configured to be visible via ultrasound visible by comprising a lipid-coated microbubble. In some embodiments, the radio-opaque label is configured to be visible via MRI via a super paramagnetic agent (e.g., iron oxide, gadolinium).

[0075] Accordingly, in some embodiments, the radio-opaque label may be present in the injectable composition in a concentration sufficient to allow for imaging of the injectable composition during injection, e.g., by a CT scan, MRI, or ultrasound, but not in too high of a concentration to result in a burst release of the label and / or the immunomodulatory oligonucleotide from the micellar structures. In some embodiments, the radio-opaque label may be present in the injectable composition in an amount of greater than 0 mg / mL (e.g., mg of the radio-opaque label per mL of the injectable composition), greater than or equal to 0.1 mg / mL, greater than or equal to 0.5 mg / mL, greater than or equal to 1 mg / mL, greater than or equal to 2 mg / mL, greater than or equal to 3 mg / mL, greater than or equal to 5 mg / mL, greater than or equal to 8 mg / mL, greater than or equal to 10 mg / mL, greater than or equal to 15 mg / mL, greater than or equal to 18 mg / mL, greater than or equal to 20 mg / mL, greater than or equal to 22 mg / mL, greater than or equal to 25 mg / mL, greater than or equal to 28 mg / mL, greater than or equal to 35 mg / mL, greater than or equal to 50 mg / mL, greater than or equal to 100 mg / mL, greater than or equal to 150 mg / mL, greater than or equal to 200 mg / mL, or greater than or equal to 250 mg / mL. In some embodiments, the radio-opaque label may be present in the injectable composition in an amount of less than or equal to 300 mg / mL, less than or equal to 250 mg / mL, less than or equal to 200 mg / mL, less than or equal to 150 mg / mL, less than or equal to 100 mg / mL, less than or equal to 50 mg / mL, less than or equal to 35 mg / mL, less than or equal to 28 mg / mL, less than or equal to 25 mg / mL, less than or equal to 22 mg / mL, less than or equal to 20 mg / mL, less than or equal to 18 mg / mL, less than or equal to 15 mg / mL, less than or equal to 10 mg / mL, less than or equal to 8 mg / mL, less than or equal to 5 mg / mL, less than or equal to 3 mg / mL, less than or equal to 2 mg / mL, less than or equal to 1 mg / mL, less than or equal to 0.5 mg / mL, or less than or equal to 0.1 mg / mL. Combinations of the foregoing ranges are possible (e.g., greater than 0 andless than or equal to 300 mg / mL, greater than 0 and less than or equal to 100 mg / mL, greater than or equal to 15 mg / mL and less than or equal to 50 mg / mL, greater than or equal to 5 mg / mL and less than or equal to 25 mg / mL). Other ranges are also possible.

[0076] In some embodiments, the injectable composition further comprises an immunoadjuvant. In some embodiments, the injectable composition comprises a hydrophobic immunoadjuvant. In some embodiments, the hydrophobic immunoadjuvant comprises a small molecule having a molecular weight of less than about 2500 Daltons, less than about 2000 Daltons, less than about 1500 Daltons, less than about 1000 Daltons, less than about 750 Daltons, less than about 500 Daltons, less or than about 400 Daltons. In some cases, the hydrophobic immunoadjuvant is a small molecule having a molecular weight between 200 Daltons and 400 Daltons, between 400 Daltons and 1000 Daltons, or between 500 Daltons and 2500 Daltons.

[0077] The hydrophobic immunoadjuvant may be any of a variety of small molecules, according to some embodiments. Non-limiting examples of suitable hydrophobic immunoadjuvants include imidazoquinoline derivatives and immunoadjuvant drug imiquimod (e.g., TLR 7 agonist), Motolimod (TLR 8 agonist), Vesatolimod (TLR 7 agonist), 852-A, azd8848, Selgantolimod, resiquimod, and 2,3 cGAMP, and derivatives thereof. In some embodiments, the hydrophobic immunoadjuvant may be a small tolllike receptor (TLR) agonist. In some such embodiments, the small TLR agonist is selected from the group consisting of imiquimod, resiquimod, 852-A, vesitolimod, azd8848, motolimod, and selgantolimod, and derivatives thereof. Other hydrophobic immunoadjuvants are also possible.

[0078] Additional non-limiting examples of suitable immunoadjuvants include NKTR-262, RG-7854, DSP-0509, BDB-001, LHC-165, BDC-1001, SHR-2150, JNJ-4964, RO-7119929, DN-1508052, VTX-1463, BNT-411, APR-003, PF-4878691, GSK-2245035, RG-7795 (ANA 773, RO 6864018), Epitirimod (R-851), DSP-3025 (AZD-8848), Sotirimod (R-850, S-30594), Telratolimod (3M-052, MEDL9197), Isatoribine (ANA-245), Loxoribine, ANA-971, ANA-975, and RG-7863 (RO6870868).

[0079] According to some embodiments, as described above, the injectable composition includes a micellar structure comprising PLGA-PEG-PLGA. In some embodiments, the injectable compositions described herein may be useful as a general platform for delivery of a wide variety of immunomodulatory oligonucleotides (or other immunomodulatorynucleic acids) that may not be typically delivered via injection due to their poor aqueous solubility. In some embodiments, the PLGA-PEG-PLGA micellar structure is capable of encapsulating a immunomodulatory oligonucleotide in high concentrations. For example, referring to FIG. IB, one or more active substances 120 (e.g., an immunomodulatory oligonucleotide) may be encapsulated within a micellar structure of the injectable composition. Encapsulating the immunomodulatory oligonucleotide within the micellar structures of the injectable composition may be desirable, in some embodiments, to facilitate the solubilization and / or delivery of the immunomodulatory oligonucleotide in high concentrations to a location internal of a subject that may otherwise not be possible.

[0080] For instance, in some embodiments, the PLGA components of a PLGA-PEG-PLGA micellar structure forms a relatively hydrophobic region when compared to an aqueous solution, e.g., indiscriminate of the LA to GA ratio present in the PLGA blocks. In some embodiments, the hydrophobic nature of the PLGA block (e.g., compared to an aqueous solution) facilitates a relatively high solubility and thus loading of a immunomodulatory oligonucleotide into the micellar structures of the injectable compositions when compared to the aqueous solubility of the immunomodulatory oligonucleotide.

[0081] For instance, in some embodiments, an immunomodulatory oligonucleotide may be hydrophobic and thus may not be substantially soluble in aqueous solutions. In some embodiments, immunomodulatory oligonucleotide may have a solubility in an aqueous solution of less than or equal to 0.1 mg / mL, less than or equal to 0.05 mg / mL, less than or equal to 0.01 mg / mL, less than or equal to 0.005 mg / mL, or less than or equal to 0.002 mg / mL. In contrast, the solubility of the immunomodulatory oligonucleotide may be relatively high in the micellar structures described herein. For example, the micellar structure may be capable of encapsulating, may be configured to encapsulate, and / or may encapsulate the immunomodulatory oligonucleotide in a high concentration. According to some embodiments, the micellar structure may be capable of encapsulating, may be configured to encapsulate, and / or may encapsulate the immunomodulatory oligonucleotide in a concentration of greater than or equal to 0.1 mg / mL (e.g., 0.1 mg of immunomodulatory oligonucleotide per mL of injectable composition), greater than or equal to 0.5 mg / mL, greater than or equal to 1 mg / mL, greater than or equal to 2 mg / mL, greater than or equal to 3 mg / mL, greater than or equal to 4 mg / mL, greater than or equalto 5 mg / mL, greater than or equal to 6 mg / mL, or greater than or equal to 7 mg / mL. In some embodiments, the micellar structure may be capable of encapsulating, may be configured to encapsulate, and / or may encapsulate the immunomodulatory oligonucleotide in a concentration of less than or equal to 8 mg / mL, less than or equal to 7 mg / mL, less than or equal to 6 mg / mL, less than or equal to 5 mg / mL, less than or equal to 4 mg / mL, less than or equal to 3 mg / mL, less than or equal to 2 mg / mL, less than or equal to 1 mg / mL, or less than or equal to 0.5 mg / mL. Combinations of the foregoing ranges are possible (e.g., greater than or equal to 0.1 mg / mL and less than or equal to 8 mg / mL, greater than or equal to 5 mg / mL and less than or equal to 6 mg / mL). Following encapsulation of the immunomodulatory oligonucleotide by the micellar structures, the micellar structures may then be delivered to a subject to deliver the immunomodulatory oligonucleotide contained therein.

[0082] In some embodiments, high concentrations of the immunomodulatory oligonucleotide in the injectable composition may be desirable. However, in other embodiments, a low concentration of the immunomodulatory oligonucleotide in the injectable composition may be desirable if the immunomodulatory oligonucleotide has a high activity.

[0083] In some embodiments, the composition of the thermoresponsive, injectable gel, the presence and concentration of the immunomodulatory oligonucleotide, the presence and concentration of a radio-opaque label, the presence of the immunomodulatory oligonucleotide, and / or the desired release profile (e.g., of the immunomodulatory oligonucleotide) are interconnected, making the parameter space and related design of experiments unexpectedly complex. Advantageously, the compositions described herein were formulated to provide injectable compositions having suitable gelling properties, retention properties of the gel, concentration of the immunomodulatory oligonucleotide, and / or concentration of the radio-opaque label, with certain release profiles, according to some embodiments.

[0084] Some aspects of the present disclosure are generally related to methods, for example, for treating tumors. The injectable compositions described herein may, in some cases, be administered to a subject, e.g., such that the immunomodulatory oligonucleotide is delivered to the subject. For example, in some cases, the composition may be administered to the subject and an immunomodulatory oligonucleotide isreleased from the composition at a location internal to the subject. In accordance with some embodiments, administering the injectable composition comprises injecting the composition. Administration of the compositions and release of immunomodulatory oligonucleotide are described in more detail herein, in some cases.

[0085] In some embodiments, the method comprises treating a tumor by injecting a composition intratumorally (i.e., into a tumor). In some embodiments, the method comprises treating a tumor by injecting a composition peritumorally. In some embodiments, the method comprises treating a tumor by injecting a composition adjacent to a tumor. According to some embodiments, the composition may be injected into or near a tumor within a subject, e.g., in intra-abdominal, intrathoracic, intracranial or other anatomic compartments. In some embodiments, the composition may be a liquid at room temperature and a gel at 37 °C and may comprise poly(lactide-co-glycolide-b-ethylene glycol-b-lactide-co-glycolide) (PLGA-PEG-PLGA) having a lactic acid (LA) to glycolic acid (GA) ratio of greater than or equal to 3:1, wherein a number average molecular weight of each PLGA is greater than or equal to 500 and less than or equal to 2500 and a number average molecular weight of the PEG is greater than or equal to 500 and less than or equal to 2500, an immunomodulatory oligonucleotide present within the injectable composition at a concentration of greater than or equal to 0.1 mg / mL, and a polycationic polymer and / or an ionizable lipid. Accordingly, in some embodiments, the method comprises injecting a liquid composition, whereafter the composition may gel at the site of injection within the subject (e.g., at a location internal to the subject, e.g., upon reaching physiological conditions and / or temperature). According to some embodiments, the injecting may occur concurrently to another treatment of at least a portion of a tumor, for example, percutaneous cryoablation, microwave ablation, radioablation, ethanol ablation, and / or irreversible electroporation of the tumor. In some embodiments, the efficacy of injection of the injectable composition (e.g., a localized immuno stimulation) does not change in combination with second treatment, as compared to the instance without the second treatment.

[0086] The method may further include determining the position of the composition during injection using CT, in some embodiments. In some such embodiments, the composition may further include a radio-opaque label to facilitate the monitoring and / or determining of the position of the composition during injection. For example, in someembodiments, the method includes imaging a site of administration when administering a composition to a subject. In some embodiments, administering comprises any of a variety of suitable techniques, as described elsewhere herein in more detail. In some cases, determining the position of the composition during injection comprises using one or more of ultrasound, CT, visual inspection, optical microscopy, and MRI. In some cases, determining a position comprises imaging. In some embodiments, imaging comprises using MRI. In some embodiments, imaging comprises using ultrasound. In some embodiments, imaging comprises using a CT scan. In some embodiments, determining a position using visual inspection. In some embodiments, imaging comprises using optical microscopy. In some cases, visual inspection may be suitable for determining an administration site, as certain markers may be present on the subject, e.g., precancerous cervical lesions. Accordingly, in some embodiments, a radio-opaque label is absent from the composition,

[0087] As described above, in some embodiments, the composition may be a gel at temperatures above room temperature and less than or equal to 37 °C (or at other temperatures as described elsewhere herein). Accordingly, following injecting the composition, the composition may heat (e.g., due to the temperature of the body of the subject) and gel. In some embodiments, the method comprises heating and gelling the composition at the injection location. In some embodiments, the method comprises allowing the composition to gel at a location of the injecting. Following, the gel may be retained at the injection location. According to some embodiments, the method comprises retaining the gelled composition at the injection location for greater than or equal to 24 hours, greater than or equal to 2 days, and so forth as described elsewhere herein. In some embodiments, the method includes releasing the immunomodulatory oligonucleotide from the composition over at least 24 hours and no more than 30 days. Advantageously, in some such embodiments, directed injection of the composition may facilitate localized delivery and treatment of a tumor by an immunomodulatory oligonucleotide of the composition. In some embodiments, the localized delivery facilitates a local immuno stimulation with a systemic effect (e.g., an abscopal effect).

[0088] In some embodiments, localized delivery may be associated with localized uptake and / or transfection of some or all of a composition by cells local to the administrationsite. In some cases, a composition comprises an encoding nucleic acid such that, once uptaken by cells local to the administration site, may transfect the cells and expression of proteins encoded thereby occurs. In some embodiments, expressed proteins may fluoresce or otherwise produce an observable signal. In some embodiments, an observable signal associated with the transfection may only be observed a distance of less than or equal to 5 cm, less than or equal to 4 cm, less than or equal to 3 cm, less than or equal to 2 cm, less than or equal to 1 cm, less than or equal to 5 mm, or less from an administration site. In some embodiments, rather than expressing a protein with an observable signal, a composition comprises a nucleic acid having an observable signal (e.g., a nucleic acid modified with a fluorescing moiety). In some embodiments, the observable signal form the modified nucleic acid may only be seen in cells that are a distance of less than or equal to 5 cm, less than or equal to 4 cm, less than or equal to 3 cm, less than or equal to 2 cm, less than or equal to 1 cm, less than or equal to 5 mm, or less from an administration site. In some cases, localized delivery and activity of a composition comprising nucleic acid results in at least 50%, at least 75%, at least 90%, at least 95%, at least 99%, or more of the whole-body primary functional activity of the nucleic acid occurring at or near the site of administration.

[0089] For example, in some embodiments, the method comprises releasing the immunomodulatory oligonucleotide over a time of greater than or equal to 24 hours under physiological conditions. In some embodiments, the release of the immunomodulatory oligonucleotide is localized to an injection location, e.g., if the composition gelled and was retained at the injection location. The release, in some embodiments, may be logarithmic in nature. In accordance with some embodiments, the method may further include administering an immune checkpoint inhibitor (e.g., anti-PD1, anti-PDLl, and / or anti-CTLA4) and / or other immune-active agents.

[0090] Still in some embodiments, the method may further comprise degrading the composition. Degradation of the composition may occur naturally internal to the subject, for example, if the composition is biodegradable. An example degradation pathway includes hydrolysis of a polymer of the composition.

[0091] In some embodiments, the injectable compositions described herein are pharmaceutically acceptable. The phrase “pharmaceutically acceptable” as used herein generally refers to compounds, materials, compositions, and / or dosage forms which are,within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0092] In some embodiments, the injectable compositions described herein are useful for treatment of cancer. In some embodiments, the injectable compositions may be packaged in kits, optionally including instructions for use of the composition for the treatment of cancer. That is, the kit can include a description of use of the injectable composition for participation in any biological or chemical mechanism (e.g., immunomodulatory) disclosed herein associated with cancer or tumor. The kits can further include a description of activity of cancer in treating the pathology, as opposed to the symptoms of the cancer. That is, the kit can include a description of use of the injectable composition as discussed herein. The kit also can include instructions for use of a combination of two or more compositions of the invention. Instructions also may be provided for administering the injectable composition by any suitable technique, such as orally, intravenously, surgically, or via another known route of delivery to a subject. In some embodiments, promotion of the treatment of cancer according to any of the techniques and compositions and composition combinations described herein is also provided.

[0093] For example, a subject (e.g., to which the injectable composition is administered) may be a subject diagnosed with cancer or otherwise known to have cancer. In certain embodiments, a subject may be selected for treatment on the basis of a known cancer in the subject. In some embodiments, a subject may be selected for treatment on the basis of a suspected cancer in the subject. In some embodiments, a cancer may be diagnosed by detecting a mutation associate in a biological sample (e.g., urine, sputum, whole blood, serum, stool, etc., or any combination thereof. Accordingly, a compound or composition of the invention may be administered to a subject based, at least in part, on the fact that a mutation is detected in at least one sample (e.g., biopsy sample or any other biological sample) obtained from the subject. In some embodiments, a cancer may not have been detected or located in the subject, but the presence of a mutation associated with a cancer in at least one biological sample may be sufficient to prescribe or administer one or more compositions of the invention to the subject. In some embodiments, the composition may be administered to prevent the development of a cancer. However, in some embodiments, the presence of an existing cancer may be suspected, but not yetidentified, and a composition of the invention may be administered to prevent further growth or development of the cancer. It should be appreciated that any suitable technique may be used to identify or detect mutation and / or over-expression associated with a cancer. For example, nucleic acid detection techniques (e.g., sequencing, hybridization, etc.) or peptide detection techniques (e.g., sequencing, antibody-based detection, etc.) may be used. In some embodiments, other techniques may be used to detect or infer the presence of a cancer (e.g., histology, etc.). The presence of a cancer can be detected or inferred by detecting a mutation, over-expression, amplification, or any combination thereof at one or more other loci associated with a signaling pathway of a cancer.

[0094] The following examples are intended to illustrate certain embodiments of the present invention, but do not exemplify the full scope of the invention.

[0095] EXAMPLE 1

[0096] In this Example, the use of CpG-ODNs within compositions as a therapeutic are described.

[0097] Checkpoint inhibitors (anti-PDl, anti-CTLA4) have revolutionized cancer therapy, offering the possibility of durable responses and even cure to some patients with metastatic disease. However, the vast majority of patients do not respond to such treatment. Alternatively, new emerging adjunctive treatments may increase the anti-tumoral immune response and thus increase checkpoint inhibitor response rate.

[0098] One area of high interest is the use of intratumoral injection of immunotherapies at one site of metastatic disease to increase local recognition of tumor by the immune system. Local recognition of tumor as ‘foreign” induces a systemic anti-tumoral immune response at other sites of metastatic disease (also termed the 'abscopal effect'). Several types of drugs can be used to trigger this effect and activate the local immune system, for example by agonizing Toll-Like Receptor (TLR) pathways within immune cells that trigger a cascade of protein-lead signal amplifications and event such as cytokine production, proliferation or greater adaptive immunity.

[0099] TLR9 is expressed mostly in immune cells including dendritic cells, macrophages, natural killer cells, and other antigen presenting cells. TLR9 is expressed on endosomes that facilitate internalization through the plasma membrane. Besides new evidence for other recognition patterns, TLR9 binds preferentially DNA containingunmethylated CpG motifs, which are common for human-foreign sources such as bacterial or viral DNA but rare in vertebrates. Therefore, unmethylated CpG motifs are considered pathogen-associated molecular patterns (PAMPs), which trigger cascaded immune cell activations.

[0100] Synthetic CpG oligodeoxynucleotides (CpG ODNs) are short single- stranded synthetic DNA molecules that are designed to trigger a PAMP-like immune response in TLR9 receptors and thereby upregulate immune activity and the innate immune system. In humans, mostly B cells and plasmacytoid dendritic cells (pDCs) respond to this treatment by producing proinflammatory cytokines, chemokines, and immunoglobulins. Some types of CpG DNA can activate plasmacytoid dendritic cells to produce a large amount of IFN-a, all of which are relevant to boost innate immunity to a local pathogen.

[0101] Despite their promise for local immune activation, no CpG ODN-based drugs have been approved by the FDA, as systemic delivery is limited by toxicity. Another general issue why intratumoral therapy (IT) has not met the expectations is the lack of sufficient retention of the drug at the tumor site by existing injection formulations, which leads to a lack of efficacy against the target and undesired off-target toxicities.

[0102] Consequentially, IT administration is typically done over the course of 5-10 multiple IT injections, ranging from daily to weekly injections, which are challenging to implement clinically. Furthermore, TLR9 is predominantly located intracellularly. Thus, intracellular delivery of CpG would be beneficial. However, CpG is a negatively charged macromolecule, which is expected to have low cell uptake. Hence, methods for intracellular delivery are desirable.

[0103] To address these challenges and unlock the promise of intratumoral, long-acting CpG-ODN, this Example describes a prolonged-release intratumoral hydrogel that can entrap CgG-ODNs (“CPGel”) to prevent premature leakage from tumor and allow for tunable release profiles that allow for an injection schedule amenable to clinical workflow. The hydrogel is designed to be optimized for image-guided minimally invasive procedures that require the gel to be injected through a small needle under image guidance. The hydrogel is loaded with nanoparticles that facilitate intracellular delivery of CpG, thus allowing for maximizing its potency.

[0104] The composition described in this Example formulation is liquid at room temperature, due to dominant hydrophilic interactions of PEG with surrounding waterand was formulated to deliver into a tumor through needle injection, and then gelates at body temperature through increasingly dominant hydrophobic interactions of PLGA, thereby forming a solid depot that releases the drug over a period of several days, while being biodegradable itself. FIG. 2 is a schematic diagram showing CpG-ODN bearing hydrogel (“CPGel”) function, triggering TLR9 responses in immune cells to facilitate local tumor recognition that escalate to systemic killing of metastases (abscopal effect).

[0105] Drug release profile were tuned by modifying the viscosity and diffusivity of the triblock hydrogel and / or by the presence of ionized co-formulating agents. Moreover, altering the molecular architecture of the macroscopic hydrogel and thus its hydrophobicity and viscosity, the lactide-to-glycolide (LA / GA) ratio (e.g. 1:1, 2:1, 3:1, 4:1, 5:1) as well as polymer concentration in aqueous phase (saline, water or another aqueous buffer) can be varied in a frame of parameters that allow leaving the gel point between room temperature (to retain injectability) and body temperature (to allow for thermogelation upon injection). Formulation with cationic and / or amphiphilic polymers allows charge-based binding associations with the anionic nature of CpG-ODNs, while also linking hydrophobic portions of the CpG-ODNs to hydrophobic moieties in the PLGA-PEG-PLGA hydrogel network, thereby retaining CpG-ODNs in the hydrogel networkError! Reference source not found..

[0106] Intratumoral injection of CPGel improves checkpoint inhibitor co-therapy by activating dendritic cells to take up tumor antigens and subsequently to activate cytotoxic T cells to attack tumor.

[0107] FIG. 3A shows the successful gel formation of a composition including 1 mg / ml CpG-ODN directly encapsulated in 25 % w / v PLGA-PEG-PLGA in saline after incubation for 5 minutes at 37 °C. FIG. 3B shows the direct incorporation of CpG-ODN into a simple hydrogel in PLGA-PEG-PLGA leads to a rapid burst release profile in PBS at 37 °C. Relatively fast release is associated with the hydrophilicity of CpG-ODN molecules, which allows for rapid passage through and out of the hydrophilic hydrogel.

[0108] In contrast to the quick release profile shown in FIG. 3B, FIG. 4 shows the release of CPG-ODN2395-FITC in PBS at 37 °C from a composition including a cationic polymer in PLGA-PEG-PLGA hydrogel. In this composition, the release profile extends out to multiple days. The composition measured in FIG. 4 was made by adding 230 microliters of PLGA-PEG-PLGA gel and 1.8 mg DEAE-dextran cationic polymer,resulting in 7.8 mg / mL of the polymer in the gel with an amine / phosphate ratio of approximately 5. Hand- heating and vortexing was performed to dissolve the polymer in the composition. CpG-ODN in an amount of 50 micrograms was added to 50 microliters of the gel / DEAE- dextran cationic polymer solution were mixed, whereafter 50 microliters of the gel were mixed with 25 mL of 40°C- warmed PBS in a 50 mL aluminum-foiled container, and then placed in a 37 °C shaker programed at 35 rpm. Aliquots were taken from the middle of the supernatant for plate reader analysis (490 / 525 nm EX / EM, 70% gain) to collect the data for FIG. 4.

[0109] EXAMPLE 2

[0110] In this Example, a composition containing CPG-ODN with PLGA-PEG-PLGA and lipid nanoparticle formulations are described.

[0111] The composition was prepared as follows. First, a pH 3 citric buffer (i.e., prepared by mixing 400 mL of DI water, 12.85 g sodium citrate dihydrate, and 1.21 g citric acid) was titrated with 8 mL HC137% to get to obtain a pH 3.14 solution.

[0112] Following this, 50 microliters of the citric buffer were used to dissolve 50 micrograms of CpG-ODN to obtain a 1 mg CpG-ODN per 1 mL citric buffer solution.

[0113] An ionizable lipid mix was prepared in ethanol. The ionizable lipid mix can contain a mixture of ionizable lipids, helper lipids, cholesterol, and / or PEG-lipid, that may facilitate stability during residence at site of action and for intracellular delivery.

[0114] The CpG-ODN solution in an amount of 50 microliters was mixed with 50 microliters of the ionizable lipid mix on ice to form a yellow cloudy nanoparticle solution. This solution was mixed in an amount of 50 microliters with 200 microliters of ReGel resulting in approximately 0.1 mg / mL CPG, and approximately 20% PLGA-PEG-PLGA gel. This solution was placed in gel wells, whereafter 250 microliters were placed in containers with 25 mL of 40 °C PBS and then shaked at 37 °C at 35 rpm. Aliquots were taken from the middle of the supernatant for plate reader analysis (490 / 525 nm EX / EM, 70% gain).

[0115] FIG. 5 shows the release of CPG-ODN2395-FITC in PBS at 37 °C from ionizable lipid nanoparticle (LNP) formulation in PLGA-PEG-PLGA hydrogel. The release rate from the gel extended over multiple days.EXAMPLE 3

[0116] In this example, lipid nanoparticle-encapsulated mRNA delivery in vitro is described.

[0117] Two different PLGA-PEG-PLGA polymers were used to form thermogels for the delivery of lipid nanoparticle (LNP) encapsulated mRNA. LNPs containing mRNA that encode firefly luciferase (FLuc) were combined with PLGA-PEG-PLGA polymers to form homogeneous fluid compositions that formed gels at 37°C. Functional delivery of the formulated mRNA was demonstrated in vitro using a cellular bioluminescence assay. LNP generation

[0118] The composition was prepared by generating LNPs containing FLuc mRNA, diluting with additional phosphate buffered saline (PBS), hydrating PLGA-PEG-PLGA polymer into the diluted LNP suspension, and confirming reversible thermogelation of the homogeneous mixture.

[0119] Briefly, mRNA-containing LNPs were generated by combining a 12.5 mM lipid stock solution comprising a 50:38.5:10:1.5 molar ratio of SM-102 (8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester):cholesterol:DPPC (l,2-dipalmitoyl-sn-glycero-3-phosphocholine):DMG-PEG2000 (l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000) with an aqueous 0.113 mg / mL solution of FLuc mRNA in 25 mM sodium acetate buffer, pH 4.5, at a 3:1 volumetric phase ratio, to produce “free LNP.” Additionally, two different polymer-mRNA containing LNPs were generated by combining (i) Polymer 1 (PLGA-PEG-PLGA with 1:1 L:G ratio) or (ii) Polymer 2 (PLGA-PEG-PLGA with 3:1 L:G ratio), with the above described Fluc-mRNA LNP suspensions to produce “Polymer 1” and “Polymer 2”.

[0120] In vitro mRNA analysis

[0121] Free LNP, Polymer 1, and Polymer 2, as described above, were evaluated in vitro for mRNA bioactivity. Briefly, mouse colon carcinoma cell line CT26 (ATCC CRL-2638 CT26.WT) was treated with either free LNP, Polymer 1, or Polymer 2, each with the same total mRNA content. After 24 h or 48 h incubation, luciferase bioluminescence was assessed.

[0122] Polymer 1 and Polymer 2 both demonstrated cell transfection activity in vitro. Signals were observed following both 24 h and 48 h incubation periods (FIG. 6). At 48h, the polymer-gel-delivered LNP provided an equal or greater signal compared to the non-polymer-delivered LNP, suggesting controlled release.

[0123] EXAMPLE 4

[0124] In this example, lipid nanoparticle-encapsulated mRNA delivery in vitro is described.

[0125] The formulation of mRNA encoding mCherry within an LNP and PLGA-PEG-PLGA composition is described, as is the subsequent expression of mCherry using an in vitro cellular assay.

[0126] LNP generation

[0127] The composition was prepared by first generating LNPs containing mCherry mRNA and subsequently dispersing the LNPs into an aqueous PLGA-PEG-PLGA solution.

[0128] Briefly, the lipid stock solution described in Example 3 was combined at a 3:1 volumetric phase ratio with 0.114 mg / mL mCherry-mRNA in 25 mM sodium acetate buffer, pH 4.5, to generate mRNA LNP solution. Polymer- mRNA containing LNPs were then generated. Specifically, a 10% w / w polymer solution with 49 pg mRNA / mL in 15 mM Tris and 0.9% saline, pH 7.4, was generated by combining hydrated PLGA-PEG-PLGA (1:1 L:G ratio), mRNA LNP solution, and saline.

[0129] In vitro mCherry analysis

[0130] Mouse colon carcinoma cell line CT26 was treated with either mCherry-LNPs, free mRNA, lipofectamine-mRNA, lipofectamine only, LNP only, or polymer only. After 48 h incubation, mCherry fluorescence was measured. The polymer-delivered mCherry-LNPs provided transfection, as measured by mCherry fluorescence, similar to LNP-only and to lipofectamine + mRNA treatments, whereas negative controls (lipofectamine only and polymer only) were absent any signal (FIG. 7).

[0131] EXAMPLE 5

[0132] In this example, the controlled release of siRNA via a thermogelling polymer is described.

[0133] The siRNA was formulated into a thermogelling polymer system, and the controlled release of the siRNA from the gel in vitro over at least a 3-day period wasdemonstrated. A FITC-tagged siRNA was first prepared into LNPs, then incorporated into a PLGA-PEG-PLGA polymer composition. The siRNA release from a gel incubated at 37 °C into PBS was measured spectrophotometrically.

[0134] LNP generation

[0135] The lipid stock solution described in Example 3 was combined at a 3:1 volumetric phase ratio with 0.160 mg / mL FITC-siRNA (Fluorescein-labeled Negative Control Duplex, Bioneer catalog number SN-1023) in 25 mM sodium acetate buffer, pH 4.5, to generate stock siRNA LNP solution. Polymer-siRNA containing LNPs were then generated. Specifically, hydrated PLGA-PEG-PLGA (3:1 L:G ratio), stock siRNA LNP solution, and PBS were combined a 42:10:23 volume ratio to create a 14% w / w polymer solution with 25 pg siRNA / mL in PBS.

[0136] In vitro release of FITC-tagged siRNA analysis

[0137] The in vitro release profile of polymer-siRNA-LNPs was determined.

[0138] Specifically, the polymer-siRNA-LNP were distributed in tube and incubated at 37 °C to form gels. PBS was added on top of the gels and the samples were tilted in a 37 °C incubator- shaker set to 100 rpm orbital shaking. Concentrations of FITC-tagged siRNA was determined in a sample of the supernatant taken at 1 h, 4 h, 24 h, 48 h, 71 h, and 143 h after the start of incubation.

[0139] The thermogelling composition consisting of FITC-tagged siRNA encapsulated in LNP and dispersed within a PLGA-PEG-PLGA medium yielded steady release of the siRNA in PBS at 37 °C, with limited release in the first hour and the majority of the release occurring steadily over three days (FIG. 8).

[0140] EXAMPLE 6

[0141] In this example, subcutaneous in vivo delivery of lipid nanoparticle encapsulated mRNA is demonstrated.

[0142] Polymer 2 composition of FLuc-mRNA LNPs was administered into subcutaneous tumors in mice. The bioluminescent signal from the tumors of treated mice confirms the in vivo delivery and transfection of the tumor, and demonstrates at least 48 h of sustained signal specifically at the tumor site.

[0143] LNP generationThe mRNA-containing LNPs (e.g., Polymer 2-formulated FLuc-mRNA LNP solution) were formulated as described in Example 3. A polymer-only control, lacking the mRNA-LNP, was also prepared in parallel.

[0144] In vivo mRNA-LNP activity and localization analysis

[0145] Colon tumor cell line CT26.WT was expanded from ATCC, passaged, and inoculated subcutaneously into the flanks of female Balb / c mice. Polymer 2-formulated FLuc-mRNA LNP solution, or polymer-only control of same polymer content, was injected into established tumors. Bioluminescence was assessed using an IVIS imaging system at 24 h, 48 h, and 120 h following administration of the polymeric compositions. In brief, luciferin reagent was administered intraperitoneally and the mice were promptly imaged. Bioluminescent flux at the tumor site, and also the whole body, was assessed. As shown in FIGS. 9A-9B, mice receiving the polymer-only control (no mRNA-LNP content) did not show bioluminescence (FIG. 9A, top row) at either 24 h or 48 h post administration of the polymer-only control. The Polymer 2 Fluc-mRNA LNP formulation, however, shows tumor-localized bioluminescence at both 24 h and 48 h (dashed circles, in bottom row). By five days the signal was no longer evident.

[0146] Bioluminescence over time for each mice is plotted in FIG. 9B.

[0147] EXAMPLE 7

[0148] In this example, intratumoral in vivo delivery of lipid nanoparticle encapsulated mRNA is demonstrated.

[0149] A FLuc-mRNA LNP with and without thermogelling polymer formulation was administered intratumorally in mice. The bioluminescent signal from the tumors of treated mice confirms the in vivo delivery and transfection of the tumor and demonstrates 24 h and 48 h of sustained activity that is comparable to the LNP-only formulation in bioluminescence.

[0150] LNP generation

[0151] mRNA containing LNPs were generated by combining a 12.5 mM lipid stock solution comprising a 50:38.5:10:1.5 molar ratio of SM-102:cholesterol:DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine):DMG-PEG2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000) at a 3:2:1 volumetric phase ratio with 0.104 mg / mL Fluc-mRNA (Capl ml'P Genscript catalog RP-A0002301) in 25 mM sodium acetatebuffer, pH 4.5, to generate stock mRNA LNP solution having mean LNP diameter of 92nm by DLS. Polymer-mRNA LNPs were prepared to yield a final composition that was 4% w / w polymer, 65 pg-mRNA / mL, 40.8 mg / mL iopamidol, lOmM Tris, 5% sucrose, and 0.9% saline pH 7.4.

[0152] In vivo bioactivity analysis

[0153] The CT26.WT tumor subcutaneous flank tumor model described in Example 6 was used to evaluate the activity, and localization of activity, of the polymer-mRNA LNPs in vivo. Formulations mRNA-LNP-only and the polymer-formulated mRNA-LNP were administered in equal dose and volume.

[0154] When performing the IVIS bioluminescence readings, both the whole-body signal and the tumor region of interest were quantified. The ratio of tumor- specific to whole body signal was calculated for each mouse. At both 24 h and 48 h post-intratumoral administration of the LNP formulations, bioluminescence was observed. The signal from the polymer-delivered LNP was focused at the location of the tumor at both time points (FIG. 10).

[0155] PROPHETIC EXAMPLE

[0156] In this example, intratumoral delivery of CPGel for the treatment of immunotherapy resistant metastatic cancer is described.

[0157] Intratumoral injection of CPGel in any anatomical compartment via a 18-22 gauge, 15-25 cm needle under endoscopic, MRI, CT, visual, optical, or ultrasound guidance into tumors that are immunotherapy resistant are possible.

[0158] Checkpoint inhibitors (PD-1, CTLA-4) are approved for the treatment of metastatic melanoma, but response rates to therapy are 30-50% depending on exact regimen and treatment line. An example patient has multiple metastases, including in the liver and lung. The patient is started on systemic therapy with PD-1 and / or CTLA-4 inhibitors. The liver metastasis is identified using CT, and image-guided injection through an 18 gauge needle into the tumor is performed to deliver the CpG-ODN hydrogel so as to release drug locally over a controlled period of time, thereby avoiding multiple injections.While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present invention is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and / or methods, if such features, systems, articles, materials, and / or methods are not mutually inconsistent, is included within the scope of the present invention.

[0159] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0160] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to “A and / or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0161] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0162] As used herein, “wt%” is an abbreviation of weight percentage.

[0163] Some embodiments may be embodied as a method, of which various examples have been described. The acts performed as part of the methods may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include different (e.g., moreor less) acts than those that are described, and / or that may involve performing some acts simultaneously, even though the acts are shown as being performed sequentially in the embodiments specifically described above.

[0164] Use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

[0165] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims

CLAIMSWhat is claimed is:

1. An injectable composition configured for intratumoral drug delivery, the injectable composition comprising:poly(lactide-co-glycolide-b-ethylene glycol-b-lactide-co-glycolide) (PLGA-PEG-PLGA) having a lactic acid (LA) to glycolic acid (GA) ratio of greater than or equal to 1:1, wherein a number average molecular weight of each PLGA is greater than or equal to 500 and less than or equal to 2500 and a number average molecular weight of each PEG is greater than or equal to 500 and less than or equal to 2500,an immunomodulatory oligonucleotide present within the injectable composition at a concentration of greater than or equal to 0.001 mg / mL; anda polycationic polymer and / or a lipid,wherein the injectable composition is a liquid at room temperature and is a gel at 37 °C, and the immunomodulatory oligonucleotide exhibits an extended-release profile from the injectable composition over greater than or equal to 24 hours as measured in phosphate-buffered saline at 37 °C.

2. An injectable composition as in claim 1, wherein the immunomodulatory oligonucleotide comprises an oligonucleotide selected from the group consisting of single- stranded DNA (ssDNA), double- stranded DNA (dsDNA), anti-sense oligonucleotides (ASO), single- stranded RNA (ssRNA), short-hairpin RNA (shRNA), short-interfering RNA (siRNA), non-coding RNA (ncRNA), and microRNA (miRNA).

3. An injectable composition configured for intratumoral drug delivery, the injectable composition comprising:poly(lactide-co-glycolide-b-ethylene glycol-b-lactide-co-glycolide) (PLGA-PEG-PLGA) having a lactic acid (LA) to glycolic acid (GA) ratio of greater than or equal to 1:1, wherein a number average molecular weight of each PLGA is greater than or equal to 500 and less than or equal to 2500 and a number average molecular weight of the PEG is greater than or equal to 500 and less than or equal to 2500; andan immunomodulatory oligonucleotide present within the injectable composition at a concentration of greater than or equal to 0.001 mg / mL;wherein the injectable composition is a liquid at room temperature and is a gel at 37 °C and the immunomodulatory oligonucleotide exhibits an extended-release profile from the injectable composition over greater than or equal to 24 hours as measured in phosphate-buffered saline at 37 °C.

4. An injectable composition configured for intratumoral drug delivery, the injectable composition comprising:poly(lactide-co-glycolide-b-ethylene glycol-b-lactide-co-glycolide) (PLGA-PEG-PLGA) having a lactic acid (LA) to glycolic acid (GA) ratio of greater than or equal to 1:1, wherein a number average molecular weight of each PLGA is greater than or equal to 500 and less than or equal to 2500 and a number average molecular weight of the PEG is greater than or equal to 500 and less than or equal to 2500;a CpG oligodeoxynucleotide present within the injectable composition at a concentration of greater than or equal to 0.001 mg / mL; andone or more of a polycationic polymer, a lipid, and a radio-opaque label wherein the injectable composition is a liquid at room temperature and is a gel at 37 °C, and the CpG oligodeoxynucleotide exhibits an extended-release profile from the injectable composition over greater than or equal to 24 hours as measured in phosphate-buffered saline at 37 °C.

5. An injectable composition configured for intratumoral delivery, the injectable composition comprising:poly(lactide-co-glycolide-b-ethylene glycol-b-lactide-co-glycolide) (PLGA-PEG-PLGA) having a lactic acid (LA) to glycolic acid (GA) ratio of greater than or equal to 1:1, wherein a number average molecular weight of each PLGA is greater than or equal to 500 and less than or equal to 2500 and a number average molecular weight of the PEG is greater than or equal to 500 and less than or equal to 2500;an immunomodulatory nucleic acid present within the injectable composition at a concentration of greater than or equal to 0.001 mg / mL; andone or more of a polycationic polymer, a lipid, and a radio-opaque label associated with the injectable composition,wherein the injectable composition is a liquid at room temperature and is a gel at 37 °C, and the immunomodulatory nucleic acid exhibits an extended-release profile fromthe injectable composition over greater than or equal to 24 hours as measured in phosphate-buffered saline at 37 °C.

6. An injectable composition as in claim 5, wherein the immunomodulatory nucleic acid is an oligonucleotide.

7. An injectable composition as in claim 5, wherein the immunomodulatory nucleic acid comprises natural and therapeutic forms of RNA, DNA, and / or hybrids thereof, the RNA, DNA, and / or hybrids thereof comprising one or more of double-stranded DNA (dsDNA), single- stranded DNA (ssDNA), anti-sense oligonucleotide (ASO), doublestranded RNA (dsRNA), single- stranded RNA (ssRNA), short-hairpin RNA (shRNA), short-interfering RNA (siRNA), non-coding RNA (ncRNA), long non-coding RNA (IncRNA), messenger RNA (mRNA), and microRNA (miRNA).

8. A method of treating a tumor, comprising:injecting a composition intratumorally, peritumorally, into tumor draining lymph nodes, and / or near tumor draining lymph nodes, wherein the composition is a liquid at room temperature and is a gel at 37 °C, wherein the composition comprises:poly(lactide-co-glycolide-b-ethylene glycol-b-lactide-co-glycolide) (PLGA-PEG-PLGA) having a lactic acid (LA) to glycolic acid (GA) ratio of greater than or equal to 1:1, wherein a number average molecular weight of each PLGA is greater than or equal to 500 and less than or equal to 2500 and a number average molecular weight of the PEG is greater than or equal to 500 and less than or equal to 2500,an immunomodulatory oligonucleotide present within the injectable composition at a concentration of greater than or equal to 0.001 mg / mL; and a polycationic polymer and / or a lipid; anddetermining the position of the composition during injection using one or more of ultrasound, CT, visual inspection, optical microscopy, and MRI.

9. The composition of any one of the preceding claims, wherein the immunomodulatory oligonucleotide exhibits an extended-release profile from the-M-injectable composition over less than or equal to 30 days as measured in phosphate-buffered saline at 37 °C.

10. The composition of any one of the preceding claims, wherein the immunomodulatory oligonucleotide is a CpG oligodeoxynucleotide (CpG ODN).

11. The composition of any one of the preceding claims, wherein the CpG ODN is Class A (type D) CpG ODN, Class B (type K) CpG ODN, Class C CpG ODN, or Class P CpG-ODN.

12. The composition of any one of the preceding claims, wherein the CpG ODN is selected from the group comprising ODN 1585, ODN 2216, ODN 2336, ODN 1018, ODN 1668, ODN 1826, ODN 2006, ODN 2007, ODN D-SL01, ODN 2395, ODN M362, ODN D-SL03, ODN 2243, ODN 2336, ODN 2395, ODN M362, ODN BW006 (ODN 684), ODN D-SL01, CMP-001, vidutolimod, Litenimod, and CpG 685 (GNKG-168).

13. The composition of any one of the preceding claims, wherein the PLGA-PEG-PLGA has a micellar structure.

14. The composition of any one of the preceding claims, wherein the immunomodulatory oligonucleotide is encapsulated by the PLGA-PEG-PLGA micellar structure.

15. The composition of any one of the preceding claims, wherein the molecular weight of each PLGA is greater than or equal to 900 and less than or equal to 2000.

16. The composition of any one of the preceding claims, wherein the molecular weight of each PLGA is greater than or equal to 1400 and less than or equal to 1600.

17. The composition of any one of the preceding claims, wherein the molecular weight of the PEG is greater than or equal to 1400 and less than or equal to 1600.

18. The composition of any one of the preceding claims, wherein poly(lactide-co-glycolide-b-ethylene glycol-b-lactide-co-glycolide) (PLGA-PEG-PLGA) has a lactic acid (LA) to glycolic acid (GA) ratio of less than or equal to 10:1.

19. The composition of any one of the preceding claims, further comprising a radioopaque label.

20. The composition of any one of the preceding claims, wherein the radio-opaque label comprises iopamidol, ioversol, iron, iron oxide, strontium, iopromide, lipiodol, iodixanol, calcium, calcium chloride, calcium sulfate, barium, barium sulfate, zinc, gold, titanocene, iohexol, diatrizoate meglumine, diatrizoate sodium, ethiodized oil, and / or gadolinium.

21. The composition of any one of the preceding claims, wherein the radio-opaque label present in the composition in an amount greater than or equal to 0 mg / mL and less than or equal to 100 mg / mL.

22. The composition of any one of the preceding claims, wherein the radio-opaque label present in the composition in an amount greater than or equal to 15 mg / mL and less than or equal to 50 mg / mL.

23. The composition of any one of the preceding claims, further comprising a polycationic polymer and / or a lipid.

24. The composition of any one of the preceding claims, further comprising a polycationic polymer.

25. The composition of any one of the preceding claims, wherein the polycationic polymer comprises a peptide (e.g., polylysine, poly ornithine), synthetic polymer (e.g., polyethyleneimine), polysaccharide (e.g., cyclodextrin, chitosan), natural polymer (e.g., histone, collagen), composite material (e.g., DEAE (diethylaminoethyl)-dextran), and / or thickening agent (e.g., HPMC (hydroxypropyl methylcellulose)).

26. The composition of any one of the preceding claims, further comprising a lipid.

27. The composition of any one of the preceding claims, wherein the lipid is an ionizable lipid, cationic lipid, and / or zwitterionic lipid.

28. The composition of any one of the preceding claims, wherein the lipid comprises CKK-E12, C12-200, 503013, DOTAP, DODAP, DOPE, DSPC, POPE, DMPC, SM102, MC3, ALC0315, and / or DOPS.

29. The composition of any one of the preceding claims, further comprising cholesterol and / or a PEG-lipid.

30. The composition of any one of the preceding claims, further comprising arachidonic acid, oleic acid, myristic acid, and / or sodium lauryl sulfate.

31. The composition of any one of the preceding claims, wherein the polycationic polymer and / or the lipid form nanoparticles encapsulating at least some of the immunomodulatory oligonucleotide of the composition.

32. The composition of any one of the preceding claims, wherein the injectable composition is capable of encapsulating the immunomodulatory oligonucleotide at a concentration of greater than or equal to 5 mg per 1 mL of injectable composition.

33. The composition of any one of the preceding claims, wherein the injectable composition is configured to be injected intratumorally in a subject to produce a local immuno stimulation with a systemic (abscopal) effect in the subject.

34. The method of any one of the preceding claims, further comprising performing a cryoablation, microwave ablation, radioablation, ethanol ablation, and / or irreversible electroporation of the tumor concurrently to the injecting.

35. The method of any one of the preceding claims, further comprising allowing the composition to gel at a location of the injecting.

36. The method of any one of the preceding claims, further comprising releasing the immunomodulatory oligonucleotide from the composition over at least 24 hours and no more than 30 days.

37. The method of any one of the preceding claims, wherein the injecting occurs at a location internal to a subject.

38. The method of any one of the preceding claims, further comprising retaining the composition at a location of the injecting for at least 24 hours.

39. The method of any one of the preceding claims, further comprising degrading the composition at a location internal to a subject.

40. The method of any one of the preceding claims, further comprising administering an immune checkpoint inhibitors (e.g., anti-PDl, anti-PDLl, anti-CTLA4) and / or other immune-active agents.

41. The method or composition of any one of the preceding claims, wherein the immunomodulatory nucleic acid comprises mRNA.

42. The method or composition of any one of the preceding claims, wherein the immunomodulatory nucleic acid comprises siRNA.

43. The method or composition of any one of the preceding claims, wherein the composition comprises a lipid, wherein the immunomodulatory nucleic acid is encapsulated within a lipid nanoparticle formed from the lipid.

44. The method or composition of any one of the preceding claims, wherein the composition produces functional bioactivity of the immunomodulatory nucleic acid over a period of at least 24 hours when administered to cells in vitro.

45. The method or composition of any one of the preceding claims, wherein the composition produces functional bioactivity of the immunomodulatory nucleic acid over a period of at least two days when administered to cells in vitro.

46. The method or composition of any one of the preceding claims, wherein administration of the composition results in localized activity of the nucleic acid in the vicinity of the administered dose, such that at least 50% of the whole-body primary functional activity of the nucleic acid occurs at the site of administration.

47. The method or composition of any one of the preceding claims, wherein administration of the composition results in localized activity of the nucleic acid in the vicinity of the administered dose, such that at least 75% of the whole-body primary functional activity of the nucleic acid occurs at the site of administration.

48. The method or composition of any one of the preceding claims, wherein administration of the composition results in localized activity of the nucleic acid in the vicinity of the administered dose, such that at least 90% of the whole-body primary functional activity of the nucleic acid occurs at the site of administration.

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