CN111321159B

CN111321159B - Chimeric nucleic acid molecules for immunomodulation and uses thereof

Info

Publication number: CN111321159B
Application number: CN201811532707.1A
Authority: CN
Inventors: 张翼中; 江建豪; 高翊玮; 王怡婷
Original assignee: Zhongtian Shanghai Biotechnology Co ltd
Current assignee: Zhongtian Shanghai Biotechnology Co ltd
Priority date: 2018-12-14
Filing date: 2018-12-14
Publication date: 2023-12-12
Anticipated expiration: 2038-12-14
Also published as: CN111321159A

Abstract

The present application relates to chimeric nucleic acid molecules for immunomodulation and uses thereof. Specifically, the chimeric nucleic acid molecule of the application comprises an oligonucleotide with a CpG motif and an aptamer of an anti-lymphocyte activation gene 3 (LAG-3), can generate a synergistic effect of activating a toll-like receptor type nine (TLR 9), can be used for starting a systemic immune response and can improve the treatment effect of diseases.

Description

Chimeric nucleic acid molecules for immunomodulation and uses thereof

Technical Field

The present application relates to chimeric nucleic acid molecules for immunomodulation and uses thereof.

Background

T cells express inhibitory receptors (called immune checkpoint molecules) that inhibit T cell responses. Examples of immune checkpoint molecules include programmed cell death protein 1 (PD-1), lymphocyte activation gene 3 (LAG-3) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4). These immune checkpoint molecules are typically activated in cancer, resulting in inhibition of the anti-tumor immune response.

T cells also express stimulatory receptors that activate the innate immune response. Examples of stimulatory receptors include Toll-like receptors (TLRs) that are activated by unmethylated cytosine-guanosine dinucleotide (CpG) motifs embedded in certain flanking sequences.

In cancer treatment, many patients still have poor response to existing drugs, and one of the possible reasons is that the immune response of these patients is suppressed, so that effective treatment cannot be achieved. Therefore, there is still a need to provide new therapeutic strategies that can more effectively inhibit tumor growth, increase response rates, and in particular achieve systemic immune protection and therapeutic effects.

Disclosure of Invention

The present application unexpectedly found that chimeric nucleic acid molecules formed by combining an oligonucleotide having a CpG motif and an anti-LAG-3 aptamer can produce a synergistic effect in activating TLR 9. The chimeric nucleic acid molecule of the application can initiate systemic immune response to achieve better disease treatment effect.

Accordingly, in one aspect, the application provides a chimeric nucleic acid molecule comprising a backbone portion and an aptamer portion, wherein the backbone portion comprises a CpG sequence, the aptamer portion comprises an aptamer that binds to lymphocyte activation gene 3 (LAG-3), and the backbone portion is linked to the aptamer portion.

In some embodiments, the backbone portion comprises the CpG sequence in a middle portion thereof, and flanking nucleotide sequences flanking the CpG sequence, said flanking nucleotide sequences comprising a docking sequence for ligating the backbone portion to the aptamer portion.

In some embodiments, the CpG sequence comprises a palindromic sequence.

In some embodiments, the scaffold moiety comprises two nucleic acid molecules comprising complementary sequences and forming a duplex region, and flanking nucleotide sequences flanking the complementary sequences, each flanking sequence comprising a docking sequence for ligating the scaffold moiety to the aptamer moiety.

In some embodiments, the aptamer portion comprises a first anti-LAG-3 aptamer and a second anti-LAG-3 aptamer, the first anti-LAG-3 aptamer comprising a first anchoring sequence and the second anti-LAG-3 aptamer comprising a second anchoring sequence; and the backbone moiety comprises a first nucleic acid molecule comprising a first nucleotide sequence of the formula 5'-X-L1-Y-L2-Z-3', wherein Y is a first CpG sequence, L1 and L2 are each a linker, X is a nucleotide fragment comprising a first docking sequence, Z is a nucleotide fragment comprising a second docking sequence, the first docking sequence being complementary to the first anchoring sequence, and the second docking sequence being complementary to the second anchoring sequence, such that the first nucleic acid molecule of the backbone moiety is linked to the first anti-LAG-3 aptamer and the second anti-LAG-3 aptamer of the aptamer moiety.

In some embodiments, the aptamer portion further comprises a third anti-LAG-3 aptamer and a fourth anti-LAG-3 aptamer, the third anti-LAG-3 aptamer comprising a third anchor sequence and the fourth anti-LAG-3 aptamer comprising a fourth anchor sequence; the backbone moiety further comprises a second nucleic acid molecule comprising a second nucleotide sequence of the formula 5'-X' -L1'-Y' -L2'-Z' -3', wherein Y' is a second CpG sequence, L1 'and L2' are each a linker, X 'is a nucleotide fragment comprising a third docking sequence, Z' is a nucleotide fragment comprising a fourth docking sequence, the third docking sequence being complementary to the third anchoring sequence, and the fourth docking sequence being complementary to the fourth anchoring sequence, such that the second nucleic acid molecule of the backbone moiety is linked to the third anti-LAG-3 aptamer and the fourth anti-LAG-3 aptamer of the aptamer moiety; and in the backbone portion, a first CpG sequence of the first nucleic acid molecule is complementary to a second CpG sequence of the second nucleic acid molecule.

In another aspect, the application provides a pharmaceutical composition comprising a chimeric nucleic acid molecule described herein, and a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutical composition of the application further comprises an immunodetection point binding agent.

In another aspect, the application provides a method of modulating an immune response, wherein the method comprises administering to a subject in need thereof a chimeric nucleic acid molecule or a pharmaceutical composition as described herein. The application also provides the use of a chimeric nucleic acid molecule or a pharmaceutical composition as described herein for the preparation of a medicament for modulating an immune response.

In some embodiments, the individual is a human patient suffering from, suspected of suffering from, or at risk of suffering from cancer.

In some embodiments, the cancer is selected from the group consisting of lung cancer, melanoma, colorectal cancer, renal cell carcinoma, urothelial cancer, and hodgkin's lymphoma.

In some embodiments, the medicament is formulated for intramuscular or enteral administration.

The details of one or more embodiments of the application are set forth in the description below. Other features or advantages of the present application will become apparent from the following detailed description of the drawings and several specific embodiments, and from the appended claims.

Drawings

The foregoing summary, as well as the following detailed description of the application, is better understood when read in conjunction with the appended drawings. The preferred embodiments of the present application presented in the figures are for illustrative purposes only. It should be understood that the application is not limited to the precise arrangements and instrumentalities shown.

In the drawings:

FIG. 1 shows a schematic design of a chimeric nucleic acid molecule of the application.

FIG. 2 shows the sequence structure of the chimeric nucleic acid molecules of the application (C695FL_B+B4_Sr3_P16, C695d8_B+B4_Sr3_P16, C695d8' _B+B4_Sr3_P16).

FIG. 3 shows an electrophoretic analysis of chimeric nucleic acid molecules of the application. Lanes 1, 2, and 3 samples were backbone moieties (C695FL_ B, C695d8_ B, C695d8 '_B), and lanes 4, 5, and 6 were chimeric nucleic acid molecules comprising backbone moieties (C695FL_B+B4_SL3_P16, C695d8_B+B4_SL3_P16, C695d8' _B+B4_SL3_P16) comprising aptamer moieties. The results show that all three backbones can stably carry out intermolecular combination into a binary body (all are close to 100 bp), and the binary body close to 250bp is presented after the aptamer of B4_Sl3_P16 is added.

FIGS. 4A to 4C show high performance liquid chromatography assays of chimeric nucleic acid molecules of the application comprising: FIG. 4A is a combination of the C695 sequence alone (C695 FL_B) and the anti-LAG-3 aptamer (C695 FL_B+B4_SL3_P16); FIG. 4B is a combination of the C695 sequence alone (C695d8_B) and the anti-LAG-3 aptamer (C695d8_B+B4_SL3_P16); and FIG. 4C shows the C695 sequence alone (C695 d '_B) and the combination of the C695 sequence with an anti-LAG-3 aptamer (C695 d8' _B+B4_Sr3_P16). The results show that the chimeric nucleic acid molecules of the application have good binding efficiency and stability.

Figure 5 shows TLR9 activation ability assays of chimeric nucleic acid molecules of the application. The results show that the chimeric nucleic acid molecules of the application produce unexpected synergistic effects of activating TLR9, superior to the C695 sequence of the backbone moiety itself or other C695 sequences.

FIG. 6 shows LAG-3 impedance analysis of chimeric nucleic acid molecules of the application. The results showed that when the backbone portions were alone, the luminescence values were all close to the background, indicating no LAG-3 impedance effect was produced; after addition of the backbone moiety to the B4_Sl3_P16 aptamer to form the chimeric nucleic acid molecule, the luminescence increases with increasing concentration (especially from 80 nM), indicating that the chimeric nucleic acid molecule can produce LAG-3 impedance. The positive control group was LAG-3 antibody (200 nM). IC50 analysis showed that the IC50 of C695d8_B+B4_SL3_P16 was optimal, reaching 102nM.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

As described herein, the definite articles "a" and "an" refer to one or more than one (i.e., at least one) of the grammatical object of an article. By way of example, "an element" refers to one element or more than one element.

As described herein, the terms "comprises" or "comprising" are used generically to mean including, which means allowing one or more features, ingredients, or components to be present. The words "comprise" or "comprise" include "or" consist of.. "

As described herein, the terms "polynucleotide" or "nucleic acid" and the like can refer to polymers composed of nucleotide units, including naturally occurring nucleic acids, such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA"), as well as nucleic acid analogs, including those having non-naturally occurring nucleotides. The synthesis of polynucleotides may be carried out using, for example, an automated DNA synthesizer. It is understood that when the nucleotide sequence is represented as a DNA sequence (i.e., A, T, G, C), it also includes the corresponding RNA sequence (i.e., A, U, G, C), where "U" replaces "T". As used herein, "polynucleotide" or "nucleic acid" includes single-stranded or double-stranded forms.

As described herein, the term "complementary" means that the topological compatibility or active surface of the two polynucleotides match. Thus, two molecules can be described as complementary, and furthermore, the interface features are complementary to each other. If the nucleotide sequence of the first polynucleotide is identical to the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide, then the first polynucleotide is complementary to the second polynucleotide. Thus, the polynucleotide having the sequence 5'-TATAC-3' is complementary to the polynucleotide having the sequence 5 '-GTATA-3'.

As described herein, the term "substantially identical" means that the two sequences have more than 85%, preferably 85%, more preferably 90%, and most preferably 95% or 100% identity. The percentage of identity or similarity between two sequences can be determined using mathematical algorithms known in the art (e.g., BLAST and Gapped BLAST programs, NBLAST and XBLAST programs, or ALIGN programs).

As described herein, the term "chimeric nucleic acid molecule" includes polynucleotides or nucleic acids having sequences that are not naturally linked together. The means of ligation may be by combining different nucleic acid fragments together via covalent bonds (e.g., phosphodiester linkages) or base pairing. The term "chimeric nucleic acid molecule" includes a monomer (monomer, composed of a single polynucleotide) or a multimer (multiplex, composed of multiple segments of polynucleotides, e.g., a binary, ternary, or quaternary entity composed of two segments of polynucleotides, and so forth).

As described herein, "palindromic sequence", also referred to as an inverted reverse sequence, refers to a sequence of nucleotides (in the 5 'to 3' forward direction) that is identical to the sequence of its complement that reads from 5 'to 3'. Palindromic sequences tend to self-assemble to form stem-loop (hairpin) structures.

As described herein, an "immune checkpoint molecule" refers to a molecule that is expressed on immune cells and can play a role in down-regulating immune responses. Examples of immune checkpoint molecules include, but are not limited to, programmed cell death protein-1 (PD-1), programmed death ligand 1 (PD-L1), lymphocyte activation gene-3 (LAG-3), T-cell immunoglobulins and mucin domain-containing-3 (TIM-3) and B and T Lymphocyte Attenuators (BTLA).

The application provides a combined use of an oligonucleotide with a CpG motif and an aptamer resisting LAG-3, in particular to a chimeric nucleic acid molecule formed by combining the oligonucleotide with the CpG motif and the aptamer resisting LAG-3, which is prepared into a nucleic acid medicament with double effects, especially achieves the synergistic effect of activating TLR9, is beneficial to activating systemic immune response and improves the disease treatment effect.

As described herein, a "nucleic acid aptamer" refers to a nucleic acid molecule (DNA or RNA) that has binding activity to a particular target molecule (e.g., LAG-3). An aptamer may bind to a particular target molecule, thereby inhibiting the activity of the target molecule, e.g., by blocking binding of the target molecule to its natural ligand, causing a conformational change in the target molecule, and/or blocking the active center of the target molecule.

In some embodiments, the aptamer portion of the chimeric nucleic acid molecule of the application comprises an anti-LAG-3 aptamer comprising a GX1GGGX2GGTX3A (SEQ ID NO: 11) nucleotide motif, wherein X1 and X2 are each independently G, C or absent, and X3 is T or C.

In some embodiments, an anti-LAG-3 aptamer described herein may include the following nucleotide sequences, or substantially the same nucleotide sequences:

(i)5'-TGGGGGGGGTTAGTTCAATACATGCGGGCG-3'(SEQ ID NO:12)；

(ii)5'-TGGGGGGGGGTTAGACTTACACTCTTATTCG-3'(SEQ ID NO:13)；

(iii) 5'-AGAGGGGGGGGTTAGCTGCTTTAACTCATG-3' (SEQ ID NO: 14); a kind of electronic device with high-pressure air-conditioning system

(iv)5'-AGGGGGGGGGTTACTGCGCATGTATCTCAG-3'(SEQ ID NO:15)。

(i)

5'-TCCCTACGGCGCTAACTGGGGGGGGTTAGTTCAATACATGCGG GCGGCCACCGTGCTACAAC-3'(SEQ ID NO:16)；

(ii)5'-ACGGCGCTAACTGGGGGGGGTTAGTTCAATACATG-3'(SEQ ID NO:17)；

(iii) 5'-GCTAACTGGGGGGGGTTAGTTCAATACATGCGGGC-3' (SEQ ID NO: 18); a kind of electronic device with high-pressure air-conditioning system

(iv)5'-CTGGGGGGGGTTAGTTCAATACATGCGGGCGGCCA-3'(SEQ ID NO:19)。

As described herein, "CpG" refers to 5 'cytosine ("C") and 3' guanine ("G") linked by a phosphate bond ("p"). As described herein, a "CpG sequence" refers to any CpG-containing oligonucleotide capable of activating immune cells (immunostimulants). At least the C at the 5'CpG 3' must be unmethylated. Nucleic acids having CpG sequences may be prepared by chemical synthesis by conventional techniques or provided by commercial suppliers.

In some embodiments, the CpG sequences described herein comprise palindromic sequences.

In some embodiments, a CpG sequence described herein may include the following nucleotide sequences, or substantially identical nucleotide sequences:

(i) 5'-AAC GTT CGA ACG TTC GAA CGT T-3' (C695 FL core part, palindromic sequence part) (SEQ ID NO: 1);

(ii) 5'-AAC GTT CGA ACG TT-3' (C695 d8 core part, palindromic sequence part) (SEQ ID NO: 2); and

(iii) 5'-TTC GAA CGT TCG AA-3' (C695 d8' core part, palindromic sequence part) (SEQ ID NO: 3).

(i)5'-TCG AAC GTT CGA ACG TTC GAA CGT T TTT-3'(C695FL)(SEQ ID NO:4)；

(ii) 5'-TCG AAC GTT CGA ACG TT TTT-3' (C695 d 8) (SEQ ID NO: 5); and

(iii)5'-TCG TTC GAA CGT TCG AA TTT-3'(C695d8')(SEQ ID NO:6)。

according to the application, the chimeric nucleic acid molecule is formed by ligating together a backbone portion comprising a CpG sequence with an aptamer portion comprising an anti-LAG-3 aptamer. The means of attachment may be by combining the backbone moiety with the aptamer moiety via a covalent bond (e.g., phosphodiester linkage) or base pairing.

In some embodiments, the aptamer of the aptamer portion comprises an anchoring sequence for linking the aptamer to the scaffold portion. For example, the anchor sequence comprises 5'-GCC ACC GTG CTA CAA C-3' (SEQ ID NO: 21).

In some embodiments, the backbone portion comprises the CpG sequence in a middle portion thereof, and flanking nucleotide sequences flanking the CpG sequence, said flanking nucleotide sequences comprising a docking sequence for ligating the backbone portion to the aptamer portion. For example, the docking sequence comprises 5'-GTT GTA GCA CGG TGG C-3' (SEQ ID NO: 20).

In some embodiments, the backbone portion comprises two nucleic acid molecules comprising complementary sequences and forming a duplex region (CpG sequence), and flanking nucleotide sequences flanking the complementary sequences, each flanking sequence comprising a docking sequence for ligating the backbone portion to the aptamer portion.

In some embodiments, the aptamer portion comprises (SEQ ID NO:22, bold portion is the anchor sequence).

In some embodiments, the backbone portion comprises a first nucleic acid molecule comprising a nucleotide sequence of (5 '-X-L1-C695 FL-L2-Z-3') (SEQ ID NO:7, bold part is the docking sequence), and/or the backbone part comprises a second nucleic acid molecule comprising a nucleotide sequence +.> (5 '-X-L1-C695 FL-L2-Z-3') (SEQ ID NO:7, bold is the docking sequence); or (b)

The backbone portion comprises a first nucleic acid molecule comprising a nucleotide sequence of (5 '-X-L1-C695d8-L2-Z-3', bold is the docking sequence) (SEQ ID NO: 8), and/or the backbone portion comprises a second nucleic acid molecule comprising a nucleotide sequence of (5 '-X-L1-C695d 8-L2-Z-3') (SEQ ID NO:8, bold is the docking sequence); or (b)

The backbone portion comprises a first nucleic acid molecule comprising a nucleotide sequence of (5 ' -X-L1-C695d8' -L2-Z-3 ') (SEQ ID NO:9, bold is the docking sequence), and/or the backbone portion comprises a second nucleic acid molecule comprising a nucleotide sequence of (5 ' -X-L1-C695d8' -L2-Z-3 ') (SEQ ID NO:9, bold is the docking sequence).

In some embodiments, the aptamer portion comprises an additional aptamer that binds to an additional immunodetection point molecule other than LAG-3. Other examples of immune checkpoint molecules include, but are not limited to, programmed cell death protein-1 (PD-1), programmed death ligand 1 (PD-L1), T-cell immunoglobulins and mucin domain-containing-3 (TIM-3) and B and T Lymphocyte Attenuators (BTLA).

In accordance with the present application, an effective amount of the active ingredient (chimeric nucleic acid molecules of the present application) may be formulated with a pharmaceutically acceptable carrier (carrier) into a suitable form of composition for delivery and absorption. The compositions of the present application specifically comprise from about 0.1% to about 100% by weight of the active ingredient, wherein the weight percentages are calculated based on the weight of the entire composition. In some embodiments, the compositions of the present application may be pharmaceutical compositions or agents for use in therapy.

As used herein, "pharmaceutically acceptable" means that the carrier is compatible with the active ingredient of the composition, and preferably stabilizes the active ingredient, and is safe for the receiving individual. The carrier may be a diluent, carrier, excipient or matrix for the active ingredient. Some examples of suitable excipients include lactose, dextrose, sucrose, sorbose, mannose, starch, acacia, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methylcellulose. The composition may also contain lubricants such as talc, magnesium stearate and mineral oil; a wetting agent; emulsifying and suspending agents; preservatives, such as methyl and propyl hydroxybenzoates; sweetener and flavoring agents. The compositions of the present application may provide rapid, sustained or delayed release of the active ingredient upon administration to a patient.

In some embodiments, the compositions of the application comprise other active ingredients, e.g., further comprising an immunodetection point binding agent.

In another aspect, the application provides a method of modulating an immune response comprising administering to a subject in need thereof a chimeric nucleic acid molecule or composition described herein. The application also provides the use of a chimeric nucleic acid molecule or composition described herein for the preparation of a medicament for modulating an immune response. The methods of the application are useful for treating cancer.

As described herein, the terms "individual," "individual," and "patient" are used interchangeably herein and refer to a mammal being evaluated for treatment and/or being treated. The individual may be a human, but also includes other mammals, particularly those that may be used as laboratory models of human disease, e.g., mice, rats, rabbits, dogs, etc.

As described herein, the subject to be treated using the methods described herein may be a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice, and rats.

In some embodiments, the individual in need of treatment may be a human patient at risk or suspected of having a disease/disorder of interest (e.g., cancer). An individual suspected of having any of the disease/disorder of interest may exhibit one or more symptoms of the disease/disorder. An individual at risk for a disease/disorder may be an individual having one or more risk factors for the disease/disorder.

As used herein, the term "treating" refers to the application or administration of a composition comprising one or more active agents to a subject suffering from, or predisposed to, a disease or disorder of interest, a symptom of a disease/disorder, and whose purpose is to cure, treat, alleviate, alter, remedy, ameliorate, augment, or affect the disease, symptom of a disease, or predisposition to a disease or disorder.

In some embodiments, the individual in need of treatment is a human patient suffering from, suspected of suffering from, or at risk of suffering from cancer.

In some embodiments, the cancer is lung cancer, melanoma, colorectal cancer, renal cell carcinoma, urothelial cancer, or hodgkin's lymphoma.

The compositions of the application may be delivered via a physiologically acceptable route. Such as oral, parenteral (e.g., muscle, vein, subcutaneous, and peritoneal). For parenteral administration, it is preferably used in the form of a sterile aqueous solution which may contain other substances, such as salts or glucose, sufficient to render the solution isotonic with blood. The aqueous solution may be suitably buffered (e.g., at a pH of 3 to 9) as desired. Preparation of suitable non-oral compositions under sterile conditions can be accomplished by standard pharmacological techniques known to those skilled in the art.

In some embodiments, the compositions of the present application are administered via intratumoral injection (intratumoral injection). The dosage is determined according to the tumor volume, for example, 50mm ³ To a tumor of 50 μg, 100 μg or 200 μg total of active ingredient. For example, the strokes may be applied once a day for a relatively short period of time (e.g., within a week), three or more times in total.

The application is further illustrated by the following examples, which are provided for purposes of illustration and not limitation. In view of the present disclosure, those of skill in the art will understand that many variations may be made in the specific embodiments disclosed and still obtain a like or similar result without departing from the spirit and scope of the application.

Examples

In the present application, we devised a chimeric nucleic acid molecule combining an oligonucleotide having a CpG motif and an anti-LAG-3 aptamer to form a multiplex double effect nucleic acid drug. The chimeric nucleic acid molecule of the application can simultaneously activate TLR9 and inhibit LAG-3, and especially has a synergistic effect of activating TLR9, which is superior to the effect of singly using oligonucleotide with CpG motif. The chimeric nucleic acid molecules of the application can be used to initiate a systemic immune response, which is beneficial for disease treatment.

1. Materials and methods

1.1. Nucleic acid

Nucleic acids were purchased from Baili Biotech Co.Ltd or Integrated DNA Technologies. TLR9 Activity kit was purchased from InvivoGen (HEK-Blue ^TM hTLR9,Catalog:hkb-htlr9.HEK-Blue ^TM Detection, catalysis: hb-det 3). LAG-3 blockade kit was purchased from Promega (LAG-3 Blockage Bioassay,Catalog #: CS 194823).

Table 1: sequence content of nucleic acids

The bold sequence is the anchor sequence or the docking sequence.

1.2 design and Synthesis of chimeric nucleic acid molecules

The test uses three C695 nucleic acid molecules (C695FL_ B, C695d8_ B, C695d8' _B), each combined with an anti-LAG-3 aptamer, to form a chimeric nucleic acid molecule. The aptamer comprises an anchor sequence at one end such that the aptamer can be linked to the C695 nucleic acid molecule. The C695 nucleic acid molecules each have a plurality of pairs of complementary palindromic sequences (C695 sequences) and flanking sequences at the middle position that can be base-pairing with another identical C695 nucleic acid molecule to form an intermolecular (inter-molecular) linkage at the corresponding middle position to create a duplex structure (bridge), and the flanking sequences comprise docking sequences that are complementary to the anchoring sequences of the aptamer such that the C695 nucleic acid molecule is linked to the aptamer. One part of double-stranded C695 nucleic acid molecule can be finally connected with 4 aptamers to form the multi-element double-effect nucleic acid medicament. FIG. 1 shows the design of a chimeric nucleic acid molecule of the application. FIG. 1 shows the sequence structure of a chimeric nucleic acid molecule of the application.

Each DNA nucleic acid sequence was dissolved back in the buffer solution containing 40mM HEPES, 111mM NaCl, 5mM KCl, 1mM CaCl ₂ 、1mM MgCl ₂ The pH was 7.5. The final concentration is the concentration of the final product chimeric nucleic acid molecule, and can be adjusted according to the concentration of the bridge and the aptamer before the reaction. After 16 hours of reaction, electrophoresis analysis was performed using 3% agarose gel, and the results were measured by irradiation with ultraviolet light.

1.3 high Performance liquid chromatography analysis

Samples were all analyzed using a Waters Alliance e2695 liquid chromatography system, with a Shodex PROTEIN KW-802.5 molecular sieve column (Catalog #: F6989000). The buffer solution of the mobile phase is prepared from 40mM HEPES, 111mM NaCl, 5mM KCl and 1mM CaCl ₂ 、1mM MgCl ₂ The pH was 7.5. Each sample was eluted in an allelic wash (isocratic elution) mode at a flow rate of 1ml per minute for 15 minutes and the chromatographic results were finally analyzed using Empower 3 software.

1.4TLR9 activation ability analysis

Mu.l of each sample to be tested was pipetted into a 96-well plate, after which the cell concentration was pipetted to 4.5x10 ⁵ Cells/ml were added 180. Mu.l total to a 96 well plate to give a final reaction volume of 200. Mu.l. Then the mixture was placed in a constant temperature incubator at 37℃for 16 hours. After completion of the reaction, 20. Mu.l of the supernatant in each well was aspirated, and 180. Mu.l of the Quanti-Blue detection reagent was added thereto to color it, and the mixture was detected in a 655nm absorption mode by a spectroluminometer for 8 hours (data were collected every hour). Data presentation was plotted from GraphPad Prism 6.

1.5LAG-3 impedance Capacity analysis

On the first day of the assay, MHC II antigen presenting cells (antigen presenting cell, APC) were thawed and TCR activating antigen was added, and then the cells were seeded in a kit-attached 96-well plate (100. Mu.l/well) and incubated for 18 hours in a 37℃incubator.

The next day of the experiment, the culture medium (95. Mu.l/well) was removed from the 96-well plate containing MHC II APC cells, and 40. Mu.l/Kong Daice sample and LAG-3 acting cells (effector cells) were added thereto, followed by a reaction for 6 hours in an incubator at 37 ℃. After 6 hours, the reaction plate was left at room temperature for 15 minutes and warmed. Then, 80. Mu.l of the reaction agent was added to each reaction well, and after 15 minutes of coloration, the luminescence value of each reaction was detected by using GloMax Discover System. Data presentation was plotted from GraphPad Prism 6.

2. Results

2.1 production of chimeric nucleic acid molecules

FIG. 3 shows an electrophoretic analysis of chimeric nucleic acid molecules. The results show that all three independent C695 framework (bridge) parts can stably combine into a binary body (approaching 100 bp) (lanes 1, 2 and 3), a chimeric nucleic acid molecule is formed after B4_Sl3_P16 anti-LAG-3 aptamer is added, the molecular weight is approaching 250bp, the formation of a multiple body is shown, no product exists at the 100bp position, the chimeric efficiency is high, and almost no independent framework (bridge) part remains. In addition, further analysis by high performance liquid chromatography showed that the retention time of the molecular sieve column of the individual backbone moieties averaged about 7.89 minutes, and that the retention time after addition of b4_sl3_p16 anti-LAG-3 aptamer averaged about 6.77 minutes, indicating an overall increase in molecular weight, and no washout product at 7.89 minutes, indicated good binding efficiency and stability of the chimeric nucleic acid molecules of the application.

2.2 chimeric nucleic acid molecules of the application produce a synergistic effect of activating TLR9

Figure 5 shows that the chimeric nucleic acid molecules of the application exhibit excellent TLR9 activation ability. Compared with the conventional C695 (25 nucleotides in length) or C695-PS modified by phosphorothioate bonds, the chimeric nucleic acid molecules of the application have the capability of activating TLR9 by more than 2.5-3 times. The chimeric nucleic acid molecules of the application (binding aptamer moieties) also achieve a trend of increasing the ability to activate TLR9 by a factor of about 1.1 to 1.5 compared to the C695 sequence of the backbone moiety alone. The results show that the chimeric nucleic acid molecules of the application produce unexpected synergistic effects of activating TLR 9.

2.3 chimeric nucleic acid molecules of the application have LAG-3 impedance capability

FIG. 6 shows that chimeric nucleic acid molecules of the application may exhibit LAG-3 resistance capability. The results showed that the backbone portion alone did not show LAG-3 impedance capability, indicating that the C695 sequence itself did not provide LAG-3 impedance capability. In contrast, the chimeric nucleic acid molecules of the present application (backbone moiety plus anti-LAG-3 aptamer) produced LAG-3 resistance, indicating that the structure of the chimeric nucleic acid molecules of the present application did not affect the function of the anti-LAG-3 aptamer, corresponding to the effect of the LAG-3 antibody of the positive control group. IC50 analysis showed that the IC50 of C695d8_B+B4_SL3_P16 was optimal, reaching 102nM.

3. Conclusion(s)

The chimeric nucleic acid molecule provided by the application combines CpG sequences and the aptamer resisting LAG-3, has stable structure and retains the activities (TLR 9 activation capacity and LAG-3 impedance capacity) of the original CpG sequences and the aptamer resisting LAG-3. In particular, the chimeric nucleic acid molecules of the application produce unexpected synergistic effects of activating TLR9 over CpG sequences (backbone moieties) alone in the chimeric nucleic acid molecules. The chimeric nucleic acid molecule of the application achieves dual efficacy, helps to initiate systemic immune response, and improves the therapeutic effect of the disease (for example, improves the response rate of cancer patients to treatment, and achieves systemic immune protection and therapeutic effect).

Sequence listing

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<213> artificial sequence

<220>

<223> C695FL

<400> 4

tcgaacgttc gaacgttcga acgttttt 28

<210> 5

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> C695d8

<400> 5

tcgaacgttc gaacgttttt 20

<210> 6

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> C695d8'

<400> 6

tcgttcgaac gttcgaattt 20

<210> 7

<211> 60

<212> DNA

<213> artificial sequence

<220>

<223> X-L1-C695FL-L2-Z

<400> 7

gttgtagcac ggtggctcga acgttcgaac gttcgaacgt ttttgttgta gcacggtggc 60

<210> 8

<211> 52

<212> DNA

<213> artificial sequence

<220>

<223> X-L1-C695d8-L2-Z

<400> 8

gttgtagcac ggtggctcga acgttcgaac gtttttgttg tagcacggtg gc 52

<210> 9

<211> 52

<212> DNA

<213> artificial sequence

<220>

<223> X-L1-C695d8'-L2-Z

<400> 9

gttgtagcac ggtggctcgt tcgaacgttc gaatttgttg tagcacggtg gc 52

<210> 10

<211> 25

<212> DNA

<213> artificial sequence

<220>

<223> C695 (25 mers)

<400> 10

tcgaacgttc gaacgttcga acgtt 25

<210> 11

<211> 11

<212> DNA

<213> artificial sequence

<220>

<223> LAG-3 motif

<400> 11

gsgggsggty a 11

<210> 12

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> anti-LAG-3 aptamer (12)

<400> 12

tggggggggt tagttcaata catgcgggcg 30

<210> 13

<211> 31

<212> DNA

<213> artificial sequence

<220>

<223> anti-LAG-3 aptamer (13)

<400> 13

tggggggggg ttagacttac actcttattc g 31

<210> 14

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> anti-LAG-3 aptamer (14)

<400> 14

agaggggggg gttagctgct ttaactcatg 30

<210> 15

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> anti-LAG-3 aptamer (15)

<400> 15

aggggggggg ttactgcgca tgtatctcag 30

<210> 16

<211> 62

<212> DNA

<213> artificial sequence

<220>

<223> anti-LAG-3 aptamer (16)

<400> 16

tccctacggc gctaactggg gggggttagt tcaatacatg cgggcggcca ccgtgctaca 60

ac 62

<210> 17

<211> 35

<212> DNA

<213> artificial sequence

<220>

<223> anti-LAG-3 aptamer (17)

<400> 17

acggcgctaa ctgggggggg ttagttcaat acatg 35

<210> 18

<211> 35

<212> DNA

<213> artificial sequence

<220>

<223> anti-LAG-3 aptamer (18)

<400> 18

gctaactggg gggggttagt tcaatacatg cgggc 35

<210> 19

<211> 35

<212> DNA

<213> artificial sequence

<220>

<223> anti-LAG-3 aptamer (19)

<400> 19

ctgggggggg ttagttcaat acatgcgggc ggcca 35

<210> 20

<211> 16

<212> DNA

<213> artificial sequence

<220>

<223> parking sequence

<400> 20

gttgtagcac ggtggc 16

<210> 21

<211> 16

<212> DNA

<213> artificial sequence

<220>

<223> Anchor sequence

<400> 21

gccaccgtgc tacaac 16

<210> 22

<211> 51

<212> DNA

<213> artificial sequence

<220>

<223> aptamer+anchor sequence

<400> 22

gctaactggg gggggttagt tcaatacatg cgggcgccac cgtgctacaa c 51

Claims

1. A chimeric nucleic acid molecule comprising a backbone portion and an aptamer portion, wherein the backbone portion comprises a CpG sequence, the aptamer portion comprises an aptamer that binds to lymphocyte activation gene 3 (LAG-3), and the backbone portion is linked to the aptamer portion, wherein

The CpG sequence comprises a palindromic sequence;

the aptamer portion comprises a first anti-LAG-3 aptamer and a second anti-LAG-3 aptamer, the first anti-LAG-3 aptamer comprising a first anchoring sequence and the second anti-LAG-3 aptamer comprising a second anchoring sequence; and

the backbone moiety comprises a first nucleic acid molecule comprising a first nucleotide sequence of the formula 5'-X-L1-Y-L2-Z-3', wherein Y is a first CpG sequence, L1 and L2 are each a linker, X is a nucleotide fragment comprising a first docking sequence, Z is a nucleotide fragment comprising a second docking sequence, the first docking sequence being complementary to the first anchoring sequence, and the second docking sequence being complementary to the second anchoring sequence such that the first nucleic acid molecule of the backbone moiety is associated with the first anti-LAG-3 aptamer and the second anti-LAG-3 aptamer of the aptamer moiety

Wherein the first anti-LAG-3 aptamer and the second anti-LAG-3 aptamer are each composed of a nucleotide sequence of 5'-GC TAA CTG GGG GGG GTT AGT TCA ATA CAT GCG GGC GCC ACC GTG CTA CAA C-3'; and

the first nucleic acid molecule consists of the nucleotide sequence of 5'-GTT GTA GCA CGG TGG C TCG AAC GTT CGA ACG TTC GAA CGT T TT T GTT GTA GCA CGG TGG C-3'; or (b)

The first nucleic acid molecule consists of the nucleotide sequence of 5'-GTT GTA GCA CGG TGG C TCG AAC GTT CGA ACG TT TTT GTT GTA GCA CGG TGG C-3'; or (b)

The first nucleic acid molecule is comprised of the nucleotide sequence of 5'-GTT GTA GCA CGG TGG C TCG TTC GAA CGT TCG AA TTT GTT GTA GCA CGG TGG C-3'.

2. The chimeric nucleic acid molecule of claim 1, wherein

The aptamer portion further comprises a third anti-LAG-3 aptamer and a fourth anti-LAG-3 aptamer, the third anti-LAG-3 aptamer comprising a third anchoring sequence and the fourth anti-LAG-3 aptamer comprising a fourth anchoring sequence;

the backbone moiety further comprises a second nucleic acid molecule comprising a second nucleotide sequence of the formula 5'-X' -L1'-Y' -L2'-Z' -3', wherein Y' is a second CpG sequence, L1 'and L2' are each a linker, X 'is a nucleotide fragment comprising a third docking sequence, Z' is a nucleotide fragment comprising a fourth docking sequence, the third docking sequence being complementary to the third anchoring sequence, and the fourth docking sequence being complementary to the fourth anchoring sequence, such that the second nucleic acid molecule of the backbone moiety is linked to the third anti-LAG-3 aptamer and the fourth anti-LAG-3 aptamer of the aptamer moiety; and

in the backbone portion, a first CpG sequence of a first nucleic acid molecule is complementary to a second CpG sequence of a second nucleic acid molecule

Wherein the third anti-LAG-3 aptamer and the fourth anti-LAG-3 aptamer are each composed of a nucleotide sequence of 5'-GC TAA CTG GGG GGG GTT AGT TCA ATA CAT GCG GGC GCC ACC GTG CTA CAA C-3'; and

the second nucleic acid molecule consists of the nucleotide sequence of 5'-GTT GTA GCA CGG TGG C TCG AAC GTT CGA ACG TTC GAA CGT T TT T GTT GTA GCA CGG TGG C-3'; or (b)

The second nucleic acid molecule consists of the nucleotide sequence of 5'-GTT GTA GCA CGG TGG C TCG AAC GTT CGA ACG TT TTT GTT GTA GCA CGG TGG C-3'; or (b)

The second nucleic acid molecule consists of the nucleotide sequence of 5'-GTT GTA GCA CGG TGG C TCG TTC GAA CGT TCG AA TTT GTT GTA GCA CGG TGG C-3'.

3. A pharmaceutical composition for modulating an immune response in a cancer individual comprising the chimeric nucleic acid molecule of claim 1 or 2, and a pharmaceutically acceptable carrier.

4. The pharmaceutical composition of claim 3, further comprising an immunodetection point antagonist.

5. Use of the chimeric nucleic acid molecule of claim 1 or 2 or the pharmaceutical composition of claim 3 or 4 for the preparation of a medicament for modulating an immune response in a cancer individual.

6. The use of claim 5, wherein the individual is a human patient suffering from cancer.

7. The use of claim 6, wherein the cancer is selected from the group consisting of lung cancer, melanoma, colorectal cancer, renal cell carcinoma, urothelial cancer, and hodgkin's lymphoma.

8. The use of claim 6, wherein the medicament is formulated for intramuscular or enteral administration.