CN115725586A

CN115725586A - Aptamer modified structure of targeting PTK7, coupled drug thereof, preparation method and application

Info

Publication number: CN115725586A
Application number: CN202110986513.4A
Authority: CN
Inventors: 谭蔚泓; 殷红; 刘庄; 陈思凯
Original assignee: Renji Hospital Shanghai Jiaotong University School of Medicine
Current assignee: Renji Hospital Shanghai Jiaotong University School of Medicine
Priority date: 2021-08-26
Filing date: 2021-08-26
Publication date: 2023-03-03

Abstract

The invention belongs to the field of biomedicine, and discloses a modified aptamer (formula II), a PTK 7-targeted nucleic acid coupling drug SGCR-YH containing the aptamer, and a preparation method and application thereof. The nucleic acid coupling drug has a structure shown in a formula III. The nucleic acid coupled drug can target PTK 7and has strong activity of killing tumor cells. The conjugate drug disclosed by the invention shows better tumor inhibition activity, is suitable for treating tumors such as breast cancer, ovarian cancer, gastric cancer, intestinal cancer, non-small cell lung cancer, liver cancer, pancreatic cancer, triple negative breast cancer, esophageal cancer, squamous cell carcinoma, head and neck cancer or prostate cancer, and has wide application prospects.

Description

Aptamer modified structure of targeting PTK7, coupled drug thereof, preparation method and application

Technical Field

The invention belongs to the field of biomedicine, and relates to a PTK 7-targeted aptamer modified structure, a coupled drug, a preparation method and application thereof, in particular to a nucleic acid coupled drug capable of generating a targeted tumor killing effect (especially to a PTK 7-targeted nucleic acid coupled drug), a preparation method and application thereof.

Background

Receptor Tyrosine Kinases (RTKs) are now known transmembrane signaling proteins that can transmit biological signals from the extracellular environment to the interior of cells. RTKs are important for regulating cell growth, differentiation, epithelial growth, development, adhesion, migration, apoptosis, etc., and are involved in the development and progression of various forms of cancer.

Tyrosine kinase 7 (PTK 7) is a member of the RTK family. As a multifunctional co-receptor of PTK7/Otk, the protein plays a molecular switch function in a signal transduction pathway mediated by Wnt, semaphorin/Plexin and vascular endothelial growth factor, and plays an important role in promoting the growth, angiogenesis, migration and invasion of vascular endothelial cells of tumors. PTK7 is highly expressed in many human malignancies including colon cancer, lung cancer, gastric cancer and acute myeloid leukemia, among others.

Nucleic acid as a drug or a targeting molecule is easily metabolized rapidly in vivo by the action of nuclease and the like, and has problems of low stability, low specificity and the like. Therefore, there is a need to develop PTK 7-targeting drugs and methods of constructing such drugs.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects of the prior art, provide a method for improving the nucleic acid aptamer with high nucleic acid affinity, targeting property and the like, and correspondingly provide a drug conjugate containing the nucleic acid aptamer, wherein the nucleic acid aptamer can be combined with PTK7 with high specificity and high affinity, has the advantages of small molecular weight, no immunogenicity, good biocompatibility, high stability, long conjugate circulation time, easiness in storage, easiness in chemical synthesis and the like, can be applied to the fields of drug separation and purification, drug preparation of tumors with PTK7 expression and the like, and can be used as a drug carrier.

The aptamer which is obtained by screening and targeted to combine with the PTK7 protein is obtained by a chemical modification method, and the invention analyzes the stability, targeting property, affinity and the like of the sequence of the modified aptamer, thereby proving that the modified aptamer can be specifically targeted to combine with the PTK7 protein and combine with tumor cells expressing the PTK7 protein to play a role in targeted delivery.

The invention aims to provide a ribonucleic acid aptamer with the advantages of high affinity, strong targeting, long half-life period, high selectivity, small side effect and the like, and a nucleic acid aptamer drug conjugate, a drug composition and the like containing the ribonucleic acid aptamer.

The invention modifies the aptamer by using a specific modification group, so that the stability of the aptamer can be enhanced on one hand, and the anti-tumor drug can be delivered to tumor cells in a targeted manner by using the specific recognition capability of the modified aptamer to the tumor cells on the other hand, thereby improving the selectivity of the anti-tumor drug to the tumor cells. Meanwhile, the aptamer can be coupled with other anti-tumor mechanism drugs, so that tumors are treated by different action targets, and the aptamer has a synergistic effect. The conjugate drug of the invention shows better tumor inhibition activity.

In one aspect, the present invention provides a method for modifying a nucleic acid aptamer, the method comprising modifying the nucleic acid aptamer with a group represented by formula I below to obtain a modified nucleic acid aptamer:

formula I

Wherein R1 is H or PO3 ^– -CnH2n +1, n is 0-24; preferably, n is 10 to 20; further preferably, n is 12 to 18.

R2 is PO3 ^- A carboxyl, a maleimide or an azide group.

x is 1 to 24; preferably, x is 1 to 8 or 4 to 24;

y is 1 to 6; preferably, y is 2 to 3; further preferably, y is 1.

The invention provides a modified nucleic acid aptamer with a structure shown in formula II:

formula II

R2 is PO3 ^- A carboxyl, maleimide or azide group.

x is 1 to 24; preferably, x is 1 to 8 or 4 to 24;

y is 1 to 6; preferably, y is 2 to 3.

Aptamer is an Aptamer.

Further preferably, when R2 is PO3 ^- When x is 1 to 8; y is 1-6, and is connected with the aptamer through O;

when R2 is carboxyl, maleimide or azide group, Z is amino, sulfydryl or alkynyl/DBCO on Aptamer, x is 4-24, y is 1. By way of illustration, when R2 is carboxyl, the Aptamer provides an amino group, the carboxyl group reacts with the amino group, linking R2 to the Aptamer; when R2 is maleic amide, the Aptamer provides sulfydryl, the maleic amide reacts with the sulfydryl, and the R2 is connected with the Aptamer; when R2 is an azide group, the Aptamer provides alkynyl or DBCO, the azide group reacts with the alkynyl or DBCO, and the R2 is connected with the Aptamer.

Further preferably, when R is H, x is 6, y is 2, aptamer is a sulfhydryl modified SGC8 sequence shown in SEQ ID NO.1, and the 3' end of the modified aptamer is inverted T base (inverted dT), the obtained modified aptamer is labeled as 2spacer18-SGC8-inverted-dT and is labeled as SGCR4.

Further preferably, R is PO ₃ ^– -C _n H _2n+1 When n is 18, x is 6 and y is 2, the Aptamer is a sulfhydryl modified SGC8 sequence shown in SEQ ID NO.1, and the modified nucleic acid is adaptedThe 3' end of the body was terminated with an inverted T base (inverted dT) and the resulting modified aptamer was labeled C18-2spacer18-SGC8-inverted-dT and designated SGCR5.

Wherein the Aptamer Aptamer is selected from a nucleotide molecule or a nucleotide analogue.

Wherein the nucleotide molecule comprises a double-stranded nucleotide molecule and a single-stranded nucleotide molecule; the single-stranded nucleotide molecule is selected from cDNA, mRNA, tRNA, rRNA, miRNA, micromolecule RNA, telomerase RNA, antisense RNA, ribozyme, non-coding RNA and antisense oligonucleotide.

The nucleotide analogue is selected from 1) a nucleic acid analogue which can play a role in regulation and control after being combined with a nucleotide molecule or a nucleic acid structure molecule, 2) a peptide nucleic acid, a nano nucleic acid, a locked nucleic acid and a nucleic acid protein which are partially modified from the nucleotide molecule or the nucleic acid structure molecule, and 3) a thio nucleotide, a 2 '-methoxy nucleotide and a 2' -fluoro nucleotide which are obtained by substituting the nucleotide molecule or the nucleic acid structure molecule; or 4) a nucleic acid derivative formed by combining the nucleotide molecule and one or a combination of other functional nucleotides, functional polypeptides, functional proteins, antibodies and the like in any form of chemical coupling or assembly.

Preferably, the nucleotide molecule is selected from any one of the following sequences:

(1) A nucleotide sequence (SGC 8) as shown in SEQ ID No.1 or a nucleotide sequence (PDL 1) as shown in SEQ ID No. 2;

SGC8：5’-ATC TAA CTG CTG CGC CGC CGG GAA AAT ACT GTA CGG TTA GA-3’(SEQ ID NO.1)；

PD-L1：5'-TAC AGG TTC TGG GGG GTG GGT GGG GAA CCT GTT-3’(SEQ ID NO.2)。

(2) A nucleotide sequence which has an identity of 80% or more (preferably 85% or more; more preferably 90% or more; further more preferably 95% or more; further more preferably 99% or more) to the nucleotide sequence of (1) and has a function of specifically binding to human tumor cells;

(3) A truncation of the nucleotide sequence of (1), and having a nucleotide sequence that specifically binds human tumor cells with the same/similar specificity;

(4) A nucleotide sequence which can be hybridized with the nucleotide sequence in (1) under strict conditions and has the same/similar function of specifically binding human tumor cells;

(5) A nucleotide sequence complementary to the nucleotide sequence defined in any one of (1) to (4);

(6) (1) an RNA sequence transcribed from the nucleotide sequence defined in any one of (1) to (4);

(7) (1) - (4) the corresponding locked nucleic acid or peptide nucleic acid modified from said nucleotide sequence as defined in any one of (1) to (4);

(8) And (3) a nucleic acid derivative formed by combining the nucleotide sequence defined in any one of (1) to (4) with other functional nucleotides, functional polypeptides, functional proteins, antibodies and the like in any form of chemical coupling or assembly and the like.

It is a further aspect of the present invention to provide the use of an aptamer as described above, selected from one or more of the following:

for use as a pharmaceutical carrier;

for use in drug design and development;

use for studying tumor cell surface proteins;

use in the isolation and purification of drugs;

the application in preparing copolymer and biological product.

Specifically, the invention provides the use of the aptamer for preparing a medicament targeting PTK7 or a medicament for treating tumors with PTK7 expression; wherein the drug is selected from a copolymer and a pharmaceutical composition, wherein the copolymer comprises a nucleic acid conjugated drug.

One aspect of the present invention is to provide a copolymer as above, wherein the copolymer comprises the aptamer and the drug molecule as above, and the aptamer and the drug molecule are linked by one or more of coupling, polymerization, attachment, and the like.

It is another aspect of the present invention to provide the copolymer as described above, which is a nucleic acid-conjugated drug when the ribonucleic acid aptamer and the drug molecule are linked by a coupling means; wherein the nucleic acid coupling drug is a PTK 7-targeted nucleic acid coupling drug SGCR-YH, the coupling drug is composed of a modified aptamer fragment, a drug fragment and a linker, and the coupling drug has a structure shown in the following formula III:

formula III

Wherein R-Aptamer is a modified Aptamer as described above, wherein each group is as defined above;

and R3 is a drug fragment.

In the invention, the medicine is a cytotoxic medicine and is selected from one or more of nucleoside analogues and base analogues, metabolic medicines, high-activity small molecules, alkaloids, traditional Chinese medicine extracts, small molecule inhibitors, plant alkalis, anti-tumor small molecule targeted medicines, nucleic acid medicines, protein medicines and the like. Preferably, the drug is one or more of MMAE, MMAF, DM1, DM4, or PBD dimer (PBD dimer), etc.

Another aspect of the present invention is to provide the use of the nucleic acid-conjugated drug as described above for the preparation of a drug for targeted therapy of tumors with PTK7 expression.

Wherein the tumor with PTK7 expression comprises one or more of breast cancer, ovarian cancer, gastric cancer, colorectal cancer, non-small cell lung cancer, liver cancer, pancreatic cancer, triple negative breast cancer, esophageal cancer, squamous cell carcinoma, head and neck cancer, prostatic cancer and the like. Preferably, the tumor is selected from ovarian cancer, colorectal cancer or non-small cell lung cancer.

In a preferred embodiment, said tumor cell with expression of PTK7 is selected from the group consisting of ovarian cancer cell OVCAR3, colorectal cancer cell HCT116, non-small cell lung cancer cell NCI-H446, triple negative breast cancer cell MDA-MB-231, pancreatic cancer cell PANC-1, and gastric cancer cell NCI-87.

The copolymer (including aptamer coupled drug) provided by the invention can obviously inhibit the growth, migration, infiltration and bone metastasis of cancer cells, promote the apoptosis of tumor cells and the like.

It is a further aspect of the present invention to provide a pharmaceutical composition comprising an aptamer as described above or a copolymer as described above, with or without a pharmaceutically acceptable carrier or excipient or adjuvant.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Compared with the prior art, the invention has the beneficial effects that:

(1) The inventor obtains a modifying group which can effectively improve the characteristics of the nucleotide molecules or the nucleotide analogs through a large number of experimental screens, the modifying group can change the three-dimensional conformation of the nucleotide molecules or the nucleotide analogs through being connected with the nucleotide molecules or the nucleotide analogs, and further obtains the nucleic acid aptamer with high affinity, high selectivity and high specificity, and the modified nucleic acid aptamer also has the characteristics of strong targeting property, long half-life period, small side effect and the like.

(2) The modified aptamer and the conjugated drug thereof obtained by modifying the nucleotide molecules or the nucleotide analogs with the specifically screened modifying groups can be combined with tumor cells with high affinity, for example, the modified aptamer and the conjugated drug thereof have enhanced affinity for colon cancer cells HCT116 expressed by PTK 7.

(3) The aptamer and the coupled drug thereof obtained by modifying the nucleotide molecules or the nucleotide analogs with the specifically screened modification groups can specifically recognize NCI-H446 and OVCAR3, and have the highest toxicity on the two tumor cells.

(4) The aptamer obtained by modification of the specifically screened modification group is selected as a drug carrier, so that the drug can be protected from being influenced by the environment, a proper drug release speed can be obtained, the circulation time of the conjugate is prolonged, and a lasting drug effect is obtained. Wherein the carbon chain of the modifying group plays an important role in realizing the characteristics of the modifying group.

(5) The aptamer obtained by modifying the nucleotide molecule or the nucleotide analogue with the specifically screened modification group has the advantages of good chemical stability, good pharmacokinetic property, no immunogenicity, no toxicity, shorter sequence length and the like, and is beneficial to large-scale industrial production and application.

(6) The aptamer can be synthesized and modified in vitro, has the characteristic of easy modification, and can be obtained by connecting a specifically screened modification group to a nucleotide molecule or a nucleotide analogue; short synthesis period and good reproducibility.

(7) The invention utilizes the characteristic that the modified aptamer is combined with PTK7, is expected to improve the stability of the aptamer, further improves the safety and solubility of the aptamer coupled drug, prolongs the drug effect, and reduces the combination of the drug and other blood endogenous substances.

(8) The aptamer is labeled and modified, so that development of novel medicaments related to PTK7 protein is facilitated.

(9) The aptamer has wide application prospect and important academic value in the aspects of new technology development and targeted therapy of tumor cell biology.

Drawings

In order to more clearly illustrate the technical solutions and advantages of the embodiments of the present invention, the drawings used in the embodiments are briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without inventive efforts.

FIG. 1 is a graph showing a comparison of the affinity of aptamers to cells before and after modification.

FIG. 2 comparison of in vivo distribution of nucleic acid aptamers in mice before and after chemical modification; wherein the modified aptamer is SGCR4.

FIG. 3 Effect of nucleic acid conjugate drugs on the activity of different cells, IC50 curves; in this figure, the ordinate Inh% represents the inhibition ratio (inhibition).

FIG. 4 tumor suppression effect in a mouse model of NCI-H446 small cell lung cancer.

FIG. 5NCI-H446 Small cell Lung cancer mouse model weight changes during treatment.

Wherein when R is H, x is 6, y is 2, and aptamer is SGC8, the obtained modified aptamer is marked as 2spacer18-SGC8-inverted-dT and is marked as SGCR4; when the aptamer SGCR4 and the drug MMAE are linked by linker (vc), the corresponding aptamer-conjugated drug marker is 2spacer18-SGC8-inverted-dT-vc-MMAE, noted: SGCR4-vc-MMAE (YH 2502).

Wherein R is PO ₃ ^– -C _n H _2n+1 When n is 18, x is 6, y is 2 and the Aptamer is SGC8, the obtained modified Aptamer is marked as C18-2spacer18-SGC8-inverted-dT and is marked as SGCR5; when the aptamer SGCR5 is linked to the drug MMAE via vc, the corresponding aptamer-conjugated drug marker is C18-2spacer18-SGC8-inverted-dT-vc-MMAE and is designated SGCR5-vc-MMAE (YH 2504).

Detailed Description

The invention is further illustrated by the following examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. The procedures, conditions, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.

The invention provides a method for modifying a nucleic acid aptamer, which comprises the step of modifying the nucleic acid aptamer by using a group shown in the formula I to obtain the modified nucleic acid aptamer. For example, in order to improve the properties such as stability of the riboaptamer and time for effective action in vivo (e.g., increase half-life), a group represented by the following formula I is chemically modified at some of the bases at both ends or at the middle of the riboaptamer sequence to obtain a modified Aptamer R-Aptamer represented by the formula II.

Formula I

In another aspect, the present invention provides a modified aptamer, which has a structure represented by formula II:

formula II

In the formula I or the formula II, R1 is H or PO3 ^– -CnH2n +1, n is 0-24; preferably, n is 10 to 20; further preferably, n is 12 to 18.

R2 is PO3 ^- A carboxyl, maleimide or azide group.

x is 1 to 24; preferably, x is 1-8 or 4-24;

y is 1 to 6; preferably, y is 2 to 3.

An Aptamer is an Aptamer.

In a specific embodiment, in formula I or II, the R group is PO ₃ ^– -C _n H _2n+1 In the case, the number of carbon atoms n is 0 to 24 or 10 to 20 or 10 to 15 or 15 to 20 or 12 to 18 or 13 to 19 or 15 to 18 or 16 to 19 or 16 to 18 or 18 to 20. In a specific embodiment, the number of carbon atoms n may be 1, 2, 3,4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

In a specific embodiment, in formula I or II, x can be 1, 2, 3,4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24.

In a specific embodiment, in formula I or II, y can be 1, 2, 3,4,5, 6.

In the present invention, the nucleic acid aptamer suitable for the modification method includes a nucleotide molecule or a nucleotide analog.

Wherein the nucleotide molecule comprises a double-stranded nucleotide molecule, a single-stranded nucleotide molecule; the single-stranded nucleotide molecule comprises cDNA, mRNA, tRNA, rRNA, miRNA, micromolecular RNA, telomerase RNA, antisense RNA, ribozyme, non-coding RNA, antisense oligonucleotide and nucleotide molecules or fragments with certain identity with the sequences.

Wherein, the nucleotide molecule or fragment with certain identity with the sequence is obtained by the following method: a nucleotide molecule or fragment which is obtained by substituting, deleting or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) bases to the sequence shown in the foregoing, or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) bases to the 5 'end and/or the 3' end, and has the same/similar function as the foregoing sequence.

In the present invention, "sequence identity" refers to the percentage of corresponding bases that are identical by position, which is indicative of the level of similarity (also referred to as sequence homology, similarity, or identity) between two or more nucleic acids. In a specific embodiment, in the "nucleotide molecule or fragment having a certain identity to the aforementioned sequence," identity "refers to similarity to a native or known nucleic acid sequence, including nucleotide sequences having 75% or greater, or 85% or greater, or 90% or greater, or 95% or greater identity. The above-mentioned identity of 75% or more may be 75%, 80%, 85%, 90% or 95% or more; specifically, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% may be used. The above identity of 90% or more may be 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%.

Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

Wherein, the nucleotide analogue is selected from nucleic acid analogues which can combine with nucleotide molecules or nucleic acid structural molecules to play a role in regulation and control, peptide nucleic acids, nano nucleic acids and nucleic acid proteins which are partially modified from the nucleotide molecules or nucleic acid structural molecules, or nucleotides obtained by modifying part or all of the skeleton of the nucleic acid aptamer into a phosphorothioate skeleton, or nucleotides which are obtained by substituting or modifying a certain position (comprising a sugar ring at the 5 'end, the 3' end, part of the skeleton or all of the skeleton) of the nucleotide molecules or nucleic acid structural molecules and comprise thio nucleotides, 2 '-methoxy nucleotides, 2' -fluoro nucleotides, phosphorylated nucleotides, oxygen methylated nucleotides, locked nucleic acid modified nucleotides, methylated nucleotides and aminated nucleotides (such as 2-NH) ₂ Modified), thiolated nucleotides, biotinylated nucleotides, fluorescence (e.g., FITC, etc.) labeled nucleotides, enzyme-labeled nucleotides, nano-luminescent material-labeled nucleotides, folate-labeled or isotopically-labeled nucleotides, PEG-modified nucleotides, aptamers embedded with a PEG-liposome mixture.

Among these, several examples of nucleotide modifications are as follows:

formula A

In one embodiment, the aptamer is a monovalent or polyvalent thiol or disulfide-protected thiol group modified nucleotide, the structure of which includes, but is not limited to, the following, and the modification site is at the 3 'end, 5' end or in the middle of the nucleic acid:

formula B

In the formula B, x is 1-40, preferably, x is 1-6; y is 0 to 10, preferably, y is 1 to 3; z is 2 to 20, preferably z is 2 to 6; a is 2 to 20, preferably, a is 2 to 8; b is 2 to 20, preferably, b is 2 to 6; c is 1 to 20, preferably, c is 1 to 3.

The aptamer of the invention is an oligonucleotide that binds to a specific target using a self-folded higher order structure, such as a piece of DNA (deoxyribonucleic acid), RNA (ribonucleic acid), or modified DNA and RNA. The DNA is composed of a plurality of deoxynucleotides, and one deoxynucleotide molecule is formed by coupling a molecule of basic group (adenine A, thymine T, cytosine C and guanine G), a molecule of deoxyribose and a molecule of phosphate; RNA is composed of multiple ribonucleotides, one of which is formed by coupling a molecular base (adenine A, uracil U, cytosine C, guanine G), a molecular ribose and a molecular phosphate.

Preferably, the nucleotide molecule described herein is selected from any one of the following sequences:

PD-L1：5'-TAC AGG TTC TGG GGG GTG GGT GGG GAA CCT GTT-3’(SEQ ID NO.2)；

(3) A truncation of the nucleotide sequence of (1), and having a nucleotide sequence that specifically binds to human tumor cells with the same/similar specificity;

(4) A nucleotide sequence which can be hybridized with the nucleotide sequence in (1) under strict conditions and has the same/similar function of specifically binding to human tumor cells;

In the present invention, the 3' -end of the modified aptamer is synthesized from an inverted T base (inverted dT). The purpose is to resist the shearing of DNA exonuclease and raise the stability of the aptamer.

In the present invention, the nucleic acid aptamer includes a nucleotide molecule or a nucleotide analog. The full-length sequence of nucleotides in the nucleotide molecule or nucleotide analogue or a fragment thereof is selected from naturally occurring, in vitro transcribed, recombinant or artificially synthesized (obtained by solid phase synthesis or post-synthetic modification), or any other source of the same sequence.

The aptamer has the advantages of higher affinity and specificity, no immunogenicity, capability of chemical synthesis, low cost, capability of marking, good stability, easiness in storage and the like.

The modified aptamers obtained from the modified nucleotide analogs all have molecular structural properties and functions substantially the same as or similar to those of the original aptamers obtained from the modified nucleotide molecules (or basic nucleotide molecules), and thus can be used for specific binding to PTK 7.

In the present invention, the modified aptamer is capable of specifically binding to a disease-related molecule. The aptamer can be combined with a target molecule through 3D conformation complementation, has affinity and specificity, has the advantages of good water solubility, high stability, low immunogenicity, simple preparation, low cost and the like, and is safe. Moreover, since specific delivery at a cellular level can be achieved, the aptamer of the present invention can be applied to targeted delivery of drugs as a targeting molecule.

In a specific embodiment, the invention provides a use of the aptamer, any of the aptamers described above for use as a pharmaceutical carrier.

In a specific embodiment, the invention provides a use of the modified aptamer, and any one of the modified aptamers is used for drug design and development.

In a specific embodiment, the invention provides a use of the modified aptamer, and any one of the modified aptamers is used for researching a tumor cell surface protein.

In a specific embodiment, the invention provides a use of the modified aptamer, and any one of the modified aptamers is used for drug isolation and purification.

In a specific embodiment, the present invention provides a use of the modified aptamer, any of the aptamers described above is used to prepare an aptamer copolymer, and the coupling of the aptamer and the drug molecule can be performed in a manner known in the art, such as coupling, polymerization or attachment of the aptamer and the drug molecule.

In one scheme of the invention, the medicine is connected with the modified aptamer in a physical mosaic mode to obtain the aptamer-cytotoxic conjugate capable of targeted combination, and the targeting property, affinity, cytotoxic effect and the like of the conjugate are explored, so that a better target is provided for clinically screening and researching novel targeted medicines, and a new method and thought are provided for clinical treatment.

Wherein the copolymer (aptamer copolymer) is a mono-specific aptamer drug copolymer, a bi-specific aptamer drug copolymer, or a multi-specific aptamer drug copolymer.

When the aptamer and the drug molecule are coupled, the aptamer copolymer is the aptamer drug conjugate, the coupling refers to coupling of a cytotoxic drug by connecting a linker to the 5 'end or the 3' end of the aptamer, or the coupling refers to directly coupling the 5 'end or the 3' end of the aptamer with the drug molecule to obtain the coupled cytotoxic drug. The coupling may be synthesized at the 5 'end or the 3' end by solid phase synthesis or chemical synthesis methods.

In a specific embodiment, the present invention provides a use of the aptamer, a use of any of the aptamers described above for preparing a pharmaceutical composition, wherein the pharmaceutical composition may comprise any of the modified aptamers described above (preferably, in an effective amount), or a copolymer linked, conjugated or polymerized with other anti-tumor drugs. Typically, for pharmaceutical use, the pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant. Further, the pharmaceutical composition of the present invention may optionally further comprise one or more other pharmaceutically active compounds.

Preferably, the nucleic acid coupled drug is a PTK 7-targeting nucleic acid coupled drug for treating PTK 7-positive tumors.

In a specific embodiment, the invention provides the use of a nucleic acid-conjugated drug targeting PTK7 for the treatment of tumors.

In the invention, the tumor is a tumor with PTK7 expression, and comprises breast cancer, ovarian cancer, gastric cancer, colorectal cancer, non-small cell lung cancer, liver cancer, pancreatic cancer, triple negative breast cancer, esophageal cancer, squamous cell carcinoma, head and neck cancer and prostatic cancer.

In a specific embodiment, the invention provides a copolymer comprising an aptamer and a drug molecule as described above, linked by coupling, polymerization or attachment.

When the ribonucleic acid aptamer and the drug molecule are connected in a coupling mode, the copolymer is a nucleic acid coupling drug; wherein the nucleic acid-coupled drug is a nucleic acid-coupled drug targeting PTK7, designated SGCR-YH, wherein SGCR is an aptamer with a modifying group. R2 is coupled with aptamer, and is synthesized at the 5 'end or the 3' end by a solid phase synthesis or chemical synthesis method. The conjugate drug is composed of a modified aptamer fragment, a drug fragment (such as a small molecule drug) and a linker, and has a structure shown in the following formula III:

formula III

Wherein the definitions of the groups are shown above.

In the present invention, the drug is a cytotoxic drug, such as a chemotherapeutic drug (cytotoxic drug), and can be used for treating tumors, inflammation, infection, immune diseases, and the like, and for example, can include nucleoside analogs and base analogs (such as gemcitabine, pentafluorouracil, and nucleoside structures or base structures of other active small molecules), high-activity small-molecule dolastatins (or Auristatins such as MMAE and MMAF) and maytansinoids (such as DM1 and DM 4), toxin-calicheamicins (calicheamicins) interacting with DNA, duocarmycin (duocarmycin) analogs, pyrrolobenzodiazepine dimers (PBD dimers), alkaloids, herbal extracts (such as triptoresinone, and the like), small-molecule inhibitors (such as camptothecin, SN38, topotecan, doxorubicin, combretastatin, and the like). Specifically, the drugs include, but are not limited to: metabolic drugs such as methotrexate, fluorouracil, floxuridine, gemcitabine, raltitrexed, anticancer antibiotic drugs such as mitomycin C, bleomycin, doxorubicin, epirubicin, pirarubicin, plant alkaloids such as vinblastine, paclitaxel, hydroxycamptothecin, antitumor hormones such as tamoxifen, letrozole, prednisone, hybrids such as cisplatin, carboplatin, mirtaedanthraquinone, antitumor small molecule targeted drugs such as gefitinib, imatinib or lapatinib.

More preferably, the drug is selected from fluorouracil, floxuridine, MMAE, MMAFE, PBD dimer, maytansine, maytansinol, paclitaxel, methotrexate, triptolide, gemcitabine, raltitrexed, mitomycin, bleomycin, vinblastine, paclitaxel, hydroxycamptothecin, carboplatin, tamoxifen, letrozole, prednisone, gefitinib, imatinib, lapatinib, epirubicin, doxorubicin, carboplatin, cisplatin, docetaxel, oxaliplatin, epirubicin, pirarubicin, calicheamicin, tarobulin, dolastatin, auristatin F, daunomycin, cyclophosphamide, everolimus, capecitabine.

In a preferred embodiment of the present invention, the anti-tumor drug is MMAE, MMAF, PBD dimer, DM1 or DM4.

Examples of suitable several drug molecules suitable for use in the aptamers of the invention are as follows:

formula C

In the present invention, the drug can be linked to the modified aptamer by a known or conventional method, or linked to a drug molecule by linking with a linker; for example, the nucleic acid coupled drug molecule is obtained by coupling nucleic acid aptamer through a DNA solid phase synthesis method.

In the invention, when the linker is a chemical linker or a chemical connecting bond or a connecting element, the structure is as follows:

in the invention, the linker comprises an Aptamer coupling region (Y), a functional region and a drug molecule coupling region (X). The linker is a phosphoester bond (such as phosphodiester bond), a cleavable bond (vc shown in formula D, SPBD), or a non-cleavable bond (SMCC shown in formula D).

The aptamer can be coupled with the Y group of a linker, including carboxyl and active species thereof, maleimide, alkynyl, azido, amino, tetrazine, trans-cyclooctene and other functional groups through solid phase synthesis or post-synthesis modified amino, sulfhydryl, azido, alkynyl, carboxyl, trans-cyclooctene and other functional groups. Drug molecule R ₃ The amino, hydroxyl or carboxyl of the linker is coupled with the X group of the linker, including carboxyl and active species thereof, hydroxyl or amino and the like.

In a specific embodiment, R is ₃ The drug molecule is connected with the aptamer through a phosphodiester bond, or the R3 drug molecule is connected with the aptamer of which the oxygen is fully or partially substituted by sulfur, or the R3 drug molecule is embedded into the 5 'end or the 3' end or the middle of the aptamer. In a preferred embodiment, the linker of the present invention is a phosphodiester group.

In a preferred embodiment, the linker used in the present invention has a hydroxyl group or a carboxyl group at one end and a maleimide group at the other end.

Formula D

As an example of the present invention, when linker is a cleavable chemical bond, it is vc, and the structure is shown as formula e below:

formula E

In the present invention, the linker and the modified aptamer may be linked by any method known in the art, such as a phospho-oxygen bond, a phospho-sulfur bond.In one embodiment, the linker is attached to NH on the aptamer ₂ The SH groups are bonded.

In the present invention, the linker and the drug molecule may be linked by any method known in the art, such as a phosphate bond. When R3 is in a nucleoside structural form, the connecting bond of the linker and R3 in the formula is a phosphodiester bond. In one embodiment, the hydroxyl group on the linker is linked to the NH group on the drug molecule ₂ The COOH-group is attached.

Preferably, when the modified aptamer is a thiol-modified aptamer, the aptamer provides a thiol, linker is vc shown as formula E, wherein Y is maleimide, and the thiol and maleimide can be coupled to form a chemical bond; the X group of the linker provides a carboxyl group or a hydroxyl group, the R3 group of the drug provides an amino group, and the carboxyl group or the hydroxyl group and the amino group can be coupled to form a chemical bond.

In one embodiment, when the attached drug molecule R3 is in the form of a nucleoside analog, base analog structure (preferably, a nucleoside structure of gemcitabine or pentafluorouracil), the linker is a linking element such as a phosphodiester linkage.

In one embodiment, when the attached drug molecule R3 is a chemical molecule, such as a high-activity small molecule, e.g., dolastatin such as MMAE or MMAF, maytansinoid such as DM1 or DM4, etc., an alkaloid, a herbal extract, a small molecule inhibitor, etc., a linker is any chemical bond with a terminal group containing a hydroxyl group, a carboxyl group, and a maleimide group, and reacts with NH on the aptamer through a chemical reaction ₂ -, SH-etc. groups are linked;

formula F

In one embodiment, when the aptamer is a thiol-modified aptamer and the drug molecule is mMMAE, the aptamer provides a thiol, the Y group of the Linker is maleimide, and the thiol and the maleimide can be coupled to form a chemical bond; the X group of the linker provides a carboxyl group, the drug mmMAE provides an amino group, and the carboxyl group and the amino group can be coupled to form a chemical bond.

In another embodiment, when the aptamer is a thiol-modified aptamer and the drug molecule is vc-MMAE, the aptamer provides a thiol group, the Y group of Linker is maleimide, and the thiol group and the maleimide can be coupled to form a chemical bond; the X group of the linker provides a hydroxyl group, the drug vc-MMAE provides an amino group, and the hydroxyl group and the amino group can be coupled to form a chemical bond.

Formula G

In a specific embodiment, the invention also provides application of the nucleic acid coupled drug in preparing a drug for tumor targeted therapy, wherein the tumor is a tumor with PTK7 expression.

Wherein the tumor with PTK7 expression comprises one or more of breast cancer, ovarian cancer, gastric cancer, colorectal cancer or intestinal cancer, liver cancer, non-small cell lung cancer, pancreatic cancer, triple negative breast cancer, esophageal cancer, squamous cell carcinoma, head and neck cancer, prostatic cancer and the like.

In the invention, the aptamer targeted drug can target PTK7, has strong activity of killing tumor cells, can obviously reduce the growth, migration, infiltration and bone metastasis of cancer cells, promotes the apoptosis of the tumor cells and the like.

In a specific embodiment, the present invention provides a pharmaceutical composition targeting PTK7, comprising any one of the aptamers or any one of the aptamer drug co-polymers described above (e.g., aptamer drug conjugates), with or without one or more of a pharmaceutically acceptable carrier, adjuvant or diluent.

The pharmaceutical composition is selected from any one of external preparations, oral preparations and injection preparations. Wherein the external preparation is a spray or an aerosol; the oral preparation is any one of granules, capsules, tablets and vesicular agents; the injection preparation consists of the modified aptamer, a cosolvent and 0.9% sodium chloride solution or water for injection. The cosolvent is selected from any one or more of tween-80, propylene glycol, glycerol, ethanol and PEG-400.

The carrier is any one or a mixture of more than two of a slow release agent, an excipient, a filler, an adhesive, a wetting agent, a disintegrating agent, an absorption enhancer, an adsorption carrier, a surfactant and a lubricant.

In a specific embodiment, the present invention provides the use of the modified aptamers, copolymers, pharmaceutical compositions as described above for the preparation of a kit for inhibiting tumors.

In one embodiment, the invention provides a kit for preparing a medicament for inhibiting a tumor, the kit comprising:

the pharmaceutical composition described; or

The modified aptamer; or

The copolymer is described.

In a preferred embodiment, the aptamer has a longer half-life or enhanced stability than the unmodified form of the aptamer;

in a preferred embodiment, the chemotherapeutic drug is MMAE or MMAF, and the aptamer is coupled with the MMAE or MMAF to form a copolymer, wherein the molar ratio of the aptamer to the MMAE or MMAF in the copolymer is 1 (6-20); preferably 1 (8-12); more preferably 1 (9-10).

In a preferred embodiment, the drug is MMAE or MMAF.

The present invention also provides a method for treating tumors, which comprises administering an effective amount of the modified aptamer, the copolymer containing the modified aptamer, the pharmaceutical composition, etc. to an individual in need of treatment.

In one embodiment, the present invention provides a method for preparing a aptamer drug conjugate capable of targeted binding to PTK7, comprising attaching a linker to the 5' end of an aptamer, and incubating the aptamer and a drug molecule at a molar ratio of, for example, 1.

More specifically, the method comprises the steps of: dissolving 120mg of modified DNA in 24.50mL of RNA-free water, adding 24.50mL of phosphate buffer solution, uniformly mixing by using a vortex oscillator, adding dissolved vc-MMAE (8eq, 96mg,50.18mL of DMSO), reacting at room temperature overnight (16 h), precipitating by using 3M NaCl (10 mL) and absolute ethanol (500 mL) after the reaction is finished, remaining the precipitate, adding water for dissolving, passing through a membrane, and carrying out HPLC purification to obtain the aptamer drug conjugate-SGCR-YH.

In the course of research, the inventors of the present invention broken through the conventional thinking of stability modification of nucleic acids, modified nucleic acid aptamers with a group having a specific property (including a carbon chain) selected to obtain modified nucleic acid aptamers having a novel structure, which not only have significantly improved stability against nuclease, but also can bind with high specificity and high affinity to human tumors in vivo or in vitro to exert a tumor-inhibiting effect. It can also be connected, coupled or polymerized with other tumor treatment drugs to realize more excellent tumor inhibition effect. The invention also improves the selectivity of the antitumor drug on tumor cells, and the modified aptamer can release drug molecules under the action of cytoplasm after entering the tumor cells, thereby exerting the antitumor effect.

In the present invention, hybridization of polynucleotides is a technique well known to those skilled in the art, and the hybridization properties of a particular pair of nucleic acids indicate their similarity or identity. Thus, the invention also includes polynucleotides that hybridize to the nucleotide sequences of the riboaptamers of the invention and that are at least 70%, and more preferably at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, or 98%) identical between the two sequences, and which also function to specifically bind human tumor cells.

In the present invention, a polynucleotide hybridizable with the polynucleotide of the present invention under stringent conditions is particularly concerned. "stringent conditions" means: (1) Hybridization and elution at lower ionic strength and higher temperature, e.g., 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding a denaturing agent such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll,42 ℃ etc. at the time of hybridization; or (3) hybridization occurs only when two sequences have at least 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more identity, more preferably 95% or more. Furthermore, the hybridizable polynucleotide also has a function of specifically binding to the human tumor cell.

The term "pharmaceutically acceptable" means that the molecular entities and compositions do not produce adverse, allergic, or other untoward reactions when properly administered to an animal or human.

Specific examples of some substances that may serve as pharmaceutically acceptable carriers or components thereof are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose and methyl cellulose; powdered gum tragacanth; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyhydric alcohols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as wetting agents, for example sodium lauryl sulfate; a colorant; a flavoring agent; tableting agents, stabilizers; an antioxidant; a preservative; pyrogen-free water; isotonic saline solution, phosphate buffer, and the like.

When using a pharmaceutical composition, a safe and effective amount of the pharmaceutical composition of the present invention is administered to a subject in need of treatment. The term "effective amount" refers to an amount that exerts the pharmaceutical activity of the ribo-aptamer (or the antineoplastic drug linked, conjugated or polymerized thereto) to the subject receiving the drug.

In the present invention, the "subject" generally includes human, non-human primates, such as mammals, dogs, cats, horses, sheep, pigs, cows, etc., which would benefit from treatment with the above-described medicament, composition, formulation, kit or combined formulation.

In the present invention, a "therapeutically effective amount" generally refers to an amount which, after an appropriate period of administration, is capable of achieving the effect of treating the diseases as listed above.

Such effective amount will generally be an amount that can be assessed by one of skill in the art or a clinician, depending on the species, age, weight and general disease state of the subject to which the drug is to be administered, mode of administration, and the like. Of course, the particular dosage will also take into account such factors as the route of administration, the health of the patient, and the like, which are within the skill of the skilled practitioner.

In a preferred embodiment of the present invention, the nucleic acid is modified for the purpose of improving targeting property and affinity, prolonging the drug circulation time, and the like. The inventor firstly discovers that the modified RNA aptamer can specifically and obviously inhibit the proliferation, growth and the like of tumor cells, and the modified RNA aptamer provides a new strategy for treating tumors. Although the present inventors provide preferred modifications, it is to be understood that other modifications that have been used in the art to modify nucleic acids to increase their half-life or stability may also be used in the present invention.

The invention also relates to copolymers containing the ribonucleic acid aptamers. Typically, the copolymer comprises the aptamer and an anti-tumor drug linked, conjugated or polymerized to the aptamer. The inventor finds that after some antitumor drugs and the RNA aptamer form a copolymer, the RNA aptamer has good targeting property, so that the antitumor drugs can reach the tumor part and play a role of inhibiting tumors together with the RNA aptamer.

The anti-tumor drug is generally a drug with an inhibiting effect on tumors, and the anti-tumor drug can be stably connected (including coupling) with nucleic acid. The therapeutic drug does not have the targeting property of targeting human tumors, but can well reach tumor sites by being connected, coupled or polymerized with the modified ribonucleic acid aptamer. Although not specifically listed, it is contemplated that certain antineoplastic agents capable of being linked, conjugated or polymerized to the riboaptamers of the invention may be used in the invention.

In one embodiment, MMAE or DM1 is coupled with the RNA aptamer to form a nucleic acid coupled drug. In the nucleic acid conjugate drug, the molar ratio of the aptamer to MMAE or DM1 may be, for example, 1 (2 to 20).

Therefore, the inventor firstly discloses a strategy that the modified ribonucleic acid aptamer forms a copolymer with a drug molecule for treating human tumors. The copolymer can effectively inhibit the proliferation of human tumors in vitro and in vivo. The copolymer has stronger inhibitory activity on tumor proliferation than the aptamer alone and the drug alone.

In a specific embodiment, the present invention provides the use of the modified aptamer, copolymer, or pharmaceutical composition described above for the preparation of a kit for inhibiting a tumor.

the pharmaceutical composition described; or

The modified aptamer; or

The copolymer is described.

The kit may also include instructions for use or medication for the clinician and subject to use.

The invention also relates to a kit for inhibiting tumors, which can also comprise an instruction book or a medicine instruction book for facilitating the application of a clinician and a medicine object.

According to the present invention, the nucleic acid aptamer can be modified with the modifying group to improve nuclease stability, targeting property, affinity, and the like, and the modified nucleic acid aptamer is not particularly limited in the position and number of the corresponding base or a conjugate of the base and deoxyribose or ribose. According to a preferred embodiment of the invention, at least 1 of the corresponding bases in the aptamer or the conjugate of a base and deoxyribose or ribose is modified with a modifying group. According to a preferred embodiment of the invention, all such modifications are used to modify the corresponding base or the conjugate of a base and deoxyribose or ribose, as in aptamers.

In accordance with a particular embodiment of the present invention, 1, 2, 3,4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 2, 8, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, B56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 bases or conjugates of bases and deoxyribose in the aptamer as shown in formula II.

As described above, the number of the modifying group-modified aptamers is merely listed, and the number of substitutions can be adjusted as necessary. In a preferred embodiment of the invention, only one of the nucleic acid aptamers is modified with the modifying group for the corresponding base or the conjugate of a base and deoxyribose or ribose. In a preferred embodiment of the invention, the 5 'or 3' base is modified with the modifying group.

One of the beneficial effects of the invention is that: the PTK7 aptamers of the invention have: (1) Can selectively bind to PTK7 positive tumor cells, but has weak binding to PTK7 negative cells; (2) Preferably, cytotoxic drugs such as MMAE can be carried directly, selectively binding and killing PTK7 positive tumor cells.

The modified aptamer has no toxicity and excellent biocompatibility; the aptamer has small molecular weight, good cell permeability and easy absorption; the special spatial conformation of the aptamer enables the aptamer to have better structural stability and not to be easily degraded by biological enzymes in vivo. The characteristics enable the aptamer to have important application prospects in biomedical detection, tumor marker discovery and tumor clinical treatment.

In one embodiment, the modified group provided by the present invention is formed by connecting the modified group to the 5' end of the nucleotide sequence, and the modified aptamer can directly carry cytotoxic drugs (such as MMAQ) to selectively kill PTK7 positive tumor cells.

In the embodiment of the invention, the aptamer is coupled with classical chemotherapy drugs such as MMAE and DM1, the aptamer is confirmed to have better cytotoxicity, and the aptamer can selectively bind to PTK7 positive tumor cells and is weakly bound to PTK7 negative cells, so that in a further aspect of the invention, the aptamer is provided as a PTK7 positive tumor chemotherapy targeting drug carrier, the chemotherapy drugs are bound to the aptamer, and the PTK7 positive tumor cells can be selectively killed and have no toxicity to normal cells during tumor radiotherapy and chemotherapy.

According to one embodiment, the aptamer, copolymer or pharmaceutical composition of the invention may be administered as part of a combination therapy. Embodiments within the scope of the present invention therefore include co-administration of compositions and medicaments comprising additional therapeutic agents and/or active ingredients in addition to the nucleic acid aptamers, copolymers or pharmaceutical compositions of the invention as active ingredients. Such multi-drug therapy (often referred to as combination therapy) may be used for the treatment and/or prevention of any PTK7 receptor related disease or disorder, in particular those defined above.

Thus, the therapeutic methods and pharmaceutical compositions of the invention may employ the nucleic acid aptamers, copolymers or pharmaceutical compositions of the invention in the form of a monotherapy, but the methods and compositions may also be employed in the form of a combination therapy in which one or more nucleic acid aptamers, copolymers or pharmaceutical compositions are co-administered in combination with one or more other therapeutic agents.

The invention of the present application is further illustrated by the following examples, which are not intended to limit the scope of the present application.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, sambrook et al: a LABORATORY MANUAL, second edition, cold Spring Harbor LABORATORY Press,1989and Third edition,2001; ausubel et al, current PROTOCOLS IN MOLECULAR BIOLOGY, john Wiley & Sons, new York,1987and periodic updates; the series METHODS IN ENZYMOLOGY, academic Press, san Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, third edition, academic Press, san Diego,1998; (iii) METHODS IN ENZYMOLOGY, vol.304, chromatin (P.M.Wassarman and A.P.Wolffe, eds.), academic Press, san Diego,1999; and METHODS IN MOLECULAR BIOLOGY, vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, totowa,1999, etc.

Example 1: synthesis of aptamer-conjugated drug MMAE:

the structure of the thiol-modified aptamer used in this example is shown in formula II below (the 3' -end of the modified aptamer is synthesized from inverted T base):

formula II

R1 is H or PO3 ^– -CnH2n +1, n is 0-24; preferably, n is 10 to 20; further preferred isAnd n is 12-18.

R2 is PO3 ^- A carboxyl, a maleimide or an azide group.

x is 1 to 24; preferably, x is 1-8 or 4-24;

y is 1 to 6; preferably, y is 2 to 3; further preferably, y is 1.

An Aptamer is a thiol-modified nucleotide molecule (the 3' -end of a modified Aptamer is synthesized with an inverted T base).

when R2 is carboxyl, maleimide or azide, Z is amino, sulfydryl or alkynyl/DBCO on the Aptamer, x is 4-24, y is 1. By way of illustration, when R2 is carboxyl, the Aptamer provides an amino group, the carboxyl group reacts with the amino group, linking R2 to the Aptamer; when R2 is maleic amide, the Aptamer provides sulfydryl, the maleic amide reacts with the sulfydryl, and the R2 is connected with the Aptamer; when R2 is an azide group, the Aptamer provides alkynyl or DBCO, the azide group reacts with the alkynyl or DBCO, and the R2 is connected with the Aptamer.

Wherein, when R is H, x is 6, y is 2, the nucleotide sequence of the Aptamer is shown in SEQ ID NO.1 (wherein, sulfydryl is modified at the 3 'or 5' end position of the Aptamer), and the obtained modified Aptamer is marked as 2spacer18-SGC8-inverted-dT and is marked as SGCR4.

Wherein, when R is PO ₃ ^– -C _n H _2n+1 The nucleotide sequence of the aptamer, wherein n is 18, x is 6, y is 2, and the nucleotide sequence of the aptamer is shown in SEQ ID No.1 (wherein, sulfydryl is modified at the 3 'or 5' end position of the aptamer), and the obtained modified aptamer is marked as C18-2spacer18-SGC8-inverted-dT and is marked as SGCR5.

The nucleic acid aptamer is coupled with MMAE through vc, and the operation flow of the experiment is shown as follows:

the structure of the obtained aptamer-conjugated drug is as follows:

120mg of modified DNA (SGCR 4 or SGCR 5) is dissolved in 24.50mL of purified water, 24.50mL of phosphate buffer solution is added, after vortex oscillator mixing, dissolved vc-MMAE (8eq, 96mg,50.18mL DMSO) is added, reaction is carried out overnight (16 h) at room temperature, after the reaction is finished, 3M NaCl (10 mL) and absolute ethyl alcohol (500 mL) are used for precipitation, precipitate is left, water is added for dissolution, membrane passing is carried out, and HPLC purification is carried out to obtain the vc-MMAE conjugate drug (SGCR 4-vc-MMAE (YH 2502) or SGCR5-vc-MMAE (YH 2504)) with the purification yield of about 4%.

When the aptamer SGCR4 is connected with the drug MMAE through vc, the corresponding aptamer-coupled drug is marked as 2spacer18-SGC8-inverted-dT-vc-MMAE and is marked as follows: SGCR4-vc-MMAE (YH 2502).

YH2504 when the aptamer SGCR5 is linked to the drug MMAE via vc, the corresponding aptamer-conjugated drug is labeled C18-2spacer18-SGC8-inverted-dT-vc-MMAEC18-2spacer18-SGC 8-inverted-dT-dT-vc-MMAE and is designated SGCR5-vc-MMAE (YH 2504).

Example 2: synthesis of aptamer-conjugated drug DM 1:

10.42mg of DNA (SGCR 3:2spacer18-SGC 8-inverted-dT) was dissolved in 7.721mL of water, followed by 6.176mL of PB buffer (pH = 8) and then the previously dissolved drug DM1-SMCC (24.83mg, 30eq,18.53mL of DMSO) were added and reacted at 37 ℃ for 16 hours. A total of 84.37mg of DNA starting material was dosed in 8 batches in parallel, pooled and purified by direct injection in the system of 50mM TEAA in water, CH3CN. After fraction freeze-drying, desalting was performed with desalting column (glucose gel G-25), and desalting was repeated 3 times to obtain 90mg of the conjugated drug DM1-SGCR4 with a purity of 99.5%.

Example 3: comparison of cell affinity before and after aptamer modification.

After 24 hours of HCT116 cell culture, the cells were digested with enzyme-free cell digestate or 0.04% EDTA digest, collected and centrifuged (1300rpm, 5min per minute). Then, 250nM fluorescently labeled samples Random, SGC8, SGCR4 and SGCR5 were compared with 2.5X 10, respectively ⁵ Individual cells were incubated in Binding Buffer (BB). The incubation condition is 4 ℃ and the time is 45 minutes; after incubation, the cells were washed three times with washing buffer WB, detected on a flow cytometer, and the experimental data were analyzed by Flowjo software.

The Washing Buffer (WB) and Binding Buffer (BB) were formulated as follows: WB formula: DPBS 5mM MgCl ₂ 4.5mg/mL glucose (storable at 4 ℃ for 2 months); BB formulation: 0.1mg/mL tRNA and 1mg/mL bovine serum albumin (which can be stored at 4 ℃ for 1 month) were added to the wash buffer.

HCT116 is a colon cancer cell; random is a Random aptamer sequence; SGC8 is the nucleic acid sequence before chemical modification. SGCR4 and SGCR5 are two modified aptamers. The results are shown in the flow cytometric data of fig. 1, where the fluorescence peaks of SGCR4 and SGCR5 shifted more to the right, indicating an increased amount of cell binding and higher affinity, while the fluorescence of controls Random and SGC8 decreased relatively, indicating an increased affinity of the modified aptamer for the target cell HCT 116.

Example 4: before and after aptamer modification, live mice are imaged.

0.1mL (10X 10) ⁶ cells) NCI-H446 cells were inoculated subcutaneously in the right back of 6-8 week old BALB/c female mice. The tumor grows for about 10-15 days, and the average volume reaches 300-500mm ³ At the beginning of the administration, V = (a × b) is used ² ) The tumor volume was calculated by the equation/2, where V is the tumor volume, a is the tumor length, and b is the tumor width. Two groups, cy5 modified SGC8 and SGCR4, respectively. Dissolved in DPBS,10uM, 100ul/DPBS and injected via tail vein. After injection, at 2.0,3.0,4.0,5.0 hours at 4 time points, cy5 fluorescence was observed at different time points for each organ and tumor of the mouse using small animal in vivo imager (luminea XR) at the wavelength: ex:620 plus or minus 5nm; em:670 + -5 nm. At the last time point (5.0 hours)Mice were dissected and tumors, heart, liver, spleen, lung, kidney were imaged.

As a result, as shown in FIG. 2, in comparison with the case of the mouse treated with the aptamer before modification, the tumor site of the mouse treated with the aptamer after modification showed a very significant fluorescence of Cy5, and the fluorescence was less concentrated in other organs. The fluorescence metabolism of the tumor site of the aptamer-treated mice before modification disappears, and strong fluorescence can still be observed in the tumor site of the aptamer-treated mice after modification, which is observed at the three time points of 3,4 and 5 hours after injection. This indicates that the modified aptamer has stronger tumor targeting and longer retention time in the tumor. That is, the modification increases the metabolic half-life of the aptamer, allowing fluorescence to accumulate in the tumor for a long period of time.

Example 5: cytotoxicity experiments of aptamer-conjugated drugs.

Cytotoxicity experiments were carried out in 6 cell lines (NCI-H446, HCT116, MDA-MB-231, OVCAR3, PANC-1).

Cells were seeded in 96-well plates (5X 10) ⁴ one/mL), after 16 hours of incubation, the medium was removed, the nucleic acid-conjugated drug molecules prepared in example 1 (YH 2502 and YH 2504) were formulated to different concentrations with medium, with final drug concentration ranging from 1uM to 0.15nM, with a 2-fold dilution gradient, and after 48 hours of incubation with added cells, the medium was removed, CCK-8 reagent was added, incubated for 1 hour, measured on a microplate reader (absorbance at 450 nM), and the data were processed with Graphpad prism 7 to fit IC50 curves. When the concentration of the cell activity inhibitor is 50%, the lower the concentration of the drug used, the greater the toxicity. The results are shown in FIG. 3, where the toxicity of the nucleic acid-conjugated drug to NCI-H446 and OVCAR3 was the greatest and the IC50 value was the lowest.

Example 6: evaluation of in vivo anticancer Effect of nucleic acid-conjugated drugs on non-Small cell Lung cancer NCI-H446.

0.1mL (10X 10) ⁶ cells) NCI-H446 cells were inoculated subcutaneously in the right back of 6-8 week old BALB/c female mice. The tumor grows for about 10-15 days, and the average volume reaches 130mm ³ I.e., about 6 × 6mm, the administration is started by using V = (a × b) ² ) 2 publicThe tumor volume is calculated, where V is the tumor volume, a is the tumor length, and b is the tumor width. Animals were divided into 3 groups of 8 animals each, vc-MMAE, YH2502 and YH2504, where YH2502 and YH2504 represent two nucleic acid-conjugated drugs, respectively (YH 2502: SGCR4-vc-MMAE; YH2504: SGCR 5-vc-MMAE). The dose (vc-MMAE 0.266mg/kg, YH2502 and YH2504:3 mg/kg), 200ul per mouse, was administered by tail vein injection once every two days. Mice body weight and tumor volume were monitored and two weeks later, treatment was terminated. The results are shown in FIGS. 4 and 5. Changes in tumor volume in mice after treatment (FIG. 4), a reduction in tumor volume in YH2502 (SGCR 4-vc-MMAE) and YH2504 (SGCR 5-vc-MMAE) drug-treated groups, were evident compared to the reduction in tumor volume in vc-MMAE-treated mice. The change in tumor body weight in mice after treatment (fig. 5) was not significant.

The above examples are only intended to illustrate the technical solution of the present invention and not to limit it; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art will understand that: modifications of the embodiments of the invention or equivalent substitutions for parts of the technical features are possible; variations and advantages that may occur to those skilled in the art may be made without departing from the spirit and scope of the inventive concept, which is intended to be covered by the claims.

Sequence listing

<110> Shanghai university of traffic college medical school affiliated medical relief Hospital

<120> aptamer modified structure of targeting PTK7, coupled drug, preparation method and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atctaactgc tgcgccgccg ggaaaatact gtacggttag a 41

<210> 2

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

tacaggttct ggggggtggg tggggaacct gtt 33

Claims

1. A method for modifying a nucleic acid aptamer, the method comprising modifying the nucleic acid aptamer with a group of formula I to obtain a modified nucleic acid aptamer:

wherein R1 is H or PO3 ^– -C _n H _2n+1 N is 0 to 24;

r2 is PO3 ^- A carboxyl, maleimide or azide group;

x is 1 to 24; y is 1 to 6.

2. A modified aptamer having the structure of formula II:

wherein R1 is H or PO3 ^– -C _n H _2n+1 N is 0 to 24;

r2 is PO3 ^- A carboxyl, maleimide or azide group;

x is 1 to 24; y is 1 to 6; aptamer is an Aptamer.

3. The aptamer of claim 2,

when R2 is PO3 ^- When x is 1-8 and y is 1-6, R2 is connected with the Aptamer through O;

when R2 is carboxyl, maleimide or azide group, the connection is carried out through amino, sulfydryl, alkynyl or DBCO on the Aptamer respectively, x is 4-24, and y is 1;

the aptamer is selected from a nucleotide molecule or a nucleotide analogue;

wherein the nucleotide molecule is selected from a double-stranded nucleotide molecule and a single-stranded nucleotide molecule; the single-stranded nucleotide molecule is selected from cDNA, mRNA, tRNA, rRNA, miRNA, micromolecule RNA, telomerase RNA, antisense RNA, ribozyme, non-coding RNA and antisense oligonucleotide;

the nucleotide analogue is selected from nucleic acid analogues which can be combined with nucleotide molecules or nucleic acid structure molecules to play a role in regulation and control, peptide nucleic acids, nano nucleic acids, locked nucleic acids and nucleic acid proteins which are partially modified from the nucleotide molecules or the nucleic acid structure molecules, or thio nucleotides, 2 '-methoxy nucleotides, 2' -fluoro nucleotides, phosphorylated nucleotides, methylated nucleotides, aminated nucleotides, thiolated nucleotides, biotinylated nucleotides, fluorescence-labeled nucleotides, enzyme-labeled nucleotides, nano luminescent material-labeled nucleotides, folic acid-labeled nucleotides, isotopic nucleotides, PEG-modified nucleotides and nucleic acid aptamers embedded by a PEG-liposome mixture, wherein the thioacid, the aminated nucleotides, the biotinylated nucleotides, the enzyme-labeled nucleotides, the nano luminescent material-labeled nucleotides, the folic acid-labeled nucleotides, the isotopic nucleotides, the PEG-modified nucleotides and the nucleic acid aptamers are obtained by substituting the nucleotide molecules or the nucleic acid structure molecules; or a nucleic acid derivative formed by combining the nucleotide molecule and other functional nucleotides, functional polypeptides, functional proteins and antibodies in a chemical coupling or assembling mode.

4. The aptamer according to claim 2, wherein the nucleotide sequence of the aptamer is selected from any one of:

(1) A nucleotide sequence shown as SEQ ID NO.1 or a nucleotide sequence shown as SEQ ID NO. 2;

PD-L1：5'-TAC AGG TTC TGG GGG GTG GGT GGG GAA CCT GTT-3’(SEQ ID NO.2)；

(2) A nucleotide sequence which has more than 90 percent of identity with the nucleotide sequence in (1) and has the same/similar function of specifically binding human tumor cells;

(7) (1) the corresponding locked nucleic acid or peptide nucleic acid modified from the nucleotide sequence defined in any one of (1) to (4);

(8) (1) a sequence of a nucleic acid derivative formed by combining the nucleotide sequence defined in any one of (1) to (4) with other functional nucleotides, functional polypeptides, functional proteins and antibodies in a chemically coupled or assembled form.

5. Use of an aptamer according to claim 2, selected from one or more of the following:

for use as a pharmaceutical carrier;

use in drug design and development;

use for studying tumor cell surface proteins;

use in the isolation and purification of drugs;

the application in preparing copolymer and biological product.

6. Use according to claim 5, wherein the aptamer is used for the preparation of a medicament targeting PTK7 or for the treatment of a tumour with PTK7 expression.

7. A copolymer comprising the aptamer according to claim 2 and a drug molecule linked by one or more of coupling, polymerization, and attachment.

8. The copolymer according to claim 7, wherein when the nucleic acid aptamer and the drug molecule are linked by a coupling means, the copolymer is a nucleic acid-conjugated drug; wherein the nucleic acid coupling drug is a PTK 7-targeted nucleic acid coupling drug SGCR-YH, and is characterized in that the nucleic acid coupling drug is composed of a modified aptamer fragment, a drug fragment and a linker, and has a structure shown in the following formula III:

wherein R-Aptamer is a modified Aptamer according to claim 2, each group being as defined in claim 2;

r3 is a drug fragment; linker is linker.

9. The copolymer of claim 7, wherein the drug is a cytotoxic drug selected from one or more of nucleoside analogs and base analogs, metabolic drugs, high-activity small molecules, alkaloids, herbal extracts, small molecule inhibitors, plant alkaloids, anti-tumor small molecule targeted drugs, nucleic acid drugs, and protein drugs.

10. The copolymer of claim 7, wherein the drug is one or more of MMAE, MMAF, PBD dimer, DM1 or DM4.

11. Use of a copolymer according to claim 7 for the preparation of a medicament for the targeted treatment of tumors, wherein said tumors are tumors with PTK7 expression.

12. Use according to claim 11, wherein the tumor with PTK7 expression is selected from one or more of breast cancer, ovarian cancer, gastric cancer, intestinal cancer, non-small cell lung cancer, liver cancer, pancreatic cancer, triple negative breast cancer, esophageal cancer, squamous cell carcinoma, head and neck cancer or prostate cancer.

13. A pharmaceutical composition comprising the aptamer of claim 2 or the copolymer of claim 7and one or more pharmaceutically acceptable carriers, excipients, adjuvants or diluents.