Anti-cd40 antibodies and uses thereof

Abstract

Provided are anti-CD40 antibodies and uses thereof, e.g., mouse, human, chimeric or humanized antibodies or antigen-binding fragments thereof that specifically bind CD40, as well as nucleic acid molecules encoding the antibodies and antigen-binding fragments thereof, and expression vectors and host cells for expressing the antibodies or antigen-binding fragments thereof. Further provided are methods of making and using the antibodies and antigen-binding fragments thereof, including treating and preventing immune diseases.

Description

Technical Field

[0001] This disclosure belongs to the field of biotechnology, specifically relating to antibodies that specifically bind to CD40 and their antigen-binding fragments, and to methods of using such antibodies and antigen-binding fragments. Background Technology

[0002] CD40 is a type I transmembrane glycoprotein belonging to the tumor necrosis factor receptor (TNFR) superfamily. CD40 is constitutively expressed on antigen-presenting cells (including dendritic cells, B cells, and macrophages), and also expressed in small amounts on non-hematopoietic cells such as epithelial cells, endothelial cells, smooth muscle cells, fibroblasts, and keratinocytes. Furthermore, CD40 is expressed on various tumor cells, such as B lymphoma cells. The major ligand of CD40, CD40L (CD154), is a type II transmembrane glycoprotein, primarily expressed on activated CD40. + In T lymphocytes, activated B cells, memory T cells, activated NK cells, and activated platelets.

[0003] The CD40 / CD40L signaling pathway participates in humoral and cellular immune responses, playing a crucial regulatory role in T cell-dependent antibody-dependent immune responses such as B cell activation, proliferation, and differentiation; antibody production and antibody class switching; and inflammatory responses. Studies have shown that tissues and organs of patients with autoimmune diseases infiltrate large numbers of T cells and B cells targeting self-antigens, exhibiting persistently high expression of CD40 and / or CD40L. IHC staining results from patients with primary Sjögren's syndrome showed CD40 enrichment in salivary gland epithelial cells, accompanied by elevated serum levels of anti-SSA and other autoantibodies. Furthermore, the key role of the CD40 / CD40L signaling pathway in inflammatory bowel disease has been confirmed in animal models such as mice.

[0004] Therefore, antibodies that specifically bind to CD40 and inhibit the CD40 / CD40L signaling pathway have potential clinical value in the treatment of immune diseases such as inflammatory diseases and autoimmune diseases. For example, the anti-CD40 antibody Iscalimab (CFZ533) developed by Novartis is currently in clinical trials for the treatment of CD40-mediated immune diseases. Results show that when Iscalimab is administered intravenously at a dose of 10 mg / kg to patients with primary Sjögren's syndrome (inclusion criteria: positive serum autoantibodies), 62% (13 / 21) of patients had an ESSDAI disease activity score below 5, which is significantly better than the 36% (4 / 11) in the placebo group. Given the large number of patients with immune diseases worldwide, there is an urgent need to develop more anti-CD40 antibodies with better drug properties. Summary of the Invention

[0005] On one hand, this disclosure provides isolated antibodies or antigen-binding fragments thereof that bind to CD40. In some embodiments, the antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody. In some embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody, a monospecific antibody, a bispecific antibody, a trispecific antibody, a multispecific antibody, a Fab fragment, an F(ab′)2 fragment, an Fd fragment, an Fv fragment, a dAb, an isolated CDR, a single-chain Fv molecule, a recombinant polypeptide, a fusion protein, a bispecific molecule, or a combination thereof. In some embodiments, the antibody or antigen-binding fragment thereof of this disclosure binds to human CD40. In other embodiments, the antibody or antigen-binding fragment thereof of this disclosure binds to CD40 (e.g., human CD40 and cynomolgous CD40). In some embodiments, the antibody or antigen-binding fragment thereof of this disclosure blocks the interaction between CD40 and CD40L. In some embodiments, the antibody or antigen-binding fragment thereof of this disclosure inhibits CD40 activity.

[0006] In some embodiments, the anti-CD40 antibody or its antigen-binding fragment provided in this disclosure comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), wherein: (1) heavy chain CDR1 comprises the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 9, heavy chain CDR2 comprises the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 9, and heavy chain CDR3 comprises the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 9; (2) heavy chain CDR1 comprises the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 10, heavy chain CDR2 comprises the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 10, and heavy chain CDR3 comprises the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 10; (3) heavy chain CDR1 comprises the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 11, heavy chain CDR2 comprises the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 11, and heavy chain CDR3 comprises the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 11. (4) Heavy chain CDR1 contains the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 12, heavy chain CDR2 contains the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 12, and heavy chain CDR3 contains the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 12; (5) Heavy chain CDR1 contains the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 13, heavy chain CDR2 contains the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 13, and heavy chain CDR3 contains the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 13; (6) Heavy chain CDR1 contains the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 22, heavy chain CDR2 contains the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 22, and heavy chain CDR3 contains the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 22; (7) Heavy chain CDR1 contains the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 11. (8) The heavy chain CDR1 in SEQ ID NO: 30 contains the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 30 and the heavy chain CDR3 contains the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 30; (9) The heavy chain CDR1 contains the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 38, the heavy chain CDR2 contains the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 38 and the heavy chain CDR3 contains the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 38;(9) Heavy chain CDR1 contains the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 44, heavy chain CDR2 contains the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 44, and heavy chain CDR3 contains the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 44; or (10) Heavy chain CDR1 contains the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 52, heavy chain CDR2 contains the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 52, and heavy chain CDR3 contains the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 52.

[0007] In some embodiments, the anti-CD40 antibody or its antigen-binding fragment provided in this disclosure comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, wherein (1) heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 1, 2, and 5 or have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences shown in SEQ ID NOs: 1, 2, and 5; and (2) heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 1, 3, and 5 or have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 87%, 88%, 89%, 99%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences shown in SEQ ID NOs: 1, 3, and 5. NOs: 1, 3 and 5 have amino acid sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical; (3) Heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 1, 4 and 5 or are identical to those in SEQ ID NOs: 1, 4 and 5. NOs: 1, 4 and 5 have amino acid sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical; (4) Heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 18, 19 and 20 or are identical to those in SEQ ID NOs: 18, 19 and 20. The amino acid sequences shown in IDNOs: 18, 19 and 20 have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity; (5) Heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 24, 25 and 26 or are identical to those in SEQ ID NOs: 26. NOs: The amino acid sequences shown in NOs 24, 25 and 26 have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid identity.(6) Heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 32, 33, and 34, or have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences shown in SEQ ID NOs: 32, 33, and 34; (7) Heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 40, 41, and 42, or have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 87%, 88%, 89%, 99%, or 100% identity with the amino acid sequences shown in SEQ ID NOs: 32, 33, and 34; NOs: The amino acid sequences shown in NOs: 40, 41 and 42 have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity; or (8) Heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 46, 47 and 48 or are identical to those in SEQ ID NOs: 46, 47 and 48. NOs: The amino acid sequences shown in NOs 46, 47, and 48 have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity.

[0008] In one embodiment, the anti-CD40 antibody or its antigen-binding fragment provided in this disclosure comprises light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), wherein (1) light chain CDR1 comprises the amino acid sequence of light chain CDR1 in SEQ ID NO: 14, light chain CDR2 comprises the amino acid sequence of light chain CDR2 in SEQ ID NO: 14, and light chain CDR3 comprises the amino acid sequence of light chain CDR3 in SEQ ID NO: 14; (2) light chain CDR1 comprises the amino acid sequence of light chain CDR1 in SEQ ID NO: 15, light chain CDR2 comprises the amino acid sequence of light chain CDR2 in SEQ ID NO: 15, and light chain CDR3 comprises the amino acid sequence of light chain CDR3 in SEQ ID NO: 15; (3) light chain CDR1 comprises the amino acid sequence of light chain CDR1 in SEQ ID NO: 16, light chain CDR2 comprises the amino acid sequence of light chain CDR2 in SEQ ID NO: 16, and light chain CDR3 comprises the amino acid sequence of light chain CDR1 in SEQ ID NO: 16. (4) Light chain CDR1 contains the amino acid sequence of light chain CDR1 in SEQ ID NO: 17, light chain CDR2 contains the amino acid sequence of light chain CDR2 in SEQ ID NO: 17, and light chain CDR3 contains the amino acid sequence of light chain CDR3 in SEQ ID NO: 17; (5) Light chain CDR1 contains the amino acid sequence of light chain CDR1 in SEQ ID NO: 23, light chain CDR2 contains the amino acid sequence of light chain CDR2 in SEQ ID NO: 23, and light chain CDR3 contains the amino acid sequence of light chain CDR3 in SEQ ID NO: 23; (6) Light chain CDR1 contains the amino acid sequence of light chain CDR1 in SEQ ID NO: 31, light chain CDR2 contains the amino acid sequence of light chain CDR2 in SEQ ID NO: 31, and light chain CDR3 contains the amino acid sequence of light chain CDR3 in SEQ ID NO: 31; (7) Light chain CDR1 contains the amino acid sequence of light chain CDR3 in SEQ ID NO: 16. (8) The light chain CDR1 in SEQ ID NO: 39 contains the amino acid sequence of the light chain CDR2 in SEQ ID NO: 39 and the light chain CDR3 contains the amino acid sequence of the light chain CDR3 in SEQ ID NO: 39; (9) The light chain CDR1 contains the amino acid sequence of the light chain CDR1 in SEQ ID NO: 45, the light chain CDR2 contains the amino acid sequence of the light chain CDR2 in SEQ ID NO: 45 and the light chain CDR3 contains the amino acid sequence of the light chain CDR3 in SEQ ID NO: 45;Or (9) light chain CDR1 contains the amino acid sequence of light chain CDR1 in SEQ ID NO: 53, light chain CDR2 contains the amino acid sequence of light chain CDR2 in SEQ ID NO: 53, and light chain CDR3 contains the amino acid sequence of light chain CDR3 in SEQ ID NO: 53.

[0009] In some embodiments, the anti-CD40 antibody or its antigen-binding fragment provided in this disclosure comprises light chain CDR1, light chain CDR2 and light chain CDR3, wherein (1) light chain CDR1, light chain CDR2 and light chain CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 6, 7 and 8 or have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NOs: 6, 7 and 8; (2) light chain CDR1, light chain CDR2 and light chain CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 21, 7 and 8 or have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 87%, 88%, 89% or 100% identity with the amino acid sequences shown in SEQ ID NOs: 6, 7 and 8; The amino acid sequences shown in NOs: 21, 7 and 8 have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity; (3) light chain CDR1, light chain CDR2 and light chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 27, 28 and 29 or are identical to those in SEQ ID NOs: 29. The amino acid sequences shown in NOs: 27, 28 and 29 have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity; (4) light chain CDR1, light chain CDR2 and light chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 35, 36 and 37 or are identical to those in SEQ ID NOs: 35, 36 and 37. The amino acid sequences shown in NOs: 35, 36 and 37 have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity; (5) light chain CDR1, light chain CDR2 and light chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 43, 36 and 37 or are identical to those in SEQ ID NOs: 43, 36 and 37. NOs: The amino acid sequences shown in NOs 43, 36 and 37 have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid identity.Or (6) light chain CDR1, light chain CDR2, and light chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 49, 50, and 51, or have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences shown in SEQ ID NOs: 49, 50, and 51.

[0010] In some embodiments, the anti-CD40 antibody or its antigen-binding fragment provided in this disclosure comprises heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3, wherein (1) heavy chain CDR1 comprises the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 9, heavy chain CDR2 comprises the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 9, heavy chain CDR3 comprises the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 9, light chain CDR1 comprises the amino acid sequence of light chain CDR1 in SEQ ID NO: 14, light chain CDR2 comprises the amino acid sequence of light chain CDR2 in SEQ ID NO: 14, and light chain CDR3 comprises the amino acid sequence of light chain CDR3 in SEQ ID NO: 14; (2) heavy chain CDR1 comprises the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 10, heavy chain CDR2 comprises the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 10, and heavy chain CDR3 comprises the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 10. NO: 10 contains the amino acid sequence of heavy chain CDR3, light chain CDR1 contains the amino acid sequence of light chain CDR1 in SEQ ID NO: 15, light chain CDR2 contains the amino acid sequence of light chain CDR2 in SEQ ID NO: 15, and light chain CDR3 contains the amino acid sequence of light chain CDR3 in SEQ ID NO: 15; (3) heavy chain CDR1 contains the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 11, heavy chain CDR2 contains the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 11, heavy chain CDR3 contains the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 11, light chain CDR1 contains the amino acid sequence of light chain CDR1 in SEQ ID NO: 15, light chain CDR2 contains the amino acid sequence of light chain CDR2 in SEQ ID NO: 15, and light chain CDR3 contains the amino acid sequence of light chain CDR3 in SEQ ID NO: 15; (4) heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 15. The amino acid sequence of heavy chain CDR1 in NO: 10, the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 10, the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 10, the amino acid sequence of light chain CDR1 in SEQ ID NO: 16, the amino acid sequence of light chain CDR2 in SEQ ID NO: 16, and the amino acid sequence of light chain CDR3 in SEQ ID NO: 16;(5) Heavy chain CDR1 contains the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 11, heavy chain CDR2 contains the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 11, heavy chain CDR3 contains the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 11, light chain CDR1 contains the amino acid sequence of light chain CDR1 in SEQ ID NO: 16, light chain CDR2 contains the amino acid sequence of light chain CDR2 in SEQ ID NO: 16, and light chain CDR3 contains the amino acid sequence of light chain CDR3 in SEQ ID NO: 16; (6) Heavy chain CDR1 contains the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 10, heavy chain CDR2 contains the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 10, heavy chain CDR3 contains the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 10, light chain CDR1 contains the amino acid sequence of light chain CDR1 in SEQ ID NO: 17, and light chain CDR2 contains the amino acid sequence of SEQ ID NO: 17. (7) The heavy chain CDR1 contains the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 17, the heavy chain CDR2 contains the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 12, the heavy chain CDR3 contains the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 12, the light chain CDR1 contains the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 17, the light chain CDR2 contains the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 17, and the light chain CDR3 contains the amino acid sequence of the light chain CDR3 in SEQ ID NO: 17; (8) The heavy chain CDR1 contains the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 13, the heavy chain CDR2 contains the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 13, and the heavy chain CDR3 contains the amino acid sequence of the light chain CDR3 in SEQ ID NO: 17. The amino acid sequence of heavy chain CDR3 in NO: 13, the amino acid sequence of light chain CDR1 in SEQ ID NO: 17, the amino acid sequence of light chain CDR2 in SEQ ID NO: 17, and the amino acid sequence of light chain CDR3 in SEQ ID NO: 17 are all present.(9) Heavy chain CDR1 contains the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 22, heavy chain CDR2 contains the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 22, heavy chain CDR3 contains the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 22, light chain CDR1 contains the amino acid sequence of light chain CDR1 in SEQ ID NO: 23, light chain CDR2 contains the amino acid sequence of light chain CDR2 in SEQ ID NO: 23, and light chain CDR3 contains the amino acid sequence of light chain CDR3 in SEQ ID NO: 23; (10) Heavy chain CDR1 contains the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 30, heavy chain CDR2 contains the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 30, heavy chain CDR3 contains the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 30, light chain CDR1 contains the amino acid sequence of light chain CDR1 in SEQ ID NO: 31, and light chain CDR2 contains the amino acid sequence of SEQ ID NO: 22. (11) The heavy chain CDR1 contains the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 31, the heavy chain CDR2 contains the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 38, the heavy chain CDR3 contains the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 38, the light chain CDR1 contains the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 39, the light chain CDR2 contains the amino acid sequence of the light chain CDR2 in SEQ ID NO: 39, and the light chain CDR3 contains the amino acid sequence of the light chain CDR3 in SEQ ID NO: 39; (12) The heavy chain CDR1 contains the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 44, the heavy chain CDR2 contains the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 44, and the heavy chain CDR3 contains the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 31. The amino acid sequence of heavy chain CDR3 in NO:44, the amino acid sequence of light chain CDR1 in SEQ ID NO:45, the amino acid sequence of light chain CDR2 in SEQ ID NO:45, and the amino acid sequence of light chain CDR3 in SEQ ID NO:45 are all present.Or (13) Heavy chain CDR1 contains the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 52, heavy chain CDR2 contains the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 52, heavy chain CDR3 contains the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 52, light chain CDR1 contains the amino acid sequence of light chain CDR1 in SEQ ID NO: 53, light chain CDR2 contains the amino acid sequence of light chain CDR2 in SEQ ID NO: 53, and light chain CDR3 contains the amino acid sequence of light chain CDR3 in SEQ ID NO: 53.

[0011] In some embodiments, the anti-CD40 antibody or its antigen-binding fragment provided in this disclosure comprises heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3, wherein (1) heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 1, 2, 5, 6, 7, and 8 or the sequences shown in SEQ ID NOs: 8. The amino acid sequences shown in NOs: 1, 2, 5, 6, 7 and 8 have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity; (2) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 1, 3, 5, 6, 7 and 8 or are identical to those in SEQ ID NOs: 1, 3, 5, 6, 7 and 8. NOs: 1, 3, 5, 6, 7 and 8 have amino acid sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical; (3) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 1, 4, 5, 6, 7 and 8 or are identical to SEQ ID NOs: 1, 4, 5, 6, 7 and 8. NOs: 1, 4, 5, 6, 7 and 8 have amino acid sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical; (4) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 18, 19, 20, 21, 7 and 8 or are identical to those in SEQ ID NOs: 18, 19, 20, 21, 7 and 8. NOs: The amino acid sequences shown in NOs 18, 19, 20, 21, 7 and 8 have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid identity.(5) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 24, 25, 26, 27, 28, and 29 or have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences shown in SEQ ID NOs: 24, 25, 26, 27, 28, and 29; (6) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 24, 25, 26, 27, 28, and 29. (7) The amino acid sequences shown in NOs: 32, 33, 34, 35, 36 and 37 or the amino acid sequences shown in SEQ ID NOs: 32, 33, 34, 35, 36 and 37 have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NOs: 32, 33, 34, 35, 36 and 37; (8) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 40, 41, 42, 43, 36 and 37 or the amino acid sequences shown in SEQ ID NOs: 32, 33, 34, 35, 36 and 37. NOs: The amino acid sequences shown in NOs: 40, 41, 42, 43, 36 and 37 have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity; or (8) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 respectively contain the amino acid sequences shown in NOs: 46, 47, 48, 49, 50 and 51 or are identical to those in NOs: SEQ ID NOs: 46, 47, 48, 49, 50 and 51. NOs: The amino acid sequences shown in NOs 46, 47, 48, 49, 50, and 51 have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity.

[0012] In some embodiments, the anti-CD40 antibody or its antigen-binding fragment provided in this disclosure comprises a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 9, 10, 11, 12, 13, 22, 30, 38, 44 or 52, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence represented by SEQ ID NOs: 9, 10, 11, 12, 13, 22, 30, 38, 44 or 52.

[0013] In some embodiments, the anti-CD40 antibody or its antigen-binding fragment provided in this disclosure comprises a light chain variable region comprising an amino acid sequence represented by SEQ ID NOs: 14, 15, 16, 17, 23, 31, 39, 45 or 53, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence represented by SEQ ID NOs: 14, 15, 16, 17, 23, 31, 39, 45 or 53.

[0014] In some embodiments, the anti-CD40 antibody or its antigen-binding fragment provided in this disclosure comprises a heavy chain variable region and a light chain variable region, wherein (1) the heavy chain variable region and the light chain variable region respectively comprise the amino acid sequences shown in SEQ ID NOs: 9 and 14 or have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences shown in SEQ ID NOs: 9 and 14; (2) the heavy chain variable region and the light chain variable region respectively comprise the amino acid sequences shown in SEQ ID NOs: 10 and 15 or have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences shown in SEQ ID NOs: 9 and 14; NOs:10 and 15 show amino acid sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity; (3) the heavy chain variable region and the light chain variable region respectively contain the amino acid sequences shown in SEQ ID NOs:11 and 15 or the amino acid sequences shown in SEQ ID NOs:11 and 15. The amino acid sequences shown in NOs: 11 and 15 have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity; (4) the heavy chain variable region and the light chain variable region respectively contain the amino acid sequences shown in SEQ ID NOs: 10 and 16 or the amino acid sequences shown in SEQ ID NOs: 10 and 16. (5) The heavy chain variable region and the light chain variable region respectively contain the amino acid sequences shown in SEQ ID NOs: 11 and 16 or amino acid sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequences shown in SEQ ID NOs: 11 and 16;(6) The heavy chain variable region and the light chain variable region respectively contain the amino acid sequences shown in SEQ ID NOs: 10 and 17 or have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences shown in SEQ ID NOs: 10 and 17; (7) The heavy chain variable region and the light chain variable region respectively contain the amino acid sequences shown in SEQ ID NOs: 12 and 17 or have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences shown in SEQ ID NOs: 10 and 17; The amino acid sequences shown in SEQ ID NOs: 12 and 17 have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity; (8) the heavy chain variable region and the light chain variable region respectively contain the amino acid sequences shown in SEQ ID NOs: 13 and 17 or the amino acid sequences shown in SEQ ID NOs: 13 and 17. The amino acid sequences shown in NOs: 13 and 17 have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity; (9) the heavy chain variable region and the light chain variable region respectively contain the amino acid sequences shown in SEQ ID NOs: 22 and 23 or the amino acid sequences shown in SEQ ID NOs: 22 and 23. NOs: 22 and 23 show amino acid sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity; (10) the heavy chain variable region and the light chain variable region respectively contain the amino acid sequences shown in SEQ ID NOs: 30 and 31 or the amino acid sequences shown in SEQ ID NOs: 30 and 31. (11) The heavy chain variable region and the light chain variable region respectively contain the amino acid sequences shown in SEQ ID NOs: 38 and 39 or have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequences shown in SEQ ID NOs: 38 and 39;(12) The heavy chain variable region and the light chain variable region respectively contain the amino acid sequences shown in SEQ ID NOs: 44 and 45 or have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences shown in SEQ ID NOs: 44 and 45; or (13) The heavy chain variable region and the light chain variable region respectively contain the amino acid sequences shown in SEQ ID NOs: 52 and 53 or have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences shown in SEQ ID NOs: 44 and 45; NOs: The amino acid sequences shown in NOs 52 and 53 have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity.

[0015] In some embodiments, the anti-CD40 antibody or its antigen-binding fragment provided in this disclosure comprises a heavy chain and a light chain. The heavy chain comprises a heavy chain variable region and a heavy chain constant region, and the light chain comprises a light chain variable region and a light chain constant region. The heavy chain variable region and the light chain variable region each comprise the amino acid sequences of the aforementioned heavy chain variable region and light chain variable region. The heavy chain constant region comprises a human IgG1, IgG2, or IgG4 constant region, preferably IgG1 and IgG4 constant regions. The light chain constant region comprises a human κ constant region or a human λ constant region. In some specific embodiments, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NOs: 54, 55, or 56, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NOs: 54, 55, or 56. The light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 57, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to the amino acid sequence shown in SEQ ID NO: 57.

[0016] In some embodiments, the anti-CD40 antibody or its antigen-binding fragment provided in this disclosure comprises a heavy chain and a light chain, wherein (1) the heavy chain comprises the amino acid sequences shown in SEQ ID NOs: 9 and 54, and the light chain comprises the amino acid sequences shown in SEQ ID NOs: 14 and 57; (2) the heavy chain comprises the amino acid sequences shown in SEQ ID NOs: 10 and 54, and the light chain comprises the amino acid sequences shown in SEQ ID NOs: 15 and 57; (3) the heavy chain comprises the amino acid sequences shown in SEQ ID NOs: 11 and 54, and the light chain comprises the amino acid sequences shown in SEQ ID NOs: 15 and 57; (4) the heavy chain comprises the amino acid sequences shown in SEQ ID NOs: 10 and 54, and the light chain comprises the amino acid sequences shown in SEQ ID NOs: 16 and 57; (5) the heavy chain comprises the amino acid sequences shown in SEQ ID NOs: 11 and 54, and the light chain comprises the amino acid sequences shown in SEQ ID NOs: 16 and 57; (6) the heavy chain comprises the amino acid sequences shown in SEQ ID NOs: 10 and 54, and the light chain comprises the amino acid sequences shown in SEQ ID NOs: 16 and 57. (7) The heavy chain contains the amino acid sequences shown in SEQ ID NOs: 17 and 57, and the light chain contains the amino acid sequences shown in SEQ ID NOs: 12 and 54; (8) The heavy chain contains the amino acid sequences shown in SEQ ID NOs: 13 and 54, and the light chain contains the amino acid sequences shown in SEQ ID NOs: 17 and 57; (9) The heavy chain contains the amino acid sequences shown in SEQ ID NOs: 22 and 54, and the light chain contains the amino acid sequences shown in SEQ ID NOs: 23 and 57; (10) The heavy chain contains the amino acid sequences shown in SEQ ID NOs: 30 and 54, and the light chain contains the amino acid sequences shown in SEQ ID NOs: 31 and 57; (11) The heavy chain contains the amino acid sequences shown in SEQ ID NOs: 38 and 54, and the light chain contains the amino acid sequences shown in SEQ ID NOs: 39 and 57; (12) The heavy chain contains the amino acid sequences shown in SEQ ID NOs: 44 and 54, and the light chain contains the amino acid sequences shown in SEQ ID NOs: 17 and 57. NOs: 45 and 57, representing amino acid sequences; or (13) the heavy chain contains the amino acid sequences shown in SEQ ID NOs: 52 and 54, and the light chain contains the amino acid sequences shown in SEQ ID NOs: 53 and 57.

[0017] In some embodiments, the anti-CD40 antibody or its antigen-binding fragment provided in this disclosure comprises two heavy chains (H) and two light chains (L), or consists of two heavy chains and two light chains, the heavy chains and light chains being interconnected by disulfide bonds, wherein each heavy chain includes the aforementioned heavy chain variable region (VH) and heavy chain constant region, wherein the heavy chain variable region (VH) includes a backbone region (FR) and the aforementioned heavy chain complementarity-determining region (HCDR), and each light chain includes the aforementioned light chain variable region (VL) and light chain constant region, wherein the light chain variable region (VL) includes a FR and the aforementioned light chain complementarity-determining region (LCDR), wherein the C-terminus of the heavy chain variable region is connected to the N-terminus of the heavy chain constant region, and the C-terminus of the light chain variable region is connected to the N-terminus of the light chain constant region. The antibody of this disclosure may be, for example, a full-length antibody of an IgG1, IgG2, or IgG4 isotype. In other embodiments, the antibody disclosed herein may be a single-chain antibody (scFv) or an antibody fragment, such as Fab, F(ab′)2 fragment, Fd fragment, Fv fragment, dAb or isolated CDR.

[0018] On one hand, this disclosure provides an isolated antibody or antigen-binding fragment thereof that binds to CD40, said antibody or antigen-binding fragment thereof being generated by a hybridoma selected from the group consisting of hybridomas referred to herein as A01, A02, A03, B01, B02, and B03. Therefore, this disclosure also includes antibodies or antigen-binding fragments thereof generated from hybridomas A01, A02, A03, B01, B02, and B03, as well as from any hybridoma that generates the antibody disclosed herein.

[0019] On one hand, this disclosure provides antibodies or antigen-binding fragments thereof that bind to the same epitope on CD40 as any exemplary anti-CD40 antibody or antigen-binding fragment thereof disclosed herein. In some embodiments, this disclosure provides antibodies or antigen-binding fragments thereof that compete with any exemplary anti-CD40 antibody or antigen-binding fragment thereof disclosed herein for binding to CD40.

[0020] On the other hand, this disclosure also provides recombinant polypeptides or fusion proteins comprising one or more of the anti-CD40 antibodies or antigen-binding fragments of the present disclosure and at least one other functional fragment, said other functional fragment including but not limited to another peptide, protein, cytokine, or receptor ligand. These recombinant polypeptides or fusion proteins can be prepared by genetic modification, chemical methods, etc. In this disclosure, the term "fusion protein" generally refers to a new polypeptide sequence obtained by linking two or more identical or different polypeptide sequences, and particularly refers to a recombinant polypeptide sequence comprising one or more identical or different polypeptide sequences that are not naturally linked.

[0021] On the other hand, this disclosure also provides a bispecific molecule comprising one or more of the anti-CD40 antibodies or antigen-binding fragments of this disclosure, and at least some other functional portions (e.g., another antibody or antigen-binding fragment thereof) with specificities different from those of the antibodies or antigen-binding fragments of this disclosure, said bispecific molecule being capable of binding to at least two different binding sites or targets. As used herein, "bispecific molecule" encompasses molecules having two (i.e., bispecific molecules), three (i.e., trispecific molecules), four (i.e., tetraspecific molecules), or more specificities. These bispecific molecules can be prepared by genetic modification, chemical methods, etc.

[0022] On the other hand, this disclosure also provides an immunoconjugate comprising the anti-CD40 antibody of this disclosure or its antigen-binding portion thereof, wherein the anti-CD40 antibody of this disclosure or its antigen-binding portion is linked to a therapeutic agent (e.g., a cytotoxic agent or an imaging agent), such as an antibody-drug conjugate (ADC). The anti-CD40 antibody of this disclosure or its antigen-binding portion may be part of a chimeric antigen receptor (CAR). This disclosure also provides immune cells comprising the chimeric antigen receptor, such as T cells (i.e., CAR-T cells). Furthermore, this disclosure provides a gene vector comprising a gene encoding the anti-CD40 antibody of this disclosure or its antigen-binding portion thereof, and capable of entering and expressing the gene in mammalian cells (preferably human cells). These gene vectors include, but are not limited to, naked plasmid vectors, yeast vectors, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, poxvirus vectors, rod-shaped virus vectors, or baculovirus vectors. Techniques for inserting DNA into these gene vectors are well known to those skilled in the art.

[0023] In another aspect, this disclosure also provides a pharmaceutical composition comprising the anti-CD40 antibody of this disclosure or an antigen-binding fragment thereof, and one or more pharmaceutically acceptable carriers. In other embodiments, this disclosure also provides pharmaceutical compositions comprising recombinant peptides, fusion proteins, bispecific molecules, immunoconjugates, chimeric antigen receptors or gene vectors of this disclosure, and pharmaceutically acceptable carriers.

[0024] In another aspect, this disclosure also provides isolated nucleic acids encoding the anti-CD40 antibody of this disclosure or an antigen-binding fragment thereof. This disclosure also provides expression vectors comprising said nucleic acids, and host cells comprising said expression vectors.

[0025] In another aspect, this disclosure also provides a method for preparing an anti-CD40 antibody or an antigen-binding fragment thereof, comprising the steps of: (i) expressing an anti-CD40 antibody or an antigen-binding fragment thereof in a host cell, and (ii) isolating the anti-CD40 antibody or an antigen-binding fragment thereof from the host cell or its cell culture.

[0026] In other respects, this disclosure provides methods for treating or preventing immune diseases in a subject, said methods comprising administering to the subject a therapeutically effective amount of the disclosed anti-CD40 antibody or its antigen-binding moiety, encoding a nucleic acid, a pharmaceutical composition, a recombinant polypeptide, a fusion protein, a bispecific molecule, an immunoconjugate, a chimeric antigen receptor, or a gene vector. In some embodiments, the subject is a human.

[0027] In some implementations, the immune diseases include, but are not limited to, inflammatory diseases, allergic reactions, autoimmune diseases, or transplant-related diseases. In some specific implementations, the immune diseases include, but are not limited to: allergic reactions, Addison's disease, ankylosing spondylitis, spondyloarthritis, asthma, atherosclerosis, coronary heart disease, autoimmune hepatitis, autoimmune mumps, type I diabetes, epididymitis, nephritis, Reiter's syndrome, thyroiditis, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's disease, hemolytic anemia, idiopathic thrombocytopenic purpura, systemic lupus erythematosus, subacute cutaneous lupus erythematosus, multiple sclerosis, myasthenia gravis, psoriasis, scleroderma, arthritis, sarcoidosis, Sjögren's syndrome, dry eye syndrome, hidradenitis suppurativa, transplant-related diseases, vasculitis, and / or inflammatory bowel disease.

[0028] In some embodiments, the method further includes administration of a second therapeutic agent, which includes a nonsteroidal anti-inflammatory drug (NSAID), salicylates, hydroxychloroquine, sulfasalazine, corticosteroids, cytotoxic drugs, or immunosuppressive drugs and / or antibodies.

[0029] Other features and advantages of the present disclosure will become clearer based on the following specific descriptions and embodiments, which should not be construed as limiting. All documents, Genbank records, patents, and published patent applications cited in this disclosure are expressly included herein by reference. Attached Figure Description

[0030] Figure 1A-1B The purified mouse anti-CD40 antibody was shown to block the binding of 293T-hCD40-NFκB cells to CD40L protein.

[0031] Figure 2 The purified mouse anti-CD40 antibody was shown to have inhibitory activity against CD40L and IL-4-induced apoptosis in Ramos cells.

[0032] Figure 3The binding activity of chimeric anti-CD40 antibodies Chi-A01, Chi-A02, and Chi-A03 to CHO-K1-hCD40 cells was demonstrated.

[0033] Figure 4 The agonistic activity of chimeric anti-CD40 antibodies Chi-A01, Chi-A02, Chi-A03, Chi-B01, Chi-B02 and Chi-B03 on Ramos cell apoptosis is shown, and the agonistic activity of CP-870893 on Ramos cell apoptosis is shown as a positive control.

[0034] Figure 5 The binding activity of humanized anti-CD40 antibodies hzA01-3.1, hzA01-3.3, and hzA01-3.4 to 293T-hCD40-NFκB cells was demonstrated.

[0035] Figure 6 The humanized anti-CD40 antibodies hzA01-1.1, hzA01-2.1, hzA01-3.1, hzA01-3.3 and hzA01-3.4 were shown to block the binding activity of 293T-hCD40-NFκB cells to CHO-K1-hCD40L cells.

[0036] Figure 7 The humanized anti-CD40 antibodies hzA01-1.1, hzA01-2.1, hzA01-3.1, hzA01-3.3 and hzA01-3.4 were shown to inhibit CD40L and IL-4-induced apoptosis in Ramos cells.

[0037] Figure 8 The apoptotic agonistic activity of humanized anti-CD40 antibodies hzA01-1.1, hzA01-2.1, hzA01-3.1, hzA01-3.3 and hzA01-3.4 on Ramos cell apoptosis is shown, and the apoptotic agonistic activity of CP-870893 on Ramos cell apoptosis is shown as a positive control.

[0038] Figure 9 The humanized anti-CD40 antibodies hzA01-3.1, hzA01-3.3, and hzA01-3.4 were shown to inhibit the expression of CD86, a co-stimulatory molecule in human peripheral blood B lymphocytes induced by CD40L.

[0039] Figure 10 The humanized anti-CD40 antibodies hzA01-3.1, hzA01-3.3, and hzA01-3.4 were shown to inhibit the proliferation of human peripheral blood B lymphocytes induced by CD40L and IL-4.

[0040] Figure 11The agonistic activity of humanized anti-CD40 antibodies hzA01-3.1, hzA01-3.3, and hzA01-3.4 in inducing the proliferation of human peripheral blood B lymphocytes is shown, with the agonistic activity of CP-870893 in inducing the proliferation of human peripheral blood B lymphocytes shown as a positive control.

[0041] Figure 12 The amount of IFNγ released in response to the humanized anti-CD40 antibody hzA01-3.3 in the MLR reaction is shown, in which the mDC and DC (undifferentiated culture) groups were not added with anti-CD40 antibody and IgG4 isotype control antibody and are shown as negative controls;

[0042] Figure 13 The agonistic activity of humanized anti-CD40 antibody hzA01-3.3 on imDC cell maturation is shown, and the agonistic activity of CP-870893 on imDC cell maturation is shown as a positive control.

[0043] Figure 14 The ADCC effect mediated by the humanized anti-CD40 antibody hzA01-3.3 is shown, with Rituximab-mediated ADCC activity shown as a positive control.

[0044] Figure 15 The humanized anti-CD40 antibody hzA01-3.3-mediated CDC effect is shown, with Rituximab-mediated CDC activity shown as a positive control.

[0045] Figure 16A The concentration of human anti-mouse IgM antibody in the serum of NDG mice after treatment with humanized anti-CD40 antibody hzA01-3.3 is shown. Figure 16B The concentration of human anti-mouse IgG antibody in the serum of NDG mice after treatment with humanized anti-CD40 antibody hzA01-3.3 is shown.

[0046] Figure 17A This shows human CD4 infiltration in the spleen of mice after treatment with humanized anti-CD40 antibody hzA01-3.3. + T cell content, 17B indicates human CD19 infiltration in the spleen of mice after treatment with humanized anti-CD40 antibody hzA01-3.3. + B cell content was compared with the IgG4 isotype control group using a t-test. * indicates P≤0.05, ** indicates P<0.01, and *** indicates P<0.001.

[0047] Figure 18 The effect of humanized anti-CD40 antibody hzA01-3.3 on body weight in mice with Sjögren's syndrome model is shown.

[0048] Figure 19The effect of humanized anti-CD40 antibody hzA01-3.3 on salivary flow rate in a mouse model of Sjögren's syndrome is shown.

[0049] Figure 20A The image shows the submandibular gland index in mice with a Sjögren's syndrome model after treatment with the humanized anti-CD40 antibody hzA01-3.3. Image 20B shows the IL-6 content in the submandibular glands of these mice. Figure 20C The submandibular gland pathological scores of Sjögren's syndrome model mice after treatment with humanized anti-CD40 antibody hzA01-3.3 are shown. The results were compared with the blank control group and the model group by t-test. * indicates P≤0.05, ** indicates P<0.01, and *** indicates P<0.001.

[0050] Figures 21A-21D The study showed that CD3 levels in the blood of Sjögren's syndrome model mice after treatment with humanized anti-CD40 antibody hzA01-3.3 were... + T cells, CD4 + T cells, CD8 + T cells and CD19 + B cells account for 45% of live cells in CD45 + Percentage of live cells Figure 21E-21H The study showed that CD3+ levels in the spleen of a mouse model of Sjögren's syndrome were observed after treatment with humanized anti-CD40 antibody hzA01-3.3. + T cells, CD4 + T cells, CD8 + T cells and CD19 + B cells account for 45% of live cells in CD45 + Percentage of live cells. Invention Details

[0052] It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0053] The term "CD40" includes CD40 variants, homologs, orthologs, and paralogs. For example, in some embodiments, antibodies specific to human CD40 protein may cross-react with CD40 protein of another species (e.g., monkeys) under certain conditions. In other embodiments, antibodies specific to human CD40 protein may be completely specific to human CD40 protein without cross-reacting with other species or other types of proteins, or may cross-react with CD40 protein of some other species but not all other species.

[0054] CD40 is known and can be referred to as B cell surface antigen CD40, Bp50, CD40L receptor, CDW40, MGC9013, p50, and tumor necrosis factor receptor superfamily member 5 (TNFRSF5). "Human CD40" or "hCD40" are used interchangeably herein and refer to a protein having the amino acid sequence of human CD40, such as the CD40 protein containing the amino acid sequence shown in SEQ ID NO: 62. The terms "monkey CD40" or "cynoCD40," etc., are used interchangeably herein and refer to a protein having the amino acid sequence of monkey CD40, such as the CD40 protein containing the amino acid sequence shown in SEQ ID NO: 63.

[0055] As used herein, the term "antibody" refers to a binding protein having at least one antigen-binding domain (e.g., CD40). The antibodies or antigen-binding fragments thereof disclosed herein can be whole antibodies or any fragments thereof, including monoclonal antibodies or fragments thereof and antibody variants or fragments thereof. Examples of antibodies or antigen-binding fragments thereof include monospecific, bispecific, trispecific, or multispecific antibodies, Fab fragments, F(ab')2 fragments, Fv fragments, isolated CDR regions, single-chain Fv (scFv), and any other antibody fragments known in the art. The anti-CD40 antibodies or antigen-binding fragments thereof disclosed herein can be IgG1, IgG2, IgG3, or IgG4 isotypes. The term "isotype" refers to the type of antibody encoded by a heavy chain constant region gene. In some embodiments, the anti-CD40 antibodies or antigen-binding fragments thereof disclosed herein are IgG1 and IgG4 isotypes. The anti-CD40 antibodies or antigen-binding fragments thereof disclosed herein can be derived from any species, including but not limited to mice, rats, rabbits, primates, llamas, and humans. The anti-CD40 antibody or its antigen-binding fragment disclosed herein may be a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody. Unless otherwise stated, the term "antibody" in this disclosure includes full-length antibodies and any antigen-binding portion (i.e., "antigen-binding fragment") or single chains. Typically, a full-length antibody is a glycoprotein comprising two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, namely CH1, CH2, and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions may also be divided into hypervariable regions (i.e., complementarity-determining regions (CDRs)) and sequence-conserved backbone regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region of the antibody contains a binding domain that interacts with the antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various immune system cells (e.g., effector cells) and the first component (Clq) of the classical complement system. Meanwhile, as those skilled in the art understand, special “full-length antibodies,” such as nanobodies, contain only the heavy (H) chain and lack the light (L) chain.

[0056] An antibody's "antigen-binding fragment" or "antibody-binding part" refers to one or more fragments of an antibody that retain the function of specifically binding to an antigen (e.g., the CD40 protein). It has been demonstrated that the antigen-binding function of an antibody can be exercised by fragments of a full-length antibody. Examples encompassed in the term "antigen-binding part / fragment" of an antibody include: (i) Fab fragments: monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab′)2 fragments, bivalent fragments containing two Fab fragments connected by a disulfide bridge in the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VL and VH domains of a single arm of the antibody; (v) dAb fragments consisting of the VH domain (see Ward et al., Nature. 341: 544-546 (1989)); (vi) separated complementarity-determining regions (CDRs); and (vii) nanobodies, a heavy-chain variable region containing a single variable domain and two constant domains. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by different genes, VH and VL can be linked into a single protein chain via a linker using a recombination method. VL and VH pair to form a monovalent molecule called a single-chain Fv (scFv); see Bird et al., Science. 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. 85: 5879-5883 (1988). These single-chain antibodies are also covered under the term antigen-binding part / fraction. In addition, recombinant peptides, fusion proteins, and bispecific molecules containing this antigen-binding part / fraction are also covered under the term antigen-binding part / fraction. These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the fragments can be functionally screened using the same methods as full-length antibodies.

[0057] "Mouse antibody" or "mouse-derived antibody" refers to an antibody in which both the backbone region and the CDR region in the variable region are derived from mouse germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, that constant region is also derived from mouse germline immunoglobulin sequences. The mouse antibodies of this disclosure may include amino acid residues not encoded by mouse germline immunoglobulin sequences (e.g., mutations introduced through in vitro random or point mutations or through in vivo somatic mutations), but "mouse antibody" does not include antibodies with inserted CDR sequences derived from other mammalian germlines into the mouse backbone sequence.

[0058] A "chimeric antibody" is an antibody created by combining genetic material from a non-human source with genetic material from a human. More generally, a chimeric antibody is an antibody possessing genetic material from one species and genetic material from another species. For example, the variable regions of both the light and heavy chains may originate from the variable region of an antibody from one animal species (such as a mouse or rat), while the constant region is homologous to the sequence of an antibody from another species (such as a human). For instance, to obtain a chimeric antibody, the variable region can be generated using non-human B cells or hybridoma cells, while the constant region combined with it is derived from a human. In this disclosure, chimeric antibodies are also referred to as "Chi".

[0059] "Humanized antibody" is an antibody containing a complementarity-determining region (CDR) derived from a non-human antibody and a backbone region derived from a human antibody, as well as a constant region. For example, the CD40-binding humanized antibody provided herein may contain a CDR derived from one or more murine antibodies, as well as a human backbone region and a human constant region. Thus, in some embodiments, the humanized antibody provided herein binds to the same epitope on CD40 as the murine antibody from which the CDR of said antibody is derived. Exemplary humanized antibodies are provided herein. Additional CD40-binding humanized antibodies or variants thereof containing the heavy chain CDR and light chain CDR provided herein can be generated using any human backbone sequence and are also included in this disclosure. In some embodiments, backbone sequences suitable for use in this disclosure include those backbone sequences that are structurally similar to the backbone sequences provided herein. Additional modifications can be made to the backbone region to alter the properties of the antibody provided herein. Such additional backbone modifications may include chemical modifications, point mutations to reduce immunogenicity or remove T-cell epitopes, or reverting mutations to residues in the original germline sequence. In some embodiments, such modifications include those corresponding to the mutations exemplified herein, including reversion mutations to germline sequences. For example, in some embodiments, one or more amino acids in the human backbone regions of the VH and / or VL of the humanized antibody provided herein are reverted to the corresponding amino acids in the parental mouse antibody. In this disclosure, humanized antibody is also referred to as "hz".

[0060] The term "Fc domain" or "Fc region" refers to an antibody sequence that contains the constant CH2 and CH3 domains as defined by the Kabat numbering system. The Fc region can be derived from human IgG. For example, the Fc region can be derived from the human IgG1 or human IgG4 Fc region.

[0061] The term "derived" as used herein, when referring to a molecule or polypeptide relative to a reference antibody or other binding protein, means a molecule or polypeptide capable of specifically binding to the same epitope with a reference antibody or other binding protein.

[0062] "Isolated" refers to the target compound (e.g., antibody, antigen-binding fragment, or nucleic acid) that has been isolated from its natural environment.

[0063] The terms "antibody that specifically binds to an antigen" and "antibody specific to an antigen" are used interchangeably with the term "antibody that specifically binds to an antigen." An antibody that "specifically binds to human CD40" refers to an antibody that binds to human CD40 (and possibly CD40 from other non-human species) but substantially does not bind to non-CD40 antibodies. Preferably, the antibody binds to human CD40 with "high affinity," i.e., K. D 5.0×10 -8 M or smaller, 1.0 × 10 -8 M or smaller, preferably 5.0 × 10 -9 M or smaller, more preferably 1.0 × 10 -9 M or smaller.

[0064] The term "substantially non-binding" to proteins or cells refers to substances that do not bind to proteins or cells, or do not bind to them with high affinity; that is, substances that bind to proteins or cells are K. D 1.0×10 -6 M or higher, preferably 1.0 × 10 -5 M or higher, 1.0×10 -4 M or higher, more preferably 1.0 × 10 -3 M or higher, 1.0×10 -2 M or higher.

[0065] The term "high affinity" for IgG refers to the K+ binding antigen. D 5.0×10 -8 M or smaller, 1.0 × 10 -8 M or smaller, preferably 5.0 × 10 -9 M or smaller, more preferably 1.0 × 10 -9 M or smaller. However, "high affinity" binding may differ for other antibody isotypes. For example, "high affinity" binding for IgM isotypes refers to binding to the K+ of the antigen. D 1.0×10 -6 M or smaller, preferably 1.0 × 10 -7 M or smaller, more preferably 1.0 × 10 -8 M or smaller.

[0066] "Identity" refers to the similarity between two or more nucleic acid sequences or two or more polypeptide sequences. The sequence identity of this disclosure is at least 85%, 90%, or 95%, preferably at least 95%. Non-limiting examples include: 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%. Sequence comparisons and identity percentage determinations between two sequences can be performed using the default settings of the BLASTN / BLASTP algorithm on the National Center for Biotechnology Institute website.

[0067] "Competitive binding" antibodies refer to antibodies that partially or completely block the binding of other antibodies to a target. Whether two antibodies compete with each other for the binding of a target, i.e., whether one antibody blocks the binding of another antibody to the target and to what extent, can be determined using competition assays known in the art, such as solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competitive assays, etc. In some embodiments, an antibody competes with another antibody for the binding of a target and blocks the binding of the other antibody to the target by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0068] Two or more antibodies “binding to the same epitope” means that the antibodies bind to the same segment of amino acid residues, as determined by a given method. Techniques for determining whether an antibody binds to the “same epitope on CD40” as described herein include, for example, epitope mapping methods, such as X-ray analysis of crystals of antigen:antibody complexes and hydrogen / deuterium exchange mass spectrometry (HDX-MS).

[0069] The term "EC" 50 "IC50," also known as the half-maximal effective concentration, refers to the antibody concentration that achieves 50% of its maximum effect after a specific exposure time. 50 "Colonyx," also known as half-maximal inhibitory concentration, refers to the antibody concentration that inhibits specific biological or biochemical functions by 50% relative to the absence of antibodies. 50 and IC 50 Both can be measured by ELISA or FACS analysis or any other method known in the art.

[0070] “K D "Refers to the equilibrium dissociation constant, which is derived from K d Compared to K a (that is, K) d / K a The ratio of K to α is expressed as molar concentration (M). The K of the antibody DThe value can be determined using methods well-established in the art. The K value used for antibody determination... D The preferred method is to use surface plasmon resonance technology, preferably using biosensor systems such as Analysis of surface plasmon resonance systems.

[0071] "Cross-linking" refers to the higher-order polymerization of CD40 on cells induced by the binding of anti-CD40 antibodies to FcγR (e.g., cis or trans FcγRIIb), thereby leading to the induction of CD40 agonistic activity.

[0072] "Patient" or "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, preferably mammals, such as non-human primates, sheep, dogs, cats, cattle, and horses.

[0073] "Effective dose" or "effective amount" means an amount sufficient to achieve or at least partially achieve the desired effect. The therapeutic "effective amount" or "effective dose" of a drug or therapeutic agent means, when used alone or in combination with another therapeutic agent, an amount sufficient to prevent or improve symptoms of a disease or condition, preferably an amount that reduces the severity of disease symptoms, increases the frequency and duration of asymptomatic periods, or prevents damage or incapacity caused by the disease. The therapeutically effective amount is related to the disease being treated, and the actual effective amount can be readily determined by someone skilled in the art.

[0074] Unless otherwise stated, “about” in this disclosure means fluctuation within ±5% of a given specific numerical range, preferably within ±2%, more preferably within ±1%. For example, a pH value of about 5.5 means a pH of 5.5 ± 5%, preferably 5.5 ± 2%, more preferably 5.5 ± 1%. Unless otherwise specified, the use of the singular includes the plural.

[0075] Unless otherwise specified, the words “a” or “an” mean “at least one” or “at least one”, the phrase “at least one” means “one or more”, and the use of “and / or” means “and” or “or”.

[0076] As stated in this document, any percentage range, ratio range, or integer range should be understood as including the value of any integer within the enumerated range, unless otherwise indicated.

[0077] In this document, unless the context otherwise requires, the words “comprising,” “including,” and “containing” will be understood to mean including the stated steps or elements or a group of steps or elements, but not excluding any other steps or elements or a group of steps or elements. “Comprising of” means including and limited to what follows the phrase “comprising of.” Therefore, the phrase “comprising of” indicates that the listed elements are necessary or required, and no other elements may be present. “Substantially comprising” means including any elements listed following this phrase, and is limited to other elements that do not impede or contribute to the activity or function of the listed elements as detailed in this disclosure. Therefore, the phrase “substantially comprising” indicates that the listed elements are necessary or required, but other elements are optional and may be present or absent depending on whether they affect the activity or function of the listed elements.

[0078] Several aspects of this disclosure are described in more detail below.

[0079] The amino acid sequence IDs (SEQ ID NO.) of the heavy chain variable region, light chain variable region, and CDR of the exemplary antibodies or antigen-binding fragments of this disclosure are provided in Table 1 below. Some antibodies have the same CDR, and some antibodies have the same VH or VL. The heavy chain constant region of the antibody may be a human IgG1, IgG2, or IgG4 constant region, preferably IgG1 and IgG4 constant regions, for example comprising the amino acid sequence shown in SEQ ID NOs: 54, 55, or 56, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, and additions compared to the amino acid sequence shown in SEQ ID NOs: 54, 55, or 56. The light chain constant region of the antibody may be a human κ constant region or a human λ constant region, for example comprising the amino acid sequence shown in SEQ ID NO: 57, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, and additions compared to the amino acid sequence shown in SEQ ID NO: 57. These antibodies may also comprise mouse IgG1 or IgG4 heavy chain constant regions and / or mouse κ constant regions or λ constant regions.

[0080] Table 1. Amino acid sequence IDs (SEQ ID NO.) of the variable region and CDR

[0081]

[0082]

[0083] Given the amino acid sequence of the antibody variable region, those skilled in the art can determine which residues constitute a specific CDR using conventional methods. As is known to those skilled in the art, antibody CDRs can be defined using various methods, such as numbering systems / methods like Kabat, Chothia, IMGT, AbM, or Contact; or using a combination of two or more of these numbering systems / methods (e.g., HCDR1 defined by AbM, HCDR2 by Kabat or AbM, HCDR3 by IMGT or Kabat, and LCDR1-3 by Kabat); or using a combined numbering system that incorporates both Kabat and Chothia, where the combined numbering system combines the ranges defined by Kabat and Chothia, resulting in a larger range (e.g., if Kabat defines HCDR1 as H31-H35 and Chothia defines HCDR1 as H26-H32, then the combined system defines HCDR1 as H26-H35). The precise numbering and placement of CDRs vary across different numbering systems. Those skilled in the art should understand that, unless otherwise specified, the terms "CDR" and "complementarity-determining region" for a given antibody or its regions (e.g., variable regions) should be understood to encompass the complementarity-determining region as defined by any known scheme.

[0084] Although the CDRs claimed in this disclosure are based on the sequences shown in Tables 1 and 10, the amino acid sequences corresponding to other CDRs according to the definition rules should also fall within the scope of protection of this disclosure. For example, when defining CDRs using Kabat's definition rules, the amino acid sequence of HCDR1 for mouse, chimeric, and humanized A01 is: TSGVH (SEQ ID NO: 79); the amino acid sequence of HCDR2 for mouse, chimeric A01, and hzA01-3.4 is: VIWAGGDTNYNSALMS (SEQ ID NO: 2); the amino acid sequence of HCDR2 for hzA01-1.1, hzA01-2.1, and hzA01-3.1 is: VIWAGGDTNYNPSLKS (SEQ ID NO: 80); the amino acid sequence of HCDR2 for hzA01-1.2 and hzA01-2.2 is: VIWAGGDTNYADSVKG (SEQ ID NO: 81); and the amino acid sequence of HCDR2 for hzA01-3.3 is: VIWAGGDTNYNSALKS (SEQ ID NO: 79). The amino acid sequences of HCDR3 of mouse, chimeric and humanized A01 are: HGHFDV (SEQ ID NO: 82), the amino acid sequences of LCDR1 of mouse, chimeric and humanized A01 are: RSSQSLVHSSGNTYLQ (SEQ ID NO: 6), the amino acid sequences of LCDR2 of mouse, chimeric and humanized A01 are: KVSNRFS (SEQ ID NO: 7), and the amino acid sequences of LCDR3 of mouse, chimeric and humanized A01 are: SQTTHVPWT (SEQ ID NO: 8).

[0085] The VH and / or VL sequences (or CDR sequences) of other anti-CD40 antibodies that bind to human CD40 can be "mixed and paired" with the VH and / or VL sequences (or CDR sequences) of the anti-CD40 antibody of this disclosure or its antigen-binding moiety. Preferably, when the VH and VL chains (or their CDRs) are mixed and paired, the VH sequence in a particular VH / VL pair can be replaced by a structurally similar VH sequence. Similarly, it is preferable to replace the VL sequence in a particular VH / VL pair with a structurally similar VL sequence.

[0086] Therefore, in one embodiment, the antibody or antigen-binding fragment thereof disclosed herein comprises:

[0087] (a) The heavy chain variable region, which contains the amino acid sequences listed in Tables 1 and 10; and

[0088] (b) A light chain variable region comprising the amino acid sequence listed in Tables 1 and 10, or a VL of another anti-CD40 antibody, wherein the antibody or its antigen-binding portion specifically binds to human CD40.

[0089] In another embodiment, the antibody or antigen-binding fragment thereof disclosed herein comprises:

[0090] (a) HCDR1, HCDR2 and HCDR3 listed in Tables 1 and 10; and

[0091] (b) LCDR1, LCDR2 and LCDR3 listed in Tables 1 and 10, or the light chain variable region CDR of another anti-CD40 antibody, wherein the antibody or its antigen-binding portion specifically binds to human CD40.

[0092] In another embodiment, the antibodies or antigen-binding fragments thereof disclosed herein include HCDR2 listed in Tables 1 and 10, as well as CDRs of other anti-CD40 antibodies, such as HCDR1 and / or HCDR3 of other anti-CD40 antibodies, and / or LCDR1, LCDR2 and / or LCDR3 of other anti-CD40 antibodies.

[0093] Furthermore, it is known in the art that the CDR3 domain, independent of the CDR1 and / or CDR2 domains, can be used to determine the binding specificity of an antibody to the same antigen, and can predict the generation of multiple antibodies with the same binding specificity based on the CDR3 sequence.

[0094] In another embodiment, the antibody or antigen-binding fragment thereof of the present disclosure comprises HCDR2 listed in Tables 1 and 10, and HCDR3 and / or LCDR3 listed in Table 1, or HCDR3 and / or LCDR3 of another anti-CD40 antibody, wherein the antibody or its antigen-binding moiety specifically binds to human CD40. These antibodies preferably (a) compete with the anti-CD40 antibody of the present disclosure for binding to CD40; (b) retain functional characteristics; (c) bind the same epitope; and / or (d) have similar binding affinity. In another embodiment, the antibody or its antigen-binding moiety thereof of the present disclosure further comprises LCDR2 listed in Tables 1 and 10, or LCDR2 of another anti-CD40 antibody, wherein the antibody or its antigen-binding moiety specifically binds to human CD40. In another embodiment, the antibody or its antigen-binding portion thereof disclosed herein further includes HCDR1 and / or LCDR1 listed in Tables 1 and 10, or HCDR1 and / or LCDR1 of another anti-CD40 antibody, wherein the antibody or its antigen-binding portion thereof specifically binds to human CD40.

[0095] In another embodiment, the heavy chain variable region and / or light chain variable region or CDR1, CDR2, and CDR3 sequences of the antibody or its antigen-binding moiety disclosed herein may contain one or more conserved modifications. It will be understood in the art that some conserved sequence modifications will not cause the loss of antigen-binding specificity.

[0096] Therefore, in one embodiment, the antibody or its antigen-binding portion of the present disclosure comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region and the light chain variable region respectively comprise CDR1, CDR2 and CDR3, wherein:

[0097] (a) The HCDR1 sequence comprises the sequences listed in Tables 1 and 10, and / or their conservative modifications; and / or

[0098] (b) The HCDR2 sequence comprises the sequences listed in Tables 1 and 10, and / or their conservative modifications; and / or

[0099] (c) The HCDR3 sequence includes the sequences listed in Tables 1 and 10, and / or their conserved modifications; and / or

[0100] (d) The LCDR1 and / or LCDR2 and / or LCDR3 sequences contain the sequences listed in Tables 1 and 10; and / or their conservative modifications; and

[0101] (e) The antibody or its antigen-binding moiety specifically binds to human CD40.

[0102] As used herein, the term "conserved sequence modification" refers to amino acid modifications that do not significantly affect or alter antibody binding properties. Such conserved modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of this disclosure using standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conserved amino acid substitution refers to the replacement of an amino acid residue with an amino acid residue having similar structural or chemical properties (e.g., similar side chains). Families of amino acid residues with similar side chains are known in the art. These amino acid residue families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the CDR region of the anti-CD40 antibody of this disclosure or its antigen-binding moiety can be substituted with other amino acid residues from the same side chain family, and the resulting antibody can be functionally tested using the functional assays described herein.

[0103] The antibodies or antigen-binding fragments disclosed herein may contain variable regions (including CDR regions and / or backbone regions) modified, or the antibodies or antigen-binding portions of the disclosed herein may also contain Fc modifications, for example, altering the effector function of the antibody. Therefore, one embodiment of the disclosed invention provides an isolated anti-CD40 monoclonal antibody or its antigen-binding fragment comprising a heavy chain variable region and / or a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of the sequences described above in the disclosed invention, and the light chain variable region comprising LCDR1, LCDR2, and LCDR3 of the sequences described above in the disclosed invention, but comprising a backbone sequence different from the sequences described above in the disclosed invention. Such backbone sequences can be obtained from publicly available DNA databases or publicly available references including germline antibody gene sequences. Such backbone sequences are preferably those structurally similar to the backbone sequence used in the anti-CD40 antibody of the disclosed invention. The CDR1, CDR2, and CDR3 sequences can be transplanted into backbone regions having the same sequence as germline immunoglobulin genes containing such backbone sequences, or the CDR sequences can be transplanted into backbone regions having one or more mutations compared to the germline sequences. For example, in some cases, mutations in residues in the backbone region are beneficial and can maintain or enhance the antigen-binding ability of the antibody (see, for example, US Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0104] Another type of variable region modification involves mutating amino acid residues within the CDR1, CDR2, and / or CDR3 regions to improve one or more properties of the target antibody (e.g., affinity, physicochemical properties). Mutations can be introduced via point mutations or PCR-mediated mutations, and the effect of the mutation on antibody binding or other functional properties can be assessed using in vitro or in vivo assays known in the art. Amino acids can be substituted, added, or deleted, preferably substituted. Furthermore, typically no more than one, two, three, four, or five residues within each CDR region are altered.

[0105] In one embodiment, the antibody or antigen-binding fragment provided in this disclosure comprises a heavy chain variable region and a light chain variable region, said heavy chain variable region and light chain variable region comprising: (a) an HCDR1 region comprising the sequence of this disclosure, or having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions; (b) an HCDR2 region comprising the sequence of this disclosure, or having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions; and (c) an HCDR3 region comprising the sequence of this disclosure, or having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions. (d) an LCDR1 region containing the sequence of the present disclosure, or having amino acid sequences with 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions; (e) an LCDR2 region containing the sequence of the present disclosure, or having amino acid sequences with 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions; and (f) an LCDR3 region containing the sequence of the present disclosure, or having amino acid sequences with 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions.

[0106] The antibodies or antigen-binding fragments disclosed herein include backbone modifications of VH and / or VL to improve antibody properties. Typically, such backbone modifications can reduce the immunogenicity of the antibody. For example, one or more backbone residues are "reverted" to the corresponding germline sequence. These residues can be identified by comparing the antibody backbone sequence with the germline sequence from which the antibody is obtained.

[0107] Another type of backbone modification involves mutating one or more residues in the backbone region, or even one or more CDR regions, to remove T-cell epitopes, thereby reducing the immunogenicity that antibodies may cause. This method is also known as "deimmunogenicity" and is described in more detail in U.S. Patent Publication No. 20030153043.

[0108] In addition, the antibodies or antigen-binding fragments thereof disclosed herein include Fc modifications, which may be amino acid insertions, deletions or substitutions, and are typically used to alter one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding and / or antigen-dependent cytotoxicity.

[0109] Furthermore, the antibodies or antigen-binding fragments thereof disclosed herein can be chemically modified (e.g., by attaching one or more chemical functional groups), or modified to alter their glycosylation, to change one or more functional properties of the antibody. In other embodiments, the Fc region is modified by polyethylene glycol oxidative treatment (e.g., by reacting the antibody or a fragment thereof with polyethylene glycol (PEG)).

[0110] In another embodiment, the glycosylation of the antibody or its antigen-binding fragment disclosed herein is altered. Such glycosylation modification can be achieved, for example, by altering one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions can be made to eliminate glycosylation sites in the backbone region of one or more variable regions, thereby deglycosylating those sites. Such deglycosylation can increase the antibody's affinity for the antigen. See, for example, U.S. Patents 5,714,350 and 6,350,861.

[0111] The antibodies or antigen-binding fragments disclosed herein bind to CD40, thereby inhibiting CD40 activity. "CD40 activity" includes, but is not limited to, B cell activation, such as B cell proliferation, antibody production, antibody isotype conversion, or differentiation into plasma cells; T cell activation, such as T cell proliferation or cytokine secretion; dendritic cell activation, such as dendritic cell proliferation, differentiation, and maturation; and macrophage activation. CD40 activity can also be inhibited through interactions with other molecules. Furthermore, "CD40 activity" also includes inhibiting tumor cell growth and / or proliferation, and inducing tumor cell apoptosis.

[0112] In one embodiment, the anti-CD40 antibody or its antigen-binding fragment provided in this disclosure binds to CD40 and inhibits the activation of B cells. In another embodiment, the anti-CD40 antibody or its antigen-binding fragment provided in this disclosure binds to CD40 and inhibits the proliferation of B cells.

[0113] In one embodiment, the anti-CD40 antibody or its antigen-binding fragment provided in this disclosure binds to CD40 and inhibits T cell activation. In one embodiment, the anti-CD40 antibody or its antigen-binding fragment provided in this disclosure binds to CD40 and inhibits T cell proliferation and / or cytokine production. In one embodiment, the anti-CD40 antibody or its antigen-binding fragment provided in this disclosure binds to CD40 and inhibits the production of one or more cytokines selected from the group consisting of: IL-2, IFNγ, TNF, IL-1, IL-4, IL-5, IL-6, IL-12, IL-13, IL-17, and GM-CSF. In one embodiment, the anti-CD40 antibody or its antigen-binding fragment provided in this disclosure binds to CD40 and inhibits the production of the cytokine IFNγ. In one embodiment, the anti-CD40 antibody or its antigen-binding fragment provided in this disclosure binds to CD40 and inhibits the production of the cytokine IL-6.

[0114] Therefore, in one aspect, this disclosure provides a method for modulating an immune response, comprising contacting T cells and antigen-presenting cells with an anti-CD40 antibody or an antigen-binding fragment thereof disclosed herein. In one embodiment, the modulation of the immune response by the anti-CD40 antibody or an antigen-binding fragment thereof disclosed herein can be measured in a mixed lymphocyte reaction (MLR). In one embodiment, the anti-CD40 antibody or an antigen-binding fragment thereof disclosed herein inhibits the production of cytokines by lymphocytes in the MLR. In another embodiment, the anti-CD40 antibody or an antigen-binding fragment thereof disclosed herein inhibits the production of the cytokine IFNγ in the MLR.

[0115] In one embodiment, the anti-CD40 antibody or its antigen-binding fragment provided in this disclosure binds to CD40 and inhibits the activation of dendritic cells. In another embodiment, the anti-CD40 antibody or its antigen-binding fragment provided in this disclosure binds to CD40 and inhibits the differentiation and maturation of dendritic cells.

[0116] In one embodiment, the anti-CD40 antibody or its antigen-binding fragment provided in this disclosure binds to CD40 and inhibits tumor cell apoptosis. In another embodiment, the anti-CD40 antibody or its antigen-binding fragment provided in this disclosure binds to CD40 and inhibits Ramos cell apoptosis.

[0117] The anti-CD40 antibody or its antigen-binding fragment provided in this disclosure exhibits no significant agonistic activity against CD40. In one embodiment, the anti-CD40 antibody or its antigen-binding fragment provided in this disclosure exhibits no significant agonistic activity against tumor cell apoptosis. In one embodiment, the anti-CD40 antibody or its antigen-binding fragment provided in this disclosure exhibits no significant agonistic activity against Ramos cell apoptosis. In one embodiment, the anti-CD40 antibody or its antigen-binding fragment provided in this disclosure exhibits no significant agonistic activity against B cell activation. In one embodiment, the anti-CD40 antibody or its antigen-binding fragment provided in this disclosure exhibits no significant agonistic activity against B cell proliferation. In one embodiment, the anti-CD40 antibody or its antigen-binding fragment provided in this disclosure exhibits no significant agonistic activity against dendritic cell activation. In one embodiment, the anti-CD40 antibody or its antigen-binding fragment provided in this disclosure exhibits no significant agonistic activity against dendritic cell differentiation and maturation. A substance with "no significant agonistic activity" is defined as having agonistic activity detected in an assay that is no more than about 25% higher than that induced by the natural substance or negative control, preferably no more than about 20%, 15%, 10%, 5%, 1%, 0.5%, or even no more than about 0.1%, or having agonistic activity detected in an assay that is at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% lower than that induced by the positive control. In one embodiment, a nonspecific immunoglobulin that does not bind to CD40, such as an IgG4 isotype control antibody, is used as a negative control. In one embodiment, an agonistic anti-CD40 antibody, such as CP-870893, is used as a positive control.

[0118] The anti-CD40 antibody or its antigen-binding fragment provided in this disclosure has good safety. The anti-CD40 antibody or its antigen-binding fragment provided in this disclosure has good therapeutic effect on Sjögren's syndrome.

[0119] On the other hand, this disclosure provides pharmaceutical compositions comprising one or more of the anti-CD40 antibodies of this disclosure or antigen-binding fragments thereof and a pharmaceutically acceptable carrier. As used herein, a “pharmaceutically acceptable carrier” includes any and all physiologically compatible solvents, dispersion media, coatings, antimicrobial agents, isotonic agents, and combinations thereof. The selection and use of suitable “pharmaceutically acceptable carriers” are taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003).

[0120] Antibody or pharmaceutical compositions can be administered by any suitable method or route. Routes of administration include, for example, intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Based on the route of administration, the active ingredient can be coated in a material to protect it from acids and other natural conditions that may inactivate it. The phrase "parenteral administration" as used herein refers to a method of administration other than enteral and local administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, epidural, and intrasternal injections and infusions. Alternatively, pharmaceutical compositions can be administered via non-parenteral routes, such as local, epidermal, or mucosal routes, such as intranasal, oral, vaginal, rectal, sublingual, or topical administration.

[0121] For antibody administration, the dosage can be in the range of approximately 0.0001 to 100 mg / kg of host body weight.

[0122] The anti-CD40 antibody or its antigen-binding portion disclosed herein has various in vitro and in vivo applications relating to the diagnosis, treatment, and / or prevention of immune diseases. "Treatment" refers to methods for alleviating and / or stabilizing symptoms, signs, or conditions, delaying and / or halting disease progression, and / or reducing the severity of disease. The anti-CD40 antibody or its antigen-binding portion, encoding nucleic acid, pharmaceutical composition, recombinant polypeptide, fusion protein, bispecific molecule, immune conjugate, chimeric antigen receptor, or gene vector disclosed herein can be administered to human subjects to treat said subjects' immune diseases.

[0123] On the other hand, this disclosure provides a method for treating or preventing an immune disease in a subject, comprising administering to the subject a therapeutically effective amount of the disclosed anti-CD40 antibody or its antigen-binding moiety, encoding nucleic acid, pharmaceutical composition, recombinant polypeptide, fusion protein, bispecific molecule, immunoconjugate, chimeric antigen receptor, or gene vector. In some embodiments, the subject is a human.

[0124] "Immune disease" refers to any disease associated with the progression of an immune response in an individual, including cellular and / or humoral immune responses. In some specific embodiments, in the applications and methods described, the immune disease includes, but is not limited to, inflammatory diseases, allergic reactions, autoimmune diseases, or transplant-related diseases. Inflammatory diseases refer to any disease, disorder, or symptom resulting from an excessive or uncontrolled inflammatory response, characterized by excessive inflammatory symptoms, damage to host tissues, or loss of function, including allergic inflammation of the skin, kidneys, gastrointestinal tract, and respiratory tract, psoriasis, nephritis, epididymitis, inflammatory bowel disease, and asthma. Autoimmune diseases refer to any disease, disorder, or symptom caused by overactivation of the immune system, resulting in the body attacking and damaging its own tissues, such as sclerosis, arthritis, myasthenia gravis, psoriasis, scleroderma, autoimmune hepatitis, autoimmune mumps, and type I diabetes.

[0125] In some specific implementations, the immune diseases mentioned in the applications and methods include, but are not limited to: allergic reactions, Addison's disease, ankylosing spondylitis, spondyloarthritis, asthma, atherosclerosis, coronary heart disease, autoimmune hepatitis, autoimmune mumps, type I diabetes, epididymitis, nephritis, Reiter's syndrome, thyroiditis, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's disease, hemolytic anemia, idiopathic thrombocytopenic purpura, systemic lupus erythematosus, subacute cutaneous lupus erythematosus, multiple sclerosis, myasthenia gravis, psoriasis, scleroderma, arthritis, sarcoidosis, Sjögren's syndrome, dry eye syndrome, hidradenitis suppurativa, transplant-related diseases, vasculitis, and / or inflammatory bowel disease.

[0126] In some implementations, examples of transplant-related diseases include, but are not limited to, transplant immune rejection and transplant anti-host disease (GVHD).

[0127] In some implementations, examples of inflammatory bowel disease include, but are not limited to, Crohn's disease and ulcerative colitis.

[0128] In some implementations, examples of arthritis include, but are not limited to, rheumatoid arthritis, juvenile arthritis, and psoriatic arthritis.

[0129] In some implementations, examples of nephritis include, but are not limited to, lupus nephritis.

[0130] In some implementations, examples of psoriasis include, but are not limited to: psoriasis vulgaris, pustular psoriasis (such as palmoplantar psoriasis, generalized pustular psoriasis), erythrodermic psoriasis, and psoriatic arthritis.

[0131] In the applications and methods described herein, the anti-CD40 antibody or its antigen-binding portion may be administered alone or in combination with a second therapeutic agent. In some embodiments, the second therapeutic agent comprises a nonsteroidal anti-inflammatory drug (NSAID), salicylates, hydroxychloroquine, sulfasalazine, corticosteroids, cytotoxic agents, or immunosuppressive agents and / or antibodies. NSAIDs include, but are not limited to, ibuprofen, naproxen, diclofenac, indomethacin, ketotropic acid, meloxicam, naproxen, tetraprofen, and sulinda. Immunosuppressive agents and / or antibodies include, but are not limited to, cyclosporine, tacrolimus, rapamycin, mycophenolate mofetil, CTLA4-Ig fusion, anti-B lymphocyte stimulating antibodies, and anti-T cell antibodies (e.g., anti-CD-3). Cytotoxic agents include, but are not limited to, methotrexate and cyclophosphamide. Combinations of the therapeutic agents discussed herein may be administered simultaneously as a single composition in a pharmaceutically acceptable carrier or as separate compositions, wherein each agent is contained in a pharmaceutically acceptable carrier. In another implementation, the combination of therapeutic agents can be administered sequentially.

[0132] Furthermore, if multiple combination therapies are administered and the drugs are administered sequentially, the order of administration at each time point can be reversed or kept the same, and sequential administration can be combined with simultaneous administration or any combination thereof.

[0133] Although the foregoing invention has been described in considerable detail by way of example and embodiments for purposes of clarity, it will be apparent to those skilled in the art, based on the teachings of this disclosure, that certain changes and modifications may be made to this disclosure without departing from the spirit and scope of the appended claims. This disclosure is further illustrated by the following embodiments and is not intended to be limiting. Those skilled in the art will readily identify various non-critical parameters that can be changed or modified to produce substantially similar results.

[0134] Unless otherwise indicated, the practice of this disclosure will employ conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology within the scope of the art.

[0135] This disclosure also provides the following specific implementation schemes, but the scope of protection of this disclosure is not limited thereto:

[0136] Implementation Scheme 1. An isolated anti-CD40 antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising:

[0137] (i) Heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, wherein...

[0138] (1) Heavy chain CDR1 contains the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 9, heavy chain CDR2 contains the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 9, and heavy chain CDR3 contains the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 9;

[0139] (2) Heavy chain CDR1 contains the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 10, heavy chain CDR2 contains the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 10, and heavy chain CDR3 contains the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 10.

[0140] (3) Heavy chain CDR1 contains the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 11, heavy chain CDR2 contains the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 11, and heavy chain CDR3 contains the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 11.

[0141] (4) Heavy chain CDR1 contains the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 12, heavy chain CDR2 contains the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 12, and heavy chain CDR3 contains the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 12.

[0142] (5) Heavy chain CDR1 contains the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 13, heavy chain CDR2 contains the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 13 and heavy chain CDR3 contains the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 13.

[0143] (6) Heavy chain CDR1 contains the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 22, heavy chain CDR2 contains the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 22, and heavy chain CDR3 contains the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 22.

[0144] (7) Heavy chain CDR1 contains the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 30, heavy chain CDR2 contains the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 30 and heavy chain CDR3 contains the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 30.

[0145] (8) Heavy chain CDR1 contains the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 38, heavy chain CDR2 contains the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 38, and heavy chain CDR3 contains the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 38.

[0146] (9) Heavy chain CDR1 contains the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 44, heavy chain CDR2 contains the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 44, and heavy chain CDR3 contains the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 44; or

[0147] (10) Heavy chain CDR1 contains the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 52, heavy chain CDR2 contains the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 52, and heavy chain CDR3 contains the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 52; and / or

[0148] (ii) Light chains CDR1, CDR2, and CDR3, wherein...

[0149] (1) Light chain CDR1 contains the amino acid sequence of light chain CDR1 in SEQ ID NO: 14, light chain CDR2 contains the amino acid sequence of light chain CDR2 in SEQ ID NO: 14, and light chain CDR3 contains the amino acid sequence of light chain CDR3 in SEQ ID NO: 14.

[0150] (2) Light chain CDR1 contains the amino acid sequence of light chain CDR1 in SEQ ID NO: 15, light chain CDR2 contains the amino acid sequence of light chain CDR2 in SEQ ID NO: 15 and light chain CDR3 contains the amino acid sequence of light chain CDR3 in SEQ ID NO: 15;

[0151] (3) Light chain CDR1 contains the amino acid sequence of light chain CDR1 in SEQ ID NO: 16, light chain CDR2 contains the amino acid sequence of light chain CDR2 in SEQ ID NO: 16 and light chain CDR3 contains the amino acid sequence of light chain CDR3 in SEQ ID NO: 16;

[0152] (4) Light chain CDR1 contains the amino acid sequence of light chain CDR1 in SEQ ID NO: 17, light chain CDR2 contains the amino acid sequence of light chain CDR2 in SEQ ID NO: 17, and light chain CDR3 contains the amino acid sequence of light chain CDR3 in SEQ ID NO: 17.

[0153] (5) Light chain CDR1 contains the amino acid sequence of light chain CDR1 in SEQ ID NO: 23, light chain CDR2 contains the amino acid sequence of light chain CDR2 in SEQ ID NO: 23 and light chain CDR3 contains the amino acid sequence of light chain CDR3 in SEQ ID NO: 23;

[0154] (6) Light chain CDR1 contains the amino acid sequence of light chain CDR1 in SEQ ID NO: 31, light chain CDR2 contains the amino acid sequence of light chain CDR2 in SEQ ID NO: 31, and light chain CDR3 contains the amino acid sequence of light chain CDR3 in SEQ ID NO: 31.

[0155] (7) Light chain CDR1 contains the amino acid sequence of light chain CDR1 in SEQ ID NO: 39, light chain CDR2 contains the amino acid sequence of light chain CDR2 in SEQ ID NO: 39, and light chain CDR3 contains the amino acid sequence of light chain CDR3 in SEQ ID NO: 39.

[0156] (8) Light chain CDR1 contains the amino acid sequence of light chain CDR1 in SEQ ID NO: 45, light chain CDR2 contains the amino acid sequence of light chain CDR2 in SEQ ID NO: 45, and light chain CDR3 contains the amino acid sequence of light chain CDR3 in SEQ ID NO: 45; or

[0157] (9) Light chain CDR1 contains the amino acid sequence of light chain CDR1 in SEQ ID NO: 53, light chain CDR2 contains the amino acid sequence of light chain CDR2 in SEQ ID NO: 53 and light chain CDR3 contains the amino acid sequence of light chain CDR3 in SEQ ID NO: 53.

[0158] Implementation Scheme 2. The antibody or antigen-binding fragment thereof according to Implementation Scheme 1, wherein the antibody or antigen-binding fragment thereof comprises:

[0159] (i) Heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, wherein...

[0160] (1) Heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 1, 2 and 5 or have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 1, 2 and 5;

[0161] (2) Heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 1, 3 and 5 or have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 1, 3 and 5;

[0162] (3) Heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 1, 4 and 5 or have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 1, 4 and 5;

[0163] (4) Heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 18, 19 and 20 or amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 18, 19 and 20.

[0164] (5) Heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 24, 25 and 26 or amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 24, 25 and 26;

[0165] (6) Heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 32, 33 and 34 or amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 32, 33 and 34;

[0166] (7) Heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 40, 41, and 42 or amino acid sequences having at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 40, 41, and 42; or

[0167] (8) Heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 46, 47 and 48 or amino acid sequences having at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 46, 47 and 48; and / or

[0168] (ii) Light chains CDR1, CDR2, and CDR3, wherein...

[0169] (1) Light chain CDR1, light chain CDR2 and light chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 6, 7 and 8 or amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 6, 7 and 8.

[0170] (2) Light chain CDR1, light chain CDR2 and light chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 21, 7 and 8 or amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 21, 7 and 8.

[0171] (3) Light chain CDR1, light chain CDR2 and light chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 27, 28 and 29 or amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 27, 28 and 29;

[0172] (4) Light chain CDR1, light chain CDR2 and light chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 35, 36 and 37 or amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 35, 36 and 37.

[0173] (5) Light chain CDR1, light chain CDR2, and light chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 43, 36, and 37 or amino acid sequences having at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 43, 36, and 37; or

[0174] (6) Light chain CDR1, light chain CDR2 and light chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 49, 50 and 51 or amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 49, 50 and 51.

[0175] Implementation Scheme 3. The antibody or antigen-binding fragment thereof according to Implementation Scheme 1, wherein the antibody or antigen-binding fragment thereof comprises:

[0176] Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3, among which,

[0177] (1) The heavy chain CDR1 contains the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 9, the heavy chain CDR2 contains the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 9, the heavy chain CDR3 contains the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 9, the light chain CDR1 contains the amino acid sequence of the light chain CDR1 in SEQ ID NO: 14, the light chain CDR2 contains the amino acid sequence of the light chain CDR2 in SEQ ID NO: 14, and the light chain CDR3 contains the amino acid sequence of the light chain CDR3 in SEQ ID NO: 14;

[0178] (2) The heavy chain CDR1 contains the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 10, the heavy chain CDR2 contains the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 10, the heavy chain CDR3 contains the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 10, the light chain CDR1 contains the amino acid sequence of the light chain CDR1 in SEQ ID NO: 15, the light chain CDR2 contains the amino acid sequence of the light chain CDR2 in SEQ ID NO: 15, and the light chain CDR3 contains the amino acid sequence of the light chain CDR3 in SEQ ID NO: 15;

[0179] (3) The heavy chain CDR1 contains the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 11, the heavy chain CDR2 contains the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 11, the heavy chain CDR3 contains the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 11, the light chain CDR1 contains the amino acid sequence of the light chain CDR1 in SEQ ID NO: 15, the light chain CDR2 contains the amino acid sequence of the light chain CDR2 in SEQ ID NO: 15, and the light chain CDR3 contains the amino acid sequence of the light chain CDR3 in SEQ ID NO: 15;

[0180] (4) The heavy chain CDR1 contains the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 10, the heavy chain CDR2 contains the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 10, the heavy chain CDR3 contains the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 10, the light chain CDR1 contains the amino acid sequence of the light chain CDR1 in SEQ ID NO: 16, the light chain CDR2 contains the amino acid sequence of the light chain CDR2 in SEQ ID NO: 16, and the light chain CDR3 contains the amino acid sequence of the light chain CDR3 in SEQ ID NO: 16;

[0181] (5) The heavy chain CDR1 contains the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 11, the heavy chain CDR2 contains the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 11, the heavy chain CDR3 contains the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 11, the light chain CDR1 contains the amino acid sequence of the light chain CDR1 in SEQ ID NO: 16, the light chain CDR2 contains the amino acid sequence of the light chain CDR2 in SEQ ID NO: 16, and the light chain CDR3 contains the amino acid sequence of the light chain CDR3 in SEQ ID NO: 16;

[0182] (6) The heavy chain CDR1 contains the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 10, the heavy chain CDR2 contains the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 10, the heavy chain CDR3 contains the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 10, the light chain CDR1 contains the amino acid sequence of the light chain CDR1 in SEQ ID NO: 17, the light chain CDR2 contains the amino acid sequence of the light chain CDR2 in SEQ ID NO: 17, and the light chain CDR3 contains the amino acid sequence of the light chain CDR3 in SEQ ID NO: 17;

[0183] (7) The heavy chain CDR1 contains the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 12, the heavy chain CDR2 contains the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 12, the heavy chain CDR3 contains the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 12, the light chain CDR1 contains the amino acid sequence of the light chain CDR1 in SEQ ID NO: 17, the light chain CDR2 contains the amino acid sequence of the light chain CDR2 in SEQ ID NO: 17, and the light chain CDR3 contains the amino acid sequence of the light chain CDR3 in SEQ ID NO: 17;

[0184] (8) The heavy chain CDR1 contains the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 13, the heavy chain CDR2 contains the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 13, the heavy chain CDR3 contains the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 13, the light chain CDR1 contains the amino acid sequence of the light chain CDR1 in SEQ ID NO: 17, the light chain CDR2 contains the amino acid sequence of the light chain CDR2 in SEQ ID NO: 17, and the light chain CDR3 contains the amino acid sequence of the light chain CDR3 in SEQ ID NO: 17;

[0185] (9) The heavy chain CDR1 contains the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 22, the heavy chain CDR2 contains the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 22, the heavy chain CDR3 contains the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 22, the light chain CDR1 contains the amino acid sequence of the light chain CDR1 in SEQ ID NO: 23, the light chain CDR2 contains the amino acid sequence of the light chain CDR2 in SEQ ID NO: 23, and the light chain CDR3 contains the amino acid sequence of the light chain CDR3 in SEQ ID NO: 23;

[0186] (10) The heavy chain CDR1 contains the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 30, the heavy chain CDR2 contains the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 30, the heavy chain CDR3 contains the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 30, the light chain CDR1 contains the amino acid sequence of the light chain CDR1 in SEQ ID NO: 31, the light chain CDR2 contains the amino acid sequence of the light chain CDR2 in SEQ ID NO: 31, and the light chain CDR3 contains the amino acid sequence of the light chain CDR3 in SEQ ID NO: 31;

[0187] (11) The heavy chain CDR1 contains the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 38, the heavy chain CDR2 contains the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 38, the heavy chain CDR3 contains the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 38, the light chain CDR1 contains the amino acid sequence of the light chain CDR1 in SEQ ID NO: 39, the light chain CDR2 contains the amino acid sequence of the light chain CDR2 in SEQ ID NO: 39, and the light chain CDR3 contains the amino acid sequence of the light chain CDR3 in SEQ ID NO: 39;

[0188] (12) Heavy chain CDR1 contains the amino acid sequence of heavy chain CDR1 in SEQ ID NO: 44, heavy chain CDR2 contains the amino acid sequence of heavy chain CDR2 in SEQ ID NO: 44, heavy chain CDR3 contains the amino acid sequence of heavy chain CDR3 in SEQ ID NO: 44, light chain CDR1 contains the amino acid sequence of light chain CDR1 in SEQ ID NO: 45, light chain CDR2 contains the amino acid sequence of light chain CDR2 in SEQ ID NO: 45, and light chain CDR3 contains the amino acid sequence of light chain CDR3 in SEQ ID NO: 45; or

[0189] (13) The heavy chain CDR1 contains the amino acid sequence of the heavy chain CDR1 in SEQ ID NO: 52, the heavy chain CDR2 contains the amino acid sequence of the heavy chain CDR2 in SEQ ID NO: 52, the heavy chain CDR3 contains the amino acid sequence of the heavy chain CDR3 in SEQ ID NO: 52, the light chain CDR1 contains the amino acid sequence of the light chain CDR1 in SEQ ID NO: 53, the light chain CDR2 contains the amino acid sequence of the light chain CDR2 in SEQ ID NO: 53, and the light chain CDR3 contains the amino acid sequence of the light chain CDR3 in SEQ ID NO: 53.

[0190] Implementation Scheme 4. An antibody or antigen-binding fragment thereof according to any one of Implementation Schemes 1-3, wherein the antibody or antigen-binding fragment thereof comprises:

[0191] Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3, among which,

[0192] (1) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 1, 2, 5, 6, 7 and 8 or amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 1, 2, 5, 6, 7 and 8.

[0193] (2) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 1, 3, 5, 6, 7 and 8 or amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 1, 3, 5, 6, 7 and 8.

[0194] (3) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 1, 4, 5, 6, 7 and 8 or amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 1, 4, 5, 6, 7 and 8.

[0195] (4) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 18, 19, 20, 21, 7 and 8 or amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 18, 19, 20, 21, 7 and 8;

[0196] (5) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 24, 25, 26, 27, 28 and 29 or amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 24, 25, 26, 27, 28 and 29;

[0197] (6) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 32, 33, 34, 35, 36 and 37 or amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 32, 33, 34, 35, 36 and 37;

[0198] (7) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 40, 41, 42, 43, 36, and 37 or amino acid sequences having at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 40, 41, 42, 43, 36, and 37; or

[0199] (8) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 respectively contain the amino acid sequences shown in SEQ ID NOs: 46, 47, 48, 49, 50 and 51 or amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 46, 47, 48, 49, 50 and 51.

[0200] Implementation Scheme 5. An antibody or antigen-binding fragment thereof according to any one of Implementation Schemes 1-4, wherein the antibody or antigen-binding fragment thereof comprises:

[0201] (i) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NOs: 9, 10, 11, 12, 13, 22, 30, 38, 44 or 52, or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NOs: 9, 10, 11, 12, 13, 22, 30, 38, 44 or 52; and / or

[0202] (ii) A light chain variable region comprising an amino acid sequence shown in SEQ ID NOs: 14, 15, 16, 17, 23, 31, 39, 45 or 53, or an amino acid sequence having at least 80% identity with an amino acid sequence shown in SEQ ID NOs: 14, 15, 16, 17, 23, 31, 39, 45 or 53.

[0203] Implementation Scheme 6. An antibody or antigen-binding fragment thereof according to any one of Implementation Schemes 1-5, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein,

[0204] (1) The heavy chain variable region and the light chain variable region respectively contain the amino acid sequences shown in SEQ ID NOs: 9 and 14 or amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 9 and 14;

[0205] (2) The heavy chain variable region and the light chain variable region respectively contain the amino acid sequences shown in SEQ ID NOs: 10 and 15 or amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 10 and 15;

[0206] (3) The heavy chain variable region and the light chain variable region respectively contain the amino acid sequences shown in SEQ ID NOs: 11 and 15 or amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 11 and 15;

[0207] (4) The heavy chain variable region and the light chain variable region respectively contain the amino acid sequences shown in SEQ ID NOs: 10 and 16 or amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 10 and 16;

[0208] (5) The heavy chain variable region and the light chain variable region respectively contain the amino acid sequences shown in SEQ ID NOs: 11 and 16 or amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 11 and 16;

[0209] (6) The heavy chain variable region and the light chain variable region respectively contain the amino acid sequences shown in SEQ ID NOs: 10 and 17 or amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 10 and 17;

[0210] (7) The heavy chain variable region and the light chain variable region respectively contain the amino acid sequences shown in SEQ ID NOs: 12 and 17 or amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 12 and 17;

[0211] (8) The heavy chain variable region and the light chain variable region respectively contain the amino acid sequences shown in SEQ ID NOs: 13 and 17 or amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 13 and 17;

[0212] (9) The heavy chain variable region and the light chain variable region respectively contain the amino acid sequences shown in SEQ ID NOs: 22 and 23 or amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 22 and 23;

[0213] (10) The heavy chain variable region and the light chain variable region respectively contain the amino acid sequences shown in SEQ ID NOs: 30 and 31 or amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 30 and 31;

[0214] (11) The heavy chain variable region and the light chain variable region respectively contain the amino acid sequences shown in SEQ ID NOs: 38 and 39 or amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 38 and 39;

[0215] (12) The heavy chain variable region and the light chain variable region respectively contain the amino acid sequences shown in SEQ ID NOs: 44 and 45 or amino acid sequences having at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 44 and 45; or

[0216] (13) The heavy chain variable region and the light chain variable region respectively contain the amino acid sequences shown in SEQ ID NOs: 52 and 53 or amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 52 and 53.

[0217] Implementation Scheme 7. An antibody or antigen-binding fragment thereof according to any one of Implementation Schemes 1-6, wherein the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NOs: 54, 55 or 56 or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions compared to the amino acid sequence shown in SEQ ID NOs: 54, 55 or 56, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 57 or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions compared to the amino acid sequence shown in SEQ ID NO: 57.

[0218] Implementation Scheme 8. An antibody or antigen-binding fragment thereof according to any one of Implementation Schemes 1-7, wherein the antibody or antigen-binding fragment thereof: (a) binds to human CD40; (b) binds to monkey CD40; (c) blocks the interaction between CD40 and CD40L; and / or (d) inhibits CD40 activity.

[0219] Implementation Scheme 9. An antibody or antigen-binding fragment thereof according to any one of Implementation Schemes 1-8, wherein the antibody or antigen-binding fragment thereof is chimeric or humanized.

[0220] Implementation Scheme 10. An antibody or antigen-binding fragment thereof according to any one of Implementation Schemes 1-9, wherein the antibody or antigen-binding fragment thereof is an IgG1, IgG2 or IgG4 isotype.

[0221] Implementation Scheme 11. An antibody or antigen-binding fragment thereof according to any one of Implementation Schemes 1-10, wherein the antibody or antigen-binding fragment thereof is selected from monoclonal antibodies, monospecific antibodies, bispecific antibodies, trispecific antibodies, multispecific antibodies, Fab fragments, F(ab′)2 fragments, Fd fragments, Fv fragments, dAb fragments, isolated CDR regions, single-chain Fv molecules, or combinations thereof.

[0222] Implementation Scheme 12. An isolated antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof binds to the same epitope as the antibody or antigen-binding fragment thereof described in any one of Implementation Schemes 1-11.

[0223] Implementation Scheme 13. An isolated antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof competes with the antibody or antigen-binding fragment thereof of any one of Implementation Schemes 1-11 for binding to CD40.

[0224] Implementation Scheme 14. An isolated nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof, as described in any one of Implementation Schemes 1-11.

[0225] Implementation Scheme 15. An expression vector comprising the nucleic acid molecule described in Implementation Scheme 14.

[0226] Implementation Scheme 16. A host cell comprising the nucleic acid molecule described in Implementation Scheme 14 or the expression vector described in Implementation Scheme 15.

[0227] Implementation Scheme 17. A recombinant polypeptide or fusion protein comprising an antibody or an antigen-binding fragment thereof as described in any one of Implementation Schemes 1-11.

[0228] Implementation Scheme 18. A bispecific molecule comprising an antibody or an antigen-binding fragment thereof as described in any one of Implementation Schemes 1-11.

[0229] Implementation Scheme 19. An immunoconjugate comprising an antibody or an antigen-binding fragment thereof as described in any one of Implementation Schemes 1-11, and a therapeutic agent, such as a cytotoxic agent or an imaging agent, conjugated thereto.

[0230] Implementation Scheme 20. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described in any one of Implementation Schemes 1-11 and one or more pharmaceutically acceptable carriers.

[0231] Implementation Scheme 21. A method for treating or preventing an immune disease in a subject in need, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described in any one of Implementation Schemes 1-11, or a pharmaceutical composition as described in Implementation Scheme 20.

[0232] Implementation Scheme 22. The method according to Implementation Scheme 21, wherein the immune disease includes, but is not limited to, inflammatory diseases, allergic reactions, autoimmune diseases, or transplant-related diseases.

[0233] Implementation Scheme 23. The method according to Implementation Scheme 21, wherein the immune diseases include, but are not limited to, allergic reactions, Addison's disease, ankylosing spondylitis, spondyloarthritis, asthma, atherosclerosis, coronary heart disease, autoimmune hepatitis, autoimmune mumps, type I diabetes, epididymitis, nephritis, Reiter's syndrome, thyroiditis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, idiopathic thrombocytopenic purpura, systemic lupus erythematosus, subacute cutaneous lupus erythematosus, multiple sclerosis, myasthenia gravis, psoriasis, scleroderma, arthritis, sarcoidosis, Sjögren's syndrome, dry eye syndrome, hidradenitis suppurativa, transplant-related diseases, vasculitis and / or inflammatory bowel disease.

[0234] Implementation Scheme 24. The method according to any one of Implementation Schemes 21-23, wherein the method further comprises administering a therapeutically effective amount of a second therapeutic agent.

[0235] Implementation Scheme 25. The method according to Implementation Scheme 24, wherein the second therapeutic agent comprises a nonsteroidal anti-inflammatory drug, salicylate, hydroxychloroquine, sulfasalazine, corticosteroid, cytotoxic drug, or immunosuppressive drug and / or antibody. Example

[0236] Although this disclosure has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, such modifications or improvements made without departing from the spirit of this disclosure are all within the scope of protection claimed by this disclosure.

[0237] Example 1: Preparation of mouse anti-CD40 monoclonal antibody

[0238] Preparation of recombinant proteins

[0239] The cDNAs encoding recombinant human CD40 protein (hCD40-mFc, SEQ ID NO: 64) and recombinant monkey CD40 protein (cynoCD40-mFc, SEQ ID NO: 65) containing mouse antibody heavy chain Fc were synthesized and subcloned into the expression vector pcDNA3.1(+), which was then transfected into CHO cells for transient expression. The cell culture supernatant was collected, and the recombinant proteins hCD40-mFc and cynoCD40-mFc were purified using a MabSelectSure LX purification column (GE).

[0240] Construction of stable cell lines

[0241] cDNA encoding full-length hCD40 (SEQ ID NO: 62) and cynoCD40 (SEQ ID NO: 63) were synthesized and then subcloned into the expression vector pcDNA3.1(+) to obtain recombinant plasmids pcDNA3.1-hCD40 and pcDNA3.1-cynoCD40. Following the instructions of the Lipofectamin 3000 transfection reagent (Thermo, Cat: L3000015), the two recombinant plasmids were transfected into CHO-K1 cells to obtain stable cell lines CHO-K1-hCD40 and CHO-K1-cynoCD40.

[0242] The cDNAs encoding full-length hCD40L (SEQ ID NO: 66) and cynoCD40L (SEQ ID NO: 67) were synthesized and then subcloned into the expression vector pcDNA3.1(+) to obtain the recombinant plasmids pcDNA3.1-hCD40L and pcDNA3.1-cynoCD40L. Following the instructions of the Lipofectamin 3000 transfection reagent (Thermo, Cat: L3000015), the two recombinant plasmids were transfected into CHO-K1 cells to obtain stable cell lines CHO-K1-hCD40L and CHO-K1-cynoCD40L, respectively.

[0243] Following the instructions of the Lipofectamin 3000 transfection reagent (Thermo, Cat: L3000015), the plasmid pGL4.32[luc 2P / NF-κB-RE / Hygro]Vector (Promega, Cat: E8491) was transfected into HEK293T cells to obtain a stable cell line 293T-NFκB. Then, the BamHI and XhoI sites of the pcDNA3.1 / Zeo(+) vector were inserted into the coding sequence of the full-length hCD40 (SEQ ID NO: 62) to obtain the recombinant plasmid pcDNA3.1 / Zeo(+)-hCD40. Following the instructions of the lipofectamin 3000 transfection reagent (Thermo, Cat: L3000015), the recombinant plasmid pcDNA3.1 / Zeo(+)-hCD40 was transfected into 293T-NFκB cells to obtain a stable 293T-hCD40-NFκB cell line.

[0244] mouse immunization

[0245] Purified recombinant protein hCD40-mFc (SEQ ID NO: 64) was used as the antigen. hCD40-mFc was thoroughly mixed and emulsified with complete Freund's adjuvant (Sigma, Cat: F5881-10X10mL) at a volume ratio of 1:1 and administered subcutaneously to immunize mice. Two to four weeks after the initial immunization, hCD40-mFc or cynoCD40-mFc was thoroughly mixed and emulsified with Alum adjuvant (Thermo, Cat: 77161) at a volume ratio of 1:1 and administered as booster immunizations every two weeks, alternating between subcutaneous and intramuscular injections, for a total of six weeks. After immunization, serum was collected from each mouse to detect the serum titer of anti-CD40 specific antibodies. Mice with higher serum titers were selected for subsequent spleen cell fusion.

[0246] Preparation and screening of hybridomas

[0247] Three to four days prior to fusion, mice were intraperitoneally injected with hCD40-mFc for a pre-fusion shock immunization. On the day of fusion, mouse spleens were aseptically harvested, ground, and erythrocytes were lysed. The resulting cells were then resuspended in electrofusion buffer (BTX, Cat: 47-0001) to obtain a single-cell suspension. This suspension was mixed with logarithmic growth phase myeloma cells SP2 / 0 at a cell number ratio of 2:1, and cell fusion was performed using an electrofusion apparatus (BTX). The fused cells were then mixed with hybridoma medium (Gibco, Cat: 12045-076) containing 1×HAT and cultured at 37°C and 5% CO2 for 7 days to obtain hybridoma cells. The hybridoma cell culture supernatant was collected for screening mouse antibodies.

[0248] Hybridoma cells exhibiting hCD40 binding activity as detected by ELISA and FACS were selected for expansion culture. Once a certain cell number was reached, subcloning was performed using the limiting dilution method. The subcloned hybridoma cells were then incubated at 37°C and 5% CO2 for 7 days. The culture supernatant of the subcloned hybridoma cells was collected for further screening of mouse antibodies.

[0249] Expression and purification of mouse antibodies

[0250] After culturing the selected hybridoma cells for 10 days, the cell culture supernatant was collected by centrifugation. This supernatant was then loaded into a protein G column (Genscript, Cat: L00209-10). The protein G column was washed with PBS buffer, followed by elution of the antibody bound to the protein G column with 100 mM glycine (pH 2.8). The elution buffer was immediately neutralized with 1 M Tris-HC. Finally, the mouse antibody was replaced with PBS buffer using ultrafiltration.

[0251] Example 2 Characterization of mouse anti-CD40 antibody

[0252] ELISA assay to determine the binding activity of anti-CD40 antibody

[0253] Dilute hCD40-His protein (Acro, Cat: CD0-H5228) to a concentration of 0.1 μg / mL with PBS buffer (pH 7.4) and coat 100 μL / well of a 96-well plate. Incubate overnight at 4°C. Wash the 96-well plate with PBST (PBS containing 0.5% Tween-20), then add 200 μL / well of blocking buffer (PBST containing 1% BSA) and block at room temperature for 2 hours. Discard the blocking buffer, add 100 μL / well of hybridoma cell culture supernatant, and incubate at room temperature for 2 hours. After washing the 96-well plate with PBST, add HRP-conjugated goat anti-mouse IgG (H+L) antibody (Jackson Immuno, Cat: 115-035-062) diluted 1:10000 (v / v) and incubate at room temperature for 1 hour. After washing the 96-well plate with PBST, add 100 μL of TMB solution per well, incubate at room temperature in the dark for 5 minutes, and terminate the reaction with 0.5 M H₂SO₄. Read the OD450 values ​​using a Bio-rad iMark microplate reader.

[0254] FACS detection of anti-CD40 antibody binding activity

[0255] The cell concentration was 5 × 10 5 100 μL / well of CHO-K1-hCD40 cell suspension was added to each well of a 96-well U-plate, followed by 100 μL / well of hybridoma cell culture supernatant. The mixture was incubated at 4°C for 1 hour. After washing the cells with PBS containing 2% FBS, PE-conjugated goat anti-mouse IgG (H+L) antibody (Abcam, Cat: ab97041) diluted 1:500 (v / v) was added, and the mixture was incubated at 4°C for 1 hour. The cells were then washed again with PBS containing 2% FBS and resuspended in PBS. The binding of the anti-CD40 antibody to CHO-K1-hCD40 cells was analyzed using flow cytometry (BD Accuri C6) to measure the mean fluorescence intensity (MFI).

[0256] Anti-CD40 antibody blocks the binding activity of 293T-hCD40-NFκB cells to CD40L.

[0257] When hCD40 and CD40L interact on 293T-hCD40-NFκB cells, the expression of fluorescence signals is upregulated. Specifically, 293T-hCD40-NFκB cells in logarithmic growth phase are resuspended in DMEM medium containing 2% FBS, and the cell concentration is adjusted to 5 × 10⁶ cells / year. 5Add 10 μL of purified mouse anti-CD40 antibody (final antibody concentration range 0.064 ng / mL to 5000 ng / mL, serially diluted 5-fold) to each well of a 384-well plate, and then add 10 μL of the hybridoma cell culture supernatant or serially diluted purified mouse anti-CD40 antibody to each well. Incubate at 37°C with shaking for 20 minutes. Then add 10 μL of human CD40L protein (Acro, Cat: CDL-H52Db) to each well, and incubate at 37°C with shaking for 20 minutes. Transfer the 384-well plate to 37°C and 5% CO2 and incubate for 5–6 hours. After incubation, the detection reagents were added according to the instructions of the firefly luciferase kit (vazyme, Cat: DD1201-03). Fluorescence signals were read using a Thermo Varioskan Flash reader. The blocking activity of the anti-CD40 antibody against CD40L-mediated upregulation of fluorescence signal in 293T-hCD40-NFκB cells was analyzed by relative fluorescence intensity (RLU). The IC50 was calculated using Graphpad Prism. 50 Value. For example... Figure 1A and 1B As shown, multiple purified mouse anti-CD40 antibodies exhibited the activity of blocking the binding of 293T-hCD40-NFκB cells to CD40L.

[0258] Inhibitory activity of anti-CD40 antibody against Ramos cell apoptosis

[0259] The CD40 / CD40L-mediated MAPK activation pathway is a potential mechanism for inhibiting tumor cell proliferation and inducing tumor cell apoptosis. Using Ramos (Burkitt lymphoma) cells, a human B-cell lymphoma cell line endogenously expressing CD40, as a model, the inhibitory activity of anti-CD40 antibody on Ramos cell apoptosis was detected. Specifically, cells were prepared at a concentration of 1×10⁻⁶ cells / cells. 6Ramos cell suspension (cells / mL) was added at 50 μL / well to 96-well U-plates. Serially diluted purified mouse anti-CD40 antibody (final concentration range 0.02 ng / mL to 2000 ng / mL, 10-fold serial dilution) was added, along with human CD40L protein (Aero, Cat: CDL-H52Db) at a final concentration of 2 μg / mL and recombinant human IL-4 (Aero, Cat: IL4-H4218) at a final concentration of 60 ng / mL. After mixing, the cells were incubated overnight at 37°C with 5% CO2. The cells were then washed with PBS containing 2% FBS, and PE-conjugated mouse anti-human CD95 antibody (Biolegend, Cat: 305608) was added. After mixing, the cells were incubated at 4°C for 30 minutes. After washing, the cells were resuspended in PBS containing 2% FBS. Fluorescence signals were detected using a flow cytometer (Sartorius IQue3). The expression of the tumor cell apoptosis molecule CD95 was analyzed by the mean fluorescence intensity (MFI) of the staining cells, and the IC50 was calculated using Graphpad Prism. 50 Value. For example... Figure 2 As shown, several purified mouse anti-CD40 antibodies exhibited activity in inhibiting CD40L and IL-4-induced apoptosis in Ramos cells.

[0260] Example 3: Preparation of chimeric anti-CD40 antibody

[0261] Construction of chimeric antibodies

[0262] Total RNA was isolated from the hybridoma cells screened in Example 2 (e.g., A01, A02, A03, B01, B02, and B03) according to the instructions of the RNA extraction kit (Takara, Cat: 9767), and first-strand cDNA was synthesized using a reverse transcription kit (Thermo, Cat: K1652). Using the first-strand cDNA as a template, it was mixed with mouse IgG primers and Kappa primers, respectively, and cloned and sequenced using polymerase chain reaction (PCR) technology to obtain the variable region sequence of the mouse IgG antibody.

[0263] Using a chemical synthesis method, the DNA sequences of the VH and VL of mouse antibodies were ligated to the DNA sequences of the heavy chain constant region of human IgG4 (SEQ ID NO: 54) and the light chain constant region of Kappa (SEQ ID NO: 57), respectively, and then cloned into the pcDNA3.1(+) vector to construct recombinant human-mouse chimeric antibodies. These chimeric antibodies were named Chi-A01, Chi-A02, Chi-A03, Chi-B01, Chi-B02, and Chi-B03, respectively.

[0264] Reference antibody construction

[0265] Iscalimab (CFZ533) was selected as the reference antibody. The DNA sequences of the heavy and light chains of the antibody were obtained by chemical synthesis according to the amino acid sequence described in US Patent US9221913B2. The DNA sequences were cloned into the pcDNA3.1(+) vector to construct the reference antibody (the amino acid sequences of the heavy and light chains are shown in SEQ ID NOs: 58 and 59 of this disclosure, and the variable regions of the heavy and light chains are shown underlined). The reference antibody is also referred to herein as BM (an abbreviation for Benchmark).

[0266] Expression and purification of chimeric antibodies and reference antibodies

[0267] Chimeric antibodies and reference antibodies were transiently transfected using the expiCHO system (Gibco, Cat: A29129) according to the instructions of the transfection kit. After transfection, cells were cultured at 37°C with shaking for 6 days. Cell culture supernatant was collected by centrifugation and then loaded onto a Protein A column (GE Healthcare, Cat: 17-5474). The Protein A column was washed with 10 column volumes of PBS buffer, followed by elution with acetate buffer (300 mM acetic acid, pH 3.6) to remove the antibody bound to the Protein A column. The elution buffer was immediately neutralized with 1 M Tris-HCl. The chimeric antibody was then ultrafiltered into the PBS buffer.

[0268] Example 4: Characterization of chimeric anti-CD40 antibody

[0269] FACS detection of anti-CD40 antibody binding activity

[0270] The specific method is described in Example 2, but with some slight modifications: the hybridoma cell culture supernatant was replaced with serially diluted chimeric anti-CD40 antibody and BM (final concentration range of 0.064 ng / mL to 5000 ng / mL, serially diluted 5 times); and PE-conjugated donkey anti-human IgG (H+L) (Abcam, Cat: ab102439) was replaced with PE-conjugated goat anti-mouse IgG (H+L) antibody.

[0271] Figure 3 The binding activity of the chimeric antibody to CHO-K1-hCD40 cells is shown, with Chi-A01 and Chi-A02 binding EC2 to CHO-K1-hCD40 cells. 50 Comparable to BM.

[0272] Inhibitory activity of anti-CD40 antibody against Ramos cell apoptosis

[0273] The specific method is described in Example 2, but with some slight modifications: the serially diluted purified mouse antibody (final concentration range of 0.02 ng / mL to 2000 ng / mL, serially diluted 5 times) was replaced with serially diluted chimeric anti-CD40 antibody, BM and IgG4 isotype control antibody (final concentration range of 0.064 ng / mL to 1000 ng / mL, serially diluted 5 times).

[0274] Tables 2 and 3 show the inhibitory activity of chimeric antibodies against CD40L and IL-4-induced apoptosis in Ramos cells. Specifically, Chi-A01 and Chi-A02 inhibited 50% of CD40L and IL-4-induced tumor cell apoptosis at the concentrations (IC50) of these antibodies. 50 The value is approximately 5pM, while BM's IC 50 The concentration was 18.32 pM (approximately 3-4 times that of Chi-A01 and Chi-A02), indicating that Chi-A01 and Chi-A02 have better inhibitory activity against apoptosis in Ramos cells than BM.

[0275] Inhibition % = (MFI induced by apoptosis after incubation of Ramos with CD40L and IL-4 - MFI induced by apoptosis after incubation of Ramos with CD40L and IL-4 and anti-CD40 antibody) / (MFI induced by apoptosis after incubation of Ramos with CD40L and IL-4 - MFI of spontaneous apoptosis after incubation of Ramos with culture medium only) × 100%. According to the formula, the average maximum inhibition rate of Chi-A01 and Chi-A02 is comparable to that of BM.

[0276] Table 2. Inhibitory activities of chimeric antibodies Chi-A01, Chi-A02, and Chi-A03 on Ramos cell apoptosis.

[0277]

[0278] Table 3. Inhibitory activities of chimeric antibodies Chi-B01, Chi-B02, and Chi-B03 on apoptosis in Ramos cells.

[0279]

[0280] Note: NA indicates that IC was not fitted. 50 value.

[0281] Induced apoptosis-inducing activity of anti-CD40 antibody in Ramos cells

[0282] It is known that the agonistic anti-CD40 antibody CP-870893 (internally prepared, heavy and light chain sequences are shown in SEQ ID NOs: 60 and 61 of this disclosure) can generate a stimulatory signal that induces Ramos apoptosis upon binding to Ramos cells. The agonistic activity of the chimeric anti-CD40 antibody was detected using Ramos cells; specifically, cells were prepared at a concentration of 5 × 10⁶ cells / cells. 5 Ramos cell suspension (cells / mL) was added at 100 μL / well to a 96-well U-plate, and serially diluted anti-CD40 antibody (final concentration range 0.01 ng / mL to 10000 ng / mL, 10-fold serial dilution) was added. The mixture was incubated overnight at 37°C with 5% CO2. Cells were washed with PBS containing 2% FBS, and then PE-conjugated mouse anti-human CD95 antibody (Biolegend, Cat: 305608) was added. The mixture was incubated at 4°C for 30 minutes. After washing, cells were resuspended in PBS containing 2% FBS, and fluorescence signals were detected using flow cytometry (Sartorius IQue3). The expression of the tumor cell apoptosis molecule CD95 was analyzed by the mean fluorescence intensity (MFI). Results are shown below. Figure 4 Chimeric antibodies Chi-A02, Chi-B02, and Chi-B03 exhibit weak agonistic activity against Ramos cell apoptosis, while chimeric antibodies Chi-A01, Chi-A03, Chi-B01, and BM do not produce agonistic activity against Ramos cell apoptosis.

[0283] Example 5: Design and preparation of humanized anti-CD40 antibody

[0284] Design of humanized antibodies

[0285] Using the CDR-graft method, the heavy chain and light chain variable region sequences of mouse antibody A01 were compared and analyzed against a protein database to screen for human germline antibody sequences with the highest sequence homology. The complementarity-determining region (CDR) of the mouse antibody was grafted into the backbone of the screened human germline antibody sequence, and further mutations were made in the CDR and / or amino acid residues in the backbone to obtain more candidate variable region sequences.

[0286] Light chain VL: The combination of human embryonic skeleton sequences IGKV1-39*01 and IGKJ1*01 was used for humanization sequence design to construct light chain variant hzA01L1 (SEQ ID NO: 15); the combination of human embryonic skeleton sequences IGKV3-20*01 and IGKJ1*01 was used for humanization sequence design to construct light chain variant hzA01L2 (SEQ ID NO: 16); the combination of human embryonic skeleton sequences IGKV2-30*02 and IGKJ2*02 was used for humanization sequence design to construct light chain variant hzA01L3 (SEQ ID NO: 17).

[0287] Heavy chain VH: The combination of human embryonic skeleton sequences IGHV4-4*08 and IGHJ3*01 was used for humanization sequence design to construct heavy chain variants hzA01H1, hzA01H3 and hzA01H4 (see SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 13, respectively); The combination of human embryonic skeleton sequences IGHV3-33*01 and IGHJ3*01 was used for humanization sequence design to construct heavy chain variant hzA01H2 (SEQ ID NO: 11).

[0288] Construction of humanized antibodies

[0289] The DNA sequences of VH and VL of the above-mentioned humanized antibody were chemically synthesized and ligated to the DNA sequences of the heavy chain constant region of human IgG4 (SEQ ID NO: 54) and the light chain constant region of Kappa (SEQ ID NO: 57), respectively. These sequences were then cloned into the pcDNA3.1(+) vector to construct the recombinant humanized antibody. Table 4 shows the pairing of the VH and VL sequences of the humanized antibody.

[0290] Table 4. Variable region sequences of humanized antibodies

[0291] hzA01-1.1 hzA01L1 hzA01H1 hzA01-1.2 hzA01L1 hzA01H2 hzA01-2.1 hzA01L2 hzA01H1 hzA01-2.2 hzA01L2 hzA01H2 hzA01-3.1 hzA01L3 hzA01H1 hzA01-3.3 hzA01L3 hzA01H3 hzA01-3.4 hzA01L3 hzA01H4

[0292] Expression and purification of humanized antibodies

[0293] Humanized antibodies were transiently transfected using the expiCHO expression system (Gibco, Cat: A29129) according to the instructions of the transfection kit. After transfection, cells were cultured at 37°C with 8% CO2 for 6 days with shaking. The cell culture supernatant was collected by centrifugation and then incubated with protein A magnetic beads (GenScript, Cat: L00273) at room temperature with shaking for 2 hours. After washing with PBS, the antibody bound to the protein A magnetic beads was eluted with acetate buffer (300 mM acetic acid, pH 3.6), and the elution buffer was immediately neutralized with 1 M Tris-HCl. Finally, the humanized antibody was replaced with PBS buffer using ultrafiltration.

[0294] Example 6 Characterization of humanized anti-CD40 antibody

[0295] Affinity assay of anti-CD40 antibody

[0296] The affinity of humanized anti-CD40 antibody for hCD40-His protein (Aero, Cat: CD0-H5228) and CynoCD40-His protein (Acro, Cat: CD0-C52H6) was detected using a ForteBio molecular interaction analyzer. First, the AHC biosensor (Fortebio, Cat: 18-5060) was activated, and then immersed in an anti-CD40 antibody solution at a final concentration of 5 μg / mL to capture the antibody. The AHC biosensor then bound to serially diluted hCD40-His and CynoCD40-His proteins (final concentration range 1.56 nM–100 nM, 2-fold serial dilutions), respectively. The reaction signals were monitored in real time using a Fortebio molecular interaction analyzer (Fortebio Octet RED96e) to obtain binding and dissociation curves. After each cycle of dissociation, the AHC biosensor is regenerated for the next capture, and this cycle is repeated to determine the affinity of different antibodies for CD40. The data obtained are analyzed using Fortebio Data Analysis 11.0 software with a 1:1 (Langmuir) binding model to determine K. a (K on ) and K d (K dis ) value, and through K D =K d / K a Calculate the dissociation constant K D For more detailed detection methods and related information, please refer to www.fortebio.com. The results are shown in Table 5. The binding affinity K of humanized antibodies hzA01-3.1, hzA01-3.3, and hzA01-3.4 to human or cynomolgus monkey CD40 antigen is shown.D Between 0.1 nM and 1 nM, it has an affinity comparable to that of chimeric Chi-A01.

[0297] Table 5. Fortebio's detection of the affinity between humanized anti-CD40 antibody and CD40.

[0298]

[0299] ND: Indicates not detected.

[0300] FACS detection of anti-CD40 antibody binding activity

[0301] The specific method is described in Example 2, but with some slight modifications: 293T-hCD40-NFκB cells were used instead of CHO-K1-hCD40 cells, serially diluted anti-CD40 antibody (final concentration range 0.006 ng / mL to 1000 ng / mL, serially diluted 3 times) was used instead of hybridoma cell culture supernatant; PE-conjugated donkey anti-human IgG (H+L) antibody (Abcam, Cat: ab102439) was used instead of PE-conjugated goat anti-mouse IgG (H+L) antibody, and a flow cytometer (Sartorius IQue3) was used instead of a flow cytometer (BD Accuri C6).

[0302] See results Figure 5 Within the concentration range of 10–100 ng / mL, the humanized antibodies hzA01-3.1, hzA01-3.3, and hzA01-3.4 reached saturation in binding with 293T-hCD40-NFκB cells, and the MFI value corresponding to the saturation concentration was at the same level as that of BM.

[0303] Anti-CD40 antibodies block the interaction between CHO-K1-hCD40L cells and hCD40-mFc, and CHO-K1-CynoCD40L cells and... cynoCD40-mFc binding activity

[0304] The cell concentration was 3×10 5100 μL of CHO-K1-hCD40L cell suspension and 100 μL of CHO-K1-CynoCD40L cell suspension were added to each well of a 96-well U-plate. Then, hCD40-mFc protein (internal preparation, SEQ ID NO: 64) or cynoCD40-mFc protein (internal preparation, SEQ ID NO: 65) at a final concentration of 2 μg / mL were added accordingly. Next, serially diluted anti-CD40 antibody (final concentration range 312.5 ng / mL to 40000 ng / mL, serially diluted 2-fold) was added, and the mixture was incubated at 4°C for 1 hour. Cells were washed with PBS containing 2% FBS, and PE-conjugated goat anti-mouse IgG (H+L) antibody (Abcam, Cat: ab97041) diluted 1:500 (v / v) was added, and the mixture was incubated at 4°C for 0.5 hours. Cells were washed with PBS containing 2% FBS, then resuspended in PBS containing 2% FBS. Fluorescence signals were detected using a flow cytometer (Sartorius IQue3). The mean fluorescence intensity (MFI) of the staining was used to analyze the activity of anti-CD40 antibody in blocking the binding of CHO-K1-hCD40L cells to hCD40-mFc protein and CHO-K1-cynoCD40L cells to cynoCD40-mFc protein. The IC50 was calculated using GraphPadPrism software. 50 Values. The results are shown in Table 6, showing the blocking IC50 values ​​of humanized antibodies hzA01-3.1 and hzA01-3.4 at the cellular level. 50 Comparable to BM.

[0305] Table 6. Activity of humanized anti-CD40 antibody in blocking CD40 binding in CHO-K1-hCD40L and CHO-K1-CynoCD40L cells.

[0306]

[0307] Anti-CD40 antibody blocks the binding activity of 293T-hCD40-NFκB cells to CHO-K1-hCD40L cells.

[0308] The cell concentration was 2.5 × 10⁻⁶. 520 μL of a 293T-hCD40-NFκB cell suspension (cells / mL) was added to each well of a 96-well plate. Then, serially diluted anti-CD40 antibody (final concentration range 0.007 ng / mL to 2000 ng / mL, serially diluted 6-fold) was added. Simultaneously, CHO-K1-hCD40L cell suspension was added at a 1:1 ratio. After mixing, the plates were incubated at room temperature for 20 minutes. The 96-well plates were then transferred to a 37°C environment with 5% CO2 and incubated for 5–6 hours. After incubation, the detection reagents were added according to the instructions of the firefly luciferase kit (vazyme, Cat: DD1201-03). Cell fluorescence signals were detected using a ThermoVarioskan Flash reader. The blocking activity of the anti-CD40 antibody against CD40L-mediated upregulation of 293T-hCD40-NFκB cell fluorescence signal was analyzed by relative fluorescence intensity (RLU). Results are shown below. Figure 6 Within the concentration range of 0–2 μg / mL, humanized antibodies hzA01-1.1, hzA01-2.1, hzA01-3.1, hzA01-3.3, and hzA01-3.4 all showed significant blocking activity.

[0309] Inhibitory activity of anti-CD40 antibody against Ramos cell apoptosis

[0310] For specific methods, please refer to Example 2, but with some minor modifications: replace the serially diluted purified mouse anti-CD40 antibody (final concentration range 0.02 ng / mL to 2000 ng / mL, serially diluted 6 times) with serially diluted anti-CD40 antibody (final concentration range 0.007 ng / mL to 2000 ng / mL, serially diluted 10 times).

[0311] See results Figure 7 Within the concentration range of 0–2 μg / mL, humanized antibodies hzA01-3.1, hzA01-3.3, and hzA01-3.4 all exhibited sustained inhibitory activity, with an inhibitory IC50 value of [missing value]. 50 It is 3 to 4 times lower than BM.

[0312] Induced apoptosis-inducing activity of anti-CD40 antibody in Ramos cells

[0313] For specific methods, please refer to Example 4, but with some slight modifications: replace the serially diluted anti-CD40 antibody (final concentration range 0.01nM to 2000nM, serially diluted 5 times) with serially diluted anti-CD40 antibody (final concentration range 0.01ng / mL to 10000ng / mL, serially diluted 10 times).

[0314] See results Figure 8Humanized antibodies hzA01-1.1 and hzA01-2.1 exhibit weak agonistic activity, while humanized antibodies hzA01-3.1, hzA01-3.3, and hzA01-3.4 do not produce significantly greater agonistic activity than BM in the concentration range of 0.01 nM to 2000 nM.

[0315] Inhibitory activity of anti-CD40 antibody against peripheral blood B lymphocyte activation

[0316] CD40 expressed on human peripheral blood-derived B lymphocytes (PBMC-B) can bind to CD40L, inducing B cell activation and thereby upregulating cell surface activation signals (e.g., CD86). B lymphocytes from healthy human PBMCs were isolated and purified using a human B lymphocyte purification kit (Stemcell, Cat: 17954), with a cell concentration of 1 × 10⁻⁶ cells / cells. 6 Peripheral blood B lymphocyte suspension (50 μL / well) was added to 96-well U-type plates, followed by serially diluted anti-CD40 antibody (final concentration range 0.002 ng / mL to 2000 ng / mL, 10-fold serial dilution). Then, CHO-K1-hCD40L cell suspension was added at a 1:1 cell ratio. The plates were incubated overnight at 37°C with 5% CO2. Cells were washed with PBS containing 2% FBS, and then PE-conjugated mouse anti-human CD86 antibody (Biolegend, Cat: 305438) was added and mixed with the cells. The plates were incubated at 4°C for 30 minutes. Cells were washed again with PBS containing 2% FBS and resuspended. The mean fluorescence intensity (MFI) of the co-stimulatory molecule CD86 expressed by B cells was detected using flow cytometry (Sartorius IQue3), and the IC50 was calculated using Graphpad Prism analysis. 50 Value. See results. Figure 9 Humanized antibodies hzA01-3.1, hzA01-3.3, and hzA01-3.4 all exhibited inhibitory activity against CD40L-induced CD86 expression in human peripheral blood B lymphocytes, and their IC50 values ​​were significantly higher than those of CD40L-induced CD86 expression in human peripheral blood B lymphocytes. 50 It is about 2 to 4 times lower than BM.

[0317] Inhibitory activity of anti-CD40 antibody against peripheral blood B lymphocyte proliferation

[0318] CD40 expressed on human peripheral blood-derived B lymphocytes (PBMC-B) can bind to CD40L, inducing B cell proliferation. A cell concentration of 1×10⁻⁶ cells was used. 6Human peripheral blood B lymphocyte suspension at 50 μL / well was added to a 96-well U-plate. Serially diluted anti-CD40 antibody (final concentration range 0.000035 nM–35 nM, 10-fold serial dilution) was added, along with human CD40L protein (Acro, Cat: CDL-H52Db) at a final concentration of 2 μg / mL and recombinant human IL-4 protein (Acro, Cat: IL4-H4218) at a final concentration of 60 ng / mL. The mixture was incubated at 37°C with 5% CO2 for 5 days. Cells were collected, washed with PBS buffer, and resuspended. Cell fluorescence was detected using a CellTiter Glo luciferase assay kit (Promega, Cat: G7572) and measured using a Thermo Varioskan Flash reader. Human peripheral blood B lymphocyte proliferation was analyzed by relative fluorescence intensity (RLU), and IC50 was calculated using Graphpad Prism. 50 Value. See results. Figure 10 Humanized antibodies hzA01-3.1, hzA01-3.3, and hzA01-3.4 inhibited the IC50 of CD40L and IL-4-induced proliferation of human peripheral blood B lymphocytes. 50 It is about 3 to 6 times lower than BM.

[0319] The activity of anti-CD40 antibody in inducing peripheral blood B lymphocyte proliferation

[0320] The cell concentration was 1×10 6 A suspension of healthy human peripheral blood B lymphocytes (cells / mL) was added at 50 μL / well to a 96-well U-plate. Serially diluted anti-CD40 antibody (final concentration range 0.00035 nM–350 nM, 10-fold serial dilution) was added, and the plates were incubated at 37°C with 5% CO2 for 5 days. Cells were collected, washed with PBS buffer, and resuspended. Cell activity assay kits (Promega, Cat: G7572) were then added, and cell fluorescence signals were detected using a Thermo Varioskan Flash reader. Peripheral blood B lymphocyte proliferation was analyzed by relative fluorescence intensity (RLU). Results are shown below. Figure 11 The humanized antibody hzA01-3.1 induced weak proliferation at high concentrations (350 nM), while the humanized antibodies hzA01-3.3 and hzA01-3.4 did not produce significantly greater agonistic activity than BM in the concentration range of 0–350 nM.

[0321] Inhibitory effect of anti-CD40 antibody on mixed lymphocyte response (MLR)

[0322] Mononuclear cells were isolated from healthy human PBMCs according to the instructions of the mononuclear cell isolation kit (Stemcell, Cat: 19359), and then the cell concentration was adjusted to 1×10⁻⁶. 6 DC cells / mL. DC cell differentiation inducer was added according to the instructions of the DC cell culture kit (Stemcell, Cat: 10985), and the cells were incubated at 37°C with 5% CO2 for 5 days. Then DC cell maturation stimulant was added, and the cells were cultured for another 2 days to allow monocytes to differentiate into fully mature DC cells (mDC).

[0323] According to EasySep TM Instructions for using the CD4-positive T-cell isolation kit (Stemcell, Cat: 17952) to isolate CD4 from healthy human PBMCs. + T cells, then adjust the cell concentration to 2 × 10⁻⁶. 6 mDC cells / mL. Mix mDC cells at 1×10⁻⁶ 4 Add one sample per well to a 96-well U-plate, and add serially diluted anti-CD40 antibody or IgG4 isotype control antibody (final concentration range 10 ng / mL to 10000 ng / mL, serially diluted 10-fold). Mix well and incubate at room temperature for 30 minutes, then add mDC and CD4... + Add CD4 at a ratio of 1:20 for T cells + The T cell suspension was mixed and cultured at 37°C with 5% CO2 for 96 hours. The culture supernatant was then collected by centrifugation.

[0324] Following the instructions of the IFNγ detection kit (R&D, Cat: DY285B), IFNγ in the culture supernatant was detected by ELISA. The OD450 absorbance was read using a Bio-rad iMark microplate reader, and the IFNγ concentration was calculated and analyzed using Graphpad Prism. Results are as follows: Figure 12 As shown, the humanized antibody hzA01-3.3 inhibited mixed lymphocyte responses in a concentration-dependent manner within the range of 10 ng / mL to 10000 ng / mL, and the IFNγ release at saturation concentrations (1000 ng / mL to 10000 ng / mL) was lower than that of BM, indicating that hzA01-3.3 has a better inhibitory effect on mixed lymphocyte responses than BM at saturation concentrations.

[0325] The agonistic activity of anti-CD40 antibody on dendritic cell maturation

[0326] Monocytes were isolated from PBMCs according to the instructions of the monocyte isolation kit (Stemcell, Cat: 19359), and cultured for 5 days with a dendritic cell differentiation inducer (Stemcell, Cat: 10988). Incompletely mature dendritic cells (imDCs) were collected and the cell concentration was adjusted to 1×10⁻⁶. 6 Cells / mL. 50 μL of imDC cell suspension was added to each well of a 96-well plate, followed by serially diluted anti-CD40 antibody or IgG4 isotype control antibody (final concentration range 100 ng / mL to 10000 ng / mL, 10-fold serial dilution). The plates were incubated overnight at 37°C with 5% CO2. Cells were collected, washed with PBS containing 2% FBS, and resuspended. APC-conjugated mouse anti-human CD83 antibody (BD, Cat: 551073) was added, mixed, and incubated at 4°C for 30 minutes. Cells were washed with PBS containing 2% FBS and resuspended. Cell fluorescence signals were detected using flow cytometry (Sartorius IQue3), and the expression of CD83 on the cell surface was analyzed by mean fluorescence intensity (MFI). CD83 expression is a specific marker of mature DC cells; results are shown in [Figure 1]. Figure 13 CP-870893 has a significant stimulatory effect on imDC cell maturation, while humanized antibodies hzA01-3.3 and BM did not produce significant stimulatory activity against imDC cell maturation.

[0327] Example 7: Fc effect of anti-CD40 antibody

[0328] Human B cells, DC cells, and PBMCs express Fc receptors (FcRs) on their surface. When the Fc terminus of an anti-CD40 antibody binds to an FcR, it may mediate antibody-Fc crosslinking to produce agonistic activity, or mediate antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) leading to B cell exhaustion.

[0329] The cell concentration was 1×10 6Ramos cell suspension (50 μL / well) was added to 96-well U-plates, and anti-CD40 antibody (final concentration range 1 μg / mL to 10 μg / mL, serially diluted 10-fold) was added. After mixing, the mixture was incubated at room temperature for 20 minutes. Then, anti-human IgG Fcγ antibody (Jackson Immuno, Cat: 109-005-190) was added to a final concentration of 2 μg / mL, and the mixture was incubated overnight at 37°C with 5% CO2. Cells were collected by centrifugation, washed with PBS containing 2% FBS, and resuspended. Cell fluorescence signals were detected using a flow cytometry instrument (Sartorius IQue3), and the expression of the apoptosis molecule CD95 on Ramos cells was analyzed by mean fluorescence intensity (MFI). The results are shown in Table 7. Within the concentration range of 1 μg / mL to 10 μg / mL, the humanized antibody hzA01-3.3 did not produce cross-linking stimulatory activity significantly higher than that of BM, and its cross-linking agonist activity was much lower than that of CP-870893.

[0330] Table 7. Determination of Fc cross-linking activity of anti-CD40 antibody

[0331]

[0332] Using Ramos cells as target cells and Jurkat-NFAT-Luc cells (Promega) as effector cells, the ADCC effect mediated by anti-CD40 antibody was evaluated. Specifically, the cell concentration was 1×10⁻⁶ cells / cells. 6 Ramos cell suspension (50 μL / well) was added to each well of a 96-well plate. Serially diluted anti-CD40 antibody or Rituximab (final concentration range 0.46 ng / mL to 1000 ng / mL, 3-fold serial dilution) was then added, mixed, and incubated at room temperature for 20 minutes. The cell concentration was then increased to 5 × 10⁶ cells / well. 6 Jurkat-NFAT-Luc cell suspension (target cells: effector cells) was added at a rate of 100 μL / well to 96-well plates, with a target cell to effector cell ratio of 1:10. After mixing, the cells were incubated at 37°C for 5 hours. Cells were collected by centrifugation, washed with PBS, and then Luc fluorescence detection reagent (Suzhou Ruian, Cat: RA-GL03) was added. Cell fluorescence signals were detected using a Thermo Varioskan Flash microplate reader, and the relative fluorescence intensity (RLU) value was used to analyze the anti-CD40 antibody-mediated ADCC effect. Results are shown below. Figure 14 Rituximab exhibits a significant ADCC effect, while the humanized antibodies hzA01-3.3 and BM did not show ADCC activity.

[0333] The cell concentration was 1×10 6Ramos cell suspension (number of cells / mL) was added at 50 μL / well to a 96-well plate, along with 50 μL / well of serially diluted anti-CD40 antibody or Rituximab (final concentration range 2.4 ng / mL to 40000 ng / mL, serially diluted 4-fold). Simultaneously, 25 μL / well of human serum complement protein (Quidel, Cat: A112) was added. The mixture was incubated overnight at 37°C with 5% CO2. The assay kit (CellTiter Glo luciferase cell activity assay kit, Promega, Cat: G7572) was followed, and cell fluorescence signals were detected using a Thermo Varioskan Flash reader. Cell viability was analyzed using relative fluorescence intensity (RLU). Results showed that Rituximab exhibited significant CDC-killing activity, while the humanized antibody hzA01-3.3, similar to BM, did not show CDC-killing activity. Figure 15 ), CDC kill rate % = (target cell fluorescence value - antibody group fluorescence value) ÷ (target cell fluorescence value - target cell fluorescence value treated with detection reagent) × 100%.

[0334] Example 8: In vivo efficacy of humanized anti-CD40 antibody

[0335] Human PBMC cells from healthy individuals were transplanted into NDG-immunodeficient mice for immune reconstitution, establishing a mouse model that produces human antibodies. On one hand, human PBMCs respond to mouse antigens by producing human anti-mouse IgG and human anti-mouse IgM antibodies. On the other hand, human PBMC cells in mice, upon stimulation with exogenous proteins such as KLH, produce anti-KLH-specific antibodies. The method for constructing the mouse model producing human antibodies is as follows: Six- to eight-week-old female NDG-immunodeficient mice (purchased from Biocytogen) were selected. On day 0, each mouse was injected intravenously with 1×10-... 7 Healthy human PBMC cells were collected. From day 0 to day 5, each mouse was intraperitoneally injected with 10 μg of recombinant human IL-4 (Acro, Cat: IL4-H4218) daily. On day 0 and day 7, each mouse was intraperitoneally injected with 50 μg of KLH protein (Signa, Cat: H7017-20MG). Mice were grouped according to Table 8. Starting from day 0, each mouse received the antibody drug via intraperitoneal injection three times at a frequency of once a week.

[0336] Blood samples were collected from each mouse on days 8, 14, and 21, and serum was obtained. The concentrations of human anti-mouse IgG and human anti-mouse IgM antibodies in mouse serum were measured according to the instructions of the Human IgG ELISA kit (Thermo, Cat: BMS2091) and the Human IgM ELISA kit (Novus, Cat: NBP2-60477). OD450 absorbance was read using a Bio-rad iMark reader, and the concentrations of human anti-mouse IgG and human anti-mouse IgM antibodies in serum were calculated and analyzed using Graphpad Prism. Additionally, the titer of anti-KLH antibodies in mouse serum was determined as follows: 100 μL / well of KLH protein (Sigma, Cat: H7017-20MG) at a concentration of 2 μg / mL was added to each well of a 96-well plate and incubated overnight at 4°C. After washing the 96-well plates with PBST, 200 μL / well of blocking buffer (PBST containing 1% BSA) was added, and the plates were blocked at room temperature for 2 hours. The blocking buffer was discarded, and then 100 μL / well of serially diluted mouse serum was added, and the plates were incubated at room temperature for 2 hours. After washing the 96-well plates with PBST, HRP-conjugated goat anti-human IgG (H+L) antibody (Jackson Immuno, Cat: 109-035-088) diluted 1:10000 (v / v) was added, and the plates were incubated at room temperature for 1 hour. After washing the 96-well plates with PBST, 100 μL / well of TMB solution was added, and the plates were incubated at room temperature in the dark for 5 minutes. The reaction was terminated with 0.5 M H2SO4, and the absorbance was read at 450 nM using a Bio-rad iMark microplate reader. The results showed that, compared with the IgG4 isotype group, the humanized antibody hzA01-3.3 significantly inhibited the production of human anti-mouse IgG antibody and human anti-mouse IgM antibody. Figure 16A and 16B Furthermore, the hzA01-3.3 treatment group did not produce specific antibodies against KLH (data not shown).

[0337] On day 22, the spleens of the mice were removed after sacrifice, weighed, and the spleen coefficient of each group was analyzed (spleen coefficient = spleen weight / mouse body weight × 1000). The results are shown in Table 8. The spleens of the IgG4 isotype control group mice were significantly enlarged and the spleen coefficient was significantly increased; while the spleen enlargement of the mice in the hzA01-3.3 (0.5 mg / kg) group and the hzA01-3.3 (5 mg / kg) group was significantly alleviated, and hzA01-3.3 dose-dependently reduced the spleen coefficient of the mice; the spleen coefficients of the hzA01-3.3 (5 mg / kg) group and the BM (5 mg / kg) group were comparable.

[0338] Table 8. Determination of spleen coefficient in NDG mice

[0339] Blank control, n=3 0.04±0.01 23.33±2.71 1.81±0.40 IgG4 isotype control, n=4 0.24±0.03 22.50±1.27 10.82±1.14 hzA01-3.3 (0.5 mg / kg), n = 5 0.15±0.03 23.14±1.65 6.31±1.06 hzA01-3.3 (5 mg / kg), n = 5 0.13±0.04 22.56±2.16 5.50±1.42 BM (5 mg / kg), n = 5 0.13±0.03 24.04±0.38 5.58±1.09

[0340] Note: The blank control was an NDG mouse that had not undergone immune reconstitution and drug administration; the IgG4 isotype control was an Anti-HEL-HumanIgG4 (S228P L235E) Isotype-control (Biointron, Cat: B109805).

[0341] In addition, flow cytometry was used to analyze the content of human T lymphocytes and B lymphocytes infiltrating the spleen of mice in each group. Specifically, mouse spleens were used to prepare single-cell suspensions, and the cell concentration was adjusted to 1×10⁻⁶. 6 Cells were added at a rate of 100 μL / well to 96-well U-type plates. PE-conjugated mouse anti-human CD4 antibody (Biolegend, Cat: 980804) and APC-conjugated mouse anti-human CD19 antibody (BD, Cat: 555415) were added separately. After mixing, the cells were incubated at 4°C for 30 minutes. Cells were washed and resuspended in PBS containing 2% FBS. Cell fluorescence signals were detected using flow cytometry (Thermo Attune Nxt), and the percentage (%) of stained cells was used to analyze the infiltration of human CD4+ in the mouse spleen. + T cells and CD19 + B cell content. The results (Figure 17) showed that on day 22, the spleens of mice in the IgG4 isotype control group contained a large amount of human CD4. + T cells and human CD19 + B cell infiltration; while the number of T cells and B cells infiltrating the spleen of mice in the hzA01-3.3 (0.5 mg / kg) and hzA01-3.3 (5 mg / kg) groups was significantly lower than that in the IgG4 isotype group, and hzA01-3.3 reduced the infiltration content of human T cells and B cells in a dose-dependent manner.

[0342] Example 9: Pharmacodynamic evaluation of anti-CD40 antibody in a mandibular gland protein-induced hCD40 / hCD40L transgenic mouse model of Sjögren's syndrome.

[0343] The antigen was prepared as follows: Twenty female C57BL / 6 mice (6-8 weeks old; Biocytogen Pharmaceuticals Co., Ltd.) were used. Submandibular glands were collected, and the capsule and connective tissue were separated. The tissue was washed with PBS (Servicebio, Cat: G4202-500ML) at 4°C. The tissue was mechanically homogenized at low temperature, then centrifuged to obtain the supernatant, which was used as the antigen.

[0344] The hCD40 / hCD40L mouse model of Sjögren's syndrome was constructed as follows: On days 0 and 7, the antigen was mixed with complete Freund's adjuvant (Sigma, Cat: F5881) at a ratio of 1:1 (v / v) to prepare a submandibular gland protein emulsion, which was injected subcutaneously at two points on the back of hCD40 / hCD40L mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.), with a total volume of 200 μL; on day 14, the antigen was mixed with incomplete Freund's adjuvant (Sigma, Cat: F5506) at a ratio of 1:1 (v / v) to prepare a submandibular gland protein emulsion, which was injected subcutaneously at two points on the back of hCD40 / hCD40L mice, with a total volume of 200 μL.

[0345] hCD40 / hCD40L mice were divided into 5 groups according to their initial average saliva volume. Starting from day 0, the mice were administered the drug twice a week for a total of 10 weeks (administered on days 0, 4, 7, 11, 14, 18, 21, 25, 28, and 32), and the experiment ended on day 35. The mice were weighed every two weeks during the experiment. The grouping and administration regimens are shown in Table 9.

[0346] Table 9 Grouping and Dosing Regimens

[0347]

[0348] Note: Blank control is hCD40 / hCD40L mice that have not been immunized with the antigen; N / A, not applicable; iv, intravenous injection; BIW, twice a week.

[0349] The efficacy of hzA01-3.3 was evaluated through salivary flow rate measurement, submandibular gland index calculation, submandibular gland IL-6 detection, blood and spleen flow cytometry, and submandibular gland HE pathological staining. The specific methods are as follows:

[0350] On days -3, 6, 13, 19, 27, and 34, mice were anesthetized with 1% sodium pentobarbital solution at a dose of 60 mg / kg and intraperitoneally injected with pilocarpine (Aladdin, Cat: P129614) at a dose of 1.25 mg / kg. Saliva was collected within 15 minutes after injection, and the salivary flow rate was calculated as [wet weight of cotton ball (mg) - dry weight of cotton ball (mg)] / 15 minutes. The salivary flow rate data were analyzed by a T-test using GraphPad Prism.

[0351] On day 35, 100 μL of blood was collected from each mouse; PE-labeled anti-mouse CD45 antibody (Biolegend, Cat: 103106) and Brilliant Violct 510 were added to the blood samples. TMLabeled anti-mouse CD3 antibody (Biolegend, Cat: 100234), FITC-labeled anti-mouse CD4 antibody (Biolegend, Cat: 100510), PE / Cyanine7-labeled anti-mouse CD8a antibody (Biolegend, Cat: 100722), and APC-labeled anti-mouse CD19 antibody (Biolegend, Cat: 115512) were incubated at 4°C in the dark for 30 minutes. Then, erythrocyte lysis buffer (Biolegend, Cat: 420301) was added, and the cells were lysed at room temperature in the dark for 30 minutes. After that, the cells were washed with PBS and resuspended, and the fluorescence signal was detected by flow cytometry.

[0352] On day 35, the spleen of the mice was removed and prepared into a single-cell suspension, with the cell density adjusted to 1×10⁻⁶. 8 Cells / mL, 100 μL was added to erythrocyte lysis buffer (Biolegend, Cat: 420301) and lysed in the dark for 30 minutes. After washing with PBS, PE-labeled anti-mouse CD45 antibody (Biolegend, Cat: 103106) and Brilliant Violet 510 were added. TM Labeled anti-mouse CD3 antibody (Biolegend, Cat: 100234), FITC-labeled anti-mouse CD4 antibody (Biolegend, Cat: 100510), PE / Cyanine7-labeled anti-mouse CD8a antibody (Biolegend, Cat: 100722), and APC-labeled anti-mouse CD19 antibody (Biolegend, Cat: 115512) were incubated at 4°C in the dark for 30 minutes. The cells were then washed with PBS and resuspended, and the fluorescence signal was detected by flow cytometry.

[0353] On day 35, the submandibular glands of mice were removed, weighed, and the submandibular gland index was calculated as: submandibular gland weight (mg) / mouse body weight (g). One submandibular gland was fixed in 10% neutral formalin for pathological HE staining, and the submandibular gland pathological score was calculated. The submandibular gland pathological score data were analyzed by T-test using GraphPad Prism. Another submandibular gland was homogenized mechanically at low temperature, centrifuged at low temperature, and the supernatant was collected. The IL-6 content in the supernatant was detected according to the instructions of the mouse IL-6 ELISA kit (Abcam, Cat: ab222503).

[0354] The results of weight change showed ( Figure 18 The weight gain of animals in each group was comparable to that of the blank control group, indicating that hzA01-3.3 had no obvious toxic side effects at any dose.

[0355] Saliva flow rate results showed ( Figure 19 The salivary flow rate in both the hzA01-3.3 (5 mg / kg) and hzA01-3.3 (25 mg / kg) groups showed an increasing trend. Compared with the model group, the salivary flow rate in the hzA01-3.3 (25 mg / kg) group was significantly increased on days 27 and 34 (P < 0.05), indicating that hzA01-3.3 can improve the salivary flow rate in mice.

[0356] The results of submandibular gland index, submandibular gland IL-6 content, and submandibular gland pathology score showed that ( Figures 20A-20C The indicators of mice in the hzA01-3.3 (1 mg / kg), hzA01-3.3 (5 mg / kg), and hzA01-3.3 (25 mg / kg) groups all showed a decreasing trend. Compared with the model group, the indicators of mice in the hzA01-3.3 (5 mg / kg) and hzA01-3.3 (25 mg / kg) groups were significantly reduced, indicating that hzA01-3.3 can improve the submandibular gland index, inhibit the secretion of IL-6 in the submandibular gland, and reduce the pathological score of the submandibular gland.

[0357] Blood and spleen flow cytometry results showed ( Figures 21A-21H CD3 levels in the blood and spleen of animals in the hzA01-3.3 drug administration group were [data missing]. + T cells, CD4 + T cells, CD8 + T cells and CD19 + The percentage of B cells did not change significantly compared to the blank control group, indicating that hzA01-3.3 has no specific effect on the changes in the aforementioned cell numbers. It is expected that hzA01-3.3 will demonstrate good safety and / or good therapeutic efficacy in clinical practice.

[0358] The sequence information disclosed herein is summarized in Table 10.

[0359] Table 10 Sequence Information

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

Claims

1. An isolated anti-CD40 antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising: (1) The heavy chain CDR1 amino acid sequence, heavy chain CDR2 amino acid sequence, and heavy chain CDR3 amino acid sequence shown in SEQ ID NO: 12, and the light chain CDR1 amino acid sequence, light chain CDR2 amino acid sequence, and light chain CDR3 amino acid sequence shown in SEQ ID NO:

17. (2) The heavy chain CDR1 amino acid sequence, heavy chain CDR2 amino acid sequence, and heavy chain CDR3 amino acid sequence shown in SEQ ID NO: 9, and the light chain CDR1 amino acid sequence, light chain CDR2 amino acid sequence, and light chain CDR3 amino acid sequence shown in SEQ ID NO:

14. (3) The heavy chain CDR1 amino acid sequence, heavy chain CDR2 amino acid sequence, and heavy chain CDR3 amino acid sequence shown in SEQ ID NO: 10, and the light chain CDR1 amino acid sequence, light chain CDR2 amino acid sequence, and light chain CDR3 amino acid sequence shown in SEQ ID NO:

15. (4) The heavy chain CDR1 amino acid sequence, heavy chain CDR2 amino acid sequence, and heavy chain CDR3 amino acid sequence shown in SEQ ID NO: 11, and the light chain CDR1 amino acid sequence, light chain CDR2 amino acid sequence, and light chain CDR3 amino acid sequence shown in SEQ ID NO:

15. (5) The heavy chain CDR1 amino acid sequence, heavy chain CDR2 amino acid sequence, and heavy chain CDR3 amino acid sequence shown in SEQ ID NO: 10, and the light chain CDR1 amino acid sequence, light chain CDR2 amino acid sequence, and light chain CDR3 amino acid sequence shown in SEQ ID NO:

16. (6) The heavy chain CDR1 amino acid sequence, heavy chain CDR2 amino acid sequence, and heavy chain CDR3 amino acid sequence shown in SEQ ID NO: 11, and the light chain CDR1 amino acid sequence, light chain CDR2 amino acid sequence, and light chain CDR3 amino acid sequence shown in SEQ ID NO:

16. (7) The heavy chain CDR1 amino acid sequence, heavy chain CDR2 amino acid sequence, and heavy chain CDR3 amino acid sequence as shown in SEQ ID NO: 10, and the light chain CDR1 amino acid sequence, light chain CDR2 amino acid sequence, and light chain CDR3 amino acid sequence as shown in SEQ ID NO: 17; or (8) The heavy chain CDR1 amino acid sequence, heavy chain CDR2 amino acid sequence, and heavy chain CDR3 amino acid sequence shown in SEQ ID NO: 13, and the light chain CDR1 amino acid sequence, light chain CDR2 amino acid sequence, and light chain CDR3 amino acid sequence shown in SEQ ID NO:

17. in, The amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are defined according to the Kabat numbering method.

2. An isolated anti-CD40 antibody or its antigen-binding fragment thereof, said antibody or its antigen-binding fragment comprising heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3, wherein, (1) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 are shown in SEQ ID NOs: 1, 4, 5, 6, 7 and 8; (2) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 are shown in SEQ ID NOs: 1, 2, 5, 6, 7 and 8; (3) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 are shown in SEQ ID NOs: 1, 3, 5, 6, 7 and 8; (4) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 are shown in SEQ ID NOs: 79, 4, 82, 6, 7 and 8; (5) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 are as shown in SEQ ID NOs: 79, 80, 82, 6, 7 and 8; (6) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 as shown in SEQ ID NOs: 79, 81, 82, 6, 7 and 8; or (7) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3 are shown in SEQ ID NOs: 79, 2, 82, 6, 7 and 8.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises: (i) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NOs: 12, 9, 10, 11 or 13, or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NOs: 12, 9, 10, 11 or 13; and / or (ii) A light chain variable region comprising the amino acid sequence shown in SEQ ID NOs: 17, 14, 15 or 16, or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NOs: 17, 14, 15 or 16.

4. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein, (1) The heavy chain variable region and the light chain variable region respectively contain the amino acid sequences shown in SEQ ID NOs: 12 and 17 or amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 12 and 17; (2) The heavy chain variable region and the light chain variable region respectively contain the amino acid sequences shown in SEQ ID NOs: 9 and 14 or amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 9 and 14; (3) The heavy chain variable region and the light chain variable region respectively contain the amino acid sequences shown in SEQ ID NOs: 10 and 15 or amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 10 and 15; (4) The heavy chain variable region and the light chain variable region respectively contain the amino acid sequences shown in SEQ ID NOs: 11 and 15 or amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 11 and 15; (5) The heavy chain variable region and the light chain variable region respectively contain the amino acid sequences shown in SEQ ID NOs: 10 and 16 or amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 10 and 16; (6) The heavy chain variable region and the light chain variable region respectively contain the amino acid sequences shown in SEQ ID NOs: 11 and 16 or amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 11 and 16; (7) The heavy chain variable region and the light chain variable region respectively contain the amino acid sequences shown in SEQ ID NOs: 10 and 17 or amino acid sequences having at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 10 and 17; or (8) The heavy chain variable region and the light chain variable region respectively contain the amino acid sequences shown in SEQ ID NOs: 13 and 17 or amino acid sequences that have at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 13 and 17.

5. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NOs: 54, 55 or 56, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:

57.

6. An isolated anti-CD40 antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising a heavy chain and a light chain, wherein, (1) The heavy chain contains the amino acid sequences shown in SEQ ID NOs: 12 and 54, and the light chain contains the amino acid sequences shown in SEQ ID NOs: 17 and 57; (2) The heavy chain contains the amino acid sequences shown in SEQ ID NOs: 9 and 54, and the light chain contains the amino acid sequences shown in SEQ ID NOs: 14 and 57; (3) The heavy chain contains the amino acid sequences shown in SEQ ID NOs: 10 and 54, and the light chain contains the amino acid sequences shown in SEQ ID NOs: 15 and 57; (4) The heavy chain contains the amino acid sequences shown in SEQ ID NOs: 11 and 54, and the light chain contains the amino acid sequences shown in SEQ ID NOs: 15 and 57; (5) The heavy chain contains the amino acid sequences shown in SEQ ID NOs: 10 and 54, and the light chain contains the amino acid sequences shown in SEQ ID NOs: 16 and 57; (6) The heavy chain contains the amino acid sequences shown in SEQ ID NOs: 11 and 54, and the light chain contains the amino acid sequences shown in SEQ ID NOs: 16 and 57; (7) The heavy chain comprises the amino acid sequences shown in SEQ ID NOs: 10 and 54, and the light chain comprises the amino acid sequences shown in SEQ ID NOs: 17 and 57; or (8) The heavy chain contains the amino acid sequences shown in SEQ ID NOs: 13 and 54, and the light chain contains the amino acid sequences shown in SEQ ID NOs: 17 and 57.

7. The antibody or antigen-binding fragment thereof according to claim 1, 2 or 6, wherein the antibody or antigen-binding fragment thereof: (a) binds to human CD40; (b) binds to monkey CD40; (c) blocks the interaction between CD40 and CD40L; and / or (d) inhibits CD40 activity.

8. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is chimeric or humanized.

9. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is an IgG1, IgG2 or IgG4 isotype.

10. The antibody or antigen-binding fragment thereof according to claim 1, 2 or 6, wherein the antibody or antigen-binding fragment thereof is selected from monoclonal antibodies, monospecific antibodies, Fab fragments, F(ab')2 fragments, Fv fragments, single-chain Fv molecules or combinations thereof.

11. An isolated nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1-10.

12. An expression vector comprising the nucleic acid molecule of claim 11.

13. A host cell comprising the nucleic acid molecule of claim 11 or the expression vector of claim 12.

14. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1-10 and one or more pharmaceutically acceptable carriers.

15. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-10 or the pharmaceutical composition of claim 14 in the preparation of a medicament for treating an immune disease, wherein the immune disease is Sjögren's syndrome.

16. The use according to claim 15, wherein the medicament further comprises a second therapeutic agent.

17. The use according to claim 16, wherein the second therapeutic agent comprises a nonsteroidal anti-inflammatory drug, salicylate, hydroxychloroquine, sulfasalazine, corticosteroid, cytotoxic drug, or immunosuppressive drug and / or antibody.

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