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CN-120737214-B - Long-acting medicine for treating allergic diseases and chimeric antigen receptor of active ingredient thereof

CN120737214BCN 120737214 BCN120737214 BCN 120737214BCN-120737214-B

Abstract

The application provides chimeric antigen receptors comprising five domains of antigen specific binding, hinge, transmembrane, costimulatory and CD3 zeta signaling, antigen comprising immunoglobulin IgE EMPD domain specifically bound by antigen specific binding domain, and long-acting therapeutic use thereof for IgE mediated allergic diseases. Compared with the prior art, the application combines the anti-IgE monoclonal antibody technology with the CAR technology, changes the generated IgE protein from targeting to targeting IgE-producing B cells, and has proved the killing effect of IgE CAR-T on mIgE + B cells in vitro and in vivo experiments, thereby solving the problems of short half-life period and high administration frequency of monoclonal antibodies. In addition, the CAR can effectively avoid interference of free sIgE, and the treatment effect of the CAR-T is successfully verified by constructing a humanized mouse model to carry out in-vivo experiments.

Inventors

  • ZHAO YONG
  • LI DI

Assignees

  • 深圳先进技术研究院

Dates

Publication Date
20260512
Application Date
20250703

Claims (13)

  1. 1. The chimeric antigen receptor is characterized by comprising an antigen specific binding domain, a hinge domain, a transmembrane domain, a costimulatory domain and a CD3 zeta signaling domain, wherein the antigen specific binding domain can specifically bind to an antigen comprising an immunoglobulin IgE EMPD domain, and the antigen specific binding domain comprises a single-chain variable fragment scFv consisting of an amino acid sequence shown as SEQ ID No.1 or SEQ ID No. 2; the chimeric antigen receptor targets B cells or plasma cells expressing mIgE; The chimeric antigen receptor structure consists of a leader chain domain CD8-SP, an antigen specific binding domain, a hinge domain CD 8-range, a transmembrane domain CD8-TM, a costimulatory domain 4-1BB or CD28, a CD3zeta signaling domain CD3zeta and a tracer structure T2A EGFP which are connected in sequence.
  2. 2. The chimeric antigen receptor according to claim 1, wherein the costimulatory domain is selected from the group consisting of OX40, CD28, CD30, CD40, CD70, CD134, 4-1 BB (CD 137), PD1, dap10, CDs, ICAM-1, and polypeptides in combination thereof.
  3. 3. The chimeric antigen receptor according to any one of claims 1 or 2, wherein the hinge domain is selected to be the amino acid sequence shown in SEQ ID No.4, the transmembrane domain is selected to be the amino acid sequence shown in SEQ ID No.5, the CD3 zeta signaling domain is selected to be the amino acid sequence shown in SEQ ID No.6, and the costimulatory domain is selected to be the amino acid sequence shown in SEQ ID No.7 or SEQ ID No. 8.
  4. 4. The chimeric antigen receptor according to claim 3, wherein the hinge domain is encoded by the nucleic acid sequence shown in SEQ ID No.10, the transmembrane domain is encoded by the nucleic acid sequence shown in SEQ ID No.11, the CD3 zeta signaling domain is encoded by the nucleic acid sequence shown in SEQ ID No.12, and the costimulatory domain is encoded by the nucleic acid sequence shown in SEQ ID No.13 or SEQ ID No. 14.
  5. 5. An isolated immune cell modified to express a chimeric antigen receptor comprising an antigen binding domain linked to a costimulatory domain and a CD3 zeta signaling domain, said antigen binding domain being capable of specifically binding to a single chain variable fragment scFv comprising an immunoglobulin IgE EMPD domain, said single chain variable fragment scFv having the amino acid sequence shown in SEQ ID No.1 or SEQ ID No. 2.
  6. 6. The isolated immune cell of claim 5, wherein the immune cell is a T cell.
  7. 7. The isolated immune cell of claim 6, wherein the costimulatory domain is selected as a CD28 or 41BB polypeptide.
  8. 8. The isolated immune cell of any one of claims 5-7, wherein the chimeric antigen receptor is selected as the chimeric antigen receptor of claim 3 or claim 4.
  9. 9. The isolated immune cell of claim 8, wherein the T cell is selected from the group consisting of a CD4 + T cell, a CD8 + T cell, and a CD4 + cell and a CD8 + T cell mixed in any ratio.
  10. 10. An expression vector encoding the chimeric antigen receptor according to any one of claims 1-4.
  11. 11. A host cell, characterized in that, the host cell comprising the expression vector of claim 10.
  12. 12. Use of the chimeric antigen receptor of any one of claims 1-4, the isolated immune cell of any one of claims 5-9, the expression vector of claim 10, and the host cell of claim 11 in the manufacture of a long-acting medicament for the treatment of an allergic disease selected as IgE-mediated allergic asthma.
  13. 13. A long-acting pharmaceutical composition for the treatment of allergic diseases, comprising the chimeric antigen receptor according to any one of claims 1 to 4, the isolated immune cell according to any one of claims 5 to 9, the expression vector according to claim 10 and the host cell according to claim 11.

Description

Long-acting medicine for treating allergic diseases and chimeric antigen receptor of active ingredient thereof Technical Field The application relates to the technical field of biological medicines, in particular to a long-acting medicine for treating allergic diseases and an active ingredient chimeric antigen receptor thereof. Background The prevalence of allergic diseases continues to rise worldwide, including food allergies, allergic asthma, atopic dermatitis, urticaria, allergic rhinitis, conjunctivitis, and chronic sinusitis, among others. Immunoglobulin IgE is a key molecule in allergic reactions. Various forms of allergic diseases associated with IgE affect about 30% of the world population. When a patient is exposed to an allergen, B cells are stimulated to differentiate and IgE antibodies are produced by the combined action of dendritic cells, th2, tfh and other cells, and IL-4, IL-5, IL-13 and other cytokines. These IgE antibodies bind to mast cells and basophils and mediate their degranulation reactions to release inflammatory mediators, which in turn trigger a range of allergic symptoms. Therefore, igE becomes an important target for development of allergic disease drugs, and the anti-IgE monoclonal antibody Omalizumab also becomes a first target drug, and blocks downstream allergic inflammatory reactions by neutralizing free IgE and the like. Omalizumab was approved by the FDA for the treatment of severe allergic asthma in 2003, was approved for the treatment of urticaria in 2014, and has also been shown to have therapeutic effects on allergic diseases such as allergic rhinitis and food allergy in some studies. The Omalizumab treatment can effectively improve symptoms of allergic asthma and urticaria, and improve the life quality of patients. However, omalizumab as an IgE neutralizing antibody has a main effect of neutralizing the produced free IgE with less effect on IgE production. Omalizumab is difficult to effectively maintain free IgE levels at low levels for long periods of time. After this, genentech developed monoclonal antibody 47H4, which recognizes the EMPD domain of mIgE, and was designated Quilizumab after humanization. IgE is a membrane-bound form of IgE, expressed on the surface of B cells and plasma cells, and mlige + plasma cells and B cells are generally considered to be the major sources of IgE in allergic reactions. 47H4 mab is capable of inducing apoptosis in mIgE + B cells. Quilizumab was effective in reducing IgE levels in patients in phase I clinical trials for allergic rhinitis and phase II clinical trials for allergic asthma, but was less effective in improving clinical symptoms of asthma. At the same time, an increase in the dosage and frequency of administration has a positive effect on symptom improvement. Quilizumab monoclonal antibodies are directed against mIgE, and although the monoclonal antibodies can induce apoptosis of mIgE + B cells to a certain extent and reduce IgE production, the action of the monoclonal antibodies also depends on a certain dosage and frequency due to the short half-life of the monoclonal antibodies. To date, igE-targeted allergic disease treatment regimens have been based on monoclonal antibodies. However, the half-life of the monoclonal antibody is short, the therapeutic effect depends on a certain use frequency, and great limitation is brought, so that more effective therapeutic means are still needed. Chimeric antigen receptor T cell (CAR-T) treatment is to modify human T cells in vitro by genetic engineering means to identify specific target antigens, amplify and reinject the human T cells into human bodies for treating diseases. CAR-T technology currently achieves a remarkable effect in the treatment of hematological malignancies, and the memory of T cells enables long-term inhibition of tumors. Thus, it is also possible to apply CAR-T to the treatment of allergy-related long-term chronic diseases. Therefore, none of the current therapies targeting IgE achieve long-term inhibition of IgE levels in vivo, long-term relief of allergic symptoms. Disclosure of Invention In view of the technical limitations, the application provides a long-acting drug for treating allergic diseases and an active ingredient chimeric antigen receptor thereof, which overcome the defects and shortcomings in the background art. In order to achieve the above purpose, the present application adopts the following technical scheme: The application provides a chimeric antigen receptor, which comprises an antigen specific binding domain, a hinge domain, a transmembrane domain, a costimulatory domain and a CD3 zeta signaling domain, wherein the antigen specific binding domain can specifically bind an antigen comprising an immunoglobulin IgEEMPD domain, and the antigen specific binding domain comprises a single-chain variable fragment scFv consisting of an amino acid sequence shown as SEQ ID No.1 or comprises a single-chain variable fragment scFv which has more than