CN-121991214-A - Nanometer antibody of pre-F protein of targeting respiratory syncytial virus and application thereof

Abstract

The invention discloses a nanometer antibody of pre-F protein of targeting respiratory syncytial virus and application thereof. The nanobody comprises a heavy chain variable region comprising heavy chain complementarity determining regions CDR1, CDR2, and CDR3. The nano antibody can be specifically combined with RSV, and has good application prospect in the aspect of RSV infection related diseases.

Inventors

• LIU XIANGLEI
• HAN SHU
• LI XIAOQING

Assignees

• 上海医药工业研究院有限公司
• 中国医药工业研究总院有限公司

Dates

Publication Date

20260508

Application Date

20260205

Claims (13)

1. 1. A nanobody targeting a pre-F protein of respiratory syncytial virus comprising a heavy chain variable region comprising heavy chain complementarity determining regions CDR1, CDR2 and CDR3, characterized in that, The amino acid sequence of the CDR1 is shown as SEQ ID NO. 3, the amino acid sequence of the CDR2 is shown as SEQ ID NO. 4, the amino acid sequence of the CDR3 is shown as SEQ ID NO. 5, or, The amino acid sequence of the CDR1 is shown as SEQ ID NO. 6, the amino acid sequence of the CDR2 is shown as SEQ ID NO. 7, and the amino acid sequence of the CDR3 is shown as SEQ ID NO. 8.
2. 2. The nanobody of claim 1, wherein the heavy chain variable region further comprises a framework region, wherein the framework region is a camel or human framework region; Preferably, the heavy chain variable region has an amino acid sequence as set forth in SEQ ID NO. 1 or 2 or at least 80%, at least 85%, at least 90% or at least 95% identity to the amino acid sequence as set forth in SEQ ID NO. 1 or 2; The amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity does not involve alterations in CDR region sequences and maintains antigen binding function equivalent to the original sequence.
3. 3. A fusion protein of a pre-F protein targeting respiratory syncytial virus, wherein the fusion protein comprises the nanobody of claim 1 or 2; Preferably, the fusion protein further comprises an Fc, the N-terminus of which is preferably linked to the C-terminus of the nanobody; More preferably, the Fc is an IgG1 Fc, an IgG2 Fc or an IgG4 Fc, and/or the Fc is derived from a mouse, an alpaca or a human; even more preferably, the Fc is a human IgG1 Fc, and the amino acid sequence of the human IgG1 Fc is shown, for example, in SEQ ID NO. 9.
4. 4. An isolated nucleic acid encoding the nanobody of claim 1 or 2, or the fusion protein of claim 3.
5. 5. A recombinant expression vector, which is characterized in that, the recombinant expression vector comprising the isolated nucleic acid of claim 4; Preferably, the recombinant expression vector is a phage or plasmid; more preferably, the backbone of the plasmid is pComb3x.
6. 6. A transformant comprising the isolated nucleic acid of claim 4 or the recombinant expression vector of claim 5, wherein the host cell of the transformant is a eukaryotic cell or a prokaryotic cell; Preferably, the prokaryotic cell is E.coli, for example E.coli TG1.
7. 7. A method of producing a nanobody or fusion protein targeting the pre-F protein of respiratory syncytial virus, comprising culturing the transformant of claim 6 and isolating the nanobody or fusion protein from the culture.
8. 8. A method for detecting respiratory syncytial virus for non-diagnostic and/or therapeutic purposes, comprising contacting the nanobody of claim 1 or 2, or the fusion protein of claim 3, with a sample to be tested; preferably, the respiratory syncytial virus is RSV-A2 subtype.
9. 9. A pharmaceutical composition comprising a nanobody according to claim 1 or 2 or a fusion protein according to claim 3, and a pharmaceutically acceptable carrier.
10. 10. A kit comprising one or more selected from the nanobody of claim 1 or 2, the fusion protein of claim 3 and the pharmaceutical composition of claim 9.
11. 11. A kit of parts comprising a kit a and a kit B, wherein: The kit A contains one or more selected from the nanobody as claimed in claim 1 or 2, the fusion protein as claimed in claim 3 and the pharmaceutical composition as claimed in claim 9, the kit B contains other antibodies targeting respiratory syncytial virus or antibody drug conjugates or pharmaceutical compositions containing the other antibodies targeting respiratory syncytial virus, and/or the kit B contains a drug for treating respiratory syncytial virus infection or respiratory disease; preferably, the respiratory syncytial virus is RSV-A2 subtype.
12. 12. A drug delivery device comprising one or more selected from the group consisting of nanobody according to claim 1 or 2, fusion protein according to claim 3 and pharmaceutical composition according to claim 9; Preferably, the drug delivery device further comprises means for administering the nanobody, the fusion protein or the pharmaceutical composition to a subject, such as a syringe or an infusion device.
13. 13. Use of one or more of the nanobody of claim 1 or 2, the fusion protein of claim 3 and the pharmaceutical composition of claim 9 for the preparation of a medicament for the diagnosis, prevention and/or treatment of diseases of respiratory syncytial virus infection; preferably, the respiratory syncytial virus is RSV-A2 subtype.

Description

Nanometer antibody of pre-F protein of targeting respiratory syncytial virus and application thereof Technical Field The invention relates to the technical field of nanobody medicines, in particular to a nanobody of pre-F protein of targeting respiratory syncytial virus and application thereof. Background Respiratory syncytial virus (Respiratory syncytical virus, RSV) is the most common cause of respiratory illness in infants. RSV infection is associated with a range of respiratory diseases, from mild upper respiratory disease to life threatening bronchiolitis and pneumonia. Approximately 70% of infants are infected with RSV in the first year of birth, almost all children (90%) are infected in the first two years of birth, with up to 40% of children developing Lower Respiratory Tract Infections (LRTIs) at the time of initial onset. There is no specific drug currently available for the treatment of RSV infection, ribavirin is the only approved therapeutic drug by the united states Food and Drug Administration (FDA), but its safety and efficacy remain controversial. Patients are primarily supportive care and adjunctive therapy by interferon, bronchodilators, glucocorticoid therapy, and the like. RSV infection places a considerable burden on health and economy, making prophylaxis of RSV infection a major priority for global hygiene. The G protein and the F protein are two main protective antigens of RSV, wherein the G protein has different strains and lower conservation than the F protein, so that the F protein becomes a drug target of most RSV antibodies. Monoclonal antibody Nirsevimab is the first and only prophylactic means in China to protect infant populations against RSV infection. Compared with the traditional monoclonal antibody, the single domain antibody is a heavy chain antibody variable region with a natural deletion light chain, and the molecular weight is only 15KD which is 1/10 of that of the monoclonal antibody. Single domain antibodies tend to bind significantly smaller and recessed epitopes, including cryptic sites that may be more difficult to target by monoclonal antibodies. And the single domain antibody has the characteristics of higher affinity and stability, microbial expression, low immunogenicity, good solubility, strong penetrability and the like. The common single domain antibodies are mainly of animal origin, such as alpaca, shark and the like, are difficult to obtain, have the defects of animal dependence, long immunization time and the like, and are directly used for human administration and have immunogenicity risks. To overcome these problems with animal-derived antibodies, humanization engineering is often required, however, antibodies typically have reduced affinity after humanization. In conventional IgG antibodies, VH has a critical role in specifically binding to an antigen, and its structure is very similar to nanobody VHH, so that it is also possible to bind antigen alone, and fully human single domain antibodies can be developed using VH. The human immune system has the ability to produce high affinity, high specificity antibodies against any pathogen, making the human antibody repertoire highly diverse and diverse, thus coping with infections by a variety of different pathogens. In reality, however, the diversity of antibody libraries for a particular individual at a particular time is relatively limited. Therefore, constructing a natural fully human VH single-domain antibody phage display library based on a human antibody library is one of important strategies for preventing and treating serious diseases such as infectious diseases, tumors and the like. The specific antibody screened by the phage display library of the fully human VH single-domain antibody has the remarkable advantages of no dependence on animals, no immunogenicity risk and the like. The fully human single domain antibody has simple structure, can be expressed in large quantity by microorganisms such as yeast, escherichia coli and the like, and greatly reduces the development cost compared with the traditional monoclonal antibody Currently, single domain antibodies are commonly used directly for human administration, because they are mainly of animal origin, and present an immunogenic risk. As a medication for children, especially infants, low immunogenicity and safety have been non-negligible factors. Compared with single domain antibodies such as nano antibody VHH found in camels and cartilaginous fish bodies, the fully human single domain antibody has better safety and low adverse reaction and toxicity to organisms. Thus, the development of fully human single domain antibodies has significant advantages. The fully human VH single-domain antibody targeting the RSV pre-F protein with better patency is obtained by screening, and a new way is opened up for the development of a safer and effective novel anti-RSV virus immune preparation. Disclosure of Invention In order to enrich the variety of pre-F na