CN111321159A

CN111321159A - Chimeric nucleic acid molecules for immunomodulation and uses thereof

Info

Publication number: CN111321159A
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: 2020-06-23
Anticipated expiration: 2038-12-14
Also published as: CN111321159B

Abstract

The present invention relates to chimeric nucleic acid molecules for immunomodulation and uses thereof. Specifically, the chimeric nucleic acid molecule of the present invention includes an oligonucleotide having a CpG motif and an aptamer against lymphocyte activation gene 3(LAG-3), can produce a synergistic effect of activating toll-like receptor type nine (TLR 9), and can be used to initiate a systemic immune response and improve a disease treatment effect.

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 usually activated in cancer, resulting in suppression of the anti-tumor immune response.

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

In cancer therapy, there are still many patients who respond poorly to existing drugs, perhaps because the immune response of these patients is suppressed, such that an effective treatment cannot be achieved. Therefore, there is still a need to provide new therapeutic strategies that can more effectively inhibit tumor growth and increase response rate, especially to achieve systemic immune protection and therapeutic effects.

Disclosure of Invention

The present inventors have unexpectedly discovered that a chimeric nucleic acid molecule formed by combining an oligonucleotide having a CpG motif and an anti-LAG-3 aptamer together produces a synergistic effect in activating TLR 9. The chimeric nucleic acid molecule of the invention can start systemic immune response, and achieve better disease treatment effect.

Accordingly, in one aspect, the present invention provides a chimeric nucleic acid molecule comprising a backbone portion and an aptamer portion, wherein the backbone portion comprises CpG sequences, 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 scaffold moiety comprises the CpG sequence in its mid-portion, and flanking nucleotide sequences flanking the CpG sequence, the flanking nucleotide sequences comprising a docking sequence for linking the scaffold moiety to the aptamer moiety.

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

In some embodiments, the backbone moiety comprises two nucleic acid molecules comprising complementary sequences and forming a double-stranded region, and flanking nucleotide sequences flanking the complementary sequences, each flanking sequence comprising a docking sequence for attaching the backbone 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 anchor sequence and the second anti-LAG-3 aptamer comprising a second anchor sequence; and the scaffold 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 linkers, X is a nucleotide fragment comprising a first docking sequence, Z is a nucleotide fragment comprising a second docking sequence, the first docking sequence is complementary to the first anchor sequence, and the second docking sequence is complementary to the second anchor sequence, such that the first nucleic acid molecule of the scaffold moiety is linked to the first and second anti-LAG-3 aptamers 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 scaffold 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 linkers, X 'is a nucleotide fragment comprising a third docking sequence, Z' is a nucleotide fragment comprising a fourth docking sequence, the third docking sequence is complementary to the third anchor sequence, and the fourth docking sequence is complementary to the fourth anchor sequence, such that the second nucleic acid molecule of the scaffold moiety is linked to the third and fourth anti-LAG-3 aptamers of the aptamer moiety; and in the backbone portion, the first CpG sequence of the first nucleic acid molecule is complementary to the second CpG sequence of the second nucleic acid molecule.

In another aspect, the invention 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 invention further comprises an immunodetection point antagonist.

In another aspect, the invention 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 pharmaceutical composition described herein. The invention 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 subject is a human patient having, suspected of having, or at risk of having 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 invention are set forth in the description below. Other features and advantages of the invention will be apparent from the following drawings and detailed description of several specific embodiments, and from the appended claims.

Drawings

The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. The preferred embodiments of the present invention are presented in the drawings for purposes of illustration only. It should be understood that the invention 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 present invention.

FIG. 2 shows the sequence structure of the chimeric nucleic acid molecule of the present invention (C695FL _ B + B4_ SL3_ P16, C695d8_ B + B4_ SL3_ P16, C695d8' _ B4_ SL3_ P16).

FIG. 3 shows an electrophoretic analysis of the chimeric nucleic acid molecule of the present invention. The samples of

lanes

1, 2, and 3 are backbone moieties (C695FL _ B, C695d8_ B, C695d8'_ B), and

lanes

4, 5, and 6 are chimeric nucleic acid molecules comprising a backbone moiety and an aptamer moiety (C695FL _ B + B4_ SL3_ P16, C695d8_ B + B4_ SL3_ P16, C695d8' _ B4_ SL3_ P16). The results show that all three skeletons can stably carry out intermolecular binding to form binary bodies (all are close to 100bp), and the aptamer added with B4_ SL3_ P16 presents a multi-element body close to the position of 250 bp.

FIGS. 4A to 4C show high performance liquid chromatography analysis of the chimeric nucleic acid molecule of the invention, comprising: FIG. 4A is the C695 sequence alone (C695FL _ B) and the combination of the C695 sequence with an anti-LAG-3 aptamer (C695FL _ B + B4_ SL3_ P16); FIG. 4B is the C695 sequence alone (C695d8_ B) and the combination of the C695 sequence with an anti-LAG-3 aptamer (C695d8_ B + B4_ SL3_ P16); and fig. 4C is a C695 sequence alone (C695d '_ B) and a combination of the C695 sequence with an anti-LAG-3 aptamer (C695d8' _ B + B4_ SL3_ P16). The results show that the chimeric nucleic acid molecules of the invention have good binding efficiency and stability.

FIG. 5 shows TLR9 activation ability analysis of a chimeric nucleic acid molecule of the invention. The results show that the chimeric nucleic acid molecules of the invention produce unexpected synergistic effects of activating TLR9, superior to the C695 sequence itself or other C695 sequences of the backbone portion.

FIG. 6 shows LAG-3 impedance analysis of the chimeric nucleic acid molecules of the invention. The results show that, when the skeleton portions are alone, the luminescence values are close to the background value, indicating that no LAG-3 impedance effect is produced; after the B4_ SL3_ P16 aptamer is added into the framework part to form the chimeric nucleic acid molecule, the value of cold light is increased along with the increase of the concentration (particularly, the value is remarkably increased from 80 nM), which indicates that the chimeric nucleic acid molecule can generate the LAG-3 impedance effect. The positive control group was LAG-3 antibody (200 nM). Analysis of IC50 showed that IC50 for C695d8_ B + B4_ SL3_ P16 was optimal to 102 nM.

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 invention belongs.

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

As used herein, the terms "comprises," "comprising," "includes," "including," "contains," "containing," "including," and the like are generally used in an inclusive/inclusive sense, which is intended to mean that one or more features, ingredients, or components are allowed to be present. The terms "comprising" or "comprising" include "or" consisting of "

As described herein, the terms "polynucleotide" or "nucleic acid" can refer to a polymer composed of nucleotide units, including naturally occurring nucleic acids, such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA"), and nucleic acid analogs, including those having non-naturally occurring nucleotides. The synthesis of polynucleotides can be carried out, for example, using an automated DNA synthesizer. It is understood that when a 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) in which "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 the interaction surfaces of the two polynucleotides match. Thus, the two molecules can be described as complementary, and in addition, the interface features are complementary to each other. A first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is identical to the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide. Thus, a polynucleotide having the sequence 5 '-TATAC-3' is complementary to a polynucleotide having the sequence 5 '-GTATA-3'.

As used herein, the term "substantially identical" means that two sequences have more than 85%, preferably 85%, more preferably 90%, and most preferably 95% or 100% identity. The percent identity or similarity between two sequences can be determined using mathematical algorithms known in the art, such as the BLAST and Gapped BLAST programs, NBLAST and XBLAST programs, or the ALIGN program.

As described herein, the term "chimeric nucleic acid molecule" includes polynucleotides or nucleic acids having sequences that are not naturally linked together. The manner of linkage may be via covalent bonds (e.g., phosphodiester linkages) or base pairing to combine different nucleic acid fragments together. The term "chimeric nucleic acid molecule" includes a monomer (which consists of a single polynucleotide) or a multimer (which consists of multiple segments of a polynucleotide, e.g., a dimer consisting of two segments of a polynucleotide, a trimer consisting of three segments of a polynucleotide, or a tetrad consisting of four segments of a polynucleotide, and so forth).

As described herein, a "palindromic sequence," also referred to as a reverse reversal sequence, refers to a nucleotide sequence (5 'to 3' forward) that is identical to the sequence read 5 'to 3' from its complementary sequence. 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 an immune cell and can act to down-regulate an immune response. 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 immunoglobulin and mucin domain-containing-3 (TIM-3) and B and T lymphocyte attenuating agents (BTLA).

The invention provides a combination use of oligonucleotide with CpG motif and anti-LAG-3 aptamer, in particular to a chimeric nucleic acid molecule formed by combining the oligonucleotide with CpG motif and the anti-LAG-3 aptamer together, 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 improving the disease treatment effect.

As described herein, a "nucleic acid aptamer" refers to a nucleic acid molecule (DNA or RNA) that has binding activity for a particular target molecule (e.g., LAG-3). Aptamers can bind to a particular target molecule, thereby inhibiting the activity of the target molecule, for example by blocking the 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 molecules of the invention comprises an anti-LAG-3 aptamer comprising the nucleotide motif of GX1GGGX2GGTX3A (SEQ ID NO:11), wherein X1 and X2 are each independently G, C, or absent, and X3 is T or C.

In some embodiments, the anti-LAG-3 aptamers described herein can comprise the following nucleotide sequences, or substantially identical 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); and

(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); and

(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, "CpG sequence" refers to any CpG-containing oligonucleotide capable of activating immune cells (immune stimulators). At least the C at the 5'CpG 3' must be unmethylated. The nucleic acid having the CpG sequence can be prepared by chemical synthesis by a conventional technique or provided by a commercial supplier.

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

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

(i)5'-AAC GTT CGA ACG TTC GAA CGT T-3' (C695FL core part, palindromic sequence part) (SEQ ID NO: 1);

(ii)5'-AAC GTT CGA ACG TT-3' (C695d8 core part, palindromic sequence part) (SEQ ID NO: 2); and

(iii)5'-TTC GAA CGT TCG AA-3' (C695d8' 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' (C695d8) (SEQ ID NO: 5); and

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

according to the present invention, a chimeric nucleic acid molecule is formed by ligating together a backbone portion comprising a CpG sequence and an aptamer portion comprising an anti-LAG-3 aptamer. The linking means may be by combining the scaffold moiety and 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 anchor 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 scaffold moiety comprises the CpG sequence in its mid-portion, and flanking nucleotide sequences flanking the CpG sequence, the flanking nucleotide sequences comprising a docking sequence for linking the scaffold moiety to the aptamer moiety. For example, the docking sequence comprises 5'-GTT GTA GCA CGG TGG C-3' (SEQ ID NO: 20).

In some embodiments, the scaffold moiety comprises two nucleic acid molecules comprising complementary sequences and forming a double-stranded region (CpG sequence), and flanking nucleotide sequences flanking the complementary sequences, each flanking sequence comprising a docking sequence for attaching the scaffold moiety to the aptamer moiety.

In some embodiments, the aptamer moiety comprises

(SEQ ID NO:22, bold type part is the anchor sequence).

In some embodiments, the scaffold moiety comprises a first nucleic acid molecule comprising a nucleotide sequence of

(5'-X-L1-C695FL-L2-Z-3') (SEQ ID NO:7, with the bold part being the docking sequence), and/or the scaffold moiety comprises a second nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO

(5'-X-L1-C695FL-L2-Z-3') (SEQ ID NO:7, with the docking sequence in bold); or

The scaffold moiety comprises a first nucleic acid molecule comprising a nucleotide sequence of

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

(5'-X-L1-C695d8-L2-Z-3') (SEQ ID NO:8, with the docking sequence in bold); or

(5' -X-L1-C695d8' -L2-Z-3') (SEQ ID NO:9, with the bold part being the docking sequence), and/or the scaffold moiety comprises a second nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO

(5' -X-L1-C695d8' -L2-Z-3') (SEQ ID NO:9, with the docking sequence in bold).

In some embodiments, the aptamer portion comprises an additional aptamer that binds to an 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 immunoglobulin and mucin domain-containing-3 (TIM-3) and B and T lymphocyte attenuating agents (BTLA).

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

As used herein, "pharmaceutically acceptable" means that the carrier is compatible with, and preferably stabilizes, the active ingredient of the composition and is safe for the recipient subject. 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, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methyl cellulose. The compositions may also contain lubricating agents, such as talc, magnesium stearate, and mineral oil; a wetting agent; emulsifying and suspending agents; preservatives, such as methyl and propyl hydroxybenzoate; sweetening agents and flavouring agents. The compositions of the present invention may provide rapid, sustained or delayed release of the active ingredient upon administration to a patient.

In some embodiments, the compositions of the invention comprise other active ingredients, e.g., further comprise an immunodetection point antagonist.

In another aspect, the invention provides a method of modulating an immune response comprising administering to an individual in need thereof a chimeric nucleic acid molecule or composition described herein. The invention 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 invention are useful for treating cancer.

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

As described herein, the subject to be treated using the methods described herein can 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 subject in need of treatment can be a human patient at risk or suspected of having a disease/disorder of interest (e.g., cancer). An individual suspected of having any one of the diseases/disorders of interest may exhibit one or more symptoms of the disease/disorder. An individual at risk for a disease/disorder may be an individual with one or more risk factors for the disease/disorder.

As used herein, the term "treatment" refers to the application or administration of a composition comprising one or more active agents to an individual who has the disease or condition of interest, a symptom of the disease/condition, or a predisposition toward the disease/condition, and whose purpose is to cure, treat, alleviate, alter, remedy, ameliorate, augment, or otherwise affect the disease, disease symptom, or predisposition toward the disease or condition.

In some embodiments, the subject in need of treatment is a human patient having, suspected of having, or at risk of having 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 invention may be delivered via a physiologically acceptable route. E.g., oral, parenteral (e.g., intramuscular, intravenous, 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 as required (e.g., by using a pH of 3 to 9). The preparation of suitable parenteral compositions under sterile conditions can be accomplished by standard pharmacological techniques well known to those skilled in the art.

In some embodiments, the compositions of the invention are administered via intratumoral injection (intratumoral injection). The dose is determined according to the size of the tumor, for example, 50mm³The tumor of (2) is administered with a total of 50. mu.g, 100. mu.g or 200. mu.g of active ingredient. For example, the stroke may be administered three or more times every other day over a relatively short period of time (e.g., within a week).

The invention is further illustrated by the following examples, which are provided for purposes of illustration and not for purposes of limitation. In view of the present disclosure, it will be apparent to those skilled in the art that many changes can be made in the specific embodiments disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Examples

In the invention, a chimeric nucleic acid molecule is designed, and oligonucleotide with CpG motif and anti-LAG-3 aptamer are combined together to form the multi-element double-effect nucleic acid drug. The chimeric nucleic acid molecule can simultaneously activate TLR9 and inhibit LAG-3, particularly has a synergistic effect of activating TLR9, and is superior to the effect of singly using an oligonucleotide with a CpG motif. The chimeric nucleic acid molecules of the invention can be used to initiate a systemic immune response, which is beneficial to disease treatment. 1. Materials and methods

1.1. Nucleic acids

Nucleic acids are commercially available from Baili Biotechnology, Inc. or Integrated DNA Technologies. TLR9 Activity kit was purchased from Invivogen (HEK-Blue)^TMhTLR9,Catalog:hkb-htlr9.HEK-Blue^TMDetection, Catalog: hb-det 3). LAG-3blockade kitAvailable from Promega (LAG-3Block Bioassay, Catalog #: CS 194823).

Table 1: sequence content of nucleic acids

Bold sequences are anchor or docking sequences.

1.2 design and Synthesis of chimeric nucleic acid molecules

Three C695 nucleic acid molecules (C695 FL-B, C695d 8-B, C695d8' _ B) were used in this assay, each in combination with an anti-LAG-3 aptamer, to form a chimeric nucleic acid molecule. The aptamer includes an anchor sequence at one end, allowing the aptamer to be linked to the C695 nucleic acid molecule. The C695 nucleic acid molecules each have pairs of complementary palindromic sequences (C695 sequences) at the central portion and flanking sequences that form intermolecular bonds with another identical C695 nucleic acid molecule in the corresponding central portion on the basis of base pairing to create a double-stranded structure (bridge), and the flanking sequences include docking sequences that are complementary to the anchor sequences of the aptamer, such that the C695 nucleic acid molecule is linked to the aptamer. One double-stranded C695 nucleic acid molecule can be finally grafted with 4 aptamers to form the multi-element double-effect nucleic acid medicine. FIG. 1 shows the design of a chimeric nucleic acid molecule of the invention. FIG. 1 shows the sequence structure of the chimeric nucleic acid molecule of the present invention.

Each DNA nucleic acid sequence was back-dissolved in buffer containing 40mM HEPES, 111mM NaCl, 5mM KCl, 1mM CaCl₂、1mM MgCl₂The 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 reaction. After 16 hours of reaction, electrophoresis analysis was performed using 3% agar gel, and the results were measured by irradiation with ultraviolet light.

1.3 high Performance liquid chromatography analysis

Samples were analyzed using a Waters Alliance e2695 liquid chromatography system with the Shodex ProteinKW-802.5 protocolMolecular sieve columns (Catalog #: F69800). The mobile phase buffer solution formula is 40mM HEPES, 111mM NaCl, 5mM KCl and 1mM CaCl₂、1mM MgCl₂The pH was 7.5. Each sample was eluted in an allelic elution (isocratic elution) mode at a flow rate of 1ml per minute for 15 minutes, and the results of the chromatography were finally analyzed by the Empower 3 software.

1.4TLR9 activation Capacity assay

Pipette 20. mu.l of each sample to be tested into a 96-well plate, followed by pipetting cells at a concentration of 4.5X10⁵Cells/ml 180. mu.l were added to a 96-well plate to a final reaction volume of 200. mu.l. Then, the reaction mixture was placed in a 37 ℃ incubator and reacted for 16 hours. After the reaction, 20. mu.l of the supernatant in each well was taken up and added to 180. mu.l of Quanti-Blue detection reagent to form a color, and the color was detected by a spectrophotometer in the 655nm absorption mode for 8 hours (data was collected every hour). Data presentation is plotted by GraphPad Prism 6.

1.5LAG-3 impedance capability analysis

On the first day of the experiment, MHC II Antigen Presenting Cells (APC) were thawed and TCR-activating antigen was added, followed by plating the cells in a 96-well plate (100. mu.l/well) attached to a kit, and culturing was carried out for 18 hours in a 37 ℃ incubator.

The following day of the experiment, the culture solution (95. mu.l/well) was removed from the 96-well plate containing MHC II APC cells, 40. mu.l/well of the sample to be tested and LAG-3 effector cells (effector cells) were added thereto, followed by 6-hour reaction in an incubator at 37 ℃. After 6 hours, the reaction plate was left at room temperature for 15 minutes to warm up. Then, 80. mu.l of a reagent was added to each reaction well, and after coloring for 15 minutes, the luminescence value of each reaction was detected using a GloMax Discover System. Data are presented plotted by GraphPad Prism 6.

2. Results

2.1 Generation of chimeric nucleic acid molecules

FIG. 2 shows electrophoretic analysis of the chimeric nucleic acid molecule. The results show that three separate C695 scaffold (bridge) parts can stably carry out intermolecular association to form binary bodies (all approximate to 100bp) (

lanes

1, 2 and 3), and a chimeric nucleic acid molecule is formed after the B4_ SL3_ P16 anti-LAG-3 aptamer is added, the molecular weight is approximate to 250bp, the formation of the multi-elements is shown, no product exists at the position of 100bp, the chimeric efficiency is high, and almost no separate scaffold (bridge) parts remain. In addition, further analysis by high performance liquid chromatography showed that the retention time of the molecular sieve column of the single framework part was about 7.89 minutes on average, and after addition of the B4_ SL3_ P16 anti-LAG-3 aptamer, the retention time was about 6.77 minutes on average, indicating that the molecular weight was increased as a whole, and no elution product at 7.89 minutes, indicating that the chimeric nucleic acid molecule of the present invention was excellent in binding efficiency and stability.

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

Figure 3 shows that the chimeric nucleic acid molecules of the invention exhibit excellent TLR9 activation capacity. Compared with the conventional C695 (the length is 25 nucleotides) or C695-PS modified by phosphorothioate bond, the chimeric nucleic acid molecule of the invention has the capability of activating TLR9 by more than 2.5-3 times. The chimeric nucleic acid molecules of the invention (binding aptamer moiety) also have a tendency to increase 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 invention produce unexpected synergistic effects of activating TLR 9.

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

FIGS. 4A-4C show that the chimeric nucleic acid molecules of the invention can exhibit LAG-3 impedance capability. The results show that the backbone portion alone does not exhibit LAG-3 impedance capability, indicating that the C695 sequence itself does not provide LAG-3 impedance capability. In contrast, the chimeric nucleic acid molecules of the invention (backbone moieties plus anti-LAG-3 aptamers) produced LAG-3 impedance capacity, indicating that the structure of the chimeric nucleic acid molecules of the invention did not affect the function of the anti-LAG-3 aptamers, which is equivalent to the effect of LAG-3 antibodies in the positive control group. Analysis of IC50 showed that IC50 for C695d8_ B + B4_ SL3_ P16 was optimal to 102 nM.

3. Conclusion

The chimeric nucleic acid molecule provided by the invention combines the CpG sequence and the anti-LAG-3 aptamer, has a stable structure, and retains the activity (TLR9 activation capability and LAG-3 impedance capability) of the original CpG sequence and the anti-LAG-3 aptamer. In particular, the chimeric nucleic acid molecules of the invention produce unexpected synergistic effects of activating TLR9, over CpG sequences alone (backbone moieties) in the chimeric nucleic acid molecule. The chimeric nucleic acid molecule of the invention achieves dual effects, which is helpful for starting systemic immune response and improving the treatment effect of diseases (for example, improving the response rate of cancer patients to treatment and achieving the systemic immune protection and treatment effect).

Sequence listing

<110> Zhongtian (Shanghai) Biotechnology Ltd

<120> chimeric nucleic acid molecule for immunomodulation and uses thereof

<130>FB0007CN

<160>22

<170>PatentIn version 3.5

<210>1

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> C695FL core

<400>1

aacgttcgaa cgttcgaacg tt 22

<210>2

<211>14

<212>DNA

<213> Artificial sequence

<220>

<223> C695d8 core

<400>2

aacgttcgaa cgtt 14

<210>3

<211>14

<212>DNA

<213> Artificial sequence

<220>

<223> C695d8' core

<400>3

ttcgaacgtt cgaa 14

<210>4

<211>28

<212>DNA

<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> docking 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.

2. The chimeric nucleic acid molecule of claim 1, wherein the backbone moiety comprises the CpG sequence in its central portion and flanking nucleotide sequences flanking the CpG sequence, said flanking nucleotide sequences comprising a docking sequence for linking the backbone moiety to the aptamer moiety.

3. The chimeric nucleic acid molecule of claim 1, wherein said CpG sequence comprises a palindromic sequence.

4. The chimeric nucleic acid molecule of claim 1, wherein said aptamer portion comprises one or more anti-LAG-3 aptamers, each aptamer comprising an anchor sequence for linking said aptamer to said scaffold portion.

5. The chimeric nucleic acid molecule of claim 1, wherein the backbone moiety comprises two nucleic acid molecules comprising complementary sequences and forming a double-stranded region, and flanking nucleotide sequences flanking the complementary sequences, each flanking sequence comprising a docking sequence for linking the backbone moiety to the aptamer moiety.

6. The chimeric nucleic acid molecule of claim 1, wherein the aptamer moiety comprises 2,3, or 4 anti-LAG-3 aptamers.

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

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 anchor sequence and the second anti-LAG-3 aptamer comprising a second anchor sequence; and

the scaffold 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 linkers, X is a nucleotide fragment comprising a first docking sequence, Z is a nucleotide fragment comprising a second docking sequence, the first docking sequence is complementary to the first anchor sequence, and the second docking sequence is complementary to the second anchor sequence, such that the first nucleic acid molecule of the scaffold moiety is linked to the first and second anti-LAG-3 aptamers of the aptamer moiety.

8. The chimeric nucleic acid molecule of claim 7, 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 anchor sequence and the fourth anti-LAG-3 aptamer comprising a fourth anchor sequence;

the scaffold 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 linkers, X 'is a nucleotide fragment comprising a third docking sequence, Z' is a nucleotide fragment comprising a fourth docking sequence, the third docking sequence is complementary to the third anchor sequence, and the fourth docking sequence is complementary to the fourth anchor sequence, such that the second nucleic acid molecule of the scaffold moiety is linked to the third and fourth anti-LAG-3 aptamers of the aptamer moiety; and

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

9. The chimeric nucleic acid molecule of any one of claims 1 to 8, wherein the CpG sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:

(i)5'-AAC GTT CGA ACG TTC GAA CGT T-3' (C695FL core portion) (SEQ ID NO: 1);

(ii)5'-AAC GTT CGA ACG TT-3' (C695d8 core portion) (SEQ ID NO: 2); and

(iii)5'-TTC GAA CGT TCG AA-3' (C695d8' core portion) (SEQ ID NO: 3).

10. The chimeric nucleic acid molecule of any one of claims 1-8, wherein the backbone portion comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:

(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' (C695d8) (SEQ ID NO: 5); and

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

11. the chimeric nucleic acid molecule of any one of claims 1 to 8, wherein the aptamer portion comprises an anti-LAG-3 aptamer comprising the nucleotide motif of GX1GGGX2GGTX3A (SEQ ID NO:11), wherein X1 and X2 are each independently G, C or absent, and X3 is T or C.

12. The chimeric nucleic acid molecule of claim 11, wherein the anti-LAG-3 aptamer comprises a nucleotide sequence selected from the group consisting of seq id nos:

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

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

(iii)5'-AGAGGGGGGGGTTAGCTGCTTTAACTCATG-3' (SEQ ID NO: 14); and

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

13. the chimeric nucleic acid molecule of claim 11, wherein the anti-LAG-3 aptamer comprises a nucleotide sequence selected from the group consisting of seq id nos:

(i)5'-TCCCTACGGCGCTAACTGGGGGGGGTTAGTTCAATACATGCGGGCGGCCACCGTGCTACAAC-3'(SEQ ID NO:16)；

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

(iii)5'-GCTAACTGGGGGGGGTTAGTTCAATACATGCGGGC-3' (SEQ ID NO: 18); and

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

14. the chimeric nucleic acid molecule of claim 2, wherein the docking sequence comprises 5'-GTT GTA GCA CGG TGGC-3' (SEQ ID NO: 20).

15. The chimeric nucleic acid molecule of claim 4, wherein the anchor sequence comprises 5'-GCC ACC GTG CTA CAAC-3' (SEQ ID NO: 21).

16. The chimeric nucleic acid molecule of claim 1, wherein the aptamer comprises 5'-GC TAA CTG GGG GGG GTTAGT TCA ATA CAT GCG GGC GCC ACC GTG CTA CAA C-3' (SEQ ID NO: 22).

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

The backbone part comprises a first nucleic acid molecule comprising the nucleotide sequence 5'-GTT GTA GCA CGG TGG C TCGAAC GTT CGA ACG TTC GAA CGT T TT T GTT GTA GCA CGG TGG C-3' (5'-X-L1-C695FL-L2-Z-3') (SEQ ID NO:7) and/or the backbone part comprises a second nucleic acid molecule comprising the nucleotide sequence 5'-GTT GTA GCA CGG TGG C TCG AAC GTT CGA ACG TTC GAA CGT T TT T GTT GTA GCA CGGTGG C-3' (5'-X-L1-C695FL-L2-Z-3') (SEQ ID NO: 7); or

The backbone part comprises a first nucleic acid molecule comprising the nucleotide sequence 5'-GTT GTA GCA CGGTGG C TCGAAC GTT CGA ACG TT TTT GTT GTA GCA CGG TGG C-3' (5'-X-L1-C695d8-L2-Z-3') (SEQ ID NO:8) and/or the backbone part comprises a second nucleic acid molecule comprising the nucleotide sequence 5'-GTT GTA GCA CGGTGG C TCG AAC GTT CGA ACG TT TTT GTT GTA GCA CGG TGG C-3' (5'-X-L1-C695d8-L2-Z-3') (SEQ ID NO: 8); or

The backbone part comprises a first nucleic acid molecule comprising the nucleotide sequence 5'-GTT GTA GCA CGG TGG C TCGTTC GAA CGT TCG AA TTT GTT GTA GCA CGG TGG C-3' (5'-X-L1-C695d8' -L2-Z-3') (SEQ ID NO:9) and/or the backbone part comprises a second nucleic acid molecule comprising the nucleotide sequence 5'-GTT GTA GCA CGGTGG C TCG TTC GAA CGT TCG AA TTT GTT GTA GCA CGG TGG C-3'(5' -X-L1-C695d8'-L2-Z-3') (SEQ ID NO: 9).

18. The chimeric nucleic acid molecule of claim 1, wherein the aptamer moiety comprises an additional aptamer that binds to an additional immunodetection point molecule other than LAG-3.

19. A pharmaceutical composition comprising the chimeric nucleic acid molecule of any one of claims 1 to 17, and a pharmaceutically acceptable carrier.

20. The pharmaceutical composition of claim 1, further comprising an immunodetection point-antibody agent.

21. A method of modulating an immune response, wherein the method comprises administering the chimeric nucleic acid molecule of any one of claims 1 to 17 or the pharmaceutical composition of claim 19 or 20 to an individual in need thereof.

22. The method of claim 21, wherein the individual is a human patient having, suspected of having, or at risk of having cancer.

23. The method of claim 22, wherein the cancer is selected from the group consisting of lung cancer, melanoma, colorectal cancer, renal cell carcinoma, urothelial cancer, and hodgkin's lymphoma.

24. The method of claim 22, wherein the chimeric nucleic acid molecule or the pharmaceutical composition is administered intramuscularly or enterally.

25. Use of the chimeric nucleic acid molecule of any one of claims 1 to 17 or the pharmaceutical composition of claim 19 or 20 for the preparation of a medicament for modulating an immune response.

26. The use of claim 25, wherein the subject is a human patient having, suspected of having, or at risk of having cancer.

27. The use of claim 26, wherein the cancer is selected from the group consisting of lung cancer, melanoma, colorectal cancer, renal cell carcinoma, urothelial cancer, and hodgkin's lymphoma.

28. The use of claim 26, wherein the medicament is formulated for intramuscular or enteral administration.