CN113018432B - Preparation method of immune medicine - Google Patents

Abstract

The invention provides a preparation method of an immune drug, which comprises the steps of firstly preparing fusion protein containing specific binding protein and covalent link protein; preparing a nucleic acid structure containing the aptamer and the covalently linked DNA; and then incubating the fusion protein and the nucleic acid structure together, and enabling the covalent link protein and the covalent link DNA to be covalently combined to obtain the immune drug. The invention broadens the types of immunotherapy drugs, and the current protein bispecific antibody is evolved into a chimeric form with DNA involved. The preparation method of the invention overcomes the defect that the traditional bispecific antibody must be subjected to genetic engineering, shortens the synthesis preparation time of the immune drug, and reduces the production cost. The invention takes the aptamer as a target cell recognition element, and relies on the advantage that the aptamer can be combined in a multivalent way, so that the tumor recognition types are various, the binding affinity and specificity with tumor cells are improved, and the recognition range is expanded.

Description

A kind of preparation method of immunomedicine

technical field

The invention relates to the technical field of tumor immunotherapy drugs, and specifically provides a novel immunotherapy drug that mediates T cells to target and kill tumor cells, its preparation method and its application in tumor immunotherapy.

Background technique

Cancer immunotherapy, which activates the patient's immune system to prevent immune evasion by cancer, has great potential to reduce cancer metastasis and recurrence in the long-term. Unlike traditional chemotherapy or radiotherapy, tumor immunotherapy has higher selectivity and lower toxicity in tumor treatment, while providing long-term immunity through immune memory. Recent studies have shown that T cells can be redirected to recognize and eliminate tumors through bispecific molecules that bind tumor-specific antigens. Among them, due to the advantages of Bispecific T-cell engager (BiTE) antibody-mediated killing, it can bypass MHC restrictions and selectively kill tumor cells, which has attracted special attention. BiTE is essentially a polypeptide chain containing single-chain (sc) Fv domains of two different antibodies, one of which is used to recognize CD3e expressed on any T cell, and the other is used to bind tumor cells expressed tumor-associated antigens. Although they have achieved remarkable success, since BiTEs belong to protein drugs, BiTEs used to treat different tumor cells must rely on existing DNA sequences for genetic engineering, which is time-consuming and labor-intensive and its stability and activity cannot be achieved. Guaranteed. At the same time, it is impossible to quickly realize customized medical treatment according to the actual situation of the patient. In addition, the body is a multicellular environment, and the surface molecules of tumor cells and normal cells are the same, only the expression level is different. At present, due to the fixed structure and composition of BiTE, it will kill normal cells during immunotherapy, and there are certain side effects. From this point of view, nucleic acid aptamers (aptamers) can solve the existing problems of BiTE. Aptamers are called "chemical antibodies", because of their small size, easy preparation, and good targeting, they are considered ideal for tumor-targeted therapy. In this way, the scope of cancer treatment can be expanded and the drug production and development process can be accelerated. In addition, nucleic acid aptamers can also rely on the principle of complementary base pairing to quickly assemble DNA nanostructures with other DNA in test tubes. DNA nanostructures are widely used in biomedicine because of their programmability, easy modification, and high biocompatibility. And its different structures can be combined with different numbers of nucleic acid aptamers, thereby improving the ability of tumor recognition and reducing the side effects of immunotherapy.

Therefore, there is an urgent need in this field to develop an immune drug that uses nucleic acid to recognize tumor cell surface molecules and its preparation method to solve the problems of time-consuming and labor-intensive research and development of existing immunotherapy, limited therapeutic objects and side effects.

Contents of the invention

In the present invention, a nucleic acid aptamer is used to replace an antibody domain in the current BiTE to recognize tumor cell surface molecules. The antibody-nucleic acid chimera developed by us can solve the problems in the prior art as a new type of bispecific drug .

Therefore, the present invention firstly provides an immune drug, which is a bispecific immune drug (1) comprising a specific binding protein (11) and a nucleic acid aptamer (12), and the specific binding protein (11) Connect with the nucleic acid aptamer (12) directly or indirectly, the specific binding protein (11) itself is a protein structure, the nucleic acid aptamer (12) itself is a nucleic acid structure, the specific The binding protein (11) is used for specifically binding to immune cell receptors or target cell receptors, and correspondingly, the nucleic acid aptamer (12) is used for specifically binding to target cell receptors or immune cell receptors.

In a specific embodiment, a covalently linked protein (13) and a covalently linked DNA (14) are linked between the specific binding protein (11) and the nucleic acid aptamer (12), and the covalently linked The connexin (13) is connected to the specific binding protein (11), the covalently connected DNA (14) is directly or indirectly connected to the nucleic acid aptamer (12), and the covalently connected protein (13) and the covalently connected DNA (14) are covalently bonded.

In a specific embodiment, a connecting structure is also connected between the covalently linked DNA (14) and the nucleic acid aptamer (12), and the connecting structure is also composed of nucleotides.

In a specific embodiment, the covalent linking protein (13) is a HUH family protein, preferably a DCV protein.

In a specific embodiment, the immune drug contains one nucleic acid aptamer (12) or multiple identical or different nucleic acid aptamers (12), preferably the immune drug contains 3 to 100 identical or different nucleic acid aptamers.

In a specific embodiment, the specific binding protein (11) is αCD3.

In a specific embodiment, the nucleic acid aptamer (12) is one or more of sgc8, TE02, SYL3C, MUC1 and C-MET.

In a specific embodiment, the sequence of the covalently linked DNA (14) is aagtattaccagaaa.

The present invention also provides an application of the above immunomedicine in tumor immunotherapy.

In a specific embodiment, the immune drug targets and inhibits the growth of tumor cells in vivo.

The present invention also provides a method for preparing an immunomedicine, the immunomedicine is a bispecific immunomedicine (1) comprising a specific binding protein (11) and a nucleic acid aptamer (12), and the specific binding protein (11 ) and the nucleic acid aptamer (12), the specific binding protein (11) itself is a protein structure, the nucleic acid aptamer (12) itself is a nucleic acid structure, and the specific binding protein (11) ) is used to specifically bind to immune cell receptors or target cell receptors, and correspondingly, the nucleic acid aptamer (12) is used to specifically bind to target cell receptors or immune cell receptors; the specific binding A covalently linked protein (13) and a covalently linked DNA (14) are connected between the protein (11) and the nucleic acid aptamer (12), and the covalently linked protein (13) is directly connected to the specific binding protein (11). or indirectly connected, the covalently linked DNA (14) is directly or indirectly connected to the nucleic acid aptamer (12), and covalently binds between the covalently linked protein (13) and the covalently linked DNA (14); the The preparation method comprises the following steps,

Step A: preparing a fusion protein containing the specific binding protein (11) and a covalently linked protein (13);

Step B: preparing a nucleic acid structure containing the nucleic acid aptamer (12) and covalently linked DNA (14);

Step A and step B can be carried out simultaneously or arbitrarily sequentially;

Step C: co-incubating the fusion protein prepared in step A and the nucleic acid structure obtained in step B, so that the covalently linked protein (13) and the covalently linked DNA (14) are covalently combined to obtain the immune drug.

In a specific embodiment, firstly, the nucleic acid sequence of the fusion protein is synthesized completely and a recombinant plasmid is constructed, and then the active fusion protein is expressed by prokaryotic Escherichia coli.

In a specific embodiment, after step A, purification of the obtained fusion protein is also included.

In a specific embodiment, in step B, the nucleic acid structure also includes a linking structure for linking the nucleic acid aptamer (12) and covalently linking the DNA (14), and the linking structure is also composed of nucleotides constitute.

In a specific embodiment, the nucleic acid structure includes any one of single-stranded aptamers, Y-shaped DNA nanoparticles and dendritic DNA nanoparticles, wherein the single-stranded aptamers contain one nucleic acid Aptamers (12), the Y-shaped DNA nanoparticles contain 2 nucleic acid aptamers (12), the dendritic DNA nanoparticles contain 3 to 100 nucleic acid aptamers (12), the Y The nucleic acid aptamers (12) contained in the type DNA nanoparticles and the dendritic DNA nanoparticles are the same or different.

In a specific embodiment, Y-shaped DNA nanoparticles are prepared through an enzyme-free self-assembly process, and the dendritic DNA nanoparticles are synthesized in vitro by polymerization chain reaction.

In a specific embodiment, the incubation method is to mix the fusion protein prepared in step A and the nucleic acid structure obtained in step B, and then incubate at 36-38°C for more than 10 minutes to obtain an immunomedicine. The obtained immunomedicine is stored under the condition of 2-8°C for future use.

In a specific embodiment, the covalently linked protein (13) is a HUH family protein, preferably a DCV protein, and the sequence of the covalently linked DNA (14) is aagtattaccagaaa.

In a specific embodiment, the specific binding protein (11) is αCD3.

In a specific embodiment, the nucleic acid aptamer (12) is one or more of sgc8, TE02, SYL3C, MUC1 and C-MET.

The present invention has at least the following beneficial effects:

1) The present invention utilizes antibody single-chain-DCV fusion protein and nucleic acid aptamer to covalently combine to prepare bispecific antibody-nucleic acid chimera, which broadens the types of immunotherapeutic drugs, and evolves from current bispecific antibody to DNA participating chimeric form.

2) The present invention uses antibody-nucleic acid chimeras to mediate T cells to kill tumor cells. The generation process of the chimeras overcomes the shortcomings of traditional bispecific antibodies that must undergo genetic engineering, shortens the synthesis and preparation time of immune drugs, and reduces the production time. cost.

3) The present invention uses nucleic acid aptamers as target cell recognition elements, relying on the advantages of many nucleic acid aptamer targets and the ability to programmatically assemble into nanostructures, the chimera has various types of tumor recognition, which improves the affinity and ability to bind to tumor cells. Specificity, expanding the range of specific recognition of different tumors.

4) The present invention rapidly assembles a DNA nanostructure with a specific structure and a specific valence state based on the principle of complementary base pairing. Chimeras formed by different DNA nanostructures and antibody single chains can achieve different therapeutic effects. It is possible to become a customized immune reagent.

Description of drawings

Fig. 1 is a schematic diagram of the combination of the immune drug of the present invention with T cells (ie, immune cells) and target cells (ie, tumor cells).

Fig. 2 is the detection of the effect of the antibody-sgc8 aptamer chimera on tumor killing at the cell level.

Figures 3 to 4 all verify the universality of the application of antibody-nucleic acid chimeras in the field of immunotherapy.

5 to 8 are schematic diagrams of the antibody-nucleic acid chimera targeting and killing tumor cells in vivo. Among them, Fig. 5 and Fig. 6 are the statistical results of continuous measurement of tumor size, and Fig. 7 and Fig. 8 are the statistical results of continuous measurement of mouse body weight.

In Figure 1: immune drug 1, specific binding protein 11, nucleic acid aptamer 12, covalently linked protein 13, covalently linked DNA 14, T cell 2, T cell receptor 21, target cell 3, target cell receptor 31 .

The features and advantages of the present invention can be further understood through the following detailed description in conjunction with the accompanying drawings. The examples provided are only illustrative of the method of the present invention and do not limit the rest of the present disclosure in any way.

detailed description

In order to facilitate the understanding of the present invention, the following will describe the present invention more fully and give preferred embodiments of the present invention. However, the present invention can be implemented in many different combinations and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

The present invention provides a novel immune drug for tumor immunotherapy. The drug includes two modules: a protein module and a nucleic acid module. The two modules can be covalently connected to form a chimera, and the chimera can mediate the specificity between T cells and target cells. Sex recognition, targeted inhibition of tumor cell growth in vitro and in vivo. The antibody-nucleic acid chimera has the structure as shown in FIG. 1 .

Another object of the present invention is to provide a method for preparing the above-mentioned antibody-nucleic acid chimera, which includes the following steps: the antibody is illustrated by taking DCV-αCD3 fusion protein as an example;

1) Preparation of antibodies;

1) Whole gene synthesis of recombinant plasmids containing antibody sequences, such as pET28a-DCV-αCD3.

2) Prokaryotic expression of an active DCV-αCD3 fusion protein (that is, a fusion protein formed by linking the specific binding protein 11 and the covalent linking protein 13).

Preferably, the expression and purification method of the DCV-αCD3 fusion protein is as follows: pET28a-DCV-αCD3 recombinant plasmid is heat-shocked to transform Escherichia coli BL21 (DE3) competent, 1mM IPTG induces its expression of the target protein, collects the bacterial liquid, and after ultrasonic disruption, 6*His-DCV-αCD3 fusion protein was purified by NI column.

2) Preparation of nucleic acid;

Nucleic acids include three forms: single-stranded aptamers, Y-shaped DNA nanoparticles and dendritic DNA nanoparticles.

Optionally, the single-chain aptamer of the present invention is illustrated by taking the sgc8 aptamer as an example, and its sequence is as follows:

aagtattaccagaaaaaaaaaaaaaaaATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA. Wherein said aagtattaccagaaaaaaaaaaaaaaa is the covalently linked DNA in the present invention (14).

The number of nucleic acid aptamers (aptamers) connected to the Y-shaped DNA nanoparticles is 2, and the DNA nanoparticles can be the same or different. The Y-shaped DNA nanoparticle described in the present invention is assembled with three nucleic acid strands, and its sequence is as follows:

Y1-DCV: aagtattaccagaaa TTTTTGAC CGA TGG ATG ACT TAC GAC GCA CAA GGAGAT CAT GAG;

Y2-sgc8: ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGATTTTTGAC CGA TGG ATGACC TGT CTG CCT AAT GTG CGT CGT AAG;

Y3-sgc8: ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGATTTTTGAC CGA TGG ATGACT CAT GAT CTC CTT TAG GCA GAC AGG.

Wherein said aagtattaccagaaa is the covalently linked DNA in the present invention (14).

The number n of nucleic acid aptamers linked to the dendritic DNA nanoparticles is less than 100, the specific number n can be any integer, and the DNA nanoparticles can be the same or different. The present invention exemplifies that the structure used to assemble dendritic DNA nanoparticles is assembled with five nucleic acid strands, and its sequence is as follows:

H1: TTAACCCACGCCGAATCCTAGACTCAAAGTAGTCTAGGATTCGGCGTGAAAAAGTGAGCACGGACG;

H2: GACGTGCAGGCTGTTTAAAGTCTAGGATTCGGCGTGGGTTAACACGCCGAATCCTAGACTACTTTG;

Sgc8c-SH2: CAGCCTGCACGTCATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA;

DNA1(C1)NC: CGTCCGTGCTCAC;

DCV Initiator (I): aagtattaccagaaaAGTCTAGGATTCGGCGTGGGTTAA.

Among them, the above sequence aagtattaccagaaa is the DCV protein targeting DNA sequence, that is, the covalently linked DNA (14) in the present invention, which is used for covalently linking the antibody to the nucleic acid structure, and this sequence is the covalently linked DNA (14) in the present invention .

3) preparing antibody-nucleic acid aptamer chimera;

Incubate the antibody in step 1) and the nucleic acid in step 2) to obtain the antibody-nucleic acid chimera.

The present invention also provides the application of the antibody-nucleic acid chimera in tumor immunotherapy.

In one of the embodiments of the present invention, the effect of the bispecific antibody-nucleic acid chimera sgc8-DCV-αCD3 mediating the killing of tumor cells by T cells was detected in vitro, and the CCK8 method was used to detect its effect on CCRF-CEM human acute lymphoblastic leukemia cells at the cell level targeted killing effect. Results: The prepared sgc8-DCV-αCD3 chimera has the ability to mediate T cells to kill tumor cells, and with the increase of the drug concentration of sgc8-DCV-αCD3 chimera, the killing effect gradually increases.

In one embodiment of the present invention, the antibody-nucleic acid chimera prepared by replacing sgc8 in the above embodiment with any other nucleic acid aptamer (TE02, SYL3C, MUC1 and C-MET used in this embodiment), the cellular level utilizes The CCK8 method was used to detect the killing effect of chimera-mediated T cells on different types of tumor cells. The results show that the chimeras connected with different nucleic acid aptamers can all mediate T cells to kill tumor cells, which shows that the present invention has universality in the field of immunotherapy.

In one embodiment of the present invention, the effect of antibody-nucleic acid aptamer nanostructure chimera mediating T cells to kill tumor cells in vitro was studied. Using the antibody-aptamer nanostructure chimera as a drug, the CCK8 method was used to detect its targeted killing effect on MCF-7 human breast cancer at the cellular level. The results show that the prepared antibody-dendrimer nanostructure chimera has the best effect in mediating T cells to kill tumor cells, followed by the antibody-Y-type aptamer nanostructure. Antibody-Y aptamer nanostructures represent bivalent DNA nanoparticles. In addition to the Y-shaped structure in this embodiment, other nanostructures connected with a certain number of nucleic acid aptamers may also be used. In addition dendritic DNA nanostructures represent DNA nanostructures synthesized in vitro by polymerization chain reaction.

In a specific embodiment of the present invention, a nude mouse model was used to conduct an in vivo antibody-nucleic acid chimera targeting experiment on subcutaneous tumor-bearing CEM in nude mice. Taking the sgc8 aptamer as an example, the antibody-nucleic acid chimera with different structures was explored. Effects of Synthetic Tumor Immunotherapy. The effect of immunotherapy was reflected by continuous measurement of tumor size and measurement of mouse body weight. The results showed that the tumors in the experimental group injected with the antibody-nucleic acid chimera were significantly smaller than those in the control group. In the experimental group, injection of antibody-dendrimer aptamer nanostructure chimera significantly inhibited tumor growth, followed by antibody-Y-type aptamer nanostructure chimera. By constructing DNA nanoparticles with different structures, the effect of immunotherapy can be improved, and it also provides a new idea for tumor treatment.

All in all, the antibody-nucleic acid chimera provided by the present invention is a new type of immunotherapeutic drug, which is first applied to T cell-mediated immunotherapy. The drug includes an antibody module and a nucleic acid module. The antibody module can be any antibody single chain that can recognize immune cell surface molecules, and the nucleic acid module can also be any aptamer that can recognize tumor cell surface molecules. In addition, the present invention constructs DNA nanoparticles with different structures on the basis that antibodies can form chimeras with nucleic acids, and applies functionalized DNA nanoparticles to the field of immunotherapy. DNA nanoparticles include nanostructures connected with a specific number of aptamers (such as the number of aptamers in a Y-shaped structure n = 2, etc.) and nanostructures of multiple aptamers formed by polymerization chain hybridization (HCR) (such as The number of aptamers in the dendritic structure is n<100), wherein the multivalent DNA nanoparticles can be the same DNA sequence or different DNA sequences.

The bispecific immune drug in the present invention means that the immune drug has specificity for both T cells (immune cells) and target cells (tumor cells). In the prior art, the bispecific immune drugs used to connect T cells and target cells either have both modules as proteins or both modules as nucleic acids. However, the present invention first proposes a bispecific immune drug containing a protein module and a nucleic acid module. Specific immune drugs.

Regarding the preparation method of the immune drug of the present invention: In 2018, there was a literature report on the covalent binding of covalently linked protein 13 and covalently linked DNA14. The present invention prepared a covalently linked protein 13 according to the literature, and then prepared The binding substance of specific binding protein 11 and covalent linking protein 13, and the covalently linking DNA 14 matching the covalent linking protein 13 and the nucleic acid aptamer 12 are first connected to form a nucleic acid structure (the nucleic acid structure can be customized by a nucleic acid manufacturer), and then Utilize the covalent linkage of covalently linking protein 13 and covalently linking DNA14 to obtain the immune drug comprising specific binding protein 11, nucleic acid aptamer 12, covalently linking protein 13 and covalently linking DNA14, the immune drug The structure includes specific binding protein 11, covalently linked protein 13, covalently linked DNA 14 and nucleic acid aptamer 12 from one end to the other in sequence. In the present invention, the covalent linking protein 13 can specifically be HUH protein, more specifically DCV protein, and of course it can also be proteins of many families other than HUH, which can realize covalent linking of proteins and DNA.

The specific binding protein 11 in the present invention can bind to the receptor of T cells, and at this time, the nucleic acid aptamer 12 binds to the receptor of the target cell, or vice versa, that is, the nucleic acid aptamer 12 binds to the receptor of the T cell Binding, while specific binding protein 11 binds to the receptor of the target cell.

The nucleic acid aptamer 12 in the present invention is indispensable. The essence of the nucleic acid aptamer is nucleic acid, and ordinary DNA is also nucleic acid, but ordinary DNA does not have the ability to specifically recognize receptors that nucleic acid aptamers have, so It cannot be used in the present invention.

In addition, because it is difficult to directly connect ordinary proteins and ordinary nucleic acid chains, covalently linking protein 13 and covalently linking DNA 14 are specifically used in the present invention to connect specific binding protein 11 and nucleic acid aptamer 12, as reported in the literature The covalent connection protein 13 is the DCV protein of the HUH family, which belongs to the protein member of duck ring virus. Compared with other similar proteins, such as: PCV (porcine virus), DCV has the highest connection efficiency with DNA. Therefore, in the present invention, first The corresponding DCV protein was expressed as indicated in the literature.

Example 1

This example is the preparation of DNA nanoparticles.

One, single-stranded aptamers.

A DCV protein recognition sequence was added to the 5' end of the published aptamer sequence, named dcv-aptamer. Sent to the nucleic acid synthesis facility in Shanghai to synthesize single-stranded aptamers.

Second, prepare Y-shaped DNA nanoparticles.

1. Order three nucleotide chains: YA-sgc8, Yb-DCV and YC-sgc8.

2. Synthesis of Y-shaped DNA nanoparticles: Y-shaped DNA macromolecules were prepared through an enzyme-free self-assembly process. First, a Y-shaped junction scaffold (Y-DNA) was prepared as a building block. In the experiment, the above three oligonucleotide chains contained aptamers and DCV protein target sequences (YA-sgc8, Yb-DCV, YC-sgc8 sequences), and the above sequences were treated with PBS-MgCl ₂ solution (10mM, pH 7.4 , containing 150mM NaCl and 2mM MgCl ₂ ) dissolved at a concentration of 10mM. The DNA mixed solution was heated in boiling water for 5 min, then slowly cooled to room temperature, and kept in a refrigerator at 4°C for later use.

Third, the preparation of dendritic DNA nanoparticles.

1. Order the five nucleotide chains required to build the structure, namely H1, H2, sgc8c-SH2, DNA1(C1)NC and DCV Initiator(I) as described above.

2. Dissolve and dilute the above five chains with PBS-MgCl ₂ solution to a final concentration of 10mM. First, mix and dilute equal amounts of H1, H2, sgc8c-SH2 and DNA1(C1)NC to a final concentration of 20μM. DNA The mixed solution was heated in boiling water for 5 min, then slowly cooled to room temperature. After cooling down, add 4 μM DCV initiator (I) to the DNA mixture, and put it in 37° C. for overnight reaction. The assembled DNA nanoparticles were placed in a 4°C refrigerator for later use.

Example 2

This example is the preparation of antibody-nucleic acid chimera.

The DCV-αCD3 fusion protein was added to the dcv-sgc8 nucleic acid aptamer and mixed in equal amounts, and incubated at 37°C for 15 minutes. Then store at 4°C for later use.

Example 3

This example is an application detection of antibody-nucleic acid chimera in vitro and in vivo for targeted killing of tumors. Take dcv-sgc8 (nucleic acid aptamer for human acute lymphoblastic leukemia) as an example to prepare "nucleic acid aptamer-DCV-αCD3 chimera".

1. The CCK8 method at the cellular level detects the targeted killing effect of T cells mediated by "nucleic acid aptamer-DCV-αCD3 chimera".

1. CCRF-CEM cells (5% FBS+1640) and PBMC cells (5% FBS+1640+IL2) in good condition were raised in advance;

2. The above CEM cells (2*10 ⁴ cells) were incubated with PBMC cells (human peripheral blood mononuclear cells) (1*10 ⁵ cells) and different concentrations of sgc8-DCV-αCD3 at 37°C for 24 hours.

3. Use the CCK-8 kit (product number: C0039, Biyuntian) to detect the reduction in the number of cells when sgc8-DCV-αCD3 mediates T cells to target and lyse CEM cells, so as to judge its killing effect. CCRF-CEM human acute lymphoblastic leukemia was placed in each well of a 96-well plate. A control group was set to include only T cells and CEM cells (human acute lymphoblastic leukemia cells) and negative cells Ramos. Simultaneously evaluate the effect of the ratio of tumor cell and effector cell numbers, CEM cells (2*10 ⁴ cells) with different numbers of PBMC cells (0, 1*10 ⁵ , 2*10 ⁵ ) and 100nm sgc8-DCV-αCD3 chimera Incubate at 37°C for 24 hours.

4. After 24 hours, add 20ul CCK-8 solution to each well. After incubation at 37°C for 2 hours, the absorbance at 570 nm was measured with Bio-Tek and SYNERGYMx. The calculation method of cytotoxicity is cell killing rate (%)=100 (experimental value-experimental low control)/(high control-low control).

Results: The new immune drug prepared by the above method (sgc8-DCV-αCD3 as an example) has the ability to mediate T cells to kill tumor cells, and as the concentration of sgc8-DCV-αCD3 increases, the killing effect gradually increases. as shown in picture 2. In Figure 2, CEM is human acute lymphoblastic leukemia cells, and Ramos is negative cells.

Figure 3 and Figure 4 both verify the universality of the application of antibody-nucleic acid chimeras in the field of immunotherapy. Among them, NC, sgc8, TE02, SYL3C, MUC1 and C-MET all belong to nucleic acid aptamers, while MCF-7 in Figure 4 is a human breast cancer cell, Hela is a cervical cancer cell, and HepG2 cell is a liver cancer cell. LO2 is normal human liver cells, and SH-SY5Y is human neuroblastoma cells.

Second, detect the targeted killing effect of sgc8-DCV-αCD3 chimera on CCRF-CEM human acute lymphoblastic leukemia at the living level.

1. Subcutaneous tumor-bearing CCRF-CEM in female nude mice aged 4-6 weeks on day 0, 1*10 ⁶ cells/mouse;

2. On the 7th day, the tumor-bearing nude mice were randomly divided into two groups, group A was the PBS control group, group B was the T cell injection group, and group B was further divided into 5 groups: blank control group, DCV-αCD3 antibody control group , sgc8-DCV-αCD3 experimental group, bivalent sgc8-DCV-αCD3 experimental group and multivalent sgc8-DCV-αCD3 experimental group.

3. Throughout the experiment, use a vernier caliper to measure the tumor size every 2 days and record it;

4. Throughout the experiment, the body weight of the tumor-bearing nude mice was recorded;

5. End the experiment until 32 days.

The experimental results are shown in Figures 5-8. Among them, Fig. 5 and Fig. 6 are the statistical results of continuous measurement of tumor size, and the results show that the tumors in the sgc8-DCV-αCD3 experimental group were significantly smaller than those in the protein-injected experimental group. It also showed that the multivalent sgc8-DCV-αCD3 (dendritic DNA nanostructure) experimental group had significantly smaller tumors than the bivalent sgc8-DCV-αCD3 (Y-shaped DNA nanostructure) experimental group and sgc8-DCV-αCD3 (linear single-stranded) experimental group. The improvement of valence can improve the effect of immunotherapy. Figure 7 and Figure 8 are the statistical results of continuous measurement of the body weight of the tumor-bearing nude mice, and the results show that the injection of drugs does not affect the life activities of the tumor-bearing nude mice.

In summary, the antibody-nucleic acid chimera constructed in the present invention can be used as a drug to mediate T cells to target and kill tumors. And by constructing DNA nanostructures in vitro, the effect of immunotherapy can be customized and adjusted.

The above content is a further detailed description of the present invention in combination with specific preferred embodiments, and it cannot be assumed that the specific implementation of the present invention is limited to these descriptions. For those of ordinary skill in the technical field of the present invention, without departing from the concept of the present invention, some simple deductions and substitutions can be made, which should be regarded as belonging to the protection scope of the present invention.

Claims (7)

1. A method for preparing an immune drug, the immune drug is a bispecific immune drug (1) comprising a specific binding protein (11) and a nucleic acid aptamer (12), and the specific binding protein (11) and Indirect connection between nucleic acid aptamers (12), the specific binding protein (11) itself is a protein structure, the nucleic acid aptamer (12) itself is a nucleic acid structure, and the specific binding protein (11) uses To specifically bind to immune cell receptors or target cell receptors, correspondingly, the nucleic acid aptamer (12) is used to specifically bind to target cell receptors or immune cell receptors; the specific binding protein ( 11) and the nucleic acid aptamer (12) are connected with a covalent linking protein (13) and a covalently linking DNA (14), and the covalent linking protein (13) is directly or indirectly linked to the specific binding protein (11) The covalently linked DNA (14) is directly or indirectly linked to the nucleic acid aptamer (12), and the covalently linked protein (13) is covalently bound to the covalently linked DNA (14); the nucleic acid adapter The nucleic acid structure at the ligand (12) includes any one of Y-shaped DNA nanoparticles and dendritic DNA nanoparticles, the Y-shaped DNA nanoparticles contain two nucleic acid aptamers (12), and the dendritic The DNA nanoparticles contain 3-100 nucleic acid aptamers (12), and the nucleic acid aptamers (12) contained in the Y-shaped DNA nanoparticles and the dendritic DNA nanoparticles are the same or different; the specific The sex-binding protein (11) is αCD3, the covalently linked protein (13) is DCV protein, and the sequence of the covalently linked DNA (14) is aagtattaccagaaa; the preparation method includes the following steps, Step A: preparing a fusion protein containing the specific binding protein (11) and a covalently linked protein (13); Step B: preparing a nucleic acid structure containing the nucleic acid aptamer (12) and covalently linked DNA (14); Step A and step B can be carried out simultaneously or arbitrarily sequentially; Step C: co-incubating the fusion protein prepared in step A and the nucleic acid structure obtained in step B, so that the covalently linked protein (13) and the covalently linked DNA (14) are covalently combined to obtain the immune drug. 2. The preparation method according to claim 1, characterized in that, in step A, firstly, the nucleic acid sequence of the fusion protein is fully genetically synthesized and a recombinant plasmid is constructed, and then the active fusion protein is expressed by prokaryotic Escherichia coli. 3. The preparation method according to claim 1, characterized in that, after step A, further comprising purifying the resulting fusion protein. 4. The preparation method according to claim 1, characterized in that, in step B, the nucleic acid structure further includes a linking structure for linking nucleic acid aptamers (12) and covalently linking DNA (14), the Linking structures are likewise composed of nucleotides. 5. The preparation method according to claim 1, characterized in that, in step B, the Y-shaped DNA nanoparticles are prepared through an enzyme-free self-assembly process, and the dendritic DNA nanoparticles are synthesized in vitro by polymerization chain reaction. 6. The preparation method according to claim 1, wherein in step C, the incubation method is to mix the fusion protein prepared in step A with the nucleic acid structure obtained in step B at 36-38°C Incubate for more than 10 minutes to obtain the immune drug, and then store the obtained immune drug at 2-8°C for future use. 7. The preparation method according to any one of claims 1 to 6, characterized in that, the nucleic acid aptamer (12) comprises one or more of sgc8, TE02, SYL3C, MUC1 and C-MET kind.

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