Chimeric receptor ligand targeting cd20 and use thereof

Abstract

The application provides a chimeric receptor ligand targeting CD20 and an application thereof, and the HR14-TACI specific nucleic acid aptamer is obtained by constructing and preparing a human recombinant CD20 protein aptamer and a TACI protein aptamer. The cytotoxicity experiment shows that the immunological killing of the HR14-TACI specific nucleic acid aptamer on Daudi cells is strong, and the cell survival rate is only about 35%, and the killing ability of the immunocyte on the tumor cell is enhanced.

Description

Technical Field

[0001] This invention relates to the field of tumor treatment technology, and in particular to a chimeric receptor ligand targeting CD20 and its application. Background Technology

[0002] CD20 (B-lymphocyte antigen CD20) is an antigen expressed on the surface of all B cells. Non-Hodgkin's lymphoma (NHL) is the most common malignant tumor of the lymphatic system, with high incidence and mortality rates. Targeted therapy is commonly used to treat this tumor, achieving remission and improving treatment outcomes. Targeted therapy requires target proteins, and CD20 molecules are specifically expressed in over 95% of B-cell lymphomas. Since 85% of non-Hodgkin's lymphomas originate from B cells, CD20 is an ideal target for treating non-Hodgkin's lymphoma. Currently, there is no known ligand for CD20, but the CD20 molecule plays a crucial role in the immune response of B cells.

[0003] Nucleic acid aptamers are a class of short-chain nucleic acid molecules that bind to targets with high specificity and affinity, making them a class of tumor-targeting molecules with significant application potential. Constructing bispecific nucleic acid aptamers can be superior to monospecific nucleic acid aptamers. Current technology has not yet been able to construct bispecific nucleic acid aptamers targeting CD20 and TACI. Summary of the Invention

[0004] Therefore, the purpose of this invention is to propose a chimeric receptor ligand targeting CD20 and its application, and to construct and prepare a bispecific nucleic acid aptamer using CD20 and TACI.

[0005] The technical solution of this invention is implemented as follows:

[0006] This invention provides a chimeric receptor ligand targeting CD20, which is a bispecific nucleic acid aptamer; the bispecific nucleic acid aptamer is constructed and prepared by targeting recombinant human CD20 protein aptamer and TACI protein aptamer.

[0007] To further illustrate, the nucleotide sequence of the target human recombinant CD20 protein aptamer is shown in SEQ ID NO.2.

[0008] Furthermore, the bispecific nucleic acid aptamer also incorporates short-chain nucleotides.

[0009] To further clarify, the short-chain nucleotide is 20 nt in length.

[0010] This further illustrates the application of this chimeric receptor ligand targeting CD20 in the preparation of antitumor drugs.

[0011] Preferably, the tumor is a lymphoma.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0013] This invention constructs and prepares HR14-TACI specific nucleic acid aptamers by targeting recombinant human CD20 protein aptamers and TACI protein aptamers. Cytotoxicity experiments show that it has strong immune killing effect on Daudi cells, with a cell survival rate of only about 35%, thus enhancing the killing ability of immune cells against tumor cells.

[0014] Furthermore, inserting a 20nt nucleotide into the HR14-TACI specific nucleic acid aptamer enhances the killing effect of the bispecific nucleic acid aptamer on tumor cells. This may be because the inserted short-chain nucleotide avoids the interaction between monomeric aptamers, enhances the affinity of the aptamer for target cells, and thus enhances the killing effect on tumor cells. Attached Figure Description

[0015] Figure 1 The binding rate of ssDNA to CD20 protein;

[0016] Figure 2 This is a diagram showing the cytotoxicity results of this invention. Detailed Implementation

[0017] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0018] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0019] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0020] The random ssDNA library was synthesized by Shanghai Yingjun Biotechnology Co., Ltd., with the intermediate N(40) being a random sequence; PBS was a phosphate buffer solution; CBS was a carbonate buffer solution; and IPTG was an inducer (isopropyl-β-D-thiogalactoside).

[0021] Example 1 - In vitro screening of nucleic acid aptamers

[0022] (1) Recombinant human CD20 protein coupled with magnetic beads

[0023] Using recombinant human CD20 protein as the target, in vitro screening of nucleic acid aptamers was performed. Five × 10⁵ magnetic beads were washed three times with PBS buffer, resuspended in CBS solution, and 1 μg of recombinant human CD20 protein was added. The mixture was incubated at 25°C with shaking for 4 h, washed three times with PBS buffer, resuspended in PBS buffer, and stored at 4°C. Immunoglobulin IgG was prepared and conjugated to the magnetic beads using the same method.

[0024] (2) Screening of single-stranded DNA libraries

[0025] A 200 pmol random ssDNA library was diluted to 200 μL with PBS buffer, denatured at 95°C for 5 min, immediately incubated on ice for 15 min, and then CD20 protein-coated magnetic beads were added. The mixture was incubated at 25°C with shaking for 30 min. The magnetic beads were separated, and the supernatant was discarded. The magnetic beads were washed three times with PBS buffer, resuspended in PBS buffer, and denatured at 95°C for 5 min. The supernatant was collected. Using this supernatant as a PCR template, double-stranded DNA was amplified using FITC-labeled primer P1 and biotin-labeled primer P2. The product was separated by magnetic beads to obtain a single-stranded DNA library for further screening.

[0026] (3) Detection of candidate nucleic acid aptamers

[0027] Using the final enriched ssDNA library as a template, and unlabeled primers P1 and P2 as primers, PCR amplification of dsDNA was performed. An appropriate amount of PCR product and 1 μL of monoclonal vector were mixed, incubated at 25°C for 5 min, immediately placed on ice for 15 min, and DH5α competent cells were added. The mixture was then incubated with a 42°C water shock for 30 s, followed by an ice incubation for 15 min. LB medium was added, and the mixture was incubated at 37°C and 200 rpm for 1 h. The mixture was then plated and incubated overnight. White colonies were picked and transferred to LB medium, and incubated overnight at 37°C and 200 rpm. Using the bacterial culture as a template, double-stranded DNA was amplified using FITC-labeled primer P1 and biotin-labeled primer P2. The PCR product was added to CD20 protein-coated magnetic beads, and the mixture was incubated at 25°C with shaking for 30 min. The magnetic beads were separated, the supernatant was discarded, and the beads were washed three times with PBS buffer. ssDNA labeled with fluorescent groups was quantitatively analyzed using a TBS-380 fluorescence quantitative analyzer. A standard curve was plotted, and the fluorescence value of ssDNA that did not bind to CD20 was measured in each round of screening. The binding rate was calculated as follows: Binding rate (%) = Amount of ssDNA that did not bind to CD20 / Total amount of input library × 100.

[0028] Depend on Figure 1 It can be seen that after the 7th round of screening, the binding rate of the library to CD20 gradually stabilized and basically reached a plateau, which is conducive to the rapid enrichment of CD20 aptamers with high specificity and strong affinity.

[0029] Recombinant human CD20 protein was coated to a concentration of 5 μg / mL and incubated overnight at 4°C. Different concentrations of candidate nucleic acid aptamers were added, and the affinity of the aptamers for CD20 was detected using an enzyme-linked oligonucleotide assay. Absorbance values ​​were measured at 450 nm. Nonlinear fitting curve analysis was performed using Origin 8.0 software, and the dissociation constant Kd was calculated using the formula: Y = Bmax × X / (Kd + X), where Bmax is the maximum absorbance of the target binding to the aptamer, X is the concentration of the aptamer, Kd is the dissociation constant of the aptamer, and Y is the average absorbance of the aptamer binding to the target at the corresponding concentration. The results showed that the Kd value of HR14 was 12.66 nmol / L, which is in the nanomolar range and indicates good affinity. The nucleotide sequence of the HR14 aptamer is shown in SEQ ID NO. 2.

[0030] Example 2 - Construction and preparation of bispecific nucleic acid aptamers

[0031] (1) Construction of nucleic acid aptamer HR14 and TACI protein

[0032] The nucleic acid aptamer HR14 and the TACI protein aptamer, whose nucleotide sequence is shown in SEQ ID NO. 1, were amplified by high-fidelity PCR using primers P3 and P4 containing short-chain nucleotide Q2. The sequence of primer P3 containing short-chain nucleotide Q2 is shown in SEQ ID NO. 3, and the sequence of primer P4 containing short-chain nucleotide Q2 is shown in SEQ ID NO. 4. The nucleotide sequence of short-chain nucleotide Q2 is shown in SEQ ID NO. 9. The amplification length is 20 nt. The PCR products were then transformed into DH5α competent cells using pCZN1 plasmid as a vector. Positive clones were identified by PCR colony screening. Clones without short-chain nucleotide Q2 were used as control samples, with the sequence of primer P5 (shown in SEQ ID NO. 5) and primer P6 (shown in SEQ ID NO. 6).

[0033] Positive clone bacterial cultures and control samples were taken and amplified by PCR using primers P7 and P8 containing EcoRI restriction endonuclease sites. The sequence of primer P7 containing EcoRI restriction endonuclease sites is shown in SEQ ID NO.7, and the sequence of primer P8 containing EcoRI restriction endonuclease sites is shown in SEQ ID NO.8. The DNA fragments were then recovered by gel electrophoresis.

[0034] (2) Preparation of HR14-TACI specific nucleic acid aptamers

[0035] The pUC118 plasmid was digested with HindIII restriction endonuclease. After gel electrophoresis, the DNA fragments were recovered. The digested products were then added back in, and homologous fragments from the digested products were mixed with the recovered vector DNA using a homologous recombination kit for recombination. The reaction product was transformed into DH5α competent cells, incubated on ice for 15 min, heat-shocked at 42°C for 30 s, incubated on ice for 3 min, and then added to LB medium. The cells were incubated at 37°C and 200 rpm for 1 h. The cells were then plated onto LB solid medium (containing IPTG inducer and kanamycin) and incubated overnight at 37°C. Positive clones were then identified by PCR colony screening.

[0036] Positive clone bacterial suspensions were collected and added to LB medium (containing kanamycin), incubated overnight at 37°C, centrifuged at 8000 rpm for 5 min, the supernatant was discarded, lysis buffer was added to lyse the cells, centrifuged at 8000 rpm for 5 min, the supernatant was collected, DNA was extracted, and MC1061 competent cells were added. The cells were placed on ice for 15 min, heat-shocked at 42°C for 30 s, placed on ice for 3 min, added to LB medium, and incubated at 37°C and 200 rpm for 1 h. The culture was then plated onto LB solid medium (containing kanamycin) and incubated overnight at 37°C. Positive clones were then identified by PCR colony screening.

[0037] Positive clone bacterial suspensions were inoculated into LB liquid medium (containing kanamycin) and cultured at 37°C and 220 rpm until the OD600 value reached 0.4–0.5. VCSM13 phage was added and cultured at 37°C and 220 rpm for 2 h. Kanamycin was added and cultured overnight at 37°C and 220 rpm. After centrifugation at 8000 rpm for 5 min, the supernatant was collected. Phage was precipitated using PEG, collected, and lysed. DNA was extracted, EcoRI restriction endonuclease was added, and gel electrophoresis was performed to recover DNA fragments. The DNA fragments were purified to obtain HR14-TACI specific aptamers and HR14-TACI specific aptamer comparison samples.

[0038] Example 3 - In vitro antitumor effect of specific nucleic acid aptamers

[0039] Daudi cells were used as the target cells, with a cell density of 5 × 10⁶ cells per well. 5 The effector cells used were PBMC cells (PB009C-2), with an effector-to-target ratio of 5:1 and a killing time of 48 hours.

[0040] Experimental group setup: Group 1 consisted of samples containing HR14-TACI specific nucleic acid aptamers and PBMC cell culture medium; Group 2 consisted of control samples containing HR14-TACI specific nucleic acid aptamers and PBMC cell culture medium; Group 3 consisted of samples containing HR14 aptamers and PBMC cell culture medium; Group 4 consisted of samples containing TACI aptamers and PBMC cell culture medium; Group 1 consisted of samples containing HR14-TACI specific nucleic acid aptamers; and Group 2 consisted of samples containing PBMC cell culture medium.

[0041] Cell viability was determined using the MTS Cell Proliferation and Cytotoxicity Assay Kit. The calculation formula was: Cell viability (%) = (A 样品 -A 校正 ) / (A 对照 -A 校正 )×100, where A 样品 A represents the OD value of the sample. 对照 The OD value of the negative control group, A 校正 The OD value is the value of an equal volume of liquid added without the addition of cells.

[0042] Depend on Figure 2 It is known that the HR14-TACI specific nucleic acid aptamer has the strongest immune killing effect on Daudi cells, with a cell survival rate of only about 35%, while the cell survival rate of the HR14 aptamer monomer is about 70%, and it cannot enhance the killing ability of immune cells against tumor cells. Moreover, the short-chain nucleotides embedded in the HR14-TACI specific nucleic acid aptamer can further enhance this killing effect. It is possible that the embedded short-chain nucleotides avoid the interaction between monomeric aptamers, enhance the affinity of the aptamer for target cells, and thus enhance the killing effect on tumor cells.

[0043] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A bispecific nucleic acid aptamer, characterized in that, The bispecific nucleic acid aptamer was constructed and prepared by combining a target human recombinant CD20 protein aptamer with a TACI protein aptamer. The nucleotide sequence of the TACI protein aptamer is shown in SEQ ID NO.1; The nucleotide sequence of the target human recombinant CD20 protein aptamer is shown in SEQ ID NO.2; The bispecific nucleic acid aptamer also incorporates short-chain nucleotides; The short chain nucleotide is 20 nt in length, and its nucleotide sequence is shown in SEQ ID NO.

9.

2. The use of the bispecific nucleic acid aptamer as described in claim 1 in the preparation of an anti-lymphoma drug.

Images