CN118176216A

CN118176216A - Anti-ICOSL antibody fusion proteins and uses

Info

Publication number: CN118176216A
Application number: CN202280073112.2A
Authority: CN
Inventors: 毛浪勇; 应华; 李玲玲; 黄旋; 金薪盛; 陶维康
Original assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Current assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Priority date: 2021-11-11
Filing date: 2022-11-11
Publication date: 2024-06-11
Also published as: TW202334225A; WO2023083298A1

Abstract

Relates to an anti-ICOSL antibody fusion protein and uses thereof. In particular, it relates to anti-ICOSL antibodies, fusion proteins thereof and uses thereof.

Description

Anti-ICOSL antibody fusion proteins and uses

The present application claims priority from patent application filed 11/2021 (application number CN 202111330908.5).

Technical Field

The present disclosure is in the field of biotechnology, and more specifically, the disclosure relates to an anti-ICOSL antibody and fusion proteins and uses thereof.

Background

The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art.

Systemic lupus erythematosus (systemic lupus erythematosus, SLE) is an autoimmune disease that affects multiple organs. Under the action of genetic factors, environmental factors and the like, the human body loses immune tolerance, generates autoantibodies and nucleotide immune complexes to enter a blood circulation system, activates TLR receptors, and stimulates pDC cells to secrete a large amount of interferon alpha. The interferon further stimulates myeloid lineage cells to secrete a variety of cytokines, promoting activation of B and T cells, respectively (Curr Opin Rheumatol.2017Mar;29 (2): 178-186.). BAFF and APRIL are known to be activators of two B cells, which promote differentiation and maturation of B cells and antibody production by receptor interaction with the surface of B cells (Nat Rev Rheumatol.2016Nov22;12 (12): 716-730.). Whereas the signaling pathways of ICOSL and ICOS play a very important role in T cell activation and T-B cell interaction. The release of pro-inflammatory cytokines upon T cell activation results in differentiation of pro-inflammatory T cells and disruption of Treg cell homeostasis. In addition, the interaction between T-B cells further promotes the activation of T cells and B cells and produces pro-inflammatory cytokines to form a sustained immune response cycle (Nat Rev Rheumatol.2014Jan;10 (1): 23-34.). In the pathogenesis of SLE, the 2 immune pathways of T cells and B cells are involved in the SLE, and the signaling pathways of T cell and B cell activation and T-B cell interaction are inhibited, so that the SLE has better action effect. Other developments have also found that the ICOS/ICOSL signaling pathway plays an important role in the development of other immune responses such as inflammatory responses, allergic reactions, autoimmune diseases, transplant rejection, and the like, and B-cell or T-cell disorders (e.g., cancer).

ICOSL (Inducible costimulator-ligand, also known as B7RP1, B7H2, CD 275) is an immune co-stimulatory molecule belonging to the B7 family. ICOSL is expressed primarily on antigen presenting cell surfaces, including B cells, macrophages, monocytes and dendritic cells (biotugs.2013feb; 27 (1): 1-13.). The receptor for ICOSL is currently known to be ICOS, which is expressed primarily on the surface of T cells, and ICOSL and ICOS mediated signaling pathways play a major role in T cell differentiation and T cell dependent B cell activation (Nat Rev cancer.2012mar 22;12 (4): 252-64.). Activation of the ICOSL-ICOS signaling pathway can promote secretion of IL-4, ifnγ, IL-23R and IL-21 by Th2, th1, th17 and Tfh cells, respectively, which are involved in maintaining the activity of multiple T cell subtypes and promoting their further expansion (Curr Opin immunol 2010jun;22 (3): 326-32.). Furthermore, ICOSL also plays a very important role in the process of Tfh cell migration and T-B cell interaction.

TACI (Transmembrane activator and CAML interactor, also known as TNFRSF 13B) is a membrane-bound receptor with an extracellular domain containing two cysteine-rich pseudo-repeats, a transmembrane domain and a cytoplasmic domain that interacts with CAML (calcium modulator and cyclophilin ligand). TACI is associated with one subtype of B cells and T cells. The TACI receptor binds to BAFF (B-CELL ACTIVATING factor, also known as TNFSF 13B) of the tumor necrosis factor ligand family. BAFF is a B-cell activator belonging to the TNF family. BAFF is expressed mainly on the surface of bone marrow cell membranes and exists in the form of trimers. BAFF on the surface of cell membranes is hydrolyzed by proteases to form soluble BAFF which enters the blood circulation system and has multimerization characteristics, and can form 60 polymers at most. In addition, BAFF can also act with APRIL, another protein of the same family to form a heterologous trimer. Three receptors for BAFF are currently known on B cell surfaces, BAFF-R, BCMA and TACI, respectively. BAFF acts on these three receptors and is involved in the differentiation maturation, survival and regulation of B cells. APRIL and BAFF share two common receptors, BCMA and TACI, respectively. APRIL, which interacts with these two receptors, is important for B cell survival and regulation (Samy,E.,et al.,Int Rev Immunol,2017.36:p.3-19;Kamal,A.and M.Khamashta,Autoimmun Rev,2014.13:p.1094-1101).BAFF to maintain B cell homeostasis, and excessive activation of the BAFF signaling pathway leads to survival of self-reactive B cells and production of autoantibodies to promote autoimmune responses (Cancro, m.p., d.p.d' Cruz, and m.a. khamashta, J CLIN INVEST, 2009.119:p.1066-73).

Disclosure of Invention

The present disclosure constructs an anti-ICOSL antibody fusion protein comprising an anti-ICOSL antibody and a TACI polypeptide.

In some embodiments, the anti-ICOSL antibody fusion protein, wherein the anti-ICOSL antibody comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein,

(I) The HCDR1, HCDR2 and HCDR3 of the heavy chain variable region comprise SEQ ID NOs: 42. 3, 39, 40 or 41, and LCDR1, LCDR2 and LCDR3 of the light chain variable region, respectively, comprises the amino acid sequences of HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 38. 4, 32, 33, 34, 35, 36 or 37, LCDR1, LCDR2 and LCDR3; or (b)

(Ii) The HCDR1, HCDR2 and HCDR3 of the heavy chain variable region comprise SEQ ID NOs: 30. 1, 28 or 29, and LCDR1, LCDR2 and LCDR3 of the light chain variable region, respectively, comprises the amino acid sequences of HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 22. 2, 20, 21, 23, 24, 25, 26 or 27, LCDR1, LCDR2 and LCDR3.

In some embodiments, the anti-ICOSL antibody fusion protein of any one of the above, wherein the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2 and LCDR3 of the light chain variable region are defined according to numbering rules selected from Kabat, IMGT, chothia, abM and contacts. In some embodiments, the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2 and LCDR3 of the light chain variable region are defined according to Kabat numbering convention. In some embodiments, the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2 and LCDR3 of the light chain variable region are defined according to IMGT numbering rules. In some embodiments, the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2 and LCDR3 of the light chain variable region are defined according to Chothia numbering rules. In some embodiments, the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2 and LCDR3 of the light chain variable region are defined according to the AbM numbering convention. In some embodiments, the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2 and LCDR3 of the light chain variable region are defined according to the Contact numbering convention.

In some embodiments, the anti-ICOSL antibody fusion protein of any one of the above, wherein:

(i) The HCDR1 of the heavy chain variable region comprises SEQ ID NO:11, HCDR2 comprises the amino acid sequence of SEQ ID NO:31 or 12, and HCDR3 comprises the amino acid sequence of SEQ ID NO:13, and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO:14, LCDR2 comprises the amino acid sequence of SEQ ID NO:15, and LCDR3 comprises the amino acid sequence of SEQ ID NO:16, an amino acid sequence of seq id no; or (b)

(Ii) The HCDR1 of the heavy chain variable region comprises SEQ ID NO:5, HCDR2 comprises the amino acid sequence of SEQ ID NO:19 or 6, and HCDR3 comprises the amino acid sequence of SEQ ID NO:7, and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO:8, LCDR2 comprises the amino acid sequence of SEQ ID NO:17 or 9, and LCDR3 comprises the amino acid sequence of SEQ ID NO:10 or 18.

In some embodiments, the anti-ICOSL antibody fusion protein of any one of the above, wherein

(I) The HCDR1 of the heavy chain variable region comprises SEQ ID NO:11, HCDR2 comprises the amino acid sequence of SEQ ID NO:31, and HCDR3 comprises the amino acid sequence of SEQ ID NO:13, and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO:14, LCDR2 comprises the amino acid sequence of SEQ ID NO:15, and LCDR3 comprises the amino acid sequence of SEQ ID NO:16, an amino acid sequence of seq id no; or (b)

(Ii) The HCDR1 of the heavy chain variable region comprises SEQ ID NO:5, HCDR2 comprises the amino acid sequence of SEQ ID NO:19, and HCDR3 comprises the amino acid sequence of SEQ ID NO:7, and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO:8, LCDR2 comprises the amino acid sequence of SEQ ID NO:17, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 10.

The HCDR1 of the heavy chain variable region comprises SEQ ID NO:11, HCDR2 comprises the amino acid sequence of SEQ ID NO:12, and HCDR3 comprises the amino acid sequence of SEQ ID NO:13, and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO:14, LCDR2 comprises the amino acid sequence of SEQ ID NO:15, and LCDR3 comprises the amino acid sequence of SEQ ID NO:16, and a sequence of amino acids.

The HCDR1 of the heavy chain variable region comprises SEQ ID NO:5, HCDR2 comprises the amino acid sequence of SEQ ID NO:6, and HCDR3 comprises the amino acid sequence of SEQ ID NO:7, and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO:8, LCDR2 comprises the amino acid sequence of SEQ ID NO:9, and LCDR3 comprises the amino acid sequence of SEQ ID NO:10, an amino acid sequence of seq id no; or (b)

The HCDR1 of the heavy chain variable region comprises SEQ ID NO:5, HCDR2 comprises the amino acid sequence of SEQ ID NO:6, and HCDR3 comprises the amino acid sequence of SEQ ID NO:7, and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO:8, LCDR2 comprises the amino acid sequence of SEQ ID NO:17, and LCDR3 comprises the amino acid sequence of SEQ ID NO:10, an amino acid sequence of seq id no; or (b)

The HCDR1 of the heavy chain variable region comprises SEQ ID NO:5, HCDR2 comprises the amino acid sequence of SEQ ID NO:19, and HCDR3 comprises the amino acid sequence of SEQ ID NO:7, and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO:8, LCDR2 comprises the amino acid sequence of SEQ ID NO:9, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 10.

In some embodiments, the anti-ICOSL antibody fusion protein of any one of the above, wherein the anti-ICOSL antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

In some embodiments, the antibody is a humanized antibody.

(i) The heavy chain variable region comprises a sequence identical to SEQ ID NO: 42. 39, 40 or 41, and said light chain variable region comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 38. 32, 33, 34, 35, 36 or 37 has an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98% or 99%) sequence identity; or (b)

(Ii) The heavy chain variable region comprises a sequence identical to SEQ ID NO: 30. 28 or 29, and the light chain variable region comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%,96%,97%,98%, or 99%) sequence identity to SEQ ID NO: 22. 20, 21, 23, 24, 25, 26, or 27 has an amino acid sequence having at least 90% (e.g., at least 90%, 95%,96%,97%,98%, or 99%) sequence identity; or (iii) the heavy chain variable region comprises a sequence identical to SEQ ID NO:3 (e.g., at least 90%, 95%,96%,97%,98%, or 99%) and the light chain variable region comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%,96%,97%,98%, or 99%) sequence identity to SEQ ID NO:4 (e.g., an amino acid sequence having at least 90% (e.g., at least 90%, 95%,96%,97%,98%, or 99%) sequence identity; or (b)

(Iv) The heavy chain variable region comprises a sequence identical to SEQ ID NO:1 (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99%) and the light chain variable region comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO:2 having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

In some embodiments, an anti-ICOSL antibody fusion protein according to any one of the preceding claims, comprising one or more amino acid mutations selected from positions 37, 43 and 49 (numbered according to the Kabat numbering system) on the framework region of the heavy chain variable region of the anti-ICOSL antibody, and/or one or more amino acid mutations selected from positions 1,2,3, 42, 43, 60 and 85 (numbered according to the Kabat numbering system) on the framework region of the light chain variable region.

In some embodiments, the anti-ICOSL antibody fusion protein, wherein HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:11, HCDR2 comprises the amino acid sequence of SEQ ID NO:31 or 12, and HCDR3 comprises the amino acid sequence of SEQ ID NO:13, and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO:14, LCDR2 comprises the amino acid sequence of SEQ ID NO:15, and LCDR3 comprises the amino acid sequence of SEQ ID NO:16, an amino acid sequence of seq id no; the heavy chain variable region of the antibody comprises a framework region selected from the group consisting of: 37I, 43E and 49A (numbered according to the Kabat numbering system), and/or comprises one or more amino acid mutations in the framework regions of the light chain variable region selected from: 1N, 2T, 3V, 42Q, 43S, 60D, 85V (numbered according to the Kabat numbering system).

In some embodiments, an anti-ICOSL antibody fusion protein according to any one of the preceding claims, comprising one or more amino acid mutations selected from positions 1, 24, 69, 71, 73, 78 (numbered according to the Kabat numbering system) on the framework region of the heavy chain variable region of the anti-ICOSL antibody, and/or one or more amino acid mutations selected from positions 1,2 and 4 (numbered according to the Kabat numbering system) on the framework region of the light chain variable region.

In some embodiments, the anti-ICOSL antibody fusion protein, wherein HCDR1 of the heavy chain variable region of the anti-ICOSL antibody comprises SEQ ID NO:5, HCDR2 comprises the amino acid sequence of SEQ ID NO:19 or 6, and HCDR3 comprises the amino acid sequence of SEQ ID NO:7, and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO:8, LCDR2 comprises the amino acid sequence of SEQ ID NO:17 or 9, and LCDR3 comprises the amino acid sequence of SEQ ID NO:10 or 18; the heavy chain variable region of the antibody comprises a framework region selected from the group consisting of: 1E, 24T, 69L, 71V, 73K and 78A (numbered according to the Kabat numbering system), and/or comprises one or more amino acid mutations in the framework regions of the light chain variable region selected from the group consisting of: 1A, 2V and 4L (numbered according to the Kabat numbering system).

In some embodiments, an anti-ICOSL antibody fusion protein as defined in any of the above, wherein,

(I) The heavy chain variable region comprises SEQ ID NO: 42. 39, 40 or 41, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 38. 32, 33, 34, 35, 36 or 37; or (b)

(Ii) The heavy chain variable region comprises SEQ ID NO: 30. 28 or 29, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 22. 20, 21, 23, 24, 25, 26 or 27; or (b)

(Iii) The heavy chain variable region comprises SEQ ID NO:1, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:2, an amino acid sequence of seq id no; or (b)

(Iv) The heavy chain variable region comprises SEQ ID NO:3, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:4, and a sequence of amino acids.

(I) The heavy chain variable region comprises SEQ ID NO:42, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:38, an amino acid sequence of seq id no; or (b)

(Ii) The heavy chain variable region comprises SEQ ID NO:30, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 22.

In some embodiments, the anti-ICOSL antibody fusion protein of any one of the above, wherein the anti-ICOSL antibody comprises an antibody heavy chain constant region and a light chain constant region. In some embodiments, the heavy chain constant region is a human IgG heavy chain constant region. In some embodiments, the heavy chain constant region is selected from the group consisting of human IgG1, igG2, igG3, and IgG4 constant regions. In some embodiments, the light chain constant region is selected from the group consisting of human antibody kappa or lambda chain constant regions. In some embodiments, the heavy chain constant region is a human IgG4 heavy chain constant region and the light chain constant region is a human kappa light chain constant region. In some embodiments, the Fc region of the heavy chain constant region has one or more amino acid substitutions that reduce binding of the Fc region to Fc receptors. In some embodiments, the Fc region has an L234A, L235A mutation, and/or an S228P mutation, and/or a YTE mutation (M252Y, S T and T256E), the mutation numbering being according to the EU index. In some embodiments, the heavy chain constant region comprises SEQ ID NO:43, and the light chain constant region comprises the amino acid sequence of SEQ ID NO:44, and a polypeptide comprising the amino acid sequence of 44.

(I) The heavy chain of the anti-ICOSL antibody comprises a sequence identical to SEQ ID NO:47, and the light chain comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO:48 (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; or (b)

(Ii) The heavy chain of the anti-ICOSL antibody comprises a sequence identical to SEQ ID NO:45, and the light chain comprises an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO:46 has an amino acid sequence having at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

In some embodiments, an anti-ICOSL antibody fusion protein as described in any of the above,

(I) The heavy chain of the anti-ICOSL antibody comprises SEQ ID NO:47, and the light chain of said anti-ICOSL antibody comprises the amino acid sequence of SEQ ID NO: 48; or (b)

(Ii) The heavy chain of the anti-ICOSL antibody comprises SEQ ID NO:45, and the light chain of the anti-ICOSL antibody comprises the amino acid sequence of SEQ ID NO: 46.

In some embodiments, the anti-ICOSL antibody fusion protein of any one of the above, wherein the TACI polypeptide has better cleavage prevention function.

In some embodiments, the anti-ICOSL antibody fusion protein of any one of the above, wherein the TACI polypeptide is a polypeptide comprising SEQ ID NO:58 from amino acid residue 48 to 85 or a variant thereof; wherein the variant has an amino acid substitution at one or more of positions 49, 52, 53, 57, 65, 82 and 83, said amino acid substitution being at a position relative to the sequence of SEQ ID NO:58, and a naturally occurring numbering of amino acid residue positions. In some embodiments, the variant of the TACI polypeptide is one or more amino acid substitutions at a position selected from the group consisting of 49T or 49R, 52S, 53E or 53Q, 57E, 65T or 65A, 82A or 82R, and 83Y, which amino acid substitutions are relative to the sequence of SEQ ID NO:58, and a naturally occurring numbering of amino acid residue positions.

In some embodiments, the anti-ICOSL antibody fusion protein of any one of the above, wherein the TACI polypeptide is as set forth in SEQ ID NO:58 or SEQ ID NO:58 or a variant thereof. The truncated fragment comprises the sequence of SEQ ID NO:58 amino acid residues 48 to 85. The variant is a variant of SEQ ID NO:58 or a truncated fragment thereof, having one or more amino acid substitutions selected from the group consisting of positions 49, 52, 53, 57, 65, 82 and 83, wherein the amino acid substitutions are at a position relative to the sequence of SEQ ID NO:58, and a naturally occurring numbering of amino acid residue positions.

In some embodiments, the anti-ICOSL antibody fusion protein of any one of the above, wherein the truncated fragment of the TACI polypeptide comprises: SEQ ID NO:58 amino acid residues 48 to 86; SEQ ID NO:58 amino acid residues 48 to 87; or SEQ ID NO:58 amino acid residues 48 to 88.

In some embodiments, the anti-ICOSL antibody fusion protein of any one of the above, wherein the TACI polypeptide has the sequence set forth in SEQ ID NO: 60-63.

In some embodiments, the anti-ICOSL antibody fusion protein of any one of the above, wherein the TACI polypeptide has the sequence of SEQ ID NO:58 variant or SEQ ID NO:58 (e.g., a truncated fragment of the sequence of SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62 or SEQ ID NO: 63), which variant is a variant of the sequence set forth in SEQ ID NO:58 or a truncated fragment thereof having a substitution at any 1, 2, 3,4, 5, 6 or 7 amino acids selected from positions 49, 52, 53, 57, 65, 82 and 83, said amino acid substitution being at a position relative to the sequence of SEQ ID NO:58, and a naturally occurring numbering of amino acid residue positions.

In some embodiments, the anti-ICOSL antibody fusion protein of any one of the above, wherein the TACI polypeptide is a polypeptide set forth in SEQ ID NO: 58. SEQ ID NO: 60. SEQ ID NO: 61. SEQ ID NO:62 or SEQ ID NO:63 has one or more amino acid substitutions (e.g., 1,2,3,4, 5,6, or 7 amino acid substitutions) on the sequence selected from the group consisting of 49T or 49R, 52S, 53E or 53Q, 57E, 65T or 65A, 82A or 82R, and 83Y, wherein the amino acid substitutions are at a position relative to the sequence of SEQ ID NO:58, and a naturally occurring numbering of amino acid residue positions.

In some embodiments, the anti-ICOSL antibody fusion protein of any one of the above, wherein the TACI polypeptide is: in SEQ ID NO: 58. SEQ ID NO: 60. SEQ ID NO: 61. SEQ ID NO:62 or SEQ ID NO:63 has any one of the amino acid substitutions selected from 49T, 52S, 53E, 53Q, 57E and 82A. In SEQ ID NO: 58. SEQ ID NO: 60. SEQ ID NO: 61. SEQ ID NO:62 or SEQ ID NO:63 has 49R and 65T amino acid substitutions in the sequence. In SEQ ID NO: 58. SEQ ID NO: 60. SEQ ID NO: 61. SEQ ID NO:62 or SEQ ID NO:63 has 49R and 65A amino acid substitutions in the sequence. In SEQ ID NO: 58. SEQ ID NO: 60. SEQ ID NO: 61. SEQ ID NO:62 or SEQ ID NO:63 has 49R, 65T and 82R amino acid substitutions in the sequence. In SEQ ID NO: 58. SEQ ID NO: 60. SEQ ID NO: 61. SEQ ID NO:62 or SEQ ID NO:63 has 53E and 57E amino acid substitutions in the sequence. In SEQ ID NO: 58. SEQ ID NO: 60. SEQ ID NO: 61. SEQ ID NO:62 or SEQ ID NO:63 has 52S, 53E and 57E amino acid substitutions in the sequence. In SEQ ID NO: 58. SEQ ID NO: 60. SEQ ID NO: 61. SEQ ID NO:62 or SEQ ID NO:63 has 49T and 82A amino acid substitutions in the sequence. In SEQ ID NO: 58. SEQ ID NO: 60. SEQ ID NO: 61. SEQ ID NO:62 or SEQ ID NO:63 has 49T and 83Y amino acid substitutions in the sequence. In SEQ ID NO: 58. SEQ ID NO: 60. SEQ ID NO: 61. SEQ ID NO:62 or SEQ ID NO:63 has 49T, 82A and 83Y amino acid substitutions in the sequence. Or in SEQ ID NO: 58. SEQ ID NO: 60. SEQ ID NO: 61. SEQ ID NO:62 or SEQ ID NO:63 has 49T, 53E, 57E and 82A amino acid substitutions in the sequence. Wherein the aforementioned amino acid substitution is at a position relative to the sequence of SEQ ID NO:58, and a naturally occurring numbering of amino acid residue positions.

In some embodiments, the anti-ICOSL antibody fusion protein of any one of the above, wherein the TACI polypeptide has an amino acid sequence as set forth in SEQ ID NO:51 to 83.

In some embodiments, the anti-ICOSL antibody fusion protein of any one of the above, wherein the TACI polypeptide has an amino acid sequence as set forth in SEQ ID NO:60-63, SEQ ID NO: 66-83.

In some embodiments, the anti-ICOSL antibody fusion protein of any one of the above, wherein the TACI polypeptide has an amino acid sequence as set forth in SEQ ID NO: 83.

In some embodiments, an anti-ICOSL antibody fusion protein of any one of the above, comprising:

(A) First chain: [ heavy chain of anti-ICOSL antibody ] - [ linker 1] - [ TACI polypeptide 1], and

Second chain: [ TACI polypeptide 2] - [ linker 2] - [ light chain of ICOSL antibody ] (fig. 1);

(B) First chain: [ heavy chain of anti-ICOSL antibody ] - [ linker 1] - [ TACI polypeptide 1], and

Second chain: the light chain of the anti-ICOSL antibody (fig. 2); or (b)

(C) First chain: [ heavy chain of anti-ICOSL antibody ] - [ linker 1] - [ TACI polypeptide 1], and

Second chain: light chain of [ anti-ICOSL antibody ] - [ linker 2] - [ TACI polypeptide 2] (fig. 3);

Wherein in (a), (B) or (C), the TACI polypeptide 1 and TACI polypeptide 2 are the same or different, and the linker 1 and linker 2 are the same or different.

In some embodiments, the anti-ICOSL antibody fusion protein of any one of the above, wherein the TACI polypeptide 1 or TACI polypeptide 2 has an amino acid sequence as set forth in SEQ ID NO:51 to 83.

In some embodiments, the anti-ICOSL antibody fusion protein of any one of the above, wherein the TACI polypeptide 1 or TACI polypeptide 2 has an amino acid sequence as set forth in SEQ ID NO:60-63, SEQ ID NO: 66-83.

In some embodiments, the anti-ICOSL antibody fusion protein of any one of the above, wherein the TACI polypeptide 1 or TACI polypeptide 2 has an amino acid sequence as set forth in SEQ ID NO: 83.

The ordinal terms "first," "second," "1," "2" (e.g., "2" in "connector 2") in this disclosure are merely used to distinguish between different features, elements, components, or steps, and are not intended to limit the number, order, or level.

In some embodiments, each of the linker 1 and linker 2 independently has a structure (G _xS) _y, wherein x is selected from an integer of 1-5, and y is selected from an integer of 0-6. In some embodiments, each of the linker 1 and linker 2 independently has a structure (G _xS) _y, wherein x is selected from an integer of 1-5, and y is selected from an integer of 1-6. In some embodiments, each of the linker 1 and linker 2 independently is GGGS (as shown in SEQ ID NO: 89) or GGGGSGGGGSGGS (as shown in SEQ ID NO: 90).

In some embodiments, the anti-ICOSL antibody fusion protein of any one of the above, wherein the anti-ICOSL antibody fusion protein has: comprising SEQ ID NO:86, and a first strand comprising the amino acid sequence of SEQ ID NO: 88. 87 or 48, and a second strand of an amino acid sequence of seq id no; or the anti-ICOSL antibody fusion protein has: comprising SEQ ID NO:84, and a first strand comprising the amino acid sequence of SEQ ID NO:85 or 46. In some embodiments, the anti-ICOSL antibody fusion protein has 2 polypeptides comprising SEQ ID NO:86, and 2 strands comprising the amino acid sequence of SEQ ID NO:88, and a second strand of an amino acid sequence of seq id no. In some embodiments, the anti-ICOSL antibody fusion protein has 2 polypeptides comprising SEQ ID NO:86, and 2 strands comprising the amino acid sequence of SEQ ID NO:87, and a second strand of the amino acid sequence of seq id no. In some embodiments, the anti-ICOSL antibody fusion protein has 2 polypeptides comprising SEQ ID NO:86, and 2 strands comprising the amino acid sequence of SEQ ID NO:48, and a second strand of an amino acid sequence of seq id no. In some embodiments, the anti-ICOSL antibody fusion protein has 2 polypeptides comprising SEQ ID NO:84, and 2 strands comprising the amino acid sequence of SEQ ID NO:85, and a second strand of the amino acid sequence of seq id no. In some embodiments, the anti-ICOSL antibody fusion protein has 2 polypeptides comprising SEQ ID NO:84, and 2 strands comprising the amino acid sequence of SEQ ID NO:46, and a second strand of an amino acid sequence of seq id no.

In another aspect, the present disclosure provides an anti-ICOSL antibody, wherein the anti-ICOSL antibody comprises a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein,

In some embodiments, the anti-ICOSL antibody of any one of the above, wherein the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2 and LCDR3 of the light chain variable region are defined according to numbering rules selected from Kabat, IMGT, chothia, abM and contacts. In some embodiments, the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2 and LCDR3 of the light chain variable region are defined according to Kabat numbering convention. In some embodiments, the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2 and LCDR3 of the light chain variable region are defined according to IMGT numbering rules. In some embodiments, the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2 and LCDR3 of the light chain variable region are defined according to Chothia numbering rules. In some embodiments, the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2 and LCDR3 of the light chain variable region are defined according to the AbM numbering convention. In some embodiments, the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2 and LCDR3 of the light chain variable region are defined according to the Contact numbering convention.

In some embodiments, the anti-ICOSL antibody of any one of the above, wherein:

In some embodiments, the anti-ICOSL antibody of any one of the above, wherein

In some embodiments, the anti-ICOSL antibody of any one of the above is a murine antibody, a chimeric antibody, or a humanized antibody. In some embodiments, the antibody is a humanized antibody.

In some embodiments, the anti-ICOSL antibody of any one of the above, wherein:

In some embodiments, an anti-ICOSL antibody according to any one of the preceding claims, comprising one or more amino acid mutations selected from positions 37, 43 and 49 (numbered according to the Kabat numbering system) on the framework region of the heavy chain variable region, and/or one or more amino acid mutations selected from positions 1,2,3, 42, 43, 60 and 85 (numbered according to the Kabat numbering system) on the framework region of the light chain variable region. In some embodiments, the anti-ICOSL antibody fusion protein, wherein HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:11, HCDR2 comprises the amino acid sequence of SEQ ID NO:31 or 12, and HCDR3 comprises the amino acid sequence of SEQ ID NO:13, and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO:14, LCDR2 comprises the amino acid sequence of SEQ ID NO:15, and LCDR3 comprises the amino acid sequence of SEQ ID NO:16, an amino acid sequence of seq id no; the heavy chain variable region of the antibody comprises a framework region selected from the group consisting of: 37I, 43E and 49A (numbered according to the Kabat numbering system), and/or comprises one or more amino acid mutations in the framework regions of the light chain variable region selected from: 1N, 2T, 3V, 42Q, 43S, 60D, 85V (numbered according to the Kabat numbering system).

In some embodiments, an anti-ICOSL antibody according to any one of the preceding claims, comprising one or more amino acid mutations selected from positions 1,24, 69, 71, 73, 78 (numbered according to the Kabat numbering system) on the framework region of the heavy chain variable region, and/or one or more amino acid mutations selected from positions 1,2 and 4 (numbered according to the Kabat numbering system) on the framework region of the light chain variable region. In some embodiments, the anti-ICOSL antibody fusion protein, wherein HCDR1 of the heavy chain variable region of the anti-ICOSL antibody comprises SEQ ID NO:5, HCDR2 comprises the amino acid sequence of SEQ ID NO:19 or 6, and HCDR3 comprises the amino acid sequence of SEQ ID NO:7, and LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO:8, LCDR2 comprises the amino acid sequence of SEQ ID NO:17 or 9, and LCDR3 comprises the amino acid sequence of SEQ ID NO:10 or 18; the heavy chain variable region of the antibody comprises a framework region selected from the group consisting of: 1E, 24T, 69L, 71V, 73K and 78A (numbered according to the Kabat numbering system), and/or comprises one or more amino acid mutations in the framework regions of the light chain variable region selected from the group consisting of: 1A, 2V and 4L (numbered according to the Kabat numbering system).

In some embodiments, an anti-ICOSL antibody according to any of the preceding claims, wherein,

In some embodiments, the anti-ICOSL antibody of any one of the above, wherein the anti-ICOSL antibody comprises an antibody heavy chain constant region and a light chain constant region. In some embodiments, the heavy chain constant region is a human IgG heavy chain constant region. In some embodiments, the heavy chain constant region is selected from the group consisting of human IgG1, igG2, igG3, and IgG4 constant regions; in some embodiments, the light chain constant region is selected from the group consisting of human antibody kappa or lambda chain constant regions. In some embodiments, the heavy chain constant region is a human IgG4 heavy chain constant region and the light chain constant region is a human kappa light chain constant region. In some embodiments, the Fc region of the heavy chain constant region has one or more amino acid substitutions that reduce binding of the Fc region to Fc receptors. In some embodiments, the Fc region has an L234A, L235A mutation, and/or an S228P mutation, and/or a YTE mutation (M252Y, S T and T256E), the mutation numbering being according to the EU index. In some embodiments, the heavy chain constant region comprises SEQ ID NO:43, and the light chain constant region comprises the amino acid sequence of SEQ ID NO:44, and a polypeptide comprising the amino acid sequence of 44.

In some embodiments, an anti-ICOSL antibody as described in any of the above,

In some embodiments, the disclosure further provides an isolated anti-ICOSL antibody or anti-ICOSL antibody fusion protein that competes with an anti-ICOSL antibody or anti-ICOSL antibody fusion protein of any of the preceding claims for binding to human ICOSL or an epitope thereof.

In some embodiments, the anti-ICOSL antibody or anti-ICOSL antibody fusion protein of any one of the preceding claims has one or more of the following properties:

A. Specifically binds to human ICOSL (or an epitope thereof) and cynomolgus ICOSL (or an epitope thereof), but not murine ICOSL; preferably, it binds to human ICOSL with a KD value of less than 5.00E-09M (e.g., less than 5.00E-09M, less than 4.00E-09M, less than 3.00E-09M, less than 2.00E-09M, less than 1.50E-09M, 9.00E-10M or less), and/or with a KD value of less than 8.00E-09M (e.g., less than 8.00E-09M, less than 7.00E-09M, less than 6.00E-09M, less than 5.00E-09M, less than 4.00E-09M, less than 3.00E-09M or less), as measured by surface plasmon resonance assay (e.g., less than 8.00E-09M) Surface plasmon resonance measurement); in some embodiments, the KD values are detected by the test example 8 method of the present disclosure;

B. Specifically binds to human APRIL (or an epitope thereof), cynomolgus APRIL (or an epitope thereof) and/or murine APRIL (or an epitope thereof); preferably, binding to the human APRIL occurs with a KD value of less than 2.00E-11M (e.g., less than 2.00E-11M, less than 1.80E-11M, less than 1.70E-11M, less than 1.60E-11M, less than 1.20E-11M, less than 4.60E-12M or less), binding to the cynomolgus monkey APRIL with a KD value of less than 2.00E-10M (e.g., less than 2.00E-10M, less than 1.80E-10M, less than 1.70E-10M, less than 1.60E-10M, less than 1.50E-10M, less than 1.40E-10M, less than 1.20E-10M or less), and/or bind to murine APRIL with a KD value of less than 2.00E-11M (e.g., less than 2.00E-11M, less than 1.80E-11M, less than 1.60E-11M, less than 1.40E-11M, less than 1.20E-11M, less than 1.00E-11M, less than 8.00E-12M, or less), as measured by surface plasmon resonance assay (e.g. Surface plasmon resonance measurement); in some embodiments, the KD values are detected by the test example 8 method of the present disclosure;

C. Specifically binds to human BAFF (or an epitope thereof), cynomolgus BAFF (or an epitope thereof) and/or murine BAFF (or an epitope thereof); preferably, the binding to human BAFF is performed with a KD value of less than 7.00E-11M (e.g., less than 7.00E-11M, less than 5.00E-11M, less than 4.00E-11M, less than 3.00E-11M, less than 2.00E-11M, less than 1.00E-11M or less), with a KD value of less than 4.00E-10M (e.g., less than 4.00E-10M, less than 3.00E-10M, less than 2.00E-10M, less than 1.30E-10M or less), and/or with a KD value of less than 6.00E-11M (e.g., less than 6.00E-11M, less than 5.00E-11M, less than 4.00E-11M, less than 3.20E-11M or less), as measured by a surface resonance method (e.g., resonance method) Surface plasmon resonance measurement); in some embodiments, the KD values are detected by the test example 8 method of the present disclosure;

D. Has the activity of blocking ICOSL from binding to ICOS; preferably, the IC ₅₀ value that blocks binding of human ICOSL to ICOS is less than 0.16nM (e.g., less than 0.16nM, less than 0.14nM, less than 0.12nM, less than 0.10nM, less than 0.09nM, less than 0.08nM, less than 0.07nM, less than 0.06nM, less than 0.05nM, less than 0.04nM or less), said IC ₅₀ value being detected by FACS methods; in some embodiments, the IC ₅₀ value is detected by the test example 3 method of the present disclosure;

E. Has the activity of blocking the binding of APRIL to BCMA; preferably, the IC ₅₀ value that blocks APRIL binding to BCMA is less than 40.00nM (e.g., less than 40.00nM, less than 35.00nM, less than 30.00nM, less than 20.00nM, less than 10.00nM, less than 5.00nM, less than 2.00nM, less than 0.10nM, less than 0.06nM, less than 0.02nM or less), said IC ₅₀ value being detected by ELISA methods; in some embodiments, the IC ₅₀ value is detected by the test example 4 method of the present disclosure;

F. Has the activity of blocking the binding of APRIL to TACI; preferably, the IC ₅₀ value that blocks APRIL binding to TACI is less than 40.00nM (e.g., less than 40.00nM, less than 37.00nM, less than 33.00nM, less than 30.00nM, less than 25.00nM, less than 10.00nM, less than 5.00nM, less than 2.00nM, less than 1.00nM, less than 0.50nM or less), the IC ₅₀ value being detected by ELISA methods; in some embodiments, the IC ₅₀ value is detected by the test example 4 method of the present disclosure;

G. Has the activity of blocking the binding of BAFF to BCMA; preferably, the IC ₅₀ value that blocks BAFF binding to BCMA is less than 2.00nM (e.g., less than 2.00nM, less than 1.50nM, less than 1.00nM, less than 0.90nM, less than 0.80nM, less than 0.70nM, less than 0.60nM, less than 0.50nM, less than 0.40nM or less), said IC ₅₀ value being detected by ELISA methods; in some embodiments, the IC ₅₀ value is detected by the test example 4 method of the present disclosure;

H. Has the activity of blocking the binding of BAFF and BAFF-R; preferably, the IC ₅₀ value that blocks BAFF binding to BAFF-R is less than 2.00nM (e.g., less than 2.00nM, less than 1.70nM, less than 1.60nM, less than 1.00nM, less than 0.80nM, less than 0.70nM or less), the IC ₅₀ value being detected by ELISA method; in some embodiments, the IC ₅₀ value is detected by the test example 4 method of the present disclosure;

I. Has the activity of blocking the binding of BAFF and TACI; preferably, the IC ₅₀ value that blocks BAFF binding to TACI is less than 1.00nM (e.g., less than 1.00nM, less than 0.50nM, less than 0.30nM, less than 0.20nM, less than 0.15nM, or less), which IC ₅₀ value is detected by ELISA methods; in some embodiments, the IC ₅₀ value is detected by the test example 4 method of the present disclosure;

J. Has the activity of inhibiting the secretion of cytokines (such as IFNgamma, IL-10 and/or IgA); in some embodiments, the IC ₅₀ value is detected by the test example 9 method of the present disclosure;

K. Has the activity of inhibiting B cell proliferation and/or T cell proliferation; in some embodiments, the anti-ICOSL antibody or anti-ICOSL antibody fusion protein inhibits ICOSL activity to induce T cell proliferation; in some embodiments, the inhibition of T cell proliferation activity is detected by the test example 6 method of the present disclosure; in some embodiments, the anti-ICOSL antibody fusion protein inhibits BAFF-induced or APRIL-induced B cell proliferation activity; in some embodiments, the inhibition of B cell proliferation activity is detected by the test example 7 method of the present disclosure.

The present disclosure also provides a pharmaceutical composition comprising an anti-ICOSL antibody fusion protein or an anti-ICOSL antibody of any preceding claim, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

The present disclosure also provides a pharmaceutical composition comprising an anti-ICOSL antibody as described in any one of the preceding claims, and a polypeptide as set forth in SEQ ID NO:51 to 83, and one or more pharmaceutically acceptable carriers, diluents or excipients;

Preferably, the TACI polypeptide sequence is as set forth in SEQ ID NO:60-63, SEQ ID NO: 66-83;

More preferably, the TACI polypeptide sequence is as set forth in SEQ ID NO: 83.

In some embodiments, the disclosure provides a nucleic acid molecule encoding an anti-ICOSL antibody fusion protein or an anti-ICOSL antibody of any one of the preceding claims.

In some embodiments, the disclosure provides an expression vector comprising the aforementioned nucleic acid molecule (DNA or RNA).

In some embodiments, the disclosure provides a host cell comprising the aforementioned nucleic acid molecule.

In some embodiments, the disclosure provides a host cell comprising the foregoing expression vector.

The present disclosure provides host cells that are incapable of developing into an animal or plant individual.

In some embodiments, the present disclosure provides a method of treating an autoimmune disease, an inflammatory disease, a B cell disorder, or a T cell disorder, the method comprising the step of administering to a subject in need thereof a therapeutically effective amount of an anti-ICOSL antibody fusion protein or an anti-ICOSL antibody or pharmaceutical composition of any of the preceding claims. In some embodiments, the presently disclosed methods are used to treat autoimmune or inflammatory diseases. In some embodiments, the methods of the disclosure are used to treat a B cell disorder or a T cell disorder.

In some embodiments, the disclosure provides the use of an anti-ICOSL antibody fusion protein or an anti-ICOSL antibody, nucleic acid molecule, or pharmaceutical composition of any of the preceding claims in the manufacture of a medicament for treating a disease.

In another aspect, the disclosure also provides an anti-ICOSL antibody fusion protein or an anti-ICOSL antibody, nucleic acid molecule or composition of any one of the preceding claims for use as a medicament. In some embodiments, the medicament is for treating an autoimmune disease, an inflammatory disease, a B cell disorder, or a T cell disorder. In some embodiments, the medicament is for treating an autoimmune disease or an inflammatory disease. In some embodiments, the medicament is for treating a B cell disorder or a T cell disorder.

In some embodiments, the disease of any of the preceding claims is a disease or disorder associated with high ICOSL expression. In some embodiments, the disease or disorder of any one of the preceding claims is an autoimmune disease, an inflammatory disease, a B cell disorder, or a T cell disorder. In some embodiments, the autoimmune or inflammatory disease is selected from: systemic lupus erythematosus, rheumatoid arthritis, graft versus host disease, asthma, immune thrombocytopenic purpura, multiple sclerosis, inflammatory diseases caused by diabetes, psoriasis, inflammatory bowel disease, crohn's disease, ulcerative colitis, graves' disease and Hashimoto thyroiditis. In some embodiments, the B cell disorder or T cell disorder is a tumor. In some embodiments, the tumor is selected from: head and neck cancer, non-small cell lung cancer, urothelial cancer, leukemia, sarcoma, melanoma, adenocarcinoma, colorectal cancer, prostate tumor, breast cancer, and small cell lung cancer. In some embodiments, the autoimmune disease is systemic lupus erythematosus.

In some embodiments, the treatment of any of the preceding claims, further comprising administering an additional therapeutic agent to the subject.

Drawings

Fig. 1: structural schematic of anti-ICOSL antibody fusion proteins such as 259H4L7-T11 and 201H3L 3-T11.

Fig. 2: structural schematic of anti-ICOSL antibody fusion proteins such as 201H3L3-T7 and 259H4L 7-T7.

Fig. 3: the structural schematic of an anti-ICOSL antibody fusion protein such as 259H4L 7-T9.

Fig. 4: anti-ICOSL antibody fusion proteins inhibited IL-10 secretion assay results.

Fig. 5: results of secretion experiments in which anti-ICOSL antibody fusion proteins inhibited ifnγ.

Fig. 6: results of secretion experiments in which anti-ICOSL antibody fusion proteins inhibited IgA.

Fig. 7: the anti-ICOSL antibody fusion protein inhibited KLH-specific IgG production experimental results.

Fig. 8: the anti-ICOSL antibody fusion protein inhibited KLH-specific IgM production experimental results.

Detailed Description

Terminology

For easier understanding of the present disclosure, certain technical and scientific terms are described below. Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

As used in the specification and in the claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

Unless the context clearly requires otherwise, in the description and the claims, the words "comprise", "have", "include" and the like are to be construed in an inclusive, but not exclusive, sense as opposed to an exclusive or exhaustive sense.

The term "cytokine" is a generic term for proteins released by one cell population that act as intercellular mediators on other cells. Examples of such cytokines include lymphokines, monokines, chemokines, and traditional polypeptide hormones. Exemplary cytokines include: IL-10, IFN-gamma, IL-6, TNF alpha, IL-17 and IL-5.

TACI described in the present disclosure is a membrane-bound receptor. Wild type human TACI extracellular domain (positions 1-165) is seen in SEQ ID NO:51. the "TACI extracellular domain" and "TACI extracellular domain" in the present disclosure may be interchangeable.

ICOSL (Inducible costimulator-ligand, also known as B7RP1, B7H2, CD 275) is an immune co-stimulatory molecule belonging to the B7 family. The receptor for ICOSL is currently known to be ICOS, which is expressed primarily on the surface of T cells, and ICOSL and ICOS mediated signaling pathways play a major role in T cell differentiation and T cell dependent B cell activation (Nat Rev cancer.2012mar 22;12 (4): 252-64.).

"ICOSL" as target for an antibody or fusion protein thereof is to be construed broadly and is intended to encompass various forms of molecules of ICOSL at various stages in the mammal, such as, but not limited to, molecules produced by ICOSL gene during amplification, replication, transcription, splicing, processing, translation, modification (e.g., precursor BCMA, mature ICOSL, membrane expressed ICOSL, ICOSL splice variants, modified ICOSL, or fragments thereof); the term also encompasses ICOSL either prepared artificially or expressed in vitro.

The term "and/or" is intended to include both "and" or "meanings. For example, the phrase "A, B and/or C" is intended to encompass any of the following aspects: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; a (alone); b (alone); and C (alone).

The amino acid three-letter codes and one-letter codes used in the present disclosure are as described in j.biol. Chem,243, p3558 (1968).

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid (i.e., an alpha carbon to which hydrogen, carboxyl, amino, and R groups are bound), e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to naturally occurring amino acids.

The term "amino acid mutation" includes amino acid substitutions (also referred to as amino acid substitutions), deletions, insertions and modifications. Any combination of substitutions, deletions, insertions, and modifications may be made to achieve the final construct, provided the final construct possesses the desired properties, e.g., reduced binding to Fc receptors. Amino acid sequence deletions and insertions may be located at the amino-and/or carboxy-terminus of the polypeptide chain. In one embodiment, the amino acid mutation is a non-conservative amino acid substitution, i.e., the substitution of one amino acid with another amino acid having a different structure and/or chemical property. Amino acid substitutions include substitutions by non-naturally occurring amino acids or by derivatives of 20 natural amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be made using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, and the like. It is contemplated that other methods than genetic engineering, such as chemical modification, may be useful to alter the amino acid side chain groups. Various expressions may be used herein to indicate amino acid mutations. Herein, the amino acid residue at a particular position may be represented by the position +amino acid residue, e.g., 366W, indicating that the amino acid residue at position 366 is W. T366W indicates that the amino acid residue at position 366 is mutated from the original T to W. As an example, when the sequence is described in the claims as 366W or T366W, it is not to be understood that the original amino acid residue T at position 366 constitutes any limitation to the scope of protection.

The term "antibody" is used in the broadest sense and covers a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies); full length antibodies and antibody fragments (or antigen-binding fragments, or antigen-binding portions) so long as they exhibit the desired antigen-binding activity. "Natural antibody" refers to a naturally occurring immunoglobulin molecule. For example, a natural IgG antibody is an iso-tetrasaccharide protein of about 150,000 daltons, consisting of 2 identical light chains and 2 identical heavy chains disulfide bonded. From N-to C-terminus, each heavy chain has a variable region (VH), also known as a variable heavy domain, a heavy chain variable region, followed by a heavy chain constant region, a natural IgG heavy chain constant region typically contains three constant domains (CH 1, CH2 and CH 3). Similarly, from N-to C-terminus, each light chain has a variable region (VL), also known as a variable light domain, or light chain variable domain, followed by a constant light domain (light chain constant region, CL). The terms "full length antibody", "whole antibody" and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to the structure of a natural antibody or a heavy chain having an Fc region as defined herein. The natural intact antibody light chain comprises a light chain variable region VL at the amino terminus of the light chain and a constant region CL comprising a kappa chain and a lambda chain; heavy chains include variable regions VH and constant regions (CH 1, CH2 and CH 3), with VH at the amino terminus and constant region at the carboxy terminus of the heavy chain, with CH3 nearest the carboxy terminus of the polypeptide, and heavy chains can be of any isotype, including IgG (including IgG1, igG2, igG3 and IgG4 subtypes), igA (including IgA1 and IgA2 subtypes), igM and IgE.

The term antibody "variable region" or "variable domain" refers to the domain of an antibody that is involved in binding an antigen by an antibody in the heavy or light chain. Herein, the antibody heavy chain variable region (VH) and light chain variable region (VL) each comprise four conserved Framework Regions (FR) and three Complementarity Determining Regions (CDRs). Wherein the term "complementarity determining region" or "CDR" refers to a region within the variable domain that primarily contributes to binding to an antigen; "framework" or "FR" refers to variable domain residues other than CDR residues. VH comprises 3 CDR regions: HCDR1, HCDR2 and HCDR3; VL comprises 3 CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus (also called N-terminus) to carboxy-terminus (also called C-terminus) in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The amino acid sequence boundaries of the CDRs can be determined by various well-known schemes, such as: "Kabat" numbering rules (see Kabat et al (1991), "Sequences of Proteins of Immunological Interest", 5 th edition, public HEALTH SERVICE, national Institutes of Health, bethesda, MD), "Chothia" numbering rules, "ABM" numbering rules, "contact" numbering rules (see Martin, ACR.protein Sequence and Structure Analysis of Antibody Variable Domains [ J ]. 2001) and ImMunoGenTics (IMGT) numbering rules (Lefranc, M.P. et al, dev.Comp. Immunol.,27, 55-77 (2003); front immunol.2018Oct 16; 9:2278), etc.; the correspondence between the various numbering systems is well known to those skilled in the art, and is exemplary, as shown in table 1 below.

TABLE 1 relationship between CDR numbering systems

CDR	IMGT	Kabat	AbM	Chothia	Contact
HCDR1	27-38	31-35	26-35	26-32	30-35
HCDR2	56-65	50-65	50-58	52-56	47-58
HCDR3	105-117	95-102	95-102	95-102	93-101
LCDR1	27-38	24-34	24-34	24-34	30-36
LCDR2	56-65	50-56	50-56	50-56	46-55
LCDR3	105-117	89-97	89-97	89-97	89-96

Unless otherwise indicated, the variable regions and CDRs in the embodiments of the disclosure apply the "Kabat" numbering convention.

The term "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of the intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to Fv, fab, fab ', fab ' -SH, F (ab ') ₂, single domain antibodies, single chain Fab (scFab), diabodies, linear antibodies, single chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of the antibody heavy chain, including the native Fc region and engineered Fc region. In some embodiments, the Fc region comprises two subunits, which may be the same or different. In some embodiments, the Fc region of a human IgG heavy chain is defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxy terminus. Suitable Fc regions for antibodies described herein include the Fc regions of human IgG1, igG2 (IgG 2A, igG B), igG3, and IgG 4. In some embodiments, the boundaries of the Fc region may also vary, such as deletion of the C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) or deletion of the C-terminal glycine and lysine of the Fc region (residues 446 and 447 according to the EU numbering system). The numbering convention for the Fc region is the EU numbering system, also known as the EU index, unless otherwise indicated.

The term "chimeric antibody" refers to an antibody in which a portion of the heavy and/or light chains in the antibody are derived from a particular source or species, while the remainder of the heavy and/or light chains are derived from another, different source or species.

The term "humanized antibody" is an antibody that retains the reactivity of a non-human antibody while having lower immunogenicity in humans. For example, this can be accomplished by retaining the non-human CDR regions and replacing the remainder of the antibody with its human counterparts (i.e., the framework regions portions of the constant and variable regions).

The term "affinity" refers to the overall strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). As used herein, unless otherwise indicated, binding "affinity" refers to an internal binding affinity that reflects the interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its ligand Y can generally be expressed by a dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein.

As used herein, the term "kassoc" or "ka" refers to the rate of association of a particular antibody-antigen interaction, and the term "kdis" or "kd" refers to the rate of dissociation of a particular antibody-antigen interaction. The term "KD" refers to the dissociation constant, which is obtained from the ratio of KD to ka (i.e., KD/ka) and is expressed as molar concentration (M). The KD values of antibodies can be determined using methods well known in the art. For example, surface plasmon resonance (e.g., biacore) is measured using a biosensing system such as a system, or affinity in solution is measured by Solution Equilibrium Titration (SET).

The term "effector functions" refers to those biological activities attributable to the Fc region of an antibody (native sequence Fc region or Fc region with amino acid sequence mutations) and which vary with the antibody isotype. Examples of antibody effector functions include, but are not limited to: c1q binding and complement dependent cytotoxicity, fc receptor binding, antibody dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

The term "monoclonal antibody" refers to a population of substantially homogeneous antibodies, i.e., the amino acid sequences of the antibody molecules comprised in the population are identical, except for natural mutations that may be present in minor amounts. In contrast, polyclonal antibody preparations typically include a plurality of different antibodies having different amino acid sequences in their variable domains, which are typically specific for different epitopes. "monoclonal" refers to the characteristics of the antibody obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. In some embodiments, the antibodies provided by the present disclosure are monoclonal antibodies.

The term "antigen" refers to a molecule or portion of a molecule that is capable of being selectively bound by, for example, an antigen binding protein (including, for example, an antibody). An antigen may have one or more epitopes that are capable of interacting with different antigen binding proteins (e.g., antibodies).

The term "epitope" refers to a region (area or region) on an antigen that is capable of specifically binding to an antibody (including antigen binding fragments thereof). Epitopes can be formed from a contiguous amino acid string (linear epitope) or comprise non-contiguous amino acids (conformational epitope), e.g., due to folding of an antigen (i.e., by tertiary folding) such that the non-contiguous amino acids are spatially close to one another. Conformational epitopes differ from linear epitopes in that: in the presence of denaturing solvents, binding of the antibody to conformational epitopes is lost. An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind a particular epitope (i.e., those that bind the same epitope) can be performed using methods routine in the art, such as, but not limited to, alanine scanning, peptide blotting, peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen (see prot. Sci.9 (2000) 487-496), and cross-blocking.

The term "capable of specifically binding", "specifically binding" or "binding" refers to an antibody that is capable of binding to an antigen or epitope thereof with a higher affinity than other antigens or epitopes. Typically, an antibody binds an antigen or epitope thereof with an equilibrium dissociation constant (KD) of about 1 x 10 ^-7 M or less (e.g., about 1 x 10 ^-8M、1×10 ^-9M、1×10 ^-10M、1×10 ^-11 M or less). In some embodiments, the antibody binds to an antigen with a KD of 10% or less (e.g., 1%) of the KD of the antibody to a non-specific antigen (e.g., BSA, casein). KD can be measured using known methods, for example, byAs measured by surface plasmon resonance. However, antibodies that specifically bind to an antigen or epitope thereof may be cross-reactive to other related antigens, for example, corresponding antigens from other species (homologous), such as humans or monkeys, e.g., cynomolgus macaque (Macaca fascicularis) (cynomolgus, cyno), chimpanzee (Pan troglodes) (chimpanzee, chimp) or marmoset (Callithrix jacchus) (commonmarmoset, marmoset).

The terms "anti-ICOSL antibody" and "ICOSL-binding antibody" refer to antibodies capable of binding ICOSL or an epitope thereof with sufficient affinity. In one embodiment, the degree of binding of an antibody to an unrelated, non-ICOSL protein, as measured by a surface plasmon resonance assay, is less than about 10% of the binding of the antibody to ICOSL (e.g.). In certain embodiments, an antibody that binds to ICOSL protein has a dissociation constant (KD) of < about 1 μm, < about 100nM, < about 10nM, < about 1nM, < about 0.1nM, < about 0.01nM, or < about 0.001nM. In certain embodiments, the anti-ICOSL antibody binds to a human or cynomolgus ICOSL epitope.

The term "Linker", "Linker" or "Linker" refers to a linking unit that connects two polypeptide fragments, typically with some flexibility, without the use of a Linker to lose the original function of the protein domain. In this context, the linkers present in the same structure may be the same or different. The linker may be a peptide linker comprising one or more amino acids, typically about 1-30, 2-24 or 3-15 amino acids. The linkers used herein may be the same or different.

The terms "cytotoxicity of antibody-dependent cells", "antibody-dependent cell-mediated cytotoxicity" or "ADCC" are mechanisms that induce cell death, which mechanisms depend on the interaction of antibody-coated target cells with effector cells having lytic activity, such as natural killer cells (NK), monocytes, macrophages and neutrophils, via fcγr expressed on the effector cells. For example, NK cells express fcyriiia, whereas monocytes express fcyri, fcyrii, and fcyriiia. ADCC activity of the antibodies provided herein can be assessed using in vitro assays using cells expressing the antigen as target cells and NK cells as effector cells. Cell lysis is detected based on a label (e.g., a radioactive substrate, fluorescent dye, or native intracellular protein) released from the lysed cells.

The term "antibody-dependent cellular phagocytosis" ("ADCP") refers to the mechanism by which antibody-coated target cells are eliminated by internalization of phagocytes, such as macrophages or dendritic cells.

The term "complement-dependent cytotoxicity" or "CDC" refers to a mechanism that induces cell death in which the Fc-effector domain of a target binding antibody binds to and activates complement components C1q, C1q in turn activating the complement cascade, resulting in target cell death. Activation of complement can also result in deposition of complement components on the target cell surface that promote CDC by binding to complement receptors (e.g., CR 3) on leukocytes.

The term "nucleic acid" is used interchangeably herein with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, have similar binding properties as the reference nucleic acid, and are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs). An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location different from its natural chromosomal location. An isolated nucleic acid encoding a polypeptide or fusion protein refers to one or more nucleic acid molecules encoding a polypeptide or fusion protein, including such one or more nucleic acid molecules in a single vector or separate vectors, and such one or more nucleic acid molecules present at one or more locations in a host cell. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues, as described in detail below.

The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are the corresponding naturally occurring amino acid or an artificial chemical mimetic thereof, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

The term "sequence identity" refers to the degree (percent) to which the amino acids/nucleic acids of 2 sequences are identical at equivalent positions when optimally aligned for 2 sequences; wherein gaps are introduced in the optimal alignment as necessary to obtain the maximum percent sequence identity, and any conservative substitutions are not considered as part of sequence identity. To determine percent sequence identity, alignment may be accomplished by techniques known in the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN-2, or Megalign (DNASTAR) software. One skilled in the art can determine parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment over the full length of the sequences compared.

In the present disclosure, for example, "relative to the sequence SEQ ID NO: the natural sequence numbered amino acid residue position "of XX means that the subject sequence is identical to SEQ ID NO: the XX sequence is optimally aligned to obtain the highest percent identity, when the subject sequence is identical to SEQ ID NO: the position corresponding to the XX sequence is the relative position of 2 sequences. For example, the extracellular region sequence of TACI SEQ ID NO:51 and SEQ ID NO:60 relative to SEQ ID NO:58 are shown in table 2:

table 2.Seq ID NO: corresponding site of 58 sites on other sequences

(Taking SEQ ID NO:51 or/and SEQ ID NO:60 as an example)

Remarks: for example SEQ ID NO:60 and the sequence of SEQ ID NO:58 (natural order) is the corresponding position.

The term "fusion" or "linkage" refers to the attachment of components (e.g., TACI polypeptides and antibody heavy/light chains) by covalent bonds, either directly or via one or more linkers. When the linker is a peptide linker, the covalent bond is a peptide bond.

The term "anti-ICOSL antibody fusion protein" refers to a protein in which an anti-ICOSL antibody is fused to an active protein. For example, TACI polypeptides are fused to the N-terminus or C-terminus of the heavy or light chain of an anti-ICOSL antibody to form a protein.

The term "vector" means a polynucleotide molecule capable of transporting another polynucleotide to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop in which additional DNA segments may be ligated. Another type of vector is a viral vector, such as an adeno-associated viral vector (AAV or AAV 2), wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, thereby replicating with the host genome. The term "expression vector" or "expression construct" refers to a vector that can transform a host cell and that contains a nucleic acid sequence that directs and/or controls (along with the host cell) the expression of one or more heterologous coding regions to which it is operably linked. Expression constructs may include, but are not limited to, sequences that affect or control transcription, translation, and, when present, RNA splicing of the coding region to which they are operably linked.

The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. The progeny may not be exactly identical in nucleic acid content to the parent cell, but may contain a mutation. Included herein are mutant progeny that have the same function or biological activity as the cells selected or selected in the original transformed cells. Host cells include prokaryotic and eukaryotic host cells, where eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, canine, monkey, pig, goat, bovine, equine, and hamster cells, including, but not limited to, chinese Hamster Ovary (CHO) cells, NSO, SP2 cells, heLa cells, baby Hamster Kidney (BHK) cells, monkey kidney Cells (COS), human hepatocellular carcinoma cells (e.g., hep G2), a549 cells, 3T3 cells, and HEK-293 cells. Fungal cells include yeast and filamentous fungal cells including, for example, pichia pastoris (Pichia pastoris), pichia pastoris (PICHIA FINLANDICA), pichia seaweed (Pichia trehalophila), colorado Ma Bichi yeast (Pichia koclamae), pichia membranaceus (Pichia membranaefaciens), pichia minuta (Ogataea minuta, PICHIA LINDNERI), pichia pastoris, Pichia pastoris (Pichiaopuntiae), pichia pastoris (Pichia thermotolerans), pichia pastoris (PICHIA SALICTARIA), pichia guercuum, pichia Pi Jiepu (Pichia pijperi), pichia pastoris (PICHIA STIPTIS), pichia methanolica (Pichia methanolica), pichia pastoris (Saccharomyces cerevisiae), Saccharomyces cerevisiae, hansenula polymorpha (Hansenula polymorpha), kluyveromyces lactis (Kluyveromyces lactis), candida albicans, aspergillus nidulans (Aspergillus nidulans), aspergillus niger (Aspergillus niger), aspergillus oryzae (Aspergillus oryzae), trichoderma reesei (Trichoderma reesei), legionella (Chrysosporium lucknowense), candida albicans, trichoderma reesei (Chrysosporium lucknowense), trichoderma reesei (A.oryzae), Fusarium sp., fusarium graminearum Fusarium gramineum, fusarium graminearum Fusarium venenatum, fusarium microphylla Physcomitrella patens, and Neurospora crassa Neurospora crassa, aspergillus sp., yarrowia lipolytica Yarrowia lipolytica.

"Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not.

The term "pharmaceutical composition" means a mixture comprising one or more of the anti-ICOSL antibody fusion proteins described herein, together with other chemical components, such as physiological/pharmaceutically acceptable carriers and excipients.

The term "pharmaceutically acceptable carrier, diluent or excipient" refers to an ingredient in a pharmaceutical formulation that is different from the active ingredient and is non-toxic to the subject; such as, but not limited to, buffers, excipients, stabilizers, or preservatives.

The term "subject" or "individual" includes both human and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians, and reptiles. The terms "patient" or "subject" are used interchangeably herein unless indicated. As used herein, the term "cynomolgus monkey (cyno)" or "cynomolgus monkey (cynomolgus)" refers to cynomolgus monkey (Macaca fascicularis). In certain embodiments, the individual or subject is a human.

"Administering" or "administering," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to contacting an exogenous pharmaceutical, therapeutic, diagnostic, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid.

The term "sample" refers to a collection of fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present in the body of a subject. Exemplary samples are biological fluids such as blood, serum, serosal fluid, plasma, lymph fluid, urine, saliva, cyst fluid, tears, fecal matter, sputum, mucous secretions of secretory tissues and organs, vaginal secretions, ascites, pleura, pericardium, peritoneal cavity and other body cavity fluids, fluids collected by bronchial lavage, synovial fluid, liquid solutions in contact with a subject or biological source, such as cell and organ culture media (including cell or organ conditioned media), lavage fluid, and the like, tissue biopsy samples, fine needle punctures, surgically excised tissues, organ cultures, or cell cultures.

"Treatment" and "treatment" (and grammatical variations thereof) refer to clinical interventions that attempt to alter the course of a treated individual, and may be performed for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, alleviating/reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and regression or improved prognosis. In some embodiments, the antibodies or fusion proteins of the disclosure are used to delay the formation of a disease or to slow the progression of a disease.

An "effective amount" is generally an amount sufficient to reduce the severity and/or frequency of symptoms, eliminate such symptoms and/or underlying etiology, prevent the appearance of symptoms and/or underlying etiology, and/or ameliorate or improve the damage caused by or associated with a disease state. In some embodiments, the effective amount is a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" is an amount sufficient to treat a disease state or condition, particularly a state or condition associated with the disease state, or otherwise prevent, hinder, delay or reverse the progression of the disease state or any other undesirable condition associated with the disease in any way. A "prophylactically effective amount" is an amount that, when administered to a subject, will have a predetermined prophylactic effect, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or related symptoms. The complete therapeutic or prophylactic effect does not necessarily occur after administration of one dose, but may occur after administration of a series of doses. Thus, a therapeutically or prophylactically effective amount may be administered in one or more administrations. The "therapeutically effective amount" and "prophylactically effective amount" may vary depending on a variety of factors: such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Exemplary indicators of effective therapeutic agents or combinations of therapeutic agents include, for example, improved health of a patient.

Anti-ICOSL antibodies of the disclosure

In one aspect, the disclosure contemplates a novel anti-ICOSL antibody. Which has one or more of the following characteristics:

A. Specifically binds to human ICOSL and cynomolgus ICOSL or an epitope thereof, but not to murine ICOSL; preferably, it binds to human ICOSL with a KD value of less than 5.00E-09M (e.g., less than 5.00E-09M, less than 4.00E-09M, less than 3.00E-09M, less than 2.00E-09M, less than 1.50E-09M, 9.00E-10M or less), and/or binds to cynomolgus monkey ICOSL with a KD value of less than 8.00E-09M (e.g., less than 8.00E-09M, less than 7.00E-09M, less than 6.00E-09M, less than 5.00E-09M, less than 4.00E-09M, less than 3.00E-09M or less), as measured by surface plasmon resonance assay (e.g.) ) ; In some embodiments, the KD values are detected by the test example 8 method of the present disclosure;

B. has the activity of blocking ICOSL from binding to ICOS; preferably, the IC ₅₀ value that blocks binding of human ICOSL to ICOS is less than 0.16nM (e.g., less than 0.16nM, less than 0.14nM, less than 0.12nM, less than 0.10nM, less than 0.09nM, less than 0.08nM, less than 0.07nM, less than 0.06nM, less than 0.05nM, less than 0.04nM or less), said IC ₅₀ value being detected by FACS methods; in some embodiments, the IC ₅₀ value is detected by the test example 3 method of the present disclosure;

C. Has the effect of inhibiting IFN gamma, IL-10 and/or IgA cytokine secretion activity; in some embodiments, the IC50 value is detected by the test example 9 method of the present disclosure;

D. Has T cell proliferation inhibiting activity; in some embodiments, the anti-ICOSL antibody or anti-ICOSL antibody fusion protein inhibits ICOSL activity to induce T cell proliferation; in some embodiments, the inhibition of T cell proliferation activity is detected by the test example 6 method of the present disclosure.

Exemplary anti-ICOSL antibodies

In one aspect, the disclosure provides an anti-ICOSL antibody, wherein the anti-ICOSL antibody comprises a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein,

In some embodiments, the anti-ICOSL antibody of any one of the above, wherein the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2 and LCDR3 of the light chain variable region are defined according to numbering rules selected from Kabat.

In some embodiments, the anti-ICOSL antibody of any one of the above, wherein:

(i) The HCDR1 of the heavy chain variable region is shown in SEQ ID NO:11, HCDR2 is as set forth in SEQ ID NO:31 or 12, and HCDR3 is as set forth in SEQ ID NO:13, and LCDR1 of the light chain variable region is set forth in SEQ ID NO:14, LCDR2 is as set forth in SEQ ID NO:15, and LCDR3 is as set forth in SEQ ID NO: shown at 16; or (b)

(Ii) The HCDR1 of the heavy chain variable region is shown in SEQ ID NO:5, HCDR2 is as shown in SEQ ID NO:19 or 6, and HCDR3 is as set forth in SEQ ID NO:7, and LCDR1 of the light chain variable region is as set forth in SEQ ID NO:8, LCDR2 is as set forth in SEQ ID NO:17 or 9, and LCDR3 is as set forth in SEQ ID NO:10 or 18.

In some embodiments, the anti-ICOSL antibody of any one of the above, wherein

(I) The HCDR1 of the heavy chain variable region is shown in SEQ ID NO:11, HCDR2 is as set forth in SEQ ID NO:31 and HCDR3 as set forth in SEQ ID NO:13, and LCDR1 of the light chain variable region is set forth in SEQ ID NO:14, LCDR2 is as set forth in SEQ ID NO:15, and LCDR3 is as set forth in SEQ ID NO: shown at 16; or (b)

(Ii) The HCDR1 of the heavy chain variable region is shown in SEQ ID NO:5, HCDR2 is as shown in SEQ ID NO:19, and HCDR3 is as set forth in SEQ ID NO:7, and LCDR1 of the light chain variable region is as set forth in SEQ ID NO:8, LCDR2 is as set forth in SEQ ID NO:17, and LCDR3 is as set forth in SEQ ID NO: shown at 10.

In some embodiments, the anti-ICOSL antibody of any one of the preceding claims is a murine, chimeric, or humanized antibody. In some embodiments, the anti-ICOSL antibody is humanized.

In some embodiments, the anti-ICOSL antibody of any one of the preceding claims is a humanized antibody. In some embodiments, the heavy chain variable region of the anti-ICOSL antibody has FR1, FR2, FR3 derived from IGHV3-74 x 03 and FR4 derived from IGHJ1 x01, and is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 37I, 43E and 49A (numbered according to the Kabat numbering system); and/or the light chain variable region has FR1, FR2, FR3 derived from IGKV4-1 x 01/IGKV1-39 x01 and FR4 derived from IGKJ2 x01 and is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 1N, 2T, 3V, 42Q, 43S, 60D, 85V (numbered according to the Kabat numbering system). In some embodiments, the anti-ICOSL antibody, wherein HCDR1 of the heavy chain variable region is set forth in SEQ ID NO:11, HCDR2 is as set forth in SEQ ID NO:31 or 12, and HCDR3 is as set forth in SEQ ID NO:13, and LCDR1 of the light chain variable region is set forth in SEQ ID NO:14, LCDR2 is as set forth in SEQ ID NO:15, and LCDR3 is as set forth in SEQ ID NO: shown at 16. The variable regions and CDRs are defined according to the Kabat numbering convention.

In some embodiments, the anti-ICOSL antibody of any one of the preceding claims is a humanized antibody, in some embodiments the heavy chain variable region of the anti-ICOSL antibody has FR1, FR2, FR3 derived from IGHV1-46 x 01 and FR4 derived from IGHJ6 x 01, and is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 1E, 24T, 69L, 71V, 73K and 78A (numbered according to the Kabat numbering system); and/or the light chain variable region has FR1, FR2, FR3 derived from IGKV2-28 x 01 and FR4 derived from IGKJ4 x 01 and is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 1A, 2V and 4L (numbered according to Kabat numbering system). In some embodiments, the anti-ICOSL antibody, wherein HCDR1 of the heavy chain variable region is set forth in SEQ ID NO:5, HCDR2 is as shown in SEQ ID NO:19 or 6, and HCDR3 is as set forth in SEQ ID NO:7, and LCDR1 of the light chain variable region is as set forth in SEQ ID NO:8, LCDR2 is as set forth in SEQ ID NO:17 or 9, and LCDR3 is as set forth in SEQ ID NO:10 or 18. The variable regions and CDRs are defined according to the Kabat numbering convention.

(I) The heavy chain variable region is shown in SEQ ID NO: 42. 39, 40 or 41, and said light chain variable region is set forth in SEQ ID NO: 38. 32, 33, 34, 35, 36 or 37; or (b)

(Ii) The heavy chain variable region is shown in SEQ ID NO: 30. 28 or 29, and said light chain variable region is set forth in SEQ ID NO: 22. 20, 21, 23, 24, 25, 26 or 27; or (b)

(Iii) The heavy chain variable region is shown in SEQ ID NO:1 and the light chain variable region is as set forth in SEQ ID NO:2 is shown in the figure; or (b)

(Iv) The heavy chain variable region is shown in SEQ ID NO:3 and the light chain variable region is set forth in SEQ ID NO: 4.

(I) The heavy chain variable region is shown in SEQ ID NO:42, and the light chain variable region is set forth in SEQ ID NO: shown at 38; or (b)

(Ii) The heavy chain variable region is shown in SEQ ID NO:30 and the light chain variable region is set forth in SEQ ID NO: shown at 22.

(I) The heavy chain of the anti-ICOSL antibody hybridizes with the heavy chain of SEQ ID NO:47 has at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity, and the light chain has a sequence as set forth in SEQ ID NO:48 has at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; or (b)

(Ii) The heavy chain of the anti-ICOSL antibody hybridizes with the heavy chain of SEQ ID NO:45 has at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity, and the light chain has a sequence as set forth in SEQ ID NO:46 has at least 90% (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

In some embodiments, an anti-ICOSL antibody as described in any of the above,

(I) The heavy chain of the anti-ICOSL antibody is shown in SEQ ID NO:47, and the light chain of said anti-ICOSL antibody is set forth in SEQ ID NO: 48; or (b)

(Ii) The heavy chain of the anti-ICOSL antibody is shown in SEQ ID NO:45 and the light chain of said anti-ICOSL antibody is set forth in SEQ ID NO: 46.

Anti-ICOSL antibody fusion proteins of the present disclosure

The present disclosure constructs an anti-ICOSL antibody fusion protein comprising an anti-ICOSL antibody and a TACI polypeptide, wherein the anti-ICOSL antibody specifically binds to human ICOSL or an epitope thereof.

In some embodiments, the anti-ICOSL antibody fusion protein of any preceding claim, having one or more of the following properties:

A. Specifically binds to human ICOSL and cynomolgus ICOSL, but not to murine ICOSL; preferably, it binds to human ICOSL with a KD value of less than 5.00E-09M (e.g., less than 5.00E-09M, less than 4.00E-09M, less than 3.00E-09M, less than 2.00E-09M, less than 1.50E-09M, 9.00E-10M or less), and/or binds to cynomolgus monkey ICOSL with a KD value of less than 8.00E-09M (e.g., less than 8.00E-09M, less than 7.00E-09M, less than 6.00E-09M, less than 5.00E-09M, less than 4.00E-09M, less than 3.00E-09M or less), as measured by surface plasmon resonance assay (e.g.) ) ; In some embodiments, the KD values are detected by the test example 8 method of the present disclosure;

B. Specifically binds to human APRIL, cynomolgus APRIL and/or murine APRIL; preferably, binding to the human APRIL occurs with a KD value of less than 2.00E-11M (e.g., less than 2.00E-11M, less than 1.80E-11M, less than 1.70E-11M, less than 1.60E-11M, less than 1.20E-11M, less than 4.60E-12M or less), binding to the cynomolgus monkey APRIL with a KD value of less than 2.00E-10M (e.g., less than 2.00E-10M, less than 1.80E-10M, less than 1.70E-10M, less than 1.60E-10M, less than 1.50E-10M, less than 1.40E-10M, less than 1.20E-10M or less), and/or bind to murine APRIL with a KD value of less than 2.00E-11M (e.g., less than 2.00E-11M, less than 1.80E-11M, less than 1.60E-11M, less than 1.40E-11M, less than 1.20E-11M, less than 1.00E-11M, less than 8.00E-12M, or less), as measured by surface plasmon resonance assay (e.g. ) ; In some embodiments, the KD values are detected by the test example 8 method of the present disclosure;

C. Specifically binds to human BAFF, cynomolgus BAFF and/or murine BAFF; preferably, the binding to human BAFF is performed with a KD value of less than 7.00E-11M (e.g., less than 7.00E-11M, less than 5.00E-11M, less than 4.00E-11M, less than 3.00E-11M, less than 2.00E-11M, less than 1.00E-11M or less), with a KD value of less than 4.00E-10M (e.g., less than 4.00E-10M, less than 3.00E-10M, less than 2.00E-10M, less than 1.30E-10M or less), and/or with a KD value of less than 6.00E-11M (e.g., less than 6.00E-11M, less than 5.00E-11M, less than 4.00E-11M, less than 3.20E-11M or less), as measured by a surface resonance method (e.g., resonance method) ) ; In some embodiments, the KD values are detected by the test example 8 method of the present disclosure;

E. has the activity of blocking the binding of APRIL and BCMA; preferably, the IC ₅₀ value that blocks APRIL binding to BCMA is less than 40.00nM (e.g., less than 40.00nM, less than 35.00nM, less than 30.00nM, less than 20.00nM, less than 10.00nM, less than 5.00nM, less than 2.00nM, less than 0.10nM, less than 0.06nM, less than 0.02nM or less), said IC ₅₀ value being detected by ELISA methods; in some embodiments, the IC ₅₀ value is detected by the test example 4 method of the present disclosure;

G. Has the activity of blocking the binding of BAFF and BCMA; preferably, the IC ₅₀ value that blocks BAFF binding to BCMA is less than 2.00nM (e.g., less than 2.00nM, less than 1.50nM, less than 1.00nM, less than 0.90nM, less than 0.80nM, less than 0.70nM, less than 0.60nM, less than 0.50nM, less than 0.40nM or less), said IC ₅₀ value being detected by ELISA methods; in some embodiments, the IC ₅₀ value is detected by the test example 4 method of the present disclosure;

J. Has the effect of inhibiting IFN gamma, IL-10 and/or IgA cytokine secretion activity; in some embodiments, the IC ₅₀ value is detected by the test example 9 method of the present disclosure;

K. Has the activity of inhibiting B cell proliferation and/or T cell proliferation; in some embodiments, the anti-ICOSL antibody or anti-ICOSL antibody fusion protein inhibits ICOSL activity to induce T cell proliferation; in some embodiments, the inhibition of T cell proliferation activity is detected by the test example 6 method of the present disclosure; in some embodiments, the anti-ICOSL antibody fusion protein inhibits BAFF or APRIL-induced B cell proliferation activity; in some embodiments, the inhibition of B cell proliferation activity is detected by the test example 7 method of the present disclosure.

Exemplary anti-ICOSL antibody fusion proteins

In some embodiments, the anti-ICOSL antibody fusion protein wherein the TACI polypeptide is less susceptible to cleavage than a wild-type TACI polypeptide (having the sequence of SEQ ID NO: 51).

In some embodiments, the anti-ICOSL antibody fusion protein of any one of the above, wherein the TACI polypeptide is as set forth in SEQ ID NO:58 or SEQ ID NO:58 or a truncated fragment of SEQ ID NO:58 or a variant of SEQ ID NO:58, a variant of a truncated fragment of 58; wherein the truncated fragment comprises the sequence of SEQ ID NO:58 from amino acid residue 48 to 85, said variant being the amino acid sequence set forth in SEQ ID NO:58 or a truncated fragment thereof, having one or more amino acid substitutions selected from the group consisting of positions 49, 52, 53, 57, 65, 82 and 83, wherein the amino acid substitutions are at a position relative to the sequence of SEQ ID NO:58, and a naturally occurring numbering of amino acid residue positions.

In some embodiments, the anti-ICOSL antibody fusion protein of any one of the above, wherein the TACI polypeptide, wherein the truncated fragment of the TACI polypeptide comprises: SEQ ID NO:58 amino acid residues 48 to 86; SEQ ID NO:58 amino acid residues 48 to 87; or SEQ ID NO:58 amino acid residues 48 to 88.

In some embodiments, the anti-ICOSL antibody fusion protein of any one of the above, wherein the TACI polypeptide has the sequence of SEQ ID NO:58 variant or SEQ ID NO:58 (e.g., SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62 or SEQ ID NO: 63), which variant is a variant of the sequence set forth in SEQ ID NO:58 or a truncated fragment thereof (e.g., SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62 or SEQ ID NO: 63) has a substitution at any 1,2, 3, 4,5, 6 or 7 amino acids selected from positions 49, 52, 53, 57, 65, 82 and 83 relative to the sequence SEQ ID NO:58, and a naturally occurring numbering of amino acid residue positions.

In some embodiments, the anti-ICOSL antibody fusion protein of any one of the above, wherein the TACI polypeptide has a sequence set forth in SEQ ID NO:60-63, SEQ ID NO: 66-83. In some embodiments, the anti-ICOSL antibody fusion protein of any one of the above, wherein the TACI polypeptide sequence is as set forth in SEQ ID NO: 83.

In some embodiments, the anti-ICOSL antibody fusion protein of any of the above, consisting of 2 identical first chains and 2 identical second chains, wherein,

First chain: the method sequentially comprises the following steps from the N end to the C end: [ heavy chain of anti-ICOSL antibody ] - [ linker 1] - [ TACI polypeptide 1], and

A second chain, which is, in order from the N-terminal to the C-terminal: [ TACI polypeptide 2] - [ linker 2] - [ light chain of ICOSL antibody ]; or a second chain comprising, in order from the N-terminus to the C-terminus, [ light chain of the anti-ICOSL antibody ] - [ linker 2] - [ TACI polypeptide 2]; or a second chain that is the light chain of an anti-ICOSL antibody;

Wherein, the TACI polypeptide 1 and TACI polypeptide 2 may be the same or different, and the linker 1 and linker 2 may be the same or different. In some embodiments, the TACI polypeptide 1 and TACI polypeptide 2 are the same. The linker 1 and linker 2 are independently (G _xS) _y linker, wherein x is an integer from 1 to 5 and y is an integer from 0 to 6 more preferably the linker 1 and linker 2 are independently a linker as shown in SEQ ID NO:89 or 90.

In some embodiments, the anti-ICOSL antibody fusion protein of any one of the above, wherein the anti-ICOSL antibody is an anti-ICOSL antibody of any one of the preceding claims.

In some embodiments, the anti-ICOSL antibody fusion protein of any one of the above, wherein the first strand of the anti-ICOSL antibody fusion protein is as set forth in SEQ ID NO:86 and the second strand is set forth in SEQ ID NO: 88. 87 or 48. In some embodiments, the anti-ICOSL antibody fusion protein of any one of the above, wherein the first strand of the anti-ICOSL antibody fusion protein is as set forth in SEQ ID NO:84, and the second strand is set forth in SEQ ID NO:85 or 46.

Variants of anti-ICOSL antibodies or fusion proteins thereof

In certain embodiments, amino acid sequence variants of the anti-ICOSL antibodies or fusion proteins thereof provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of antibodies. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into, and/or substitutions of residues within the amino acid sequence of an anti-ICOSL antibody or fusion protein thereof. Any combination of deletions, insertions, and substitutions may be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, such as antigen binding properties.

Substitution, insertion, and deletion variants

In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include CDRs and FR. Conservative substitutions are shown in table 3 under the heading of "preferred substitutions". More substantial variations are provided in table 3 under the heading of "exemplary substitutions" and are described further below with reference to the amino acid side chain class. Amino acid substitutions may be introduced into the antibody of interest and the product screened for a desired activity, such as retention/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

TABLE 3 substitution of amino acids

Original residue	Exemplary substitution	Preferred substitution
Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
Asn(N)	Gln；His；Asp,Lys；Arg	Gln
Asp(D)	Glu；Asn	Glu
Cys(C)	Ser；Ala	Ser
Gln(Q)	Asn；Glu	Asn
Glu(E)	Asp；Gln	Asp
Gly(G)	Ala	Ala
His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu; val; met; ala; phe; norleucine (N-leucine)	Leu
Leu(L)	Norleucine; ile; val; met; ala; phe (Phe)	Ile
Lys(K)	Arg；Gln；Asn	Arg
Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Trp；Leu；Val；Ile；Ala；Tyr	Tyr
Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile; leu; met; phe; ala; norleucine (N-leucine)	Leu

Amino acids can be grouped according to common side chain characteristics as follows:

(1) Hydrophobic: norleucine, met, ala, val, leu, ile;

(2) Neutral, hydrophilic: cys, ser, thr, asn, gin;

(3) Acidic: asp, glu;

(4) Alkaline: his, lys, arg;

(5) Residues that affect chain orientation: gly, pro;

(6) Aromatic: trp, tyr, phe.

Non-conservative substitutions may require replacement of a member of one of these classes with a member of another class.

One class of substitution variants involves substitution of one or more CDR residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further investigation will have alterations (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody, and/or will substantially retain certain biological properties of the parent antibody. One exemplary substitution variant is an affinity matured antibody, which can be conveniently produced, for example, using phage display-based affinity maturation techniques (such as those described herein). Briefly, one or more CDR residues are mutated and the variant antibody is displayed on phage and screened for a particular biological activity (e.g., binding affinity). Changes (e.g., substitutions) may be made to the CDRs, for example, to improve antibody affinity. Such changes may be made to CDR "hot spots", i.e., residues encoded by codons that undergo mutations at high frequencies during the somatic maturation process, and/or residues that contact the antigen, while the resulting variant VH or VL is tested for binding affinity. In some embodiments of affinity maturation, diversity is introduced into the variable gene selected for maturation by any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis). Then, a secondary library is created. The library is then screened to identify any antibody variants with the desired affinity. Another approach to introducing diversity involves CDR-directed approaches in which several CDR residues (e.g., 4-6 residues) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, HCDR3 and LCDR3 are often targeted.

In certain embodiments, substitutions, insertions, or deletions may be made within one or more CDRs, provided that such changes do not substantially reduce the ability of the antibody to bind to an antigen. For example, conservative changes (e.g., conservative substitutions, as provided in table 3) may be made to the CDRs that do not substantially reduce binding affinity. Such changes may be, for example, outside of the antigen-contacting residues in the CDRs. In certain embodiments of the variant VH and VL sequences provided above, each CDR is unchanged or contains no more than 1, 2, or 3 amino acid substitutions.

One method that may be used to identify residues or regions in an antibody that may be targeted for mutagenesis is called "alanine scanning mutagenesis". In this method, a residue or group of residues (e.g., charged residues such as Arg, asp, his, lys and Glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., ala or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions may be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. In addition, the contact point between the antibody and the antigen can be identified by studying the crystal structure of the antigen-antibody complex. These contact residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants may be screened to determine whether they contain the desired property.

Amino acid sequence insertions include insertions within the sequence of amino and/or carboxy terminal fusion lengths of polypeptides ranging from 1 residue to 100 or more residues, and single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of antibody molecules include fusions of the N-or C-terminus of an antibody with an enzyme (or a polypeptide that extends the half-life of an antibody).

Modification of the Fc region

In one aspect, the Fc region of an anti-ICOSL antibody or anti-ICOSL antibody fusion protein of the present disclosure comprises one or more amino acid substitutions that reduce its binding to an Fc receptor, e.g., its binding to an fcγ receptor, and reduce or eliminate effector function. A native IgG Fc region, specifically an IgG ₁ Fc region or an IgG ₄ Fc region, may result in the fusion proteins of the present disclosure targeting cells expressing Fc receptors, rather than cells expressing antigens. In some embodiments, the engineered Fc regions of the present disclosure exhibit reduced binding affinity for Fc receptors and/or reduced effector function. In some embodiments, the engineered Fc region has a 50%, 80%, 90%, or more than 95% decrease in binding affinity to Fc receptors as compared to the native Fc region. In some embodiments, the Fc receptor is an fcγ receptor. In some embodiments, the Fc receptor is a human fcγ receptor, e.g., fcγri, fcγriia, fcγriib, fcγriiia. In some embodiments, the engineered Fc region also has reduced binding affinity for complement, such as C1q, compared to the native Fc region. In some embodiments, the engineered Fc region has no reduced binding affinity for neonatal Fc receptor (FcRn) compared to the native Fc region. In some embodiments, the engineered Fc region has reduced effector functions, which may include, but are not limited to, one or more of the following: reduced Complement Dependent Cytotoxicity (CDC), reduced antibody dependent cell mediated cytotoxicity (ADCC), reduced Antibody Dependent Cellular Phagocytosis (ADCP), reduced cytokine secretion, reduced antigen uptake by immunocomplex-mediated antigen presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling-induced apoptosis, Reduced dendritic cell maturation or reduced T cell priming. For the IgG ₁ Fc region, amino acid residue substitutions at positions 238, 265, 269, 270, 297, 327 and 329 etc. may reduce effector function. In some embodiments, the Fc region is a human IgG ₁ Fc region, and the amino acid residues at positions 234 and 235 are a, numbered according to the EU index. For the IgG ₄ Fc region, substitution of the amino acid residue at position 228 et al may reduce effector function.

The anti-ICOSL antibody or anti-ICOSL antibody fusion protein may comprise different binding domains fused to two subunits of the Fc region, thus potentially resulting in undesired homodimerization. To increase yield and purity, it would be advantageous to introduce modifications in the Fc region of the fusion proteins of the present disclosure that promote heterodimerization. In some embodiments, the Fc region of the present disclosure comprises modifications according to the knob-in-hole (KIH) technique that involve the introduction of a raised structure (knob) at the interface of a first subunit and a hole structure (hole) at the interface of a second subunit. So that the protruding structures can be positioned in the pore structure, promoting the formation of heterodimers and inhibiting the production of homodimers. Raised structures are constructed by substituting small amino acid side chains from the interface of the first subunit with larger side chains (e.g., tyrosine or tryptophan). Whereas the pore structure is created in the interface of the second subunit by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). The raised structures and pore structures were prepared by altering the nucleic acid encoding the polypeptide, with optional amino acid substitutions shown in table 4 below:

TABLE 4 KIH mutant combinations

In addition to the knob technique, other techniques for modifying the CH3 domain of the heavy chain of an antibody to achieve heterodimerization are also known in the art, such as WO96/27011、WO98/050431、EP1870459、WO2007/110205、WO 007/147901、WO2009/089004、WO2010/129304、WO2011/90754、WO2011/143545、WO2012/058768、WO2013/157954 and WO 013/096291.

The antigen binding molecule may also comprise disulfide engineering, e.g., the first subunit of the Fc region comprises a 354C mutation and the second subunit comprises a 349C mutation, to create an engineered disulfide bond between the first and second subunits of the Fc region, facilitating heterodimerization of the first and second subunits of the Fc region.

The Fc region of the antigen binding molecule may further incorporate other amino acid modifications, such as mutations in allotype amino acid residues that reduce immunogenicity. In some embodiments, the Fc of IgG1 introduces 356E and 358M mutations.

The C-terminus of the Fc region may be the complete C-terminus ending with the amino acid residue PGK; or may be a shortened C-terminal, e.g. in which one or two C-terminal amino acid residues have been removed. In a preferred aspect, the C-terminus of the heavy chain is a shortened C-terminus ending with PG. Thus, in some embodiments, an intact antibody may comprise an antibody mixture with all K447 residues and/or g446+k447 residues removed. In some embodiments, the intact antibody may comprise a mixture of antibodies that do not remove the K447 residue and/or the g446+k447 residue. In some embodiments, the whole antibody has a mixture of antibodies with and without residues K447 and/or g446+k 447.

Recombination method

The anti-ICOSL antibodies or anti-ICOSL antibody fusion proteins may be produced using recombinant methods. For these methods, one or more isolated nucleic acids encoding an antibody or fusion protein are provided.

In one embodiment, the disclosure provides isolated nucleic acids encoding an anti-ICOSL antibody or an anti-ICOSL antibody fusion protein as described above. Such nucleic acids may be given from the independent encoding of any of the polypeptide chains described previously. In another aspect, the disclosure provides one or more vectors (e.g., expression vectors) comprising such nucleic acids. In another aspect, the disclosure provides host cells comprising such nucleic acids. In one embodiment, a method of making an antibody or fusion protein is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody or fusion protein, as provided above, under conditions suitable for expression, and optionally recovering the anti-ICOSL antibody or anti-ICOSL antibody fusion protein from the host cell (or host cell culture medium).

For recombinant production of an anti-ICOSL antibody or an anti-ICOSL antibody fusion protein, the nucleic acid encoding the protein is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures, or produced by recombinant methods or obtained by chemical synthesis.

Suitable host cells for cloning or expressing vectors encoding anti-ICOSL antibodies or anti-ICOSL antibody fusion proteins include prokaryotic or eukaryotic cells as described herein. For example, it may be produced in bacteria, particularly when glycosylation and Fc effector function are not required. After expression, it can be separated from the bacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding antibodies or fusion proteins, including fungal and yeast strains. Suitable host cells suitable for expression of the antibodies or fusion proteins may also be derived from multicellular organisms (invertebrates and vertebrates); examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified which can be used in combination with insect cells, in particular for transfection of Spodoptera frugiperda (Spodoptera frugiperda) cells; plant cell cultures may also be used as hosts, for example US5959177, US6040498, US6420548, US7125978 and US6417429; vertebrate cells can also be used as hosts, for example mammalian cell lines adapted to grow in suspension. Other examples of suitable mammalian host cell lines are the SV40 transformed monkey kidney CVl line (COS-7); human embryonic kidney lines (293 or 293T cells); baby hamster kidney cells (BHK); mouse sertoli (sertoli) cells (TM 4 cells); monkey kidney cells (CV 1); african green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat (buffalo rat) hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells. Other suitable mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells; and myeloma cell lines, such as Y0, NS0, and Sp2/0. For a review of certain mammalian host cell lines suitable for producing antibodies or fusion proteins see, e.g., yazaki, p. And Wu, a.m., methods in Molecular Biology, vol.248, lo, b.k.c. (ed.), humana Press, totowa, NJ (2004), pages 255-268.

Measurement

The anti-ICOSL antibodies or anti-ICOSL antibody fusion proteins provided herein can be identified, screened, or characterized for physical/chemical characteristics and/or biological activity by a variety of assays known in the art.

In one aspect, the anti-ICOSL antibodies or anti-ICOSL antibody fusion proteins of the present disclosure are tested for activity, e.g., by known methods such as ELISA, western blot, and the like.

Methods of treatment and routes of administration

Any of the anti-ICOSL antibodies or anti-ICOSL antibody fusion proteins provided herein can be used in a method of treatment.

In yet another aspect, the disclosure provides the use of an anti-ICOSL antibody or an anti-ICOSL antibody fusion protein in the manufacture or preparation of a medicament. In some embodiments, the medicament is for treating an autoimmune disease, an inflammatory disease, a B cell disorder, or a T cell disorder, which is a disease associated with ICOSL overexpression.

In some embodiments, the autoimmune or inflammatory disease is selected from: systemic lupus erythematosus, rheumatoid arthritis, graft versus host disease, asthma, immune thrombocytopenic purpura, multiple sclerosis, inflammatory diseases caused by diabetes, psoriasis, inflammatory bowel disease, crohn's disease, ulcerative colitis, graves ' disease, hashimoto's thyroiditis and the like. In some embodiments, the B cell disorder or T cell disorder is a neoplastic disease. In some embodiments, the tumor is selected from: head and neck cancer, non-small cell lung cancer, urothelial cancer, leukemia, sarcoma, melanoma, adenocarcinoma, colorectal cancer, prostate tumor, breast cancer, small cell lung cancer, and the like. In some embodiments, the autoimmune disease is systemic lupus erythematosus. In one such embodiment, the use further comprises administering to the subject an effective amount of at least one additional therapeutic agent (e.g., one, two, three, four, five, or six additional therapeutic agents). The "subject" according to any of the above embodiments may be a human.

In a further aspect, a pharmaceutical composition comprising the anti-ICOSL antibody or anti-ICOSL antibody fusion protein is provided, e.g., for use in any of the above pharmaceutical uses or methods of treatment. In one embodiment, the pharmaceutical composition comprises any of the antibodies or fusion proteins provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition further comprises at least one additional therapeutic agent.

The anti-ICOSL antibodies or anti-ICOSL antibody fusion proteins of the present disclosure may be used alone or in combination with other agents for therapy. For example, an anti-ICOSL antibody or anti-ICOSL antibody fusion protein of the present disclosure may be administered in combination with at least one additional therapeutic agent.

"Co-administration" refers to a method of providing two or more active compounds to a subject simultaneously or sequentially for therapeutic purposes. When reference is made to "co-administration", the time interval between each administration is sufficient to achieve a synergistic effect between the administered active compounds. Two or more active compounds are in the same different containers.

The anti-ICOSL antibodies or anti-ICOSL antibody fusion proteins of the present disclosure (and any additional therapeutic agents) can be administered by any suitable means, including parenterally, intrapulmonary, and intranasally, and intralesionally, if local treatment is desired. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is short-term or long-term. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations at multiple time points, bolus administration and pulse infusion.

The anti-ICOSL antibodies or anti-ICOSL antibody fusion proteins of the present disclosure will be formulated, administered, and administered in a manner consistent with good medical practice (Good Manufacturing Practice). Factors considered in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to the medical practitioner. The polypeptide or fusion protein may be formulated with or without one or more agents currently used to prevent or treat the disorder. The effective amount of such other agents depends on the amount present in the pharmaceutical composition, the type of disorder or treatment, and other factors. These are typically used at the same dosages and routes of administration as described herein, or at about 1 to 99% of the dosages described herein, or at other dosages, and by any route of empirical/clinical determination as appropriate.

The effective amount in particular embodiments may be derived from a dose-response curve from an animal model test system and allows for determination based on the judgment of the physician and the circumstances of each patient. The amount of drug required for a single administration by a subject can be conveniently obtained by calculating the weight of the subject and the unit weight dose required for a single administration. The amount of drug used can be determined by equivalent dose scaling relationships between unit body weight doses of experimental animals and humans (FREIREICH et al 1966,Cancer Chemother Rep 50:219). For example, the guidance opinions may be provided by the drug administration such as FDA and SFDA. In some embodiments, the human and murine dosages (as in test examples 9-11) may be scaled using the body surface area conversion factor for humans and mice.

For the prevention or treatment of a disease, the appropriate dosage of an anti-ICOSL antibody or anti-ICOSL antibody fusion protein of the present disclosure (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of therapeutic molecule, the severity and course of the disease, whether administered for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history and response to the therapeutic molecule, and the discretion of the attending physician. The therapeutic molecule is suitably administered to the patient at one time or over a series of treatments.

Article of manufacture

In another aspect of the disclosure, an article of manufacture (e.g., a kit or kit) is provided that comprises materials useful for treating, preventing, and/or diagnosing the aforementioned disorders. The article comprises a container and a label or package insert (PACKAGE INSERT) on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials such as glass or plastic. The container contains the antibody, alone or in combination with another component, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper). At least one active agent in the container is an anti-ICOSL antibody or an anti-ICOSL antibody fusion protein of the present disclosure. The label or package insert indicates that the antibody or fusion protein thereof is to be used to treat the selected condition.

Further, the article may comprise: (a) A first container having a composition contained therein, wherein the composition comprises an anti-ICOSL antibody or an anti-ICOSL antibody fusion protein of the present disclosure; and (b) a second container having a composition contained therein, wherein the composition comprises an additional cytotoxic agent or other therapeutic agent.

Alternatively, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer. From a commercial and user standpoint, it may further include other materials as desired, including other buffers, diluents, filters, needles and syringes.

It should be understood that the expression "10 to 100" or "at least 90%" for a range of values is a concise manner of writing. Although not every point value (including integers and fractions) within the range is explicitly disclosed herein.

Examples and test examples

The present disclosure is further described below in conjunction with examples and test examples, which are not intended to limit the scope of the present disclosure. The experimental methods of the examples and test examples of the present disclosure, in which specific conditions are not noted, are generally according to conventional conditions, such as an antibody technical laboratory manual of cold spring harbor, a molecular cloning manual; or according to the conditions recommended by the manufacturer of the raw materials or goods. The reagents of specific origin are not noted and are commercially available conventional reagents.

Example 1: preparation of mouse anti-human ICOSL monoclonal antibodies

SJL mice were immunized with human ICOSL protein (Sino Biological, 11559-H08H). And taking blood after 3 times of immunization to measure the titer of the antibody in serum, selecting a mouse with high titer in serum and tendency to have a flat titer to perform spleen cell fusion, paving the fused hybridoma cells in a 96-well cell culture plate, and culturing in a 5% CO ₂ incubator at 37 ℃. Cell culture supernatants were assayed by enzyme-linked immunosorbent assay (ELISA). And (3) amplifying, freezing and preserving the selected positive clones and subcloning for two to three times until obtaining single cell clones. The selected hybridoma clones were further prepared and purified for antibodies using serum-free cell culture. The obtained hybridoma antibodies were tested for binding of the antibodies to the cell surface human ICOSL protein and blocking of the receptor by FACS (see test examples 1 and 3 of the present disclosure), and hybridoma cell lines having good binding activity and blocking activity were selected.

The sequences of the monoclonal hybridoma cell lines mAb201 and mAb259 were obtained. The process is as follows: hybridoma cells in logarithmic growth phase were collected, RNA was extracted with Trizol (Invitrogen, cat # 15596-018), and reverse transcribed into cDNA. The amino acid sequences of antibodies corresponding to the obtained DNA sequences are shown in the following tables 5 and 6 after PCR amplification using cDNA as a template and sequencing by sequencing company:

TABLE 5 variable region sequences of murine anti-ICOSL antibodies

Remarks: the underlined parts of the table represent CDR sequences (confirmed according to the Kabat numbering system).

TABLE 6 antibody CDR sequences

Remarks: the CDRs in the table are identified according to the Kabat numbering system.

The variable region sequences of mAb201 and mAb259 candidate molecules were amplified by PCR to obtain VH/VL sequences, and subjected to homologous recombination with expression vector pHr (fragment with signal peptide and hIgG4/hkappa constant region gene (CH 1-Fc/CL)). Exemplary, the human heavy chain IgG4 constant region sequence is set forth in SEQ ID NO:43, the human light chain kappa constant region sequence is set forth in SEQ ID NO:44, a recombinant chimeric antibody full-length expression plasmid VH-CH1-Fc-pHr/VL-CL-pHr was constructed to obtain chimeric antibodies Ch201 and Ch259 thereof.

Example 2: humanization of murine anti-human ICOSL monoclonal antibodies

By comparing the germline gene databases of the heavy and light chain variable regions of IMGT human antibodies, respectively selecting the germline genes of the heavy and light chain variable regions with high homology as templates, respectively Grafted (grafting) CDRs of the murine antibodies into corresponding human templates to form variable region sequences with the sequences of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and then fusing the variable region sequences with human constant region sequences to obtain the humanized antibody. Humanization of murine antibodies to mAb201 and mAb259 is exemplarily described below, with the CDR amino acid residues of the exemplary antibodies being determined and annotated by the Kabat numbering system.

1. Humanization of murine antibody mAb201

The heavy and light chain variable region germline gene with high homology is selected as a template. The humanized light chain template of murine antibody mAb201 is selected from IGKV2-28 x 01 and IGKJ4 x 01, i.e., the FR1, FR2, FR3, and JK4 region of IGKJ4 x 01 (as FR 4) of human germline light chain IGKV2-28 x 01 are selected as humanized antibody light chain framework regions; the humanized heavy chain template is selected from IGHV1-46 x 01 and IGHJ6 x 01, i.e., the JH6 region (as FR 4) of human germline heavy chain IGHV1-46 x 01 is selected as the humanized antibody heavy chain framework region, FR1, FR2, FR3, and IGHJ6 x 01.

Firstly, the CDR of a murine antibody mAb201 is respectively transplanted into a corresponding humanized template to replace the CDR region of the humanized template; then, amino acid residues at positions 1, 2 and/or 4 (numbering according to the Kabat numbering system) of the light chain variable region of the humanized antibody are mutated, and amino acid residues at positions 1, 24, 69, 71, 73 and/or 78 (numbering according to the Kabat numbering system) of the heavy chain variable region are mutated; furthermore, LCDR2 for the light chain variable region: LVSNRFS (SEQ ID NO: 9) from L to K, to obtain a novel LCDR2: KVSNSRFS (SEQ ID NO: 17); LCDR3 for the light chain variable region: FQSNYLPLT (SEQ ID NO: 10) from L to P, to obtain a novel LCDR3: FQSNYLPPT (SEQ ID NO: 18); HCDR2 for heavy chain variable region: MIHPNGGSTNYNEKFKS (SEQ ID NO: 6) from N to H to obtain a novel HCDR2: MIHPHGGSTNYNEKFKS (SEQ ID NO: 19). The humanized sequence of murine antibody mAb201 is shown below:

Amino acid sequence of hAb201VL1 (Graft) (SEQ ID NO: 20)

Amino acid sequence of hAb201VL2 (Graft+D1A, I2V, M4L) (SEQ ID NO: 21)

Amino acid sequence of hAb201VL3 (Graft+L50K) (SEQ ID NO: 22)

Amino acid sequence of hAb201VL4 (Graft+D1A, I2V, M4L+L50K) (SEQ ID NO: 23)

Amino acid sequence of hAb201VL5 (Graft+L96P) (SEQ ID NO: 24)

Amino acid sequence of hAb201VL6 (Graft+D1A, I2V, M4L+L96P) (SEQ ID NO: 25)

Amino acid sequence of hAb201VL7 (Graft+L50K, L96P) (SEQ ID NO: 26)

Amino acid sequence of hAb201VL8 (Graft+D1A, I2V, M4L+L50K, L96P) (SEQ ID NO: 27)

The amino acid sequence of hAb201VH1 (Graft+Q1E, A24T, M69L, R71V, T73K, V78A) (SEQ ID NO: 28)

Amino acid sequence of hAb201VH2 (Graft+Q1E, R71V, T73K+N53H) (SEQ ID NO: 29)

The amino acid sequence of hAb201VH3 (Graft+Q1E, A24T, M69L, R71V, T73K, V78A+N53H) (SEQ ID NO: 30)

Note that: the sequences mentioned above are, in turn, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the underlined part of the sequence being the CDR sequence (confirmed by the Kabat numbering system) and the remainder being the FR sequence, the italic bold representing the mutation site.

2. Humanization of murine antibody mAb259

The heavy-light chain variable region germline gene with high homology is selected as a template, for example, a humanized light chain template of a murine antibody mAb259 is selected from IGKV4-1 x 01/IGKV1-39 x01 and IGKJ2 x01, namely, a human germline light chain IGKV4-1 x01 or an FR1, FR2, FR3 of IGKV1-39 x01 and a JK4 region (serving as FR 4) of IGKJ2 x01 are selected as a humanized antibody light chain framework region; the humanized heavy chain template is selected from IGHV3-74 x 03 and IGHJ1 x01, i.e., the JH1 region (as FR 4) of human germline heavy chain IGHV3-74 x 03, FR1, FR2, FR3, and IGHJ1 x01 is selected as the humanized antibody heavy chain framework region. Firstly, the CDR of a murine antibody mAb259 is respectively transplanted into a corresponding humanized template to replace the CDR region of the humanized template; amino acid residues 1,2,3, 42, 43, 60 and/or 85 (numbered according to the Kabat numbering system) of the light chain variable region of the humanized antibody are then mutated, and amino acid residues 37, 43 and/or 49 (numbered according to the Kabat numbering system) of the heavy chain variable region are mutated. Furthermore, HCDR2 for heavy chain variable region: YISSSSGKVYADAVKG (SEQ ID NO: 12) from S to D, a new HCDR2 is obtained: YIDSSSGKVYADAVKG (SEQ ID NO: 31). The humanized sequence of murine antibody mAb259 is shown below:

The amino acid sequence of hAb259VL1 (Graft (IGKV 4-1. Mu.01-IGKJ 2. Mu.01)) (SEQ ID NO: 32)

The amino acid sequence of hAb259VL2 (Graft (IGKV 4-1 x 01-IGKJ 2x 01) +D1N, I2T, P43S) (SEQ ID NO: 33)

The amino acid sequence of hAb259VL3 (Graft (IGKV 1-39 x 01-IGKJ2 x 01)) (SEQ ID NO: 34)

The amino acid sequence of hAb259VL4 (Graft (IGKV 1-39 x 01-IGKJ2 x 01+D1N, I2T, A43S, S60D, T85V) (SEQ ID NO: 35)

An amino acid sequence of > hAb259VL5 (Graft (IGKV 1-39. Times.01-IGKJ 2. Times.01+I2T) (SEQ ID NO: 36)

The amino acid sequence of hAb259VL6 (Graft (IGKV 1-39 x 01-IGKJ 2x 01+D1N, I2T, Q3V) (SEQ ID NO: 37)

The amino acid sequence of hAb259VL7 (Graft (IGKV 1-39 x 01-IGKJ2 x 01+D1N, I2T, Q3V, K42Q) (SEQ ID NO: 38)

Amino acid sequence of hAb259VH1 (Graft) (SEQ ID NO: 39)

Amino acid sequence of hAb259VH2 (Graft+V37I, S49A) (SEQ ID NO: 40)

The amino acid sequence of hAb259VH3 (Graft+V37I, K43E, S49A) (SEQ ID NO: 41)

The amino acid sequence of hAb259VH4 (Graft+V37I, K43E, S49A+S52D) (SEQ ID NO: 42)

Note that: the sequence order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein the underlined part is the CDR sequence (confirmed according to the Kabat numbering system) and the rest is the FR sequence, the italic bold indicates the mutation site.

3. Preparation of humanized antibodies

Respectively constructing expression vectors of the antibody light chain and the heavy chain, respectively cross-pairing and combining the humanized antibody light chain and the humanized antibody heavy chain, transfecting 293E cells, and collecting culture supernatant for purification to obtain the humanized full-length antibody. The humanized antibody heavy chain constant region may be selected from the group consisting of the constant regions of IgG1, igG2, igG3, igG4, and variants thereof, and illustratively, human heavy chain IgG4 constant regions (as set forth in SEQ ID NO: 43) are used to fuse with the aforementioned humanized heavy chain variable regions to form the full length heavy chain of the antibody; the humanized antibody light chain constant region may be selected from the group consisting of the constant regions of a human kappa, lambda chain or variant thereof, and exemplified by the use of a human light chain constant region kappa chain (as shown in SEQ ID NO: 44) fused to the aforementioned humanized light chain variable region to form an antibody full length light chain.

Exemplary antibody constant region sequences are as follows:

Amino acid sequence of heavy chain constant region of human IgG4 (SEQ ID NO: 43):

Amino acid sequence of the light chain constant region of the human kappa chain (SEQ ID NO: 44):

Illustratively, the humanized antibody of mAb201 shown in Table 7-1 below was obtained by connecting the carboxy-terminus of the humanized antibody heavy chain variable region of mAb201 to the amino-terminus of the human heavy chain IgG4 constant region (e.g., SEQ ID NO: 43) to form the full-length heavy chain of the antibody, and simultaneously connecting the carboxy-terminus of the humanized antibody light chain variable region to the amino-terminus of the human light chain kappa constant region (e.g., SEQ ID NO: 44) to form the full-length light chain of the antibody:

TABLE 7-1 mAb201 humanized antibodies

Remarks: in the table, for example, 201-H1L1 represents a humanized antibody in which the heavy chain variable region is hAb201VH1 (shown as SEQ ID NO: 28), the light chain variable region is hAb201VL1 (shown as SEQ ID NO: 20), the heavy chain constant region is an IgG4 constant region (shown as SEQ ID NO: 43), and the light chain constant region is a kappa constant region (shown as SEQ ID NO: 44).

In addition, the aforementioned humanized antibody heavy chain variable region carboxy-terminus from mAb259 was linked to the amino-terminus of the human heavy chain IgG4 constant region to form an antibody full-length heavy chain, and the humanized antibody light chain variable region carboxy-terminus was linked to the amino-terminus of the human light chain kappa constant region to form an antibody full-length light chain, to obtain mAb259 humanized antibodies as shown in table 7-2 below:

TABLE 7-2 mAb259 humanized antibodies

Remarks: in the table, for example, 259-H1L1 represents a humanized antibody in which the heavy chain variable region is hAb259VH1 (shown as SEQ ID NO: 39), the light chain variable region is hAb259VL1 (shown as SEQ ID NO: 32), the heavy chain constant region is an IgG4 constant region (shown as SEQ ID NO: 43), and the light chain constant region is a kappa constant region (shown as SEQ ID NO: 44).

Exemplary, the light chain/heavy chain sequences of humanized antibodies 201-H3L3 and 259-H4L7 are shown in Table 8 below:

TABLE 8 light/heavy chain sequences of humanized antibodies

Remarks: in the table, the constant region sequences are italicized and the variable region sequences are underlined.

Binding of the anti-ICOSL antibody to hICOSL-CHOK1 was detected by the test example 1 method of the present disclosure, and the results of the binding assay for blocking the ICOSL binding to ICOS by the anti-ICOSL antibody by the test example 3 method of the present disclosure are shown in tables 9 and 10 below:

TABLE 9 results of anti-ICOSL antibody hICOSL-CHOK1 binding experiments

TABLE 10 results of anti-ICOSL antibody blocking ICOSL binding to ICOS

Example 3: construction of anti-ICOSL antibody fusion proteins

The anti-ICOSL antibody was fused to TACI polypeptide to construct an anti-ICOSL antibody fusion protein. Illustratively, the anti-ICOSL antibody is humanized antibody 259-H4L7 or 201-H3L3. The TACI polypeptide may be any suitable TACI polypeptide, illustratively a TACI polypeptide described in the patent application PCT/CN2022/084256 and its priority 202110348497.6 (title: a novel TACI polypeptide, fusion protein and use; filing date: 2021/3/31) (incorporated herein by reference in its entirety), including the TACI polypeptides shown in Table 11 below:

TABLE 11 amino acid sequence of TACI polypeptides

Illustratively, a TACI polypeptide (e.g., TACI-9-15C) is fused to the C-terminus, N-terminus, or C-terminus of the heavy chain, light chain, or C-terminus of an anti-ICOSL antibody (e.g., 201-H3L3 or 259-H4L 7), and QK is added as a protective amino acid to the C-terminus of the TACI polypeptide at the C-terminus of the heavy chain or light chain of the fusion antibody to prevent cleavage of the amino acid at the TACI terminus by carboxypeptidase. Construction of various anti-ICOSL antibody fusion proteins, wherein the structural schematic diagrams of 259H4L7-T11 and 201H3L3-T11 are shown in fig. 1, the structural schematic diagrams of 201H3L3-T7 and 259H4L7-T7 are shown in fig. 2, the structural schematic diagrams of 259H4L7-T9 are shown in fig. 3, and the anti-ICOSL antibody fusion proteins comprise 2 identical first chains and 2 identical second chains, and are specifically shown as follows:

amino acid sequence of first strand of 201H3L3-T7 (SEQ ID NO: 84)

The amino acid sequence of the second chain of 201H3L3-T7 is identical to that of the light chain of 201-H3L3 (SEQ ID NO: 46).

The amino acid sequence of the first strand of 201H3L3-T11 is identical to the amino acid sequence of the first strand of 201H3L3-T7 (SEQ ID NO: 84);

Amino acid sequence of the second chain of 201H3L3-T11 (SEQ ID NO: 85)

Amino acid sequence of the first strand of >259H4L7-T7 (SEQ ID NO: 86)

The amino acid sequence of the second chain of 259H4L7-T7 is identical to that of the light chain 259-H4L7 (SEQ ID NO: 48).

The amino acid sequence of the 259H4L7-T9 first strand is identical to the amino acid sequence of the 259H4L7-T7 first strand (SEQ ID NO: 86);

the amino acid sequence of the second strand of >259H4L7-T9 (SEQ ID NO: 87)

The amino acid sequence of the 259H4L7-T11 first strand is identical to the amino acid sequence of the 259H4L7-T7 first strand (SEQ ID NO: 86);

Amino acid sequence of the second strand of 259H4L7-T11 (SEQ ID NO: 88)

Note that: of the above sequences, the TACI sequence is underlined singly, the antibody variable region sequence is underlined, the antibody constant region sequence is italicized, and the linker sequence is bold.

The control molecule used in the test examples of the present disclosure was AMG-570 (an antibody fusion protein of an anti-ICOSL antibody fused to a binding BAFF polypeptide) with the following sequence:

Amino acid sequence of heavy chain of AMG-570 (SEQ ID NO: 49)

Amino acid sequence of light chain of AMG-570 (SEQ ID NO: 50)

Remarks: the sequence is underlined singly as variable regions, double underlined as BAFF-binding polypeptides, bold as linked amino acids, and the remainder as constant regions.

Test case

Test example 1: FACS binding experiment

To detect the binding of the test molecule to ICOSL, its binding activity to CHOK1 cells (ATCC, CCL-61) hICOSL-CHOK1 overexpressing human ICOSL was detected by FACS (flow cytometry). hICOSL-CHOK1 cells were digested with 0.25% pancreatin, centrifuged at 300g for 5 min, and the cells resuspended in PBS (source culture, B320) +2% FBS (Biosun, BS-0002-500). mu.L of the cell suspension was added to a 96-well U-shaped plate (Corning, 3795) to give 10 ⁵ cells per well. 50 μl of the gradient diluted sample solution to be tested was added to each well and incubated at 4deg.C for 1 hour in the dark. Plates were washed 2 times with PBS+2% FBS, 100. Mu.L Alexa sheep anti-human IgG (H+L) FITC fluorescent secondary antibody (Thermo, A-11013,1:500 dilution) was added to each well and incubated at 4℃for 1 hour in the absence of light. After washing the plates 2 times, 100 μl of PBS was added to each well to resuspend the cells, the fluorescence values of FITC channels were read with a flow cytometer (BD Biosciences, FACS Canto II, 338962), the binding curves of the test molecules to the cell surface ICOSL were fitted with software, and EC ₅₀ values were calculated. The experimental results are shown in table 12 below.

TABLE 12 binding assay results of anti-ICOSL antibodies and fusion proteins thereof for hICOSL-CHOK1

Sample name	And (3) with hICOSL-CHOK1 Combined with EC ₅₀(nM)
mAb201	0.08912
mAb259	0.08449
Ch201	0.08911
Ch259	0.04269
201-H3L3	0.06944
259-H4L7	0.06620
201H3L3-T7	0.06174
201H3L3-T11	0.07391
259H4L7-T7	0.06625
259H4L7-T9	0.07458
259H4L7-T11	0.1083
AMG-570	0.1766

Experimental results show that the anti-ICOSL antibody constructed by the disclosure and the fusion protein thereof have strong binding activity to hICOSL-CHOK 1.

Test example 2: ELISA binding assay

The binding activity of the test molecule to the BAFF and APRIL proteins was detected by ELISA (coating the test molecule). The specific method comprises the following steps:

the molecules to be tested were diluted to 2. Mu.g/mL with PBS buffer at pH7.4 and added to 96-well ELISA plates (Corning, 3590) at a volume of 100. Mu.L/well and incubated overnight at 4 ℃. After discarding the liquid, each well was blocked by adding 300. Mu.L of 1% Casein (37528, thermo) diluted with PBS and incubated for 2 hours at 37 ℃. After blocking was completed, blocking solution was discarded and plates were washed 3 times with PBST buffer (pH 7.4PBS containing 0.1% tween-20), 100. Mu.L of a gradient diluted human BAFF (ACROBiosystems, BAF-H52D 4) or human APRIL (R & D Systems, 5860-AP-010/CF) solution was added to each well, incubated at 37℃for 1 hour, and plates were washed 3 times with PBST after completion of incubation. For detection of binding to BAFF, 100. Mu.L of anti-his-HRP secondary antibody (Sino biological,105327-MM02T-H,1:2000 dilution) was added per well, for detection of binding to APRIL, 100. Mu.L of anti-HA-HRP (Abcam, ab1190,1:4000 dilution) was added per well and incubated for 1 hour at 37 ℃. After washing the plates 3 times with PBST, 100. Mu.L of TMB chromogenic substrate (KPL, 5120-0077) was added to each well, incubated at room temperature for 10 to 15 minutes, 50. Mu.L of 1M H ₂SO ₄ was added to each well to terminate the reaction, the absorbance at 450nm was read with a microplate reader, the binding curve of the test molecule to the antigen was fitted with software, and the EC ₅₀ value was calculated. The experimental results are shown in tables 13 and 14 below.

TABLE 13 results of binding experiments of anti-ICOSL antibody fusion proteins to APRIL protein

Sample name	To human body APRIL Bonding of EC ₅₀(nM)
201H3L3-T7	1.930
201H3L3-T11	1.803
259H4L7-T7	1.682
259H4L7-T9	2.758
259H4L7-T11	2.278
AMG-570	Not combined with

TABLE 14 results of binding experiments of anti-ICOSL antibody fusion proteins to BAFF protein

Sample name	To human body BAFF Combined with EC ₅₀(nM)
201H3L3-T7	16.14
201H3L3-T11	13.75
259H4L7-T7	18.31
259H4L7-T9	27.53
259H4L7-T11	14.92

Experimental results show that the anti-ICOSL antibody fusion protein constructed by the disclosure has strong binding activity to BAFF and APRIL proteins, and the control molecule AMG-570 has no binding activity to APRIL proteins.

Test example 3: blocking ICOSL binding experiments with ICOS

Blocking activity of the test molecule against ICOSL binding to ICOS was detected by FACS. hICOSL-CHOK1 cells (same as in test example 1) were digested with 0.25% pancreatin, centrifuged at 300g for 5 minutes, and the cells resuspended in PBS (source culture, B320) +2% FBS (Biosun, BS-0002-500). mu.L of the cell suspension was added to a 96-well plate

In the U-shaped plate, the number of cells per well was set to 10 ⁵. The gradient diluted molecules to be tested are added into a 96-well plate, gently mixed and incubated at 4 ℃ for 40 minutes in the dark. Plates were washed 2 times with PBS+2% FBS, 100. Mu.L of 20nM hICOS-rabbit Fc protein (Sino Biological, 10344-H31H) was added to each well and incubated at 4℃for 1 hour in the absence of light. Plates were washed 2 times, 100 μl of goat anti-rabbit IGG FC FITC fluorescent secondary antibody (Invitrogen, a16125,1:500 dilution) was added to each well and incubated at 4 ℃ for 40 min in the absence of light. After washing the plate 2 times, 100. Mu.L of PBS was added to each well to resuspend the cells, and the fluorescence values were read by flow cytometry. The IC ₅₀ values were calculated by fitting a curve inhibiting ligand and receptor binding using software. The experimental results are shown in Table 15 below.

TABLE 15 results of experiments blocking ICOSL binding to ICOS

Sample name	Blocking ICOSL And (3) with ICOS Combined with IC ₅₀(nM)
mAb201	0.08357
mAb259	0.08117
Ch201	0.04860
Ch259	0.04708
201-H3L3	0.06683
259-H4L7	0.06006
201H3L3-T7	0.07379
201H3L3-T11	0.09500
259H4L7-T7	0.08006
259H4L7-T9	0.08572
259H4L7-T11	0.1148
AMG-570	0.1621

Experimental results show that the anti-ICOSL antibody constructed by the disclosure and the fusion protein thereof have stronger blocking activity on the combination of ICOSL and ICOS compared with a control.

Test example 4: blocking binding experiments of BAFF, APRIL and receptor

Blocking activity of anti-ICOSL antibody fusion proteins against BAFF or APRIL binding to its receptor (BAFF/BAFF-R, BAFF/BCMA, BAFF/TACI, APRIL/BCMA, APRIL/TACI) was examined by ELISA. The specific method comprises the following steps:

The receptor protein was diluted to 2. Mu.g/mL with PBS buffer at pH7.4 and added to the 96-well ELISA plate at a volume of 100. Mu.L/well and incubated overnight at 4 ℃. After discarding the liquid, 200. Mu.L of 1% casein blocking solution (Thermo, 37528) was added to each well for blocking and incubated at 37℃for 2 hours. After blocking, the blocking solution was discarded and the plate was washed 3 times with PBST buffer (pH 7.4PBS containing 0.1% tween-20) for use. The immobilized concentration of Biotin (Biotin) labeled ligand protein was mixed with the gradient diluted fusion protein, preincubated at 37℃for 30 minutes, and then added to the blocked ELISA plate, and incubated at 37℃for 1.5 hours. After incubation, plates were washed 3 times with PBST, 100. Mu.L of streptavidin-HRP (Invitrogen, 434323,1:4000 dilution) was added to each well and incubated for 1 hour at 37 ℃. The supernatant was removed, 100. Mu.L of TMB chromogenic substrate (KPL, 5120-0077) was added to each well after 3 washes with PBST, incubated at room temperature for 10 to 15 minutes, 50. Mu.L of 1M H ₂SO ₄ was added to each well to terminate the reaction, the absorbance at 450nm was read with an microplate reader, and a curve inhibiting ligand and receptor binding was fitted with software to calculate IC ₅₀ values. The source information of the ligand proteins used in this test example are as follows: BAFF (Sino biological, 10056-HNCH), APRIL (R & D Systems, 5860-AP-010/CF). The following ：BAFF-R(Sino biological,16079-H02H),BCMA(Sino biological,10620-H02H),TACI(ACROBiosystems,TAI-H5256). experiments were carried out with the following information on the sources of the receptor proteins used in table 16, table 17 and table 18:

TABLE 16 anti-ICOSL antibody fusion protein blocking APRIL binding to its receptor experimental results

TABLE 17 results of anti-ICOSL antibody fusion protein blocking BAFF binding to BCMA

Sample of	Blocking BAFF And (3) with BCMA Combined with IC ₅₀(nM)
201H3L3-T7	0.5154
201H3L3-T11	0.6549
259H4L7-T7	0.8963
259H4L7-T9	0.8871
259H4L7-T11	0.3584
AMG-570	2.051

TABLE 18 anti-ICOSL antibody fusion protein blocking BAFF binding to BAFF-R or TACI assay results

Experimental results indicate that the anti-ICOSL antibody fusion proteins constructed by the present disclosure can effectively block the binding of BAFF or APRIL to its receptor, while the positive control AMG-570 cannot effectively block the binding of APRIL to its receptor.

Test example 5: jurkat-ICOS-NFAT reporter experiments

The inhibitory activity of the test molecule on the ICOSL-ICOS signal pathway is detected by Jurkat-ICOS-NFAT reporter gene experiment. The experimental method is as follows:

CHOK1 cells hICOSL-CHOK1 stably expressing human ICOSL were resuspended and plated in 96 well cell plates at 40. Mu.L 12500 cells per well. The full length ICOS sequence was fused with the intracellular domain of CD3zeta and stably expressed on the surface of Jurkat cells (ATCC, TIB-152) to give Jurkat-NFAT-ICOS-CD3zeta cells. Jurkat-NFAT-ICOS-CD3zeta cells were resuspended and plated in 96-well cell plates containing hICOSL-CHOK1 at 40. Mu.L of 2X 10 ⁵ cells per well. The samples to be tested were diluted in a gradient, 20. Mu.L per well was added to the cell plate and incubated in an incubator at 37℃for 48 hours. The cell culture plate was removed, 50. Mu.L of One-glo luciferase assay (Promega, E6120) was added to each well, and the bioluminescence signal was detected using a microplate reader (PERKINELMER, ENVISION 2105). The fluorescence values were fitted to inhibition curves using software and IC ₅₀ values calculated. The experimental results are shown in Table 19 below.

TABLE 19 experimental results for inhibition of Jurkat-ICOS-NFAT reporter activation

Sample name	Inhibition of Jurkat-ICOS-NFAT Reporter gene activation IC ₅₀(pM)
Ch201	51.52
Ch259	47.37
201-H3L3	45.42
259-H4L7	32.96
201H3L3-T7	47.56
259H4L7-T7	52.40
259H4L7-T9	55.63
259H4L7-T11	64.31
AMG-570	103.9

Experimental results show that the anti-ICOSL antibody constructed by the disclosure and the fusion protein thereof have stronger activity of inhibiting the activation of Jurkat-ICOS-NFAT reporter gene than the control molecule AMG-570.

Test example 6: t cell proliferation assay

The activity of the anti-ICOSL antibody fusion protein in inhibiting ICOSL-induced T cell proliferation was examined by T cell proliferation assays. The experimental method is as follows:

mu.L of 0.5. Mu.g/mL anti-human CD3 antibody (Ebioscience, 16-0037-85) was added to each well of a 96-well plate (Corning, 3599), incubated overnight at 4℃and washed 3 times with PBS (source organism, B320), 100. Mu.L of 5. Mu.g/mL ICOSL protein (R & D, 165-B7-100) solution was added to each well, and incubated for 4 hours at 37 ℃. Human Pan T cell isolation kit (Pan T Cell Isolation Kit, human, miltenyi Biotec, 130-096-535) T cells were isolated from human Peripheral Blood Mononuclear Cells (PBMC) according to the kit instructions and plated in coated 96-well plates at 2X 10 ⁵ cells per well. Samples to be tested were diluted in gradient, added to the cell plates, incubated at 37℃for 48 hours, and 50. Mu.L of CTG (CELL TITER Glo) assay (Promega, G7573) was added to each well and the bioluminescence signal was detected on a microplate reader (Perkinelmer, victor 3). The IC ₅₀ values were calculated by fitting a curve inhibiting T cell proliferation with software. The experimental results are shown in table 20 below:

TABLE 20 experimental results for inhibition of T cell proliferation

Sample name	Inhibition of T Cell proliferation IC ₅₀(nM)
201H3L3-T7	0.6146
201H3L3-T11	0.6429
259H4L7-T7	0.6007
259H4L7-T9	0.3753
259H4L7-T11	0.5890

Experimental results show that the anti-ICOSL antibody fusion protein constructed by the disclosure can effectively inhibit T cell proliferation.

Test example 7: b cell proliferation assay

The activity of the anti-ICOSL antibody fusion protein in inhibiting BAFF and APRIL-induced B cell proliferation was examined by a B cell proliferation assay. The experimental method is as follows:

The spleens of mice were ground, centrifuged at 4℃for 5 minutes to collect the lower cells, washed once with washing solution (PBS+2% FBS+2mM EDTA) and centrifuged, and after removing the supernatant, red blood cell lysate (RBC Lysis Buffer, invitrogen, 00-4333-57) was added, and the mixture was allowed to stand at room temperature for 5 minutes until the red blood cells were completely lysed. The cells were again centrifuged and resuspended for counting. Cell suspensions were sorted using a B cell isolation kit (B Cell Isolation Kit, miltenyi Biotec, 130-090-862), and isolated B cells were resuspended and counted in RPMI 1640 medium (Gibco, 11875119) +10% FBS (Gibco, 10099-141) +50. Mu.M 2-mercaptoethanol (Sigma-Aldrich, M6250) and plated in 96 well cell plates for use. BAFF (R & D Systems, 7537-BF) or APRIL (R & D Systems, 5860-AP) proteins were diluted to a fixed concentration, and the gradient diluted anti-ICOSL antibody fusion protein was added to mix well, preincubated at 37℃for 30 minutes, and then added to 96-well cell plates for culturing in a cell incubator at 37℃for 48 hours. The cell culture plate was removed, 50. Mu. L CELLTITER Glo assay (Promega, G7573) was added to each well, incubated at room temperature for 10 minutes, bioluminescence signals were detected with a microplate reader, inhibition curves were fitted with software to the detection results, and IC ₅₀ values were calculated. The experimental results are shown in tables 21 and 22 below:

TABLE 21 experimental results of inhibition of BAFF-induced B cell proliferation by anti-ICOSL antibody fusion proteins

Sample of	Inhibition of BAFF Induced by B cell proliferation Activity (IC ₅₀，pM)
201H3L3-T7	43.87
201H3L3-T11	24.96
259H4L7-T7	41.42
259H4L7-T9	20.33
259H4L7-T11	17.55

TABLE 22 experimental results of anti-ICOSL antibody fusion protein inhibiting APRIL-induced B cell proliferation

Sample of	Inhibition of APRIL Induced by B cell proliferation Activity (IC ₅₀，pM)
201H3L3-T11	20.70
259H4L7-T9	29.60
259H4L7-T11	34.86
AMG-570	Has no inhibitory activity

Experimental results show that the anti-ICOSL antibody fusion protein constructed by the disclosure can inhibit B cell proliferation induced by BAFF or APRIL, and the positive control molecule AMG-570 has no inhibitory activity on APRIL.

Test example 8: affinity test

A certain amount of sample to be detected is affinity captured by using a biological sensing chip Protein A (GE, 29127556), then a series of concentration gradient antigens flow through the surface of the chip, and a reaction signal is detected in real time by using Biacore (GE, 8K) so as to obtain a binding and dissociation curve. After completion of each cycle of dissociation, the biochip was washed and regenerated with 10mM glycine-HCl solution pH 1.5 (GE, BR-1003-54). Experimental data were fitted in a 1:1 model using BIAevaluation version4.1 software to yield affinity values.

The relevant antigenic proteins used in this test are as follows: human ICOSL (Sino biological, 11559-H08H), human BAFF (Sino biological, 10056-HNCH), human APRIL (R & D Systems, 5860-AP-010/CF), cynomolgus monkey ICOSL (Sino biological, 90800-C08H), cynomolgus monkey BAFF (Kactus, BAF-CM 412), cynomolgus monkey APRIL (Kactus, APR-CM 410B), murine ICOSL (Sino biological, 50190-M08H), murine BAFF (Acro Biosystems, BAF-M521 y), murine APRIL (R & D Systems, 7907-AP/CF). The results of the affinity tests are shown in tables 23 to 30 below:

TABLE 23 affinity assay results for binding of anti-ICOSL antibody fusion proteins to human APRIL

TABLE 24 affinity assay results for binding of anti-ICOSL antibody fusion proteins to monkey APRIL

TABLE 25 affinity assay results for binding of anti-ICOSL antibody fusion proteins to murine APRIL

TABLE 26 affinity experiments for binding of anti-ICOSL antibody fusion proteins to human BAFF

Sample of	ka(1/Ms)	kd(1/s)	KD(M)
201H3L3-T11	2.28E+06	6.47E-05	2.84E-11
259H4L7-T7	8.31E+05	5.63E-05	6.77E-11
259H4L7-T9	1.10E+06	5.28E-05	4.82E-11
259H4L7-T11	1.81E+06	5.53E-05	3.06E-11
AMG-570	1.27E+06	1.54E-04	1.22E-10

TABLE 27 affinity assay results for binding of anti-ICOSL antibody fusion proteins to monkey BAFF

Sample of	ka(1/Ms)	kd(1/s)	KD(M)
201H3L3-T11	5.88E+05	7.60E-05	1.29E-10
259H4L7-T7	1.77E+05	5.93E-05	3.36E-10
259H4L7-T9	2.23E+05	5.41E-05	2.42E-10
259H4L7-T11	3.52E+05	5.86E-05	1.66E-10
AMG-570	2.25E+05	1.76E-04	7.82E-10

TABLE 28 affinity assay results for binding of anti-ICOSL antibody fusion proteins to murine BAFF

Sample of	ka(1/Ms)	kd(1/s)	KD(M)
201H3L3-T11	2.47E+06	7.56E-05	3.06E-11
259H4L7-T7	1.25E+06	6.32E-05	5.04E-11
259H4L7-T9	1.40E+06	5.39E-05	3.86E-11
259H4L7-T11	1.86E+06	5.88E-05	3.16E-11
AMG-570	1.19E+06	1.73E-04	1.45E-10

TABLE 29 affinity assay results for anti-ICOSL antibodies and fusion proteins thereof to human ICOSL

Sample of	ka(1/Ms)	kd(1/s)	KD(M)
Ch201	5.41E+05	1.31E-03	2.42E-09
Ch259	5.90E+05	4.74E-04	8.04E-10
201-H3L3	3.81E+05	9.36E-04	2.46E-09
259-H4L7	2.71E+05	4.44E-04	1.64E-09
201H3L3-T7	4.22E+05	1.25E-03	2.96E-09
201H3L3-T11	4.13E+05	1.23E-03	2.96E-09
259H4L7-T7	3.61E+05	4.87E-04	1.35E-09
259H4L7-T9	3.86E+05	4.92E-04	1.28E-09
259H4L7-T11	3.88E+05	4.62E-04	1.19E-09
AMG-570	3.49E+05	2.00E-03	5.72E-09

TABLE 30 affinity assay results of anti-ICOSL antibody fusion proteins to monkey ICOSL/murine ICOSL binding

Experimental results show that the anti-ICOSL antibody fusion protein constructed by the disclosure has strong affinity to the APRIL of human, monkey and mouse, and the positive control molecule AMG-570 does not bind to the APRIL of human, monkey and mouse. The anti-ICOSL antibody fusion proteins constructed in the present disclosure have better affinity to BAFF in humans, monkeys, and mice than the positive control molecule AMG-570. The anti-ICOSL antibodies and fusion proteins thereof constructed by the present disclosure have good binding activity to both human ICOSL and monkey ICOSL, but have no cross-binding activity to murine ICOSL.

Test example 9: evaluation of in vivo cytokine secretion inhibitory Activity

The human hICOSL-CHOK1 cell and the human BAFF protein are mixed to stimulate the mice, the mice are induced to generate cytokines such as IFNgamma, IL-10 and IgA in vivo, and the in vivo activity of the anti-ICOSL antibody fusion protein is evaluated by detecting the level of the cytokines. The experimental procedure was as follows:

SPF-class female C57BL/6 mice (Zhejiang Vitre laboratory animal technologies Co., ltd.) were randomly divided into 4 groups of 8 animals each, and hICOSL-CHOK1 cells (10 ⁶ cells/mouse) and 1mg/kg human BAFF protein (Sino biological, 10056-HNCH) were injected by intraperitoneal mixing, once daily for four days. Each group (positive control (AMG-570-12 mpk), 259H4L 7-T7.7 mpk,259H4L 7-T11.4 mpk, negative control (PBS)) was intraperitoneally injected with the test sample 1 hour before hICOSL-CHOK1 cells and BAFF protein injection on the first and third days, respectively. On the fifth day, plasma samples from each group of mice were collected and assayed for IFNγ, IL-10 and IgA levels, respectively. AMG-570 was used as positive control, PBS was used as negative control, and AMG-570-12 mpk (mpk, i.e., mg/kg) was administered at the same molar concentration of 259H4L 7-T7.7 mpk and 259H4L 7-T11.4 mpk. The test results of the source ：Mouse IFN-gamma Quantikine ELISA Kit(R&D Systems,MIF00),Mouse IL-10Quantikine ELISA Kit(R&D Systems,M1000B),Mouse IgA ELISA Kit(Abcam,ab157717). of the test kit used in this test example are shown in table 31 below and fig. 4,5, and 6.

TABLE 31 results of anti-ICOSL antibody fusion protein inhibition cytokine secretion experiments

Sample of	Percentage decrease in IFNgamma	Percent IL-10 decline	IgA decrease (%)
Negative control (PBS)	0	0	0
259H4L7-T7	79.10％	28.10％	78.80％
259H4L7-T11	78.70％	18.60％	85.10％
AMG-570	58.40％	4.70％	73.90％

Remarks: the percentages in the table are the percentage decrease in the concentration of ifnγ, IL-10, igA in the plasma of each test group compared to the negative control.

Experimental results show that the anti-ICOSL antibody fusion protein constructed by the method can remarkably inhibit secretion of IFN gamma, IL-10 and IgA, and the inhibition activity is stronger than that of a positive control molecule AMG-570.

Test example 10: mouse in vivo immunosuppression experiments

The activity of the fusion protein in suppressing immune response in vivo was evaluated by an immunosuppression experiment. The experimental procedure was as follows: SPF-class female human ICOSL/human ICOS C57BL/6 transgenic mice (Baiosema Jiangsu Gene Biotechnology Co., ltd.) weighing 15-18g,6 to 8 weeks old. Mice were immunized by emulsion mixing with 1mg/kg KLH (Keyhole Limpet Hemocyanin, hemocyanin) (Sigma, H7017) plus adjuvant (Thermo, 77161) on day 1 and 5mg/kg KLH alone on day 21. KLH is a protein with strong immunogenicity that can elicit a strong immune response in mice and produce antibodies specific for KLH. Dosing was started one day prior to KLH immunization, once every 5 days for a total of 35 days. The doses of AMG-570 15mpk,259H4L7-T7.6 mpk and 319H 4L 7-T11.6 mpk were the same in each group, AMG-570 was used as positive control and PBS was used as negative control. Serum samples from each group of mice were taken weekly and the intensity of fusion protein inhibition of the immune response was assessed by measuring KLH-specific IgG and IgM levels in the mouse serum. The following ：Mouse anti-KLH IgG ELISA Kit(Life diagnostics,KLHG-1),Mouse anti-KLH IgM ELISA Kit(Life Diagnostics,KLHM-1). test results of the detection kit used in the test example are shown in fig. 7 and 8.

The results of the KLH-specific IgG assay showed that all the groups administered were able to significantly inhibit the production of KLH-specific IgG at both the 28 th and 35 th time points, with complete absence of immune responses in the 259H4L7-T7 and 259H4L7-T11 groups, the level of KLH-specific IgG being the same as that of normal mice and greater immunosuppressive activity than AMG-570. The results of KLH-specific IgM detection indicate that AMG-570 has significant inhibitory activity only on days 7-14, while 259H4L7-T7 and 259H4L7-T11 show significant inhibitory activity at all time points, which is significantly stronger than AMG-570.

Test example 11 in vivo pharmacokinetic experiments in rats

In vivo pharmacokinetic testing was performed with SD rats. Male SD rats (Zhejiang Venetuno laboratory animal technologies Co., ltd.) were randomly grouped, 4 animals per group were administered by intravenous injection, 0.2mL of whole blood was collected 5 minutes, 8 hours, 24 hours, 48 hours, 84 hours, 9 days, 10 days, 14 days, 21 days, 28 days before and after administration, without anticoagulation, placed at 4℃for 30 minutes after blood collection, centrifuged at 1000g for 15 minutes, and the supernatant serum was placed in an EP tube and stored at-80 ℃. Serum plasma concentrations were measured by ELISA and the pharmacokinetic parameters and in vivo half-life of the test drug were calculated using Winnolin software.

The in vivo pharmacokinetic experiment results of rats are shown in table 32 below, and the experiment results show that the fusion protein obtained by the method has better stability.

TABLE 32 results of in vivo pharmacokinetic experiments in rats

Claims

An anti-ICOSL antibody fusion protein comprising an anti-ICOSL antibody and a TACI polypeptide;

Wherein the anti-ICOSL antibody comprises a heavy chain variable region and a light chain variable region, wherein,

The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3,

The light chain variable region comprises LCDR1, LCDR2 and LCDR3,

Wherein,

(I) The HCDR1, HCDR2 and HCDR3 of the heavy chain variable region comprise SEQ ID NOs: 42. 3, 39, 40 or 41, and HCDR2 and HCDR3, and

LCDR1, LCDR2 and LCDR3 of the light chain variable region comprise SEQ ID NOs: 38. 4, 32, 33, 34, 35, 36 or 37, LCDR1, LCDR2 and LCDR 3; or (b)

(Ii) The HCDR1, HCDR2 and HCDR3 of the heavy chain variable region comprise SEQ ID NOs: 30. 1, 28 or 29, and HCDR2 and HCDR3, and

LCDR1, LCDR2 and LCDR3 of the light chain variable region comprise SEQ ID NOs: 22. 2, 20, 21, 23, 24, 25, 26 or 27, LCDR1, LCDR2 and LCDR 3.
The anti-ICOSL antibody fusion protein of claim 1, wherein:

(i) The HCDR1 of the heavy chain variable region comprises SEQ ID NO:11, HCDR2 comprises the amino acid sequence of SEQ ID NO:31 or 12, and HCDR3 comprises the amino acid sequence of SEQ ID NO:13, and

LCDR1 of the light chain variable region comprises SEQ ID NO:14, LCDR2 comprises the amino acid sequence of SEQ ID NO:15, and LCDR3 comprises the amino acid sequence of SEQ ID NO:16, an amino acid sequence of seq id no; or (b)

(Ii) The HCDR1 of the heavy chain variable region comprises SEQ ID NO:5, HCDR2 comprises the amino acid sequence of SEQ ID NO:19 or 6, and HCDR3 comprises the amino acid sequence of SEQ ID NO:7, and

LCDR1 of the light chain variable region comprises SEQ ID NO:8, LCDR2 comprises the amino acid sequence of SEQ ID NO:17 or 9, and LCDR3 comprises the amino acid sequence of SEQ ID NO:10 or 18;

Preferably, the method comprises the steps of,

(I) The HCDR1 of the heavy chain variable region comprises SEQ ID NO:11, HCDR2 comprises the amino acid sequence of SEQ ID NO:31, and HCDR3 comprises the amino acid sequence of SEQ ID NO:13, and

LCDR1 of the light chain variable region comprises SEQ ID NO:14, LCDR2 comprises the amino acid sequence of SEQ ID NO:15, and LCDR3 comprises the amino acid sequence of SEQ ID NO:16, an amino acid sequence of seq id no; or (b)

(Ii) The HCDR1 of the heavy chain variable region comprises SEQ ID NO:5, HCDR2 comprises the amino acid sequence of SEQ ID NO:19, and HCDR3 comprises the amino acid sequence of SEQ ID NO:7, and

LCDR1 of the light chain variable region comprises SEQ ID NO:8, LCDR2 comprises the amino acid sequence of SEQ ID NO:17, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 10.
The anti-ICOSL antibody fusion protein of claim 1 or 2, wherein:

(i) The heavy chain variable region comprises a sequence identical to SEQ ID NO: 42. 39, 40 or 41, and said light chain variable region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 38. 32, 33, 34, 35, 36 or 37 has an amino acid sequence having at least 90% sequence identity; or (b)

(Ii) The heavy chain variable region comprises a sequence identical to SEQ ID NO: 30. 28 or 29, and said light chain variable region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 22. 20, 21, 23, 24, 25, 26, or 27 has an amino acid sequence having at least 90% sequence identity; or (b)

(Iii) The heavy chain variable region comprises a sequence identical to SEQ ID NO:3, and the light chain variable region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:4 having an amino acid sequence with at least 90% sequence identity; or (b)

(Iv) The heavy chain variable region comprises a sequence identical to SEQ ID NO:1 and the light chain variable region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2 having at least 90% sequence identity;

preferably, (i) the heavy chain variable region comprises SEQ ID NO:42, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:38, an amino acid sequence of seq id no; or (b)

(Ii) The heavy chain variable region comprises SEQ ID NO:30, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 22.
The anti-ICOSL antibody fusion protein according to any one of claims 1-3, wherein said anti-ICOSL antibody further comprises an antibody heavy chain constant region and a light chain constant region;

Preferably, the heavy chain constant region is selected from human IgG1, igG2, igG3 and IgG4 constant regions and the light chain constant region is selected from human antibody kappa or lambda chain constant regions;

More preferably, the heavy chain constant region comprises SEQ ID NO:43, and the light chain constant region comprises the amino acid sequence of SEQ ID NO:44, and a polypeptide comprising the amino acid sequence of 44.
The anti-ICOSL antibody fusion protein according to any one of claims 1 to 4, wherein,

(I) The heavy chain of the anti-ICOSL antibody comprises a sequence identical to SEQ ID NO:47, and the light chain of said anti-ICOSL antibody comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:48 having an amino acid sequence with at least 90% sequence identity; or (b)

(Ii) The heavy chain of the anti-ICOSL antibody comprises a sequence identical to SEQ ID NO:45 and the light chain of said anti-ICOSL antibody comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:46 having an amino acid sequence with at least 90% sequence identity;

Preferably, the method comprises the steps of,

(I) The heavy chain of the anti-ICOSL antibody comprises SEQ ID NO:47, and the light chain of said anti-ICOSL antibody comprises the amino acid sequence of SEQ ID NO: 48; or (b)

(Ii) The heavy chain of the anti-ICOSL antibody comprises SEQ ID NO:45, and the light chain of the anti-ICOSL antibody comprises the amino acid sequence of SEQ ID NO: 46.
The anti-ICOSL antibody fusion protein according to any one of claims 1-5, wherein said TACI polypeptide is a polypeptide comprising the amino acid sequence of SEQ ID NO:58 from amino acid residue 48 to 85 or a variant thereof; wherein the variant comprises an amino acid substitution at one or more positions selected from positions 49, 52, 53, 57, 65, 82 and 83, said amino acid substitution being at a position relative to the sequence of SEQ ID NO:58, a naturally sequentially numbered amino acid residue position;

preferably, the variant comprises one or more amino acid substitutions selected from the group consisting of 49T or 49R, 52S, 53E or 53Q, 65T or 65A, 82A or 82R, 57E and 83Y, the positions of which are relative to the sequence of SEQ ID NO:58, and a naturally occurring numbering of amino acid residue positions.
The anti-ICOSL antibody fusion protein according to any one of claims 1-6, wherein the TACI polypeptide has an amino acid sequence as set forth in SEQ ID NO:51 to 83;

preferably, the TACI polypeptide has the amino acid sequence as set forth in SEQ ID NO: 83.
The anti-ICOSL antibody fusion protein according to any one of claims 1 to 7, comprising:

(A) First chain: [ heavy chain of anti-ICOSL antibody ] - [ linker 1] - [ TACI polypeptide 1], and

Second chain: [ TACI polypeptide 2] - [ linker 2] - [ light chain of ICOSL antibody ]; or (b)

(B) First chain: [ heavy chain of anti-ICOSL antibody ] - [ linker 1] - [ TACI polypeptide 1], and

Second chain: light chain of anti-ICOSL antibody; or (b)

(C) First chain: [ heavy chain of anti-ICOSL antibody ] - [ linker 1] - [ TACI polypeptide 1], and

Second chain: light chain of [ anti-ICOSL antibody ] - [ linker 2] - [ TACI polypeptide 2];

wherein in (a), (B) or (C), the TACI polypeptide 1 and TACI polypeptide 2 are the same or different, and the linker 1 and linker 2 are the same or different;

Preferably, the linker 1 and linker 2 each independently have the structure (G _xS) _y, wherein x is selected from integers from 1 to 5, y is selected from integers from 0 to 6,

More preferably, each of the linker 1 and linker 2 is independently as set forth in SEQ ID NO:89 or 90;

optionally, in (a), (B) or (C), a protective amino acid is also included at the C-terminus of the first chain;

optionally, in (C), a protective amino acid is also included at the C-terminus of the second strand;

preferably, the protected amino acid is QK.
The anti-ICOSL antibody fusion protein according to any one of claims 1 to 8, wherein,

The anti-ICOSL antibody fusion protein has: comprising SEQ ID NO:86, and a first strand comprising the amino acid sequence of SEQ ID NO: 88. 87 or 48, and a second strand of an amino acid sequence of seq id no; or (b)

The anti-ICOSL antibody fusion protein has: comprising SEQ ID NO:84, and a first strand comprising the amino acid sequence of SEQ ID NO:85 or 46;

Preferably, the anti-ICOSL antibody fusion protein has 2 fragments comprising SEQ ID NO:86, and 2 strands comprising the amino acid sequence of SEQ ID NO:88, and a second strand of an amino acid sequence of seq id no.
An anti-ICOSL antibody comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein,

(I) The HCDR1, HCDR2 and HCDR3 of the heavy chain variable region comprise SEQ ID NOs: 42. 3, 39, 40 or 41, and HCDR2 and HCDR3, and

LCDR1, LCDR2 and LCDR3 of the light chain variable region comprise SEQ ID NOs: 38. 4, 32, 33, 34, 35, 36 or 37, LCDR1, LCDR2 and LCDR 3; or (b)

(Ii) The HCDR1, HCDR2 and HCDR3 of the heavy chain variable region comprise SEQ ID NOs: 30. 1, 28 or 29, and HCDR2 and HCDR3, and

LCDR1, LCDR2 and LCDR3 of the light chain variable region comprise SEQ ID NOs: 22. 2, 20, 21, 23, 24, 25, 26 or 27, LCDR1, LCDR2 and LCDR 3.
The anti-ICOSL antibody of claim 10, wherein:

(i) The HCDR1 of the heavy chain variable region comprises SEQ ID NO:11, HCDR2 comprises the amino acid sequence of SEQ ID NO:31 or 12, and HCDR3 comprises the amino acid sequence of SEQ ID NO:13, and

LCDR1 of the light chain variable region comprises SEQ ID NO:14, LCDR2 comprises the amino acid sequence of SEQ ID NO:15, and LCDR3 comprises the amino acid sequence of SEQ ID NO:16, an amino acid sequence of seq id no; or (b)

(Ii) The HCDR1 of the heavy chain variable region comprises SEQ ID NO:5, HCDR2 comprises the amino acid sequence of SEQ ID NO:19 or 6, and HCDR3 comprises the amino acid sequence of SEQ ID NO:7, and

LCDR1 of the light chain variable region comprises SEQ ID NO:8, LCDR2 comprises the amino acid sequence of SEQ ID NO:17 or 9, and LCDR3 comprises the amino acid sequence of SEQ ID NO:10 or 18;

Preferably, the method comprises the steps of,

(I) The HCDR1 of the heavy chain variable region comprises SEQ ID NO:11, HCDR2 comprises the amino acid sequence of SEQ ID NO:31, and HCDR3 comprises the amino acid sequence of SEQ ID NO:13, and

LCDR1 of the light chain variable region comprises SEQ ID NO:14, LCDR2 comprises the amino acid sequence of SEQ ID NO:15, and LCDR3 comprises the amino acid sequence of SEQ ID NO:16, an amino acid sequence of seq id no; or (b)

(Ii) The HCDR1 of the heavy chain variable region comprises SEQ ID NO:5, HCDR2 comprises the amino acid sequence of SEQ ID NO:19, and HCDR3 comprises the amino acid sequence of SEQ ID NO:7, and

LCDR1 of the light chain variable region comprises SEQ ID NO:8, LCDR2 comprises the amino acid sequence of SEQ ID NO:17, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 10.
The anti-ICOSL antibody according to claim 10 or 11, wherein:

(i) The heavy chain variable region comprises a sequence identical to SEQ ID NO: 42. 39, 40 or 41, and said light chain variable region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 38. 32, 33, 34, 35, 36 or 37 has an amino acid sequence having at least 90% sequence identity; or (b)

(Ii) The heavy chain variable region comprises a sequence identical to SEQ ID NO: 30. 28 or 29, and said light chain variable region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 22. 20, 21, 23, 24, 25, 26, or 27 has an amino acid sequence having at least 90% sequence identity; or (b)

(Iii) The heavy chain variable region comprises a sequence identical to SEQ ID NO:3, and the light chain variable region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:4 having an amino acid sequence with at least 90% sequence identity; or (b)

(Iv) The heavy chain variable region comprises a sequence identical to SEQ ID NO:1 and the light chain variable region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2 having at least 90% sequence identity;

preferably, (i) the heavy chain variable region comprises SEQ ID NO:42, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:38, an amino acid sequence of seq id no; or (b)

(Ii) The heavy chain variable region comprises SEQ ID NO:30, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 22.
The anti-ICOSL antibody according to any one of claims 10 to 12, wherein said anti-ICOSL antibody further comprises an antibody heavy chain constant region and a light chain constant region;

Preferably, the heavy chain constant region is selected from human IgG1, igG2, igG3 and IgG4 constant regions and the light chain constant region is selected from human antibody kappa or lambda chain constant regions;

More preferably, the heavy chain constant region comprises SEQ ID NO:43, and the light chain constant region comprises the amino acid sequence of SEQ ID NO:44, and a polypeptide comprising the amino acid sequence of 44.
The anti-ICOSL antibody according to any one of claims 10 to 13, wherein,

(I) The heavy chain of the anti-ICOSL antibody comprises a sequence identical to SEQ ID NO:47, and the light chain of said anti-ICOSL antibody comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:48 having an amino acid sequence with at least 90% sequence identity; or (b)

(Ii) The heavy chain of the anti-ICOSL antibody comprises a sequence identical to SEQ ID NO:45 and the light chain of said anti-ICOSL antibody comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:46 having an amino acid sequence with at least 90% sequence identity;

Preferably, the method comprises the steps of,

(I) The heavy chain of the anti-ICOSL antibody comprises SEQ ID NO:47, and the light chain of said anti-ICOSL antibody comprises the amino acid sequence of SEQ ID NO: 48; or (b)

(Ii) The heavy chain of the anti-ICOSL antibody comprises SEQ ID NO:45, and the light chain of the anti-ICOSL antibody comprises the amino acid sequence of SEQ ID NO: 46.
The anti-ICOSL antibody fusion protein according to any one of claims 1 to 9 or the anti-ICOSL antibody according to any one of claims 10 to 14, having one or more of the following properties:

A. Specifically binds to human ICOSL and cynomolgus ICOSL, but not to murine ICOSL; preferably, binding to human ICOSL occurs with a KD value of less than 5.00E-09M, and/or binding to cynomolgus ICOSL occurs with a KD value of less than 8.00E-09M, as measured by surface plasmon resonance assay;

B. Specifically binds to human APRIL, cynomolgus APRIL and/or murine APRIL; preferably, binding to human APRIL with a KD value of less than 2.00E-11M, binding to cynomolgus APRIL with a KD value of less than 2.00E-10M, and/or binding to murine APRIL with a KD value of less than 2.00E-11M, as measured by surface plasmon resonance assay;

C. Specifically binds to human BAFF, cynomolgus BAFF and/or murine BAFF; preferably, binding to human BAFF with a KD value of less than 7.00E-11M, binding to cynomolgus BAFF with a KD value of less than 4.00E-10M, and/or binding to murine BAFF with a KD value of less than 6.00E-11M, as measured by surface plasmon resonance assay;

D. Has the activity of blocking ICOSL from binding to ICOS; preferably, the IC ₅₀ value blocking binding of human ICOSL to ICOS is less than 0.16nM, said IC ₅₀ value being detected by FACS method;

E. Has the activity of blocking the binding of APRIL to BCMA; preferably, the IC ₅₀ value blocking APRIL binding to BCMA is less than 40.00nM, said IC ₅₀ value being detected by ELISA;

F. Has the activity of blocking the binding of APRIL to TACI; preferably, the IC ₅₀ value blocking APRIL binding to TACI is less than 40.00nM, said IC ₅₀ value being detected by ELISA;

G. has the activity of blocking the binding of BAFF to BCMA; preferably, the IC ₅₀ value blocking BAFF binding to BCMA is less than 2.00nM, said IC ₅₀ value being detected by ELISA;

H. Has the activity of blocking the binding of BAFF and BAFF-R; preferably, the IC ₅₀ value blocking BAFF binding to BAFF-R is less than 2.00nM and the IC ₅₀ value is detected by ELISA method;

I. Has the activity of blocking the binding of BAFF and TACI; preferably, the IC ₅₀ value blocking BAFF binding to TACI is less than 1.00nM, said IC ₅₀ value being detected by ELISA;

J. inhibiting secretion of a cytokine, preferably, the cytokine is selected from any one or a combination of the following: ifnγ, IL-10 and/or IgA;

K. Inhibit B cell proliferation and/or T cell proliferation.
A pharmaceutical composition comprising:

the anti-ICOSL antibody fusion protein of any one of claims 1 to 9 or 15 or the anti-ICOSL antibody of any one of claims 10 to 15, and

One or more pharmaceutically acceptable carriers, diluents or excipients.
A nucleic acid molecule encoding the anti-ICOSL antibody fusion protein of any one of claims 1 to 9 or 15 or the anti-ICOSL antibody of any one of claims 10 to 15.
A host cell comprising the nucleic acid molecule of claim 17.
A method of treating an autoimmune disease, an inflammatory disease, a B-cell disorder, or a T-cell disorder, the method comprising the step of administering to a subject in need thereof a therapeutically effective amount of the anti-ICOSL antibody fusion protein of any one of claims 1-9 or 15 or the anti-ICOSL antibody of any one of claims 10-15 or the pharmaceutical composition of claim 16;

Preferably, the autoimmune or inflammatory disease is selected from: systemic lupus erythematosus, rheumatoid arthritis, graft versus host disease, asthma, immune thrombocytopenic purpura, multiple sclerosis, inflammatory diseases caused by diabetes, psoriasis, inflammatory bowel disease, crohn's disease, ulcerative colitis, graves' disease and Hashimoto thyroiditis; the B cell disorder or T cell disorder is a tumor;

More preferably, the tumor is selected from: head and neck cancer, non-small cell lung cancer, urothelial cancer, leukemia, sarcoma, melanoma, adenocarcinoma, colorectal cancer, prostate tumor, breast cancer, and small cell lung cancer; the autoimmune disease is systemic lupus erythematosus.