CN114099535B - Toll-like receptor 9 (TLR 9) agonist hydrogel immunomodulatory compositions - Google Patents
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Abstract
The present invention relates to immunomodulatory polynucleotides and immunomodulatory compositions comprising a TLR9 agonist and a hydrogel. The invention also relates to administering the immunomodulatory compositions to a subject to modulate the subject's immune response and/or treat cancer.
Description
Technical Field
The present invention relates to immunomodulatory polynucleotides and immunomodulatory compositions comprising a TLR9 agonist and a hydrogel. The invention also relates to the administration of an immunomodulatory polynucleotide or an immunomodulatory composition comprising a TLR9 agonist and a hydrogel to modulate an immune response and/or treat cancer.
Background
Unmethylated CpG oligodeoxynucleotides (CpG ODN) are known ligands for the innate immune receptor Toll-like receptor 9 (TLR 9). DNA containing unmethylated CpG motifs activates TLR9 to generate potent Th 1-type innate and adaptive immune responses (Hemmi, et al. Nature,2000,408, 740-745). The TLR9 agonist can be applied to immunooncology, asthma allergy, infectious diseases and vaccine adjuvants. In recent years, immune checkpoint inhibitors (CPIs) have changed cancer treatment approaches. However, only a few (10-30%) patients benefit from CPI treatment, and treatment options for patients with CPI recurrence or refractory are limited. There is increasing evidence that the Tumor Microenvironment (TME) can be controlled by direct intratumoral injection of immunostimulants (e.g., TLR9 agonists) that increase T cell infiltration, resulting in the transition from "cold" tumors to "hot" tumors that respond to CPI treatment. Currently, several TLR9 agonists are used in combination with checkpoint inhibitors (CPI) in late clinical trials in cancer patients. In these experiments, TLR9 agonists are typically administered once a week for several weeks, suggesting that they may degrade in local tissues or dissipate rapidly from sites of action that require once a week administration. The present invention improves the persistence of TLR9 agonist delivery and activity by using hydrogel formulations composed of poloxamer block copolymers and other components.
Disclosure of Invention
The inventors have discovered immunomodulatory polynucleotides and methods of using these immunomodulatory polynucleotides to modulate an immune response in an individual, particularly a human. The compositions of the invention comprise immunomodulatory polynucleotides as further described herein.
Unmethylated CpG oligodeoxynucleotides (CpG ODN) are known ligands for the innate immune receptor TLR 9. The response of TLR9 to CpG ODN results in the stimulation of immune cascades, represented by the secretion of Th 1-type cytokines (including IFN- α) and chemokine environments and the activation of immune cell surface markers. Currently, several TLR9 agonists are used in combination with checkpoint inhibitors (CPI) in late clinical trials in cancer patients. In these experiments, TLR9 agonists are typically administered once a week for several weeks, suggesting that they may degrade in local tissues or dissipate rapidly from the site of action, and therefore need to be administered once a week. To improve the stability, delivery and persistence of TLR9 agonist activity with reduced dosing frequency, we encapsulated (encapsulated) the TLR9 agonist in an injectable hydrogel containing a poloxamer. The TLR9 agonist/hydrogel formulation can be used to slow the release of the TLR9 agonist following peritumoral/intratumoral injection or by other routes of administration. Intra-tumoral injections of TLR9 agonists are typically used weekly in clinical trials. Hydrogel formulations using TLR9 agonists can achieve lower dosing frequencies and ideally match the dosing frequency of checkpoint inhibitors. Hydrogel formulated TLR9 agonists may also significantly expand the patient population that can be treated with TLR9 agonists, including those tumor patients who would otherwise not be amenable to intratumoral injection. We developed TLR9 agonists, evaluated their immunostimulatory potential, and investigated their encapsulation and non-encapsulation in hydrogels to evaluate tumor growth inhibition and toxicity in mouse syngeneic tumor models.
The inventors designed a series of TLR9 agonists (see table 1) and hydrogel formulations (see table 3) in which the hydrogel formulation encapsulating the agonist referred to herein as "C-3" has shown higher cytokine induction and better anti-tumor activity, which was tested in a CT26 tumor model in mice. The TLR9 agonists of the invention showed non-inferior tumor growth inhibition compared to the clinical candidate SD-101 (see table 1).
In one aspect of the invention, the invention provides an immunomodulatory composition comprising:
(1) An immunomodulatory polynucleotide (preferably the immunomodulatory polynucleotide is a TLR9 agonist), and
(2) A hydrogel, the hydrogel comprising:
(a) One or more poloxamer block copolymers at a concentration of 18% -40% (w/w) in the hydrogel, and/or
(b) Hyaluronic acid with a molecular weight of 50-100kDa, in a concentration of 0-0.5% (w/w) in the hydrogel, and/or
(c) Cyclodextrin or a derivative thereof in a concentration of 0-16% (w/w) in the hydrogel, and/or
(d) Low viscosity methylcellulose in a concentration of 0-16% (w/w) in the hydrogel.
Wherein, preferably, the TLR9 agonist is a polynucleotide comprising a sequence shown in SEQ ID NO. 1-49.
Wherein, preferably, the poloxamer block copolymer is preferably selected from poloxamer 407 or poloxamer 188.
Among them, preferably, the cyclodextrin derivative is 2-hydroxypropyl- β -cyclodextrin.
Wherein preferably, the low viscosity methylcellulose has a viscosity of from 10 to 20mPa.s, preferably from 12 to 18mPa.s. The viscosity of the methylcellulose can be measured using methods conventional in the art, such as NF/EP/JP viscosity measurement.
More preferably, the low viscosity methylcellulose is Benecel TM A15LV PH PRM, seehttps://www.ashlandchina.com/index.phpa＝index&c＝article&id＝788The A15LV PH PRM has the nominal viscosity of 12-18mPa.s, and the viscosity is detected by adopting a NF/EP/JP viscosity detection method.
In one aspect of the invention, the invention provides an immunomodulatory composition comprising:
(1) An immunomodulatory polynucleotide (preferably the immunomodulatory polynucleotide is a TLR9 agonist), and
(2) A hydrogel, the hydrogel comprising:
poloxamer 407:20% (w/w),
hyaluronic acid with molecular weight of 50-100 kDa: 0.5% (w/w),
2-hydroxypropyl- β -cyclodextrin: 4% (w/w), and
Benecel TM A15LV PH PRM：0.5％(w/w)。
in another aspect, the TLR9 agonist of the invention is a polynucleotide comprising a sequence shown in SEQ ID No. 1-49.
In one aspect of the invention, the invention provides an immunomodulatory composition comprising
(1) An immunomodulatory polynucleotide (preferably the immunomodulatory polynucleotide is a TLR9 agonist), and
(2) A hydrogel, the hydrogel comprising:
poloxamer 407:22.9% (w/w), and
poloxamer 188:3.3% (w/w).
In another aspect, the TLR9 agonist of the invention is a polynucleotide comprising a sequence shown in SEQ ID No. 1-49.
In one aspect of the invention, the immunomodulatory composition of the invention may further comprise an effective amount of one or more immune checkpoint modulator, preferably said immune checkpoint modulator is an antagonistic antibody that binds an antigen selected from PD1, PDL1, CTLA4, KIR, LAG3, VISTA, TIM3, B7-H4 and BTLA, and/or preferably said immune checkpoint modulator is an agonist antibody that binds an antigen selected from 41BB (CD 137), OX40 (CD 134), CD28, ICOS (CD 278) and CD 40.
In one aspect of the invention, the immunomodulatory compositions of the invention may also be used for pharmaceutical use, in particular, the immunomodulatory compositions of the invention may be used for at least: (1) Use in the manufacture of a medicament for modulating an immune response in an individual; (2) Use in the manufacture of a medicament for increasing IFN- γ in a subject; (3) Use in the manufacture of a medicament for increasing IFN- α in a subject; (4) Use in the manufacture of a medicament for ameliorating a symptom of an IgE-related disorder in a subject; (5) Use in the manufacture of a medicament for ameliorating symptoms of an infectious disease in a subject, and (6) use in the manufacture of a medicament for treating cancer in a subject, wherein preferably the cancer is selected from the group consisting of: bladder cancer, urinary tract cancer, upper urinary tract cancer, kidney cancer, gastrointestinal cancer, breast cancer, pancreatic cancer, prostate cancer, colon cancer, rectal cancer, female reproductive system cancer, melanoma, head and neck squamous cell cancer, lung cancer, liver cancer, brain cancer, colon cancer, and rectal cancer.
In addition, in one aspect, the immunomodulatory compositions of the invention further comprise one or more pharmaceutically acceptable excipients.
Drawings
FIG. 1: TLR9 agonists were screened in human PBMCs at a concentration of 0.20 μ M. Where #16 is C-3.
FIG. 2 is a schematic diagram: TLR9 agonists were screened in human PBMCs at a concentration of 0.10 μ M. Where #16 is C-3.
FIG. 3: bell-shaped dose response curves of IFN- α induced in vitro by selected TLR9 agonists in human PBMCs. Where #16 is C-3.
FIG. 4: CT26 tumor growth inhibition in mice following intratumoral administration of C-1 and C-3.
FIG. 5: tumor growth curves of individual mice in the C-1 and C-3 treatment groups.
FIG. 6: in vitro release of the TLR9 agonist C-3 in a hydrogel.
FIG. 7: tumor growth inhibition curves in CT26 tumor-bearing mice treated with hydrogel-1 and hydrogel-2 encapsulating C-3.
FIG. 8: tumor growth curves of individual mice in the group treated with hydrogel-1 encapsulating C-3.
FIG. 9: study of body weight changes in mice treated with C-3 encapsulated hydrogel and C-3 without hydrogel encapsulation.
FIG. 10A: tumor growth inhibition curves.
FIG. 10B: study of body weight change in mice treated with C-1 encapsulated with hydrogel formulation of C-1 and anhydrous gel formulation.
Detailed Description
Example 1 human PBMC study of the immunostimulatory Activity of TLR9 agonists
To screen sequences in vitro and identify TLR9 agonists (at two agonist concentrations) that were able to induce human PBMC cells to produce higher IFN- α, we designed a series of TLR9 agonists (see table 1) and used SD-101 (SEQ ID No. 45) as a control, based on "C-1".
To investigate the immunostimulatory potential of TLR9 agonist oligonucleotides (table 1), oligonucleotides were synthesized and evaluated in human PBMCs for their ability to induce secretion of IFN- α by PBMCs. Freshly isolated human Peripheral Blood Mononuclear Cells (PBMCs) were purchased and used on the same day as received. PBMC were isolated, resuspended and plated at 1X 10 per well 6 Individual cells were cultured in 96-well U-shaped bottom plates. Cells were cultured in the presence of TLR9 agonist (0.1 μ M and 0.2 μ M dose in a final volume of 0.2 mL) for 48 hours. Supernatants were collected and IFN- α detected using HTRF kit (Cisbio, bedford, mass.). The results are as followsFIG. 1 is a schematic view of aAndFIG. 2As shown. The results show that most of the designed compounds showed similar levels of IFN- α induction as the standard compound SD-101, but 5 sequences (SEQ ID NO.16 (also referred to as C-3), SEQ ID NO.29, SEQ ID NO.30, SEQ ID NO.41 and SEQ ID NO. 42) were better able to induce IFN- α production by human PBMC at concentrations of 0.1 and 0.2. Mu.M, and we performed subsequent experiments using the above oligonucleotides.
TABLE 1 oligonucleotide sequences (5 'to 3') were designed and studied for their activity as TLR9 agonists
"-" indicates a Phosphorothioate (PS) bond; c-3 is 5'-TCGTTCGAACGTTCGAACGTTCGAACGAAT-3' are all PS modified (SEQ ID No.16 in Table 1) and the Trebler linker has the following chemical structure and connectivity, where R1 can be-H, or other oligonucleotide sequence. The structure of the Trebler linker is shown below.
Example 2 study of dose-dependent immunostimulatory Activity of selected TLR9 agonists on human PBMCs
To investigate the dose-dependent IFN- α induction of selected TLR9 agonists compared to SEQ ID No.45 (SD-101), we assessed them in human PBMCs for the induction of IFN- α secretion, as follows: freshly isolated human Peripheral Blood Mononuclear Cells (PBMCs) (Allcells, alameda, CA) were purchased and used on the day of receipt. Isolated PBMCs were washed twice with Phosphate Buffered Saline (PBS) containing 2% Fetal Bovine Serum (FBS) and 2mM ethylenediaminetetraacetic acid (EDTA). Cells were resuspended and cultured in 96-well U-shaped plates at 1X 10 per well 6 Cells, wherein the medium is RPMI 1640, containing 10% FBS, 2mM L-glutamine, 100U/mL penicillin and 100. Mu.g/mL streptomycin. 5% CO at 37 ℃ in the presence of a selected TLR9 agonist 2 Culturing the cells in a final volume of 0.2mL for 48 hours in a humidified incubator of (1), the TLR9The dose of agonist is from 0.025. Mu.M to 0.40. Mu.M. Supernatants were harvested and IFN- α detected using HTRF kit (Cisbio, bedford, mass.). The results are as followsFIG. 3As shown, all 5 sequences tested were shown to induce IFN-. Alpha.production at various concentrations (especially at 100 nM) higher than the reference sequence (SEQ ID NO. 45).
Example 3C-3 and C-1 Studies in CT26 tumor model
To compare the in vivo efficacy of C-1 (SD-101, SEQ ID NO. 45) and C-3 (SEQ ID NO. 16) at two doses in a symbiotic mouse model carried by CT26 tumor, CT26 tumor cells were cultured and seeded and the tumor model was studied.
Cell culture: mouse colon cancer cell lines containing CT26 tumor cells were cultured in vitro in monolayers in RPMI-1640 medium supplemented with 10% Fetal Bovine Serum (FBS) (in a humidified 5% carbon dioxide incubator at 37 ℃). Tumor cells were routinely sub-cultured twice weekly by trypsin-EDTA treatment. Cells in the exponential growth phase were harvested and counted for seeding.
Tumor inoculation and mouse grouping: cells in the exponential growth phase were harvested and seeded, centrifuged at 335x g in a refrigerated centrifuge, and the medium aspirated. The cell pellet was resuspended in 10 volumes of serum-free medium, filtered through a 70 μm nylon mesh cell filter and counted. The cell suspension was resuspended in serum-free medium to a final cell concentration for seeding by centrifugation again as described above. Each mouse was injected subcutaneously with 0.1 ml of CT 26-containing tumor cells (8X 10) 4 ) The serum-free culture medium is used for the development of tumors. Mice were lightly anesthetized during implantation. After injection any drops on the skin were carefully wiped off with a Kimwipe to reduce the chance of tumor ulceration. When the average size of the tumor reaches about 100mm 3 At the time, tumor-bearing animals were included in the study group. Each group consisted of 6 mice. The day of treatment initiation was scored as day 0, and animals of each group received Peritumoral Treatment (PT) or Intratumoral Treatment (IT), as described in table 2. When the tumor mass of the mice reaches 100mm on average 3 At size, treatment was by intratumoral injection.
As shown in fig. 1-3In the human PBMC assay, the TLR9 agonist SEQ ID NO.16 (referred to herein as C-3) showed higher levels of IFN- α induction compared to the standard compound SD-101 (SEQ ID NO. 45) (referred to herein as C-1). Based on these results, the anti-tumor activity of the TLR9 agonist C-3 compared to C-1 was further evaluated in the CT26 tumor model of BALB/C mice. Both compounds were studied at two dose levels of 30 and 60 micrograms/mouse after intratumoral administration. When the group mean tumor volume was about 100mm 3 Treatment is initiated. These compounds were administered on days 0, 3, 6, 9 and 12 and tumor growth was followed from day 0 to the study period. Both C-3 and C-1 showed dose-dependent tumor growth inhibition. In contrast to the in vitro immunostimulatory results, C-1 and C-3 showed similar levels of tumor growth inhibition at both dose levels studied (C-1 and C-3)FIG. 4)。
However, analysis of individual mouse data in each group showed that 3 of 6 were only complete responders (reponders) (exhibiting complete tumor growth inhibition) in the group of C-3-treated mice, while in the same condition, 1 of 6 mice in the group treated with C-1 was a complete responder: (FIG. 5)。These higher complete response rates indicate that C-3 cures more effectively than C-1 cures More tumor-bearing mice。
As the data show, the C-3 solution was similar in efficacy to the C-1 solution at both dose levels, and the proportion of the complete response induced at the high dose was 3 to 1, higher than the proportion of the complete response induced by C-1.
TABLE 2 treatment groups in tumor study
Example 4 preparation of hydrogels and hydrogel-encapsulated TLR9 agonists
Encouraging these results, to explore the feasibility of hydrogel encapsulation of TLR9 agonists for immunooncology applications, we decided to encapsulate TLR9 agonists in two different component hydrogels, hydrogel-1 and hydrogel-2 (table 3).
TABLE 3 hydrogel formulations
To measure the release rate of oligonucleotide C-3 in vitro, to evaluate the release rate and to determine the dose frequency in vivo, hydrogel encapsulated TLR9 agonists were prepared.
The hydrogel formulation and release test method was as follows:
a blank hydrogel was prepared by mixing the specified ingredients in PBS and the prepared hydrogel was stored at 4 ℃ until use.
120mg of C-3 was dissolved in 3ml of sterile PBS to prepare a stock solution of C-3 (40 mg/ml).
Transfer 900 μ Ι hydrogel (at 4 ℃) into a15 ml conical glass vial (n = 2) at 4 ℃.
The hydrogel-oligonucleotide solution was vortexed gently for a few seconds and then held at 4 ℃ for 15 minutes. This process was repeated twice to complete mixing.
The mixed hydrogel was taken out from the 4 ℃ environment, and then incubated at 37 ℃ for 1 hour to form a solid gel.
After 1 hour, 5ml PBS at 37 ℃ was transferred as release medium to the top of each hydrogel and the mixture was added
Incubate in shaker.
Periodically, 1.5 ml of release medium was removed for analysis and replaced with fresh 1.5 ml of PBS.
At
The release medium was measured at 260nm in a plate reader.
As a first step, we investigated hydrogels releasing TLR9 kinase in vitroThe properties of the agonists (oligonucleotides) were then evaluated in vivo in tumor models. The in vitro release results of hydrogel-1 (OH 4) and hydrogel-2 (OH-7) are shown inFIG. 6As shown. These results indicate that hydrogel-1 releases the active ingredient more rapidly than hydrogel-2. Hydrogel-2 releases its active ingredient much more slowly. Based on these results, we decided to administer TLR9 agonist encapsulated in hydrogel-1 twice and TLR9 agonist encapsulated in hydrogel-2 once in an in vivo tumor study.
These two hydrogels were selected for in vivo studies. Hydrogel-1 was selected for two Q7D doses and hydrogel-2 was selected for a single dose, depending on the release rate.
Example 5 study of C-3 and C-1 in CT26 tumor model
To evaluate hydrogel-encapsulated TLR9 agonists in CT26 tumor-bearing mice, in vivo studies of hydrogel-encapsulated C-3 were performed in the CT26 tumor model.
To compare hydrogel-1 and hydrogel-2 encapsulated C-3 with PBS formulated C-3, total injected C-3 between groups 1, 4, 6, 7 and 8 remained constant over 14 days, and tumor growth and body weight were monitored. When the tumor mass reaches an average of 100mm 3 In size, mice were treated by peri-or intratumoral injection. The antitumor activity of the two hydrogel-encapsulated TLR9 agonists C-3 (compared to C-3 without the hydrogel formulation) was then further evaluated in a CT26 tumor model in BALB/C mice. When the average tumor size of each group reached 100mm 3 The compound is administered. C-3 encapsulated in hydrogel-1 (formulation shown in Table 3) was administered peritumorally at 150 μ g/dose on days 0 and 7. C-3 encapsulated in hydrogel-2 (formulation shown in Table 3) was administered peritumorally at 300 μ g/dose only on day 0. These doses corresponded to 60 μ g per injection per week of tumor or intratumoral injection of 5 unformulated doses of C-3 on days 0, 3, 6, 9, and 12 (as shown in Table 4 below). For comparison, a group of mice injected intratumorally 5 times at 60 μ g/injection on days 0, 3, 6, 9, and 12 was also included.
Surprisingly, C-3 encapsulated hydrogel-1 was injected twice on days 0 and 7 or singly on day 0Secondary injection Hydrogel-2 encapsulating C3 caused tumor growth inhibition for up to 12 days (FIG. 7). In contrast, multiple doses (five doses every three days starting on day 0 to day 12) of the same TLR9 agonist (without hydrogel encapsulation) were required to produce similar levels of tumor growth inhibition (figure 7). We speculate that the longer duration of activity of TLR9 agonists formulated in hydrogels is due to the controlled release of TLR9 agonist from the hydrogel formulation over a longer period of time. In this study, C-3 (anhydrous gel encapsulation) administered Peritumorally (PT) and Intratumorally (IT) showed similar tumor growth with no statistically significant differences.
TABLE 4 treatment groups in tumor study
Analysis of individual mouse data for the hydrogel encapsulated C-3 treatment group showed a higher number of complete responders (complete inhibition of tumor growth)(FIG. 8). These higher tumor suppression response rates indicate that C-3 can cure more tumor-bearing mice regardless of hydrogel encapsulation. In addition, the hydrogel encapsulated TLR9 agonists showed similar little or no weight loss (indicating lower toxicity) as the control PBS vehicle compared to TLR9 agonists not injected hydrogel encapsulated, which decreased weight in the experimental course compared to control PBS-treated mice<10% (fig. 9).As shown by these results, hydrogel-encapsulated C-3 showed more than 10 days of durability and lower than the C-3 solution Toxicity of (2)。
Example 6 study of hydrogel-1 encapsulated C-1 in CT26 tumor model
To confirm that the observed improvement in the duration of hydrogel-encapsulated C-3 activity was not specific to oligonucleotide C-3In addition, and the hydrogel formulation can be applied to any TLR9 agonist, we performed the following tumor model studies using C-1, with and without encapsulation of C-1 in hydrogel-1 (the formulation is listed in Table 3). We also used CT26 tumor bearing mice. When the tumor mass reaches an average of 100mm 3 By size, mice in each group received peri-tumoral (PT) or Intratumoral (IT) treatment. Anhydrous gel encapsulated C-1 was administered peritumorally at 50 μ g/dose on days 0, 3, 6, 9 and 12. Hydrogel encapsulated C-1 was administered as a single 250 μ g dose around the tumor on day 0 (as described in Table 5). In this case, both the encapsulated hydrogel (single dose) and the hydrogel-free group (five doses) showed similar tumor growth inhibition and prolongation of tumor-bearing mouse survival time (fig. 10A). Both the C-1 solution and the hydrogel encapsulated C-1 showed similar weight loss (FIG. 10B), indicating that the toxicity of the hydrogel encapsulated C-1 was not higher than that of the unencapsulated C-1 even when administered at higher concentrations in a single administration. These results further demonstrate that the hydrogels of the present invention can be applied to any TLR9 agonist, which is typically applied to any oligonucleotide, and achieve sustained release, reduced dosing frequency, and long duration of activity without additional toxicity compared to non-hydrogel formulations (e.g., solutions). The results show that C-1 encapsulated in hydrogel-1 shows similar tumor growth inhibition and similar low toxicity to the C-1 solution.
TABLE 5 treatment of selected groups in tumor study
Taken together, these studies indicate that hydrogel-encapsulated TLR9 agonists have great therapeutic potential in immunooncology.
Sequence listing
<110> Nanjing Jimai Biotechnology Co., ltd
<120> Toll-like receptor 9 (TLR 9) agonist hydrogel immunomodulatory compositions
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<150> US 63/123956
<151> 2020-12-10
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<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(27)
<223> modification with phosphorothioate bond
<400> 3
tcgaacgttc gaacgttcga acgttcg 27
<210> 4
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(26)
<223> modification with phosphorothioate bond
<400> 4
tcgaacgttc gaacgttcga acgttc 26
<210> 5
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(25)
<223> modification with phosphorothioate bond
<400> 5
tcgaacgttc gaacgttcga acgtt 25
<210> 6
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(24)
<223> modification with phosphorothioate bond
<400> 6
tcgaacgttc gaacgttcga acgt 24
<210> 7
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(30)
<223> modification with phosphorothioate bond
<400> 7
tcgaacgttc gttcgaacga acgttcgaat 30
<210> 8
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(30)
<223> Modified with a phosphorothioate
<400> 8
tcgaacgttc gatcgatcga acgttcgaat 30
<210> 9
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(30)
<223> modification with phosphorothioate bond
<400> 9
tcgaacgttc gtacgtacga acgttcgaat 30
<210> 10
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(30)
<223> modification with phosphorothioate bond
<400> 10
tcgaacgaac gaacgttcgt tcgttcgaat 30
<210> 11
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(30)
<223> modification with phosphorothioate bond
<400> 11
tcgaacgatc gaacgttcga tcgttcgaat 30
<210> 12
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(30)
<223> modification with phosphorothioate bond
<400> 12
tcgaacgtac gaacgttcgt acgttcgaat 30
<210> 13
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(30)
<223> modification with phosphorothioate bond
<400> 13
tcgttcgttc gaacgttcga acgaacgaat 30
<210> 14
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(30)
<223> modification with phosphorothioate bond
<400> 14
tcgatcgttc gaacgttcga acgatcgaat 30
<210> 15
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(30)
<223> modification with phosphorothioate bond
<400> 15
tcgtacgttc gaacgttcga acgtacgaat 30
<210> 16
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(30)
<223> modification with phosphorothioate bond
<400> 16
tcgttcgaac gttcgaacgt tcgaacgaat 30
<210> 17
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(30)
<223> modification with phosphorothioate bond
<400> 17
tcgaacgttc gaccggtcga acgttcgaat 30
<210> 18
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(30)
<223> modification with phosphorothioate bond
<400> 18
tcgaacgttc gagcgctcga acgttcgaat 30
<210> 19
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(28)
<223> modification with phosphorothioate bond
<400> 19
tcgaacgttc gacgtcgaac gttcgaat 28
<210> 20
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(26)
<223> modification with phosphorothioate bond
<400> 20
tcgaacgttc gcgcgaacgt tcgaat 26
<210> 21
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(24)
<223> modification with phosphorothioate bond
<400> 21
tcgaacgttc cggaacgttc gaat 24
<210> 22
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(22)
<223> modification with phosphorothioate bond
<400> 22
tcgaacgttc gaacgttcga at 22
<210> 23
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(20)
<223> modification with phosphorothioate bond
<400> 23
<210> 24
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(18)
<223> modification with phosphorothioate bond
<400> 24
tcgaacgcgc gttcgaat 18
<210> 25
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(28)
<223> modification with phosphorothioate bond
<400> 25
tcgaacgttc gaattcgaac gttcgaat 28
<210> 26
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(30)
<223> modification with phosphorothioate bond
<400> 26
tcgaacgttc gaagcttcga acgttcgaat 30
<210> 27
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(26)
<223> modification with phosphorothioate bond
<400> 27
tcgaacgttc gatcgaacgt tcgaat 26
<210> 28
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(34)
<223> modification with phosphorothioate bond
<400> 28
tcgacgacgt tcgaacgttc gaacgtcgtc gaat 34
<210> 29
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(34)
<223> modification with phosphorothioate bond
<400> 29
tcgaacgtcg tcgaacgttc gacgacgttc gaat 34
<210> 30
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(34)
<223> modification with phosphorothioate bond
<400> 30
tcgaacgttc gacgacgtcg tcgaacgttc gaat 34
<210> 31
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(32)
<223> modification with phosphorothioate bond
<400> 31
tcgacgacgt tcgaacgttc gaacgttcga at 32
<210> 32
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(32)
<223> modification with phosphorothioate bond
<400> 32
tcgaacgtcg tcgaacgttc gaacgttcga at 32
<210> 33
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(32)
<223> modification with phosphorothioate bond
<400> 33
tcgaacgttc gacgacgttc gaacgttcga at 32
<210> 34
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(32)
<223> modification with phosphorothioate bond
<400> 34
tcgaacgttc gaacgtcgtc gaacgttcga at 32
<210> 35
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(32)
<223> modification with phosphorothioate bond
<400> 35
tcgaacgttc gaacgttcga cgacgttcga at 32
<210> 36
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(32)
<223> modification with phosphorothioate bond
<400> 36
tcgaacgttc gaacgttcga acgtcgtcga at 32
<210> 37
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(22)
<223> modification with phosphorothioate bond
<400> 37
acgttcgaac gttcgaacgt at 22
<210> 38
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(20)
<223> modification with phosphorothioate bond
<400> 38
<210> 39
<211> 14
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(14)
<223> modification with phosphorothioate bond
<400> 39
<210> 40
<211> 12
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(12)
<223> modification with phosphorothioate bond
<400> 40
<210> 41
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(38)
<223> modification with phosphorothioate bond
<400> 41
tcgaacgtcg tcgacgacgt cgtcgacgac gttcgaat 38
<210> 42
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(42)
<223> modification with phosphorothioate bond
<400> 42
tcgacgacgt cgtcgacgac gtcgtcgacg acgtcgtcga at 42
<210> 43
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(38)
<223> modification with phosphorothioate bond
<400> 43
tcgacgacgt cgtcgaacgt tcgacgacgt cgtcgaat 38
<210> 44
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(20)
<223> modification with phosphorothioate bond
<400> 44
<210> 45
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(30)
<223> modification with phosphorothioate bond
<400> 45
tcgaacgttc gaacgttcga acgttcgaat 30
<210> 46
<211> 14
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(14)
<223> modification with phosphorothioate bond
<400> 46
<210> 47
<211> 11
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(11)
<223> modification with phosphorothioate bond
<400> 47
tcgttcgaac g 11
<210> 48
<211> 14
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(5)
<223> modification with phosphorothioate bond
<400> 48
<210> 49
<211> 11
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Synthesis of polynucleotides
<220>
<221> misc_feature
<222> (1)..(5)
<223> modification with phosphorothioate bond
<400> 49
tcgttcgaac g 11
Claims (3)
1. An immunomodulatory composition, comprising:
(1) An immunomodulatory polynucleotide which is a TLR9 agonist, the sequence of which is shown in SEQ ID No.16, an
(2) The hydrogel is formed by the reaction of a hydrogel,
wherein the hydrogel is:
poloxamer 407:20% (w/w) of the total weight of the composition,
hyaluronic acid with molecular weight of 50-100 kDa: 0.5% (w/w),
2-hydroxypropyl- β -cyclodextrin: 4% (w/w), and
Benecel™ A15LV PH PRM：0.5% (w/w)；
alternatively, the hydrogel is:
poloxamer 407:22.9% (w/w), and poloxamer 188:3.3% (w/w).
2. Use of the immunomodulatory composition of claim 1 for the preparation of a medicament for treating cancer in a subject, wherein the cancer is colon cancer.
3. The immunomodulatory composition of claim 1 or the use of claim 2, wherein the immunomodulatory composition further comprises one or more pharmaceutically acceptable excipients.
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| US202063123956P | 2020-12-10 | 2020-12-10 | |
| US63/123,956 | 2020-12-10 |
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| CN114099535A CN114099535A (en) | 2022-03-01 |
| CN114099535B true CN114099535B (en) | 2023-01-03 |
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Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2982679A1 (en) * | 2005-10-12 | 2016-02-10 | Idera Pharmaceuticals, Inc. | Immune regulatory oligonucleotide (iro) compounds to modulate toll-like receptor based immune response |
| WO2014172532A2 (en) * | 2013-04-17 | 2014-10-23 | Lowsky Robert | Methods for cancer treatment |
| PL3240801T3 (en) * | 2014-12-31 | 2021-06-14 | Checkmate Pharmaceuticals, Inc. | Combination tumor immunotherapy |
| KR20180021700A (en) * | 2015-05-29 | 2018-03-05 | 다이나박스 테크놀로지 코퍼레이션 | Intrapulmonary administration of polynucleotide Toll-like receptor 9 agonists to treat lung cancer |
| WO2017181128A1 (en) * | 2016-04-15 | 2017-10-19 | Dynavax Technologies Corporation | Intratumoral administration of particles containing a toll-like receptor 9 agonist and a tumor antigen for treating cancer |
| WO2020081398A1 (en) * | 2018-10-14 | 2020-04-23 | Dynavax Technologies Corporation | Combination including a cpg-c type oligonucleotide and a pd-1 antagonist for treating breast cancer |
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