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Dimeric cpg oligonucleotides for use in modulating immune responses

A technology of dimeric oligonucleotides and oligonucleotides, which is applied in the field of dimeric CpG oligonucleotides for regulating immune responses, and can solve problems such as shrinking tumor volume

Pending Publication Date: 2022-02-18
MICROBIO (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] It has been reported that local injection of CpG oligonucleotides at or near the tumor site will stimulate a local antitumor immune response, leading to tumor shrinkage

Method used

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  • Dimeric cpg oligonucleotides for use in modulating immune responses
  • Dimeric cpg oligonucleotides for use in modulating immune responses
  • Dimeric cpg oligonucleotides for use in modulating immune responses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0118] Example 1 : Preparation and evaluation of CpG oligonucleotide MBS513

[0119] MBS513 is a single-stranded 25bpCpG oligodeoxynucleotide (CpG DNA) with the nucleotide sequence shown in SEQ ID NO: 1 and phosphorothioate internucleotide linkages. Dissolve CpG DNA powder in 15 mg / ml (1.87 mM) filtered distilled deionized water (ddH 2 O) MBS513 stock solutions were prepared. The stock solution of MBS513 was stored at -20°C for future use. To obtain the MBS513 working solution, the stock solution was dissolved in SELEX buffer (40mM HEPES, 5mM KCl, 111mM NaCl, 1mM CaCl 2 , 1mM MgCl 2 , pH 7.5) was further diluted to the desired concentration for subsequent experiments.

[0120] To compare the state of MBS513 in solution, MBS513 was dissolved in 2 μM ddH2O or SELEX buffer. DNA was visualized on an agarose gel to determine the amount of MBS513 in monomeric or dimer form in both solutions. Such as Figure 1A As shown, MBS513 dissolved in SELEX buffer runs on the gel at th...

Embodiment 2

[0123] Example 2 : MBS513 inhibits tumor growth in vivo

[0124] In order to evaluate the role of MBS513 in tumor growth in vivo, the following experiments were designed, as Figure 2A shown. 5×10 5 CT26 tumor cells were injected subcutaneously into the left and right sides of the abdomen of BALB / c mice. When the tumor size reaches 200cm 3 At 10, 12 and 14 days after tumor implantation, 12.5 nmol, 25 nmol or 50 nmol of MBS513 was injected into the tumor in a volume of 50 μl only on the right side of the animal. Measure the tumor volume on both sides of the abdomen every 2-3 days. According to the guidelines, when the tumor volume reaches 25000cm 3 mice were sacrificed. Such as Figure 2B and Figure 2C As shown, MBS513 not only inhibited the growth of local tumors (right) but also inhibited the growth of distant tumors on the other side of the abdomen (left). Higher doses of MBS513 (eg, 50 nmol) showed better effectiveness in inducing a systemic immune response lead...

Embodiment 3

[0131] Example 3 : Formation of dimeric CpG oligonucleotides is concentration dependent

[0132] MBS513 was dissolved in SELEX buffer, PBS buffer or saline at different concentrations (including 20 μM, 100 μM and 500 μM). The solutions thus formed were incubated at the various temperatures indicated ( image 3 ) and the content of dimer MBS513 and monomer MBS513 in the solution was analyzed by HPLC. The ratio (ds / ss) between dimer MBS513 and monomer MBS513 was calculated by dimer area (AU*min) / monomer area (AU*min) determined by HPLC.

[0133] Such as image 3 As shown, the formation of dimeric CpG complexes is concentration-dependent. CpG oligonucleotides in SELEX buffer and PBS buffer showed similar dd / ss ratios.

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Abstract

Pharmaceutical compositions comprising a CpG oligonucleotide, a buffer agent, and one or more salts having a total salt concentration of about 80-130 mM. A majority population of the CpG oligonucleotides in the composition is in dimeric form. Also provided herein are uses of the pharmaceutical compositions for modulating immune responses in subjects in need of the treatment, for example, cancer patient.

Description

Background technique [0001] CpG oligonucleotides are short synthetic nucleic acid molecules containing a "CpG" motif, where C and G represent cytidine and guanosine residues, respectively, and "p" represents a phosphodiester bond between C and G residues . Unmethylated CpG oligonucleotides were found to activate stimulatory immune receptors such as Toll-like receptors or TLRs on various immune cells such as T cells or B cells, thereby stimulating an innate immune response. [0002] It has been reported that local injection of CpG oligonucleotides at or near the tumor site will stimulate a local antitumor immune response, leading to a reduction in tumor volume. However, this antitumor activity is usually localized. See, eg, Rava et al., Science Translational Medicine, 10(426):eaan8723 (2018). Contents of the invention [0003] The present disclosure is based, at least in part, on the unexpected discovery that dimeric forms of CpG oligonucleotides successfully induce system...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/117A61K31/70A61K8/00A61P35/00
CPCC12N15/117A61P35/00C12N2310/17C12N2310/315C12N2310/51A61K31/7088A61P37/04A61K9/0019A61K47/02A61K9/08A61K31/7125
Inventor 张翼中叶士齐陈智庚江建豪彭竺英
Owner MICROBIO (SHANGHAI) CO LTD
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