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CN-121987810-A - DNA cube-sialidase conjugate, and preparation method and application thereof

CN121987810ACN 121987810 ACN121987810 ACN 121987810ACN-121987810-A

Abstract

The invention discloses a DNA cube-sialidase conjugate, a preparation method and application thereof, and belongs to the technical field of biological medicines. The main body of the conjugate is a DNA cube, and is formed by assembling DNA single chains of AB chain, AE chain, AD chain, AC chain, a chain, b chain and x chain with sequences shown as SEQ ID NO. 1-7 through base complementation, wherein the 5' end of the a chain or the b chain is complemented with a DNA cube bracket chain to form a double chain, the 3' end is free in a single chain form, so that four non-adjacent vertexes of the DNA cube extend out of a single-chain DNA segment, and the single-chain DNA segment is connected with sialidase at the 3' end. The DNA cube-sialidase conjugate is used for carrying out targeted degradation on sialic acid on the cell surface, can be used for targeted degradation of a tumor cell surface glycoimmune check point, has high targeting and action efficiency, and is expected to be used for enhancing immunotherapy.

Inventors

  • PAN QIUHUI
  • CHEN TIANSHU
  • LIU XUEXUE
  • MA JI
  • DING MIAO
  • TANG XIAOCHEN
  • GUO CHENGCHENG

Assignees

  • 上海交通大学医学院附属上海儿童医学中心

Dates

Publication Date
20260508
Application Date
20260226

Claims (7)

  1. 1. A DNA cube-sialidase conjugate is characterized in that the DNA cube-sialidase conjugate is mainly composed of a DNA cube, and is assembled by base complementation of an AB chain with a sequence shown as SEQ ID NO. 1, an AE chain with a sequence shown as SEQ ID NO. 2, an AD chain with a sequence shown as SEQ ID NO. 3, an AC chain with a sequence shown as SEQ ID NO. 4, an a chain with a sequence shown as SEQ ID NO. 5, a b chain with a sequence shown as SEQ ID NO. 6 and an x chain with a sequence shown as SEQ ID NO. 7, wherein the 5' end of the a chain or the b chain is complementary with a DNA cube bracket chain to form a double chain, the 3' end is free in a single chain form, a single-chain DNA segment extends out of four non-adjacent peaks of the DNA cube, and the 3' end of the single-chain DNA segment is connected with sialidase.
  2. 2. The method for preparing the DNA cube-sialidase conjugate of claim 1, comprising the steps of: S1, synthesizing a DNA cube, wherein the steps comprise the steps of mixing seven DNA single chains of an AB chain, an AE chain, an AD chain, an AC chain, an a chain, a b chain and an x chain in proportion by using a1 xTAE buffer solution containing 12.5 mM MgCl 2 , heating for 5 min at 95 ℃ and 3 min at 80 ℃, cooling to 60 ℃ at a speed of 2 min/°C, and slowly cooling to 4 ℃ at a speed of 3 min/°C to obtain the DNA cube; S2, connecting the DNA cube with sialidase, wherein the steps comprise that the sialidase is activated by 10 mu M of Sulfo-SMCC at 25 ℃ for 1h, then the sialidase is purified by an ultrafiltration centrifuge tube of 10 kD to obtain a sialidase-SMCC product, the DNA cube is mixed with TCEP at a concentration ratio of 1:10, 1h is pretreated at 37 ℃, the sialidase-SMCC product is mixed with the activated DNA cube, the reaction is carried out overnight and in a dark place at 4 ℃, and the crosslinked product of the DNA cube and the sialidase is purified by the ultrafiltration centrifuge tube of 100 kD to obtain the sialidase-connected DNA cube, namely the DNA cube-sialidase conjugate.
  3. 3. The method of preparing a DNA cube-sialidase conjugate according to claim 2, wherein in step S1, the mass ratio of AB strand, AE strand, AD strand, AC strand, a strand, b strand, and x strand is 1:1:1:1:2:2:4.
  4. 4. The method of preparing a DNA cube-sialidase conjugate according to claim 2, wherein in step S2, 1 μΜ DNA cube is cross-linkable with 0.01U sialidases per 100 μΜ final system.
  5. 5. Use of a DNA cube-sialidase conjugate according to claim 1 for the preparation of a medicament for targeted degradation of cell surface glycoimmune checkpoints.
  6. 6. A method of in vitro targeted degradation of a cell surface glycoimmune checkpoint, characterized by being achieved by a DNA cube-sialidase conjugate according to claim 1, comprising the steps of: Sa, cell glucose metabolism engineering and its connection with DBCO-d single-stranded DNA with sequence shown as SEQ ID NO. 8, comprising the steps of inoculating cells onto a cell culture dish with a density of 10 4 cells per well and attaching 12h, then adding 50 mu M Ac 4 ManAz, incubating the cells at 37 ℃ for 40 h, washing 3 times with PBS, incubating the cells with 2 mu M DBCO-d at 37 ℃ for 1 h until sialic acid on the cell surface is connected with DBCO-d, obtaining glucose metabolism engineering cells; Sb, the DNA cube-sialidase conjugate and the glycometabolism engineering cells are combined in a targeting way, and the glycometabolism engineering cells are washed for 3 times by PBS, 200 mu L of DNA cubes connected with sialidases are added, and incubated for 40 min at 37 ℃ in a dark place, so that degradation of the sialidases on the cell surfaces is realized.
  7. 7. The method of in vitro targeted degradation of cell surface glycoimmune checkpoints according to claim 6, characterized in that in step Sa, the cells are human cervical cancer cells and/or human mammary epithelial cells.

Description

DNA cube-sialidase conjugate, and preparation method and application thereof Technical Field The invention belongs to the technical field of biological medicine, and particularly relates to a DNA cube-sialidase conjugate, a preparation method and application thereof. Background Immune checkpoints are regulatory molecules in the immune system that play an inhibitory role, playing an important role in maintaining autoimmune tolerance. In tumors, the immune checkpoint pathway is typically activated to suppress the immune system, immune checkpoint inhibition therapy is an effective method of activating anti-tumor immunity. Among the most prominent targets to date in immune checkpoint blocking strategies are cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) and its ligand (PD-L1). However, it is evident from large clinical trials that only a small proportion of patients respond to anti-CTLA-4 and anti-PD-1/PDL 1 treatment and that a large proportion of patients relapse. At the same time, a large number of patients using checkpoint inhibitors may experience immune related adverse events that can affect each system. This is due to the reduced self-tolerance caused by the loss of T cell inhibition, is unpredictable and heterogeneous, and in some cases permanent and life threatening. In recent years, sialic acid on the surface of tumor cells has been found to be an emerging targetable glycoimmune checkpoint, and is receiving attention. It is capable of binding to sialic acid-binding immunoglobulin lectin (SIALIC ACID-binding Ig-LIKE LECTIN, siglec) on immune cells and inhibiting activation of immune cells by a variety of mechanisms. Studies have shown that sialic acid tends to be upregulated in malignant tumors and that activation of immune cells can be inhibited by a variety of mechanisms, which is associated with poor prognosis in tumor patients. Thus, sialoglyc-Siglec immunomodulating axis may be a major contributor to tumor immunosuppression and an attractive target for cancer immunotherapy. Currently, reported cancer treatment regimens for glycoimmune checkpoints primarily target sialic acid cleavage by direct sialidase action or design of tumor specific antibody-sialidase conjugates, thereby preventing Siglec-mediated inhibition signals and activating immune responses against cancer. However, these methods have limitations, on the one hand, that single protein-targeted antibodies are difficult to be applied to all types of cancers due to tumor heterogeneity, and on the other hand, that the degradation of sialidases is limited to the vicinity of the protein after the antibody-sialidase conjugate is bound to the protein, and that the efficiency of enzyme action is to be improved. Glycometabolism engineering is a powerful tool that can label cell membranes using chemical tags, followed by targeted binding of molecular cargo to the cell membranes by efficient chemical means. Non-natural monosaccharides such as chemically modified mannosamine, galactosamine, fucose or sialic acid, once in the cell, can be conjugated to proteins via glycometabolism pathways, processed in glycoprotein form, and expressed on the cell membrane, thereby making possession of the chemical modification on the cell membrane. These chemical modifications (azides, alkynes, etc.) expressed on the cell surface can then be used for targeted delivery of the target molecule by efficient chemical methods such as reagent-free "click chemistry" reactions between azides and Dibenzocyclooctyne (DBCO). This technology has been widely used today for cancer marking and targeting studies. Thus, the application of glycometabolism engineering to the degradation of cell surface sialic acid will aid in its targeted treatment of cancer. The DNA nano technology is used as an emerging field, and because of the characteristics of easy construction, controllable structure and size, multiple chemical modification approaches and the like of DNA molecules, a large amount of DNA or nano structure based on DNA can be precisely self-assembled in two dimensions or three dimensions, the size and shape of the DNA can be precisely controlled, and the DNA nano technology has controllable surface chemistry and dynamic functions. Meanwhile, due to biocompatibility, biodegradability and non-toxicity, the DNA nano structure has unique advantages and great potential in enhancing the targeting of target molecule delivery, and the maximum efficacy can be achieved with minimum toxicity. Thus, application of DNA nanotechnology to targeted delivery of sialidases will aid in the degradation of cell surface sialic acid. Disclosure of Invention In order to overcome the heterogeneity of antigen expression between tumors and efficiently target and shear sialic acid on the surface of tumor cells, so that the problem of universality brought by molecular phenotypes is solved, and a high-targeting and high-efficiency method for degrading the glycoim