GluN2B single-domain antibody and fusion protein, related biological material and application thereof

By developing a highly specific and high-affinity GluN2B single-domain antibody NB001 and fusing it with a human Fc tag to form a GluN2B single-domain antibody-Fc fusion protein, the problem of insufficient specificity and penetration ability of existing GluN2B antagonists in the treatment of cerebral ischemia was solved, and significant anti-cerebral ischemia effects were achieved.

CN120647755APending Publication Date: 2025-09-16HEBEI UNIV OF SCI & TECH
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Patent Information

Application Number
CN202510576895.1
Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Filing Date
2025-05-06
Publication Date
2025-09-16

AI Technical Summary

Technical Problem

Existing GluN2B antagonists or monoclonal antibodies have poor specificity, low affinity or weak tissue penetration in the treatment of cerebral ischemia and have failed to achieve clinical translation.

Method used

A GluN2B single-domain antibody NB001 was developed and fused with a human Fc tag to form a GluN2B single-domain antibody-Fc fusion protein, which was prepared through genetic recombination technology to enhance its specificity and affinity and improve tissue penetration ability.

Benefits of technology

GluN2B single-domain antibody-Fc fusion protein significantly alleviated NMDA-induced neuronal excitotoxic damage in vitro, significantly reduced cerebral infarction volume in vivo, improved neurological deficits, and enhanced animal motor coordination and learning and memory abilities.

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Abstract

The invention belongs to the technical field of biomedicine, and discloses a GluN2B single-domain antibody as well as a fusion protein, a related biological material and application thereof. The amino acid sequence of the GluN2B single-domain antibody disclosed by the invention comprises a sequence as shown in SEQ ID No.1 or a sequence which has at least 80% of identity with the sequence as shown in SEQ ID No.1. The GluN2B single-domain antibody has the advantages of high characteristics, high affinity, strong tissue penetrating ability and the like, and the fusion protein obtained after the GluN2B single-domain antibody is fused with a humanized tag shows a remarkable anti-cerebral ischemia effect in vivo and in vitro, for example, the GluN2B single-domain antibody can be used for remarkably reducing the cerebral infarction volume of MCAO rats, the nerve function defect score and the crawling dislocation rate in a grid experiment; the total movement distance, the central area movement distance, the central area detention time and the number of times of entering the central area of MCAO animals are remarkably increased, and the target quadrant path proportion, the detention time and the number of times of crossing the platform in the water maze experiment are remarkably increased. The invention can be applied to the field of preparation of medicaments for treating cerebral ischemia.
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Claims

1. A GluN2B single-domain antibody, characterized in that: The amino acid sequence thereof includes the sequence shown in SEQ ID No. 1 or a sequence having at least 80% identity with the sequence of SEQ ID No.

1.

2. A GluN2B single-domain antibody fusion protein, characterized in that: It comprises the GluN2B single domain antibody as claimed in claim 1 and a tag protein.

3. The GluN2B single-domain antibody fusion protein according to claim 2, wherein: The tag protein includes at least one of a human Fc tag, a CH3 domain tag, or a human albumin binding tag.

4. The GluN2B single-domain antibody fusion protein according to claim 2 or 3, wherein: including GluN2B single domain antibody-Fc fusion protein; The amino acid sequence of the GluN2B single domain antibody-Fc fusion protein includes the sequence shown in SEQ ID No. 4 or a sequence having at least 80% identity with the sequence of SEQ ID No.

4.

5. The biomaterial related to the GluN2B single-domain antibody fusion protein according to any one of claims 2 to 4, characterized in that: The biological material is any one of the following (I) to (IV): (I) a nucleic acid molecule encoding the GluN2B single-domain antibody fusion protein according to any one of claims 2 to 4; (II) an expression cassette containing the nucleic acid molecule described in (I); (III) a recombinant vector containing the nucleic acid molecule described in (I), or a recombinant vector containing the expression cassette described in (II); (IV) A recombinant cell containing the nucleic acid molecule described in (I), or a recombinant cell containing the expression cassette described in (II), or a recombinant cell containing the recombinant vector described in (III).

6. The biomaterial related to the GluN2B single-domain antibody fusion protein according to claim 5, characterized in that: The nucleic acid molecule encoding the GluN2B single domain antibody-Fc fusion protein is any one of the following genes (i) to (v): (i) the coding sequence is positions 70 to 1110 from the 5′ end of SEQ ID No. 5; (ii) the nucleotide sequence of which is positions 1 to 1110 from the 5′ end of SEQ ID No. 5; (iii) the nucleotide sequence of which is shown in SEQ ID No. 5; (iv) A gene that hybridizes with the gene represented by (i), (ii) or (iii) and encodes the GluN2B single domain antibody-Fc fusion protein according to claim 4.

7. The biomaterial related to the GluN2B single-domain antibody fusion protein according to claim 5, characterized in that: The nucleic acid molecule encoding the GluN2B single-domain antibody fusion protein according to any one of claims 2 to 4 is inserted into the pCDNA3.1 vector to construct the recombinant vector.

8. The biomaterial related to the GluN2B single-domain antibody fusion protein according to claim 5, characterized in that: The host cell of the recombinant cell includes a prokaryotic cell or a eukaryotic cell; The eukaryotic cells include any one of CHO, COS, and HEK-293; the prokaryotic cells include Escherichia coli.

9. Use of a biomaterial related to the GluN2B single-domain antibody according to claim 1, the GluN2B single-domain antibody fusion protein according to any one of claims 2 to 4, or the GluN2B single-domain antibody fusion protein according to any one of claims 5 to 8 in the preparation of a medicament for treating cerebral ischemia.

10. A drug for treating cerebral ischemia, characterized in that: It includes the GluN2B single domain antibody according to claim 1 or the GluN2B single domain antibody fusion protein according to any one of claims 2 to 4.